South Alabama Microbiology

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For what I understood V.harveyi can be considered a “cheater” because it encodes genes for the byosinthesis of amphi-enterobactin which is has a high affinity to siderophore that acquires iron formicrobial systems.

It seems that aerE provides  an adventage in the adquisition of iron and also an adventage of a superior growth.

Interesting experiment, it explained very well how the aerobactin works as a inhibitor molecule for V. harveyi and taking in count that this lead to a quantitative investigation the results can be more precise in future experiments.

After reading about FISH I was impressed with the many adventages it has, since it allows the detection of one to three orders of magnitude more bacterial cells in the samples, allows study of the spatial organization of cells and in top of that, cells do not have to be alive.

For what I investigate it seems that is a constant concentration gradient which is obtained by imposing a limited solubility on the compound.

I was also wondering the same thing about the use of icebergs to supply cities with water and it could bring more damage to the environment than it seems. The towing of icebergs can cause a change in water temperatures which could affect the aquatic life of the area creating a chain reaction that would distort the ecosystem of Antarctica.

Taking into account that the alternative of using icebergs as a source of water supply is not very viable due to the alteration that it would cause in the ecosystem, it is possible that these contaminated icebergs can be treated after bioremediation for an emergency case due to Lack of water ?, Is this “fresh water” still drinkable?

I had the same question and I have done some research. It seems that over long periods of time (two years) it can have an impact on the PH levels causing them to decrease. However, taking in count that the waiting time it was not so long it would not make much of a difference. But, I’m still not so sure why they waited two months to do the analysis.

For what I researched the agitation moves the liquid onto the walls of a vessel and it can increase the surface area which would subsequently increase the oxygen concentration allowing the bacteria to grow faster.

Situ bioremediation caught my attention and it seems that there are many methods that can be used. One of them is the biosparging that involves the injection of air under pressure below the water to increase water oxygen concentrations.

yeah that caught my attention too. Foreign organism are forbidden because of the isolates location, meaning that the local organism have not developed natural protection against foreign species.

When they say “we restricted our analyses to their free-living lifestyles using shaking liquid culture for co-incubations”, what kind of specific culture do they mean?.

It is really interesting how this species interact with each other and the fact that V. Fischeri ES114 can use a filtration method of a 10000 MWCO membrane to prevent V. Harveyi from growing.

Can this Iron chelation also affect the amounts of Magnesium if that was the case?

As stated in the introduction, nitrification only have a good performance in the lower oxic zone, this is because one of the main factors that nitrification needs its a good concentration of oxygen and as we go deeper in water this concentrations of oxygens star to decrease making it very difficult to carry out the process in the suboxic and anoxic zones.

(A). the crenarchea cells showed better performance in the suboxic area and very low or non existent performance in the lower oxic and anoxic areas.
(B). BAOB cells and mRNA performed very poorly inall the zones.
(C). YAOB cells in the suboxic zone performed their best during this time but their performance was very low in the lower oxic and anoxic zones. However, mRNA did not have fized performance where it decreased and increased between zones.

Yes, because port activies can provoke a lot of pollution with ships, transport, diesel emision, and loading of products. Also, we can add the risk of a possible oil spill. In conclusion, Mobile bay could have greater pollution due to high activities than other places that are not this active.

Good point, MSM would be a good media to start specially because of what the scientist are looking for which is a bacteria that can tolerate high concentration of salt like the ocean. After, we can exclude those who can’t biodegrade the hydrocarbons in a high salinity enviroment.

MSM media is considered a slective and differential growth media at the same time because it encourages the growth of certainbacteria while decreasing the growth of other kind of bacteria. In the other hand, we have Lurie Broth is a rich mediaum that allows the growth of a different bacteria and it consist of tryptone, yeast and sodium chloride. The reason that they used both its because by having a rich medium it is highly probable that it would grow in that medium but in the MSM there could an exclusion.

I did some investigation about the UltraClean soil DNA kit and I found it that the soil samples are added to a bead beating tube containing beads, lysis solution, bead solution and inhibitor removal solution. The obective is to lyse the microorganisms in the soil by a combination of heat, detergent, and mechanical force against specialized beads.

it is really interesting that seminole was able to use benzene as a carbon and energy source but the situation changed when the bacteria could not dregrade benze at a 4 M NaCl.

I was curious about this topic two and I did some research an catechol can be degradaded by two ways: the meta cleavage pathway or the ortho-cleavage pathway.

I think is whitin the category mentioned, correct me if I’m wrong.

I’m interested in what kind os metabolic reconstruction process is done when using “-omics”.

I look up fluorene and is PAH used to make dyes, plastics and pesticides. Also, one interesting fact that I found is that it has an aromatic odor similar to naphthalene. It is possible that compounds that have similar odors could have similar degrading bacteria, like there could be any correlation?.

interesting that they also have problems identifying the enzymes that govern the initial attack on the PAH degrador as the scientist from the previous paper.

interesting that they are using gas chromatography for the separation of mixtures. we just used column chromatography separation methos which is similar to the gas method, helped me to understand.

Yes, I agree, this article can help us as a guide for our research and give us a perspective about the process in general.

I have the same question about this topic, if I would have to make a guess I will decide that they both are the same.

I was curious about gene transfer so I ended up google in it and it says that is the movement of genetic material between organisms belonging to distinct groups of interbreeding individuals.

I googled Fluoranthene and is a molecule that can be viewed as the fussion of naphthalene and benzene unit connected by the previously said five-membered ring. However, Fluoranthene is a colorless compound.

I did some research and a study says that the Indian Ocean is the second- most polluted in the world which makes it a escenario to do this kind of research.

It is interesting with the results of yeast. Would it have been more efficient to also use yeast with this experiment at least for a comparison?

It makes sense to isolate the PAH-degrading bacteria from that specific location because it they already adapted. So that factor can be ignored.

I find it so interesting on how the PCR technique works. By just going through cycles of different temperatures resulting the the amplification off DNA is very intriguing.

I wonder why they depended on optical density for bacterial growth instead of having it grow on a Petri dish and counting the colonies.

I believe the environmental factors play a huge effect. Even with the previous paper the bacteria that is closest to that region had the best result of being the most effective in PAH degradation. I am assuming that’s the same case for this paper.

I do not understand how this technique relates to the overall goal of degrading the PAHs. Like how is isotope-stabilization a specific characteristic?

Isolating a sample from the electrophoresis gels can help determine morphology through DNA sequencing.

I know we are not supposed to question too much about the details of the technique, but I truly do not understand the treatment of the controls and what is being compared. Or how it contributes to the overall experiment.

I did some research on the neighbor-joining method, because I was curious also. and it does not seem to be the most popular to make phylogenic trees, but it is a quick way to organize a large amount of data. One advantage is that it shows how everything does not evolve at the same rate.

I was curious about the same thing. How is this information will be used to help solve the overall problem? How are they going to take this information and possibly enhance the productivity of the PAH degraders?

It is interesting how there are structural variations of Se(0) particles from different bacteria that are present. I wonder through evolution how that came to be. Like are the systems similar or just the product. Is it due to an ancestor or just environmental pressures over time?

I was curious as to why this bacteria would use this metabolic pathway over others. Why would it use its energy for this in the first place?

The purpose of adding antibiotics to the media to prevent contamination from other strains. The strains they are trying to isolate are resistant to the antibiotic so in theory its the only strain able to produce colonies. 

I believe it is more of a confirmation test. If the bacterial strain is so showing it has the ability to degrade selenium, then there must be a transcriptional pathway that gives the bacteria the tools to degrade. 

I believe your question is answered in the next paragraph. It has the characteristic ability to reduce Se(VI).

Yes this appears to be the case, showing that the cosmic clone pECL1e has a significant effect in Se(VI) degradation. This also covers any unforeseen chemical reactions that might have contaminated this test. 

That what I go from this paragraph. The purpose was to find out how Se(VI) can be degraded and for this bacterial strain it has to live in anaerobic conditions forcing the strain to use Se(VI) to the the sole electron acceptor this in turns needing FRN regulation to become active. 

So the overall result is that the environment greatly dictates the metabolic pathways. It uses “oxygen sensing proteins” in order to to decide which regulatory pathway would be most efficient. So when these proteins do not sense oxygen then that is when the FNR regulator becomes active allowing the bacteria to degrade Se(VI). 

I was reading up on the mobile chemistry plant pollution in Chickasaw and I was wondering is it possible that there are cancer possibilities through the aromatics and chemical waste. I’m asking for a family member.

What specific characteristics in the bacteria are they searching for?

Could the chloroplast-type ferrodoxins be involved the coordination of more than the two iron atoms or is this strictly for this cluster only?

Wouldn’t hydrolyzing the products   make the structures more water soluble, in which help break it down?

If aerobic non-sporulating rhodococci are great contributors to the biodegradation of environmental pollutants, would it be hazardous to introduce more into the environment to help speed up the end of pollution?

Can Rhodococcus be used to degrade other pollutants other than PAH and is there other bacteria that can degrade PAH?

Along with the diluted culture spreads, I was thinking that a concentrated culture could also be spread on a agar plate to compare between the dilute agar plate spreads.

I noticed that the experiment used MSM plates instead of TSA and R2A like we use in our experiments. What exactly are the differences between using MSM plates compared to the others?

If complete degradation of concentrated Fla is the overall goal of the experiment in this section, why not prolong the incubation period? Will it mess up the data?

I’m not quite sure I’m understanding the chart here, but is it basically saying that every relevant match with a percent identity of 86 shows a closer homology to Rhodococcus sp. CMGCZ than the relevant matches that show a  percent identity of 84 and 82?

So I’m assuming that LMW PAH has 2-3 ring structures while HMW PAH has 4-5 ring structures. I find it interesting that even though LMW PAH is more favorable than HMW PAH when it comes to degradation, some bacterial strains degrade HMW more readily than LMW, despite ring structure size.

If Nap, Phe and Fla degradation was inhibited on a Ye medium plate, would it have done the same thing if Nap, Phe and Fla were added on a R2A, TSA or MSM plate? 

I aslo think that marine bacteria are able to degrade PAH more readily because the environment they live in might be a little bit harsher compared to terrestrial bacteria, for example, saltwater compared to a forest. 

I’m also interested in how terrestrial and marine bacteria differentiate between each other when it comes to degrading PAH. If environment plays such a key role in how quickly they degrade it, then we could possibly colonize and utilize them in quicker degradation of harmful substances.

I did not know that PAH affected the surrounding environments and people that live in them to that extent. I think this is very useful in bringing awareness to the situation so ideas can be brainstormed to fix and prevent this problem.

I love how we are able to do these experiments in our lab to see how we could apply this to real world problems. Even though these experiments are a little more in depth than ours, I still enjoy learning and applying techniques to these types of problems.

For the thin layer chromatography, is there a reason why silica gel is used? I’m just curious because i know that you could also use cellulose and aluminum. 

I wonder how the results would’ve differed if the use of a different company was used for each experiment or different purities were used instead. 

Hey Jennifre. I had the same exact question that you stated. Maybe it could be due to some gene silencing or maybe it’s just expensive to keep doing multiple cloning.Not entirely sure though. Just throwing out some suggestions. 

Could the reason why the cis-dihydrodiols of the latter compounds were converted to monohydroxylated compounds due to ring stability and aromaticity? Also, could it be due to activating and deactivating groups that are present in the ting structures or the types of reagents used on them that made them become monohydroxylated?

Isn’t upper pathways how the original compound goes to a intermediate compound that still contains ring structures, but they are unstable and lower pathways are rings that are being cleaved and de-aromatized? 

Is the reason why the data for the location of the genes encoding PAH dioxygenase of A5 wasn’t shown because it was irrelevant to the paper or was it too much to be included or too complicated?

I wonder if obtaining biofilms from different extreme environments would have the same affect as the biofilm taken from the Paoha Island.

I was thinking the same thing Tim. This makes me wonder if there are other chemolithotrophs and cyanobacteria that share a symbiotic relationship to help each other and do the same thing when it comes to breaking down arsenic to use for energy.

I wonder if it had mattered whether they chose from these springs or other springs that are around the area if they all contained dissolved sulfide, ammonia, methane and arsenite.

This is very interesting to me. I wonder if they would have the same results that they obtained from this same location either now or last year and whether different steps could be taken with the new technology they possibly  have now.

I was wondering the same thing Sarah. To me it would make since that it would find an alternative way to survive if exposed to a darker region than what is was previously in. If this is true, then this is a cool way to examine how adaptation to a new environment plays a role in metabolic pathways.

Hey Lameace, I’m curious as well of whether there is more than one clone and whether this experiment was successful or not. Maybe they could specify that there are more (or just one), but because this was the more dominant arsenic cycler, they concentrated on it more for the sake of the paper.

I was wondering the same thing Jessica. If a sample was taken from a lake that contained less salt than the lake they obtained the biofilm from, would there be a drastic change or a slight change. This is very interesting to me.

Could the temperature ranges from the experiments be due to the environment that they came from (the springs being around 45C and the microbials halting activity at around 50C)? 

I agree with your question Jessica. If they already knew that sulfide and natural gases help to drive the reduction to As(V), then why was the experiments incubated with the same electron donors. Maybe they could’ve used a different electron donor to observe the reaction.

If acetate did not give the needed results for the experiment then what did it do? i think that they should include the results of what was observed with the experiment cause I am actually curious as to what happened.

I’ve always associated aromatics with being more good than harmful. I guess because of ‘aroma’ being apart of the name. I knew that their were some that could do some damage, but to the extent that it has caused in The Persian Gulf.

Is bioremediation the only strategy that can efficiently eliminate environmental pollutants?

Is there any particular scientific reason why sediment samples were taken from 12 cm below the surface and seawater samples taken from a depth of 15 cm?

What is a turbidity measurement, and why would you take such a measurement indirectly assess growth curves opposed to something else?

There seems to be a steady increase then a more dramatic decrease.

The isolates are more extensive than what I expected them to be. The phylogenetic tree gives a lot more insight and a better visual.

With this particular source being from 2014, why is this the only method to break down the crude oil?

Is there a cyclic aromatic compound that would be virtually impossible to be decomposed by bacteria?

What is LB agar?

Since the strain was incubated for 48 hours, would substantial growth have no happened within 24 hours? I am assuming that the growth rate is slower.

Phenanthrene concentration had a pretty steady decrease per the graph.

Could this be due to the compound being more easily degradable?

Since the strain was found degrade several other aromatic compounds, is this indicative of phenanthrene being more stable than naphthalene?

Because of the ability to degrade more than one compound would phenanthrene be a better aromatic to use concerning the issue of oil contamination in the gulf?

What is the possible reasoning for being unable to obtain authentic PCR results?

I was absolutely unaware of this side of arsenic. It has always been viewed as a fatally toxic compound.

Since no clones matched any of that in the database then what does that mean? Were new species possibly discovered?

Why weren’t the single clones included in the analysis? By doing that would the results still be reliable?

Is the RFLP type percentage more important than the identity percentage in determining the representative clone?

Why would the rate be less in the dark?

The PHS-1 Strain was consistent with the Arsenite range when it comes to oxidation.

It is extremely interesting to learn the different operations of both arsenite and arsenate, and their respective processes in light and dark.

My species from 314 was Burkholderia (Paraburkholderia, specifically). When I was researching it, what I found concerning PAH degradation only spoke of toluene. It interesting to read about the study of another of its degraders.

The fact that this strain was the first fluoranthene-degrading bacterium to be found with the family of Rhodobacteraceae just goes to show how much of the world had yet to be researched, studied, and discovered.

Are all PAHs about the same in regards to being harmful to the environment or are there some more detrimental than others?

I wonder why the mechanism of PAH degradation has so much unknown about it. Is it really more in depth than the basic/well known chemical reactions?

What is a conjugal donor strain and why was E. coli used?

Was 28 degrees Celsisus the ASM temperature?

Why does the entire genome need to be described?

This must mean that the P73 strain is soluble in water because of the lipids it contains, correct?

How exactly could the P73 strain using gene transfer enhance its PAH degradation ability.

Would the presence of glucose-6-phosphate have made a difference in results?

Could strain B30 also have been acquired via horizontal gene transfer?

What is the first discovered PAH? I’m sure it has been widely studied. Would that not be a good basis for studying the physiology of not just fluoranthene, but other PAHs as well?

Since in situ is low cost can it not be used as a treatment for PAHs?

Does ryegrass contain some type of chemical that makes it helpful in biodegradation?

What does the methylene chloride do in the liquid-liquid extraction?

Why was MSM supplemented with glucose used as a blank control? Or rather,why wouldn’t MSM by itself be sufficient as a blank control?

What could have caused the decrease from 24-48 hrs? Is that just a naturally occurring circumstance in this specific gene?

Planted vs unplanted soil? Is this soil that grew plants and soil that did not?

Since xylE and ahdA1b-1 had consistent results to each other when promoted by Cu(II), would this mean that both genes have similar phylogeny or genetic makeup?

So would planting more plants in rhizospheric soils be a solution to lowering the level of PAH concentration in the environment?

So fluorene degrades differently than the other PAHs?

Why is there not a lot of data concerning fluorene and gram-negative bacteria? Does that mean gram-positive is more associated with this particular PAH?

What does the phosphate buffer do?

Why did the extracts need to be dissolved in acetonitrile? Why couldn’t the dried extracts themselves be used as they are?

What does it mean that no amplification could be obtained? The PCR did not work?

I see that they used a set of 15 primers. So you are not limited to just two primers? More than two can be used at a time?

Is the reason that phenanthrene cannot be used as the sole carbon source because when the strain is in the presence of another substrate it uses up that substrate first? I may be completely off with my reasoning here.

Even though it was weakly detected, doesn’t that still mean that the strain was able to used glucose? Or would you just ignore that data because it wasn’t substantial enough?

The other papers never mentioned a half-life, if I recall correctly. Does this make carbofuran significantly different or do all PAHs have similar half-lives?

Mineralization concerning PAHs hasn’t been discussed previously in the other papers. Does this make carbofuran significantly different as well?

To make sure I am understanding each step correctly- the process discussed in this paragraph was the initial step for the mutagenesis, correct?

Here is where the random mutagenesis process was completed and they moved on to RT-PCR, correct?

Being that the pink/light red color was more intense in the medium without additional nitrogen source, this basically means that when carbofuran is able to take up the nitrogen is changes in color similar to other PAHs turning blue/black?

“The selected mutants were tentatively assigned to five different groups” So basically, one genus but 5 different species of that genus?

Being that isolate sp. BAL3 was not conserved should it just be ruled out as insignificant?

If mutations affected protein expression of other genes does that mean the experiment needs to be start over?

What is the problem with categorizing individual disrupted genes by phenotypic affects?

The formation of carbofuran phenol begins the first step in degradation. Why would the mutants show no effects?

Is phenanthrene more or less easily degradable than naphthalene?

Is phenanthrene located in oil-contaminated soil? If we wanted to pull samples from the gulf like it was done for naphthalene could we do that?

I had this same question upon reading this section. Another possibility is a novel gain-of-function mutation, with analogous protein function to other extant PAH pathways in terrestrial bacteria. I also wonder if the ‘acquisition’ could be an activation of a pathway that may be just inactive in other marine bacteria? It could mean a lot of things, and makes me want to read the cited paper.

It seems like there are going to be a lot of results to read, as indicated by this statement. I am most interested to see a comparison of similarities and dissimilarities between the C. indicus PAH degradation pathway and those characterized terrestrial bacteria. Do they involve protein homologs? Do they produce the same intermediates and ultimate products? Are the products of the degradation waste products, or metabolites, or otherwise useful molecules for the organism? What is the energy investment into the degradation? Will the organism maintain the function of the pathway under stress? I hope to see the answers to some of these questions in the results.

Hi Annie,
From what we know about PAH degradation so far, I think  it requires oxygen, because the enzyme RDH needs molecular O2 to break the aromaticity of one of the rings and start the process. There may be another enzyme that can do the same thing without O2 though, that would be interesting to learn about for the sake of comparison.

I don’t want this to count for one of my comments, but how cool would it be to get to go diving or in a submersible to collect sediment from the ocean floor for your project? That’s peak biology right there.

Does the term ‘metabolites’ refer to the products of PAH degradation only, or to intermediates in the process also? Is this technically an observational portion to the study since the strain they were growing and analyzing was all under the same conditions? 

What is the goal of this knockout? I feel like I am missing some information – is P73 a protein involved in RHD? So the knockout mutant would be unable to metabolize PAHs? It seems like this would be a lethal mutation, for a bacterium that can no longer utilize its sole source of carbon and energy.

It definitely makes you wonder what kind of acquisition it was, of the three types we discussed in class. 

So, in addition to the PAH degradation pathway, P73 can also metabolize many different sugars, but cannot initiate gluconeogenesis? I wonder what environmental factors would select for a bacteria with such a versatile ‘palate’ for carbon and energy sources? 

A quick search revealed that Acetobacter aceti is a terrestrial organism, frequently sought out and used for its production of acetic acid from alcohol in fermentation of foods and wines. Makes me wonder how a gene that originated in this A. aceti ended up in sediment in the bottom of the Indian ocean!

This is the authors’ most interesting finding. A gene coding for a new protein that can initiate the ring hydroxylating dioxygenase step of the degradation pathway for toluene and biphenyl PAHs. As they state in the next paragraph, a marine organism that could degrade toluene would be a great tool for the cleanup of oil spills, since toluene is a toxic chemical that occurs naturally in crude oil. 

BAD34447: large subunit of PAH-dioxygenase
CAG17576: ring-hydroxylating dioxygenase alpha subunit
ABW37061: Rieske domain; a cluster binding domain commonly found in Rieske non-heme iron oxygenase (RO) systems such as naphthalene and biphenyl dioxygenases
ABM11369: ring hydroxylating dioxygenase, alpha subunit
ABK27720: putative ring-hydroxylating dioxygenase large subunit
BAA21728: terminal dioxygenase component of carbazole 1,9a-dioxygenase
ABM11377: ring hydroxylating dioxygenase, alpha subunit
ABM11383: Rieske (2Fe-2S) domain protein
ABV68886: angular dioxygenase from fluorene-degrading Sphingomonas sp. strain
AF474963: Pseudomonas alcaligenes indole oxygenase-like gene, complete sequence; and putative dehydrogenase gene, partial cds
 
 

One question I could see the researchers pursuing in future studies is the control of the PAH degradation genes. Since P73 conceivably has the capacity to use multiple different carbon and energy sources, it would be logical it has some epigenetic capabilities to preferentially produce proteins to metabolize whatever substrate it was exposed to or growing on, right?

I understand that the mutants that didn’t produce a clear zone were chosen because it meant the mutagenesis process succeeded in mutating the gene. But why did they test it further with indigo if they already had what they wanted?

Why are they sequencing the plasmid at this point instead of transforming it into a host?

What factors helped determine that they should use a cosmid in stead of say a regular plasmid?

They concluded that the arhA gene was constitutively expressed because the the A4DR cells had low levels of arhA3 and arhA1 even when not induced. Could this be corrected by some of the methods we talked about in class like changing the inducer?

[These observations suggested that the genes involved in the initial oxygenation of acenaphthene did not function in strains AG2-45, AG2-48 and AG3-15.]

I get that a mutagenesis was performed and the strains were grown in different environments to determine whether they degrade acenaphthene. I don’t understand how the researchers cam to this conclusion.

I haven’t read about finding the regulator site in any of the papers we’ve read so far. How  can knowing where the regulator binds be useful in further studies?

Could the OFRs that have no apparent connection to acenaphthene degradation have an unknown role in the cell? An experiment could be designed to knock out one to see if there is a change.

The scattered nature of the the sphingomonad’s genome seems to be an obstacle in identifying genes. What could be a possible explanation for its gene dispersal?

Does the mechanism for breaking down fluoranthene work for all the other types of similar PAHs? Wouldn’t they be different for every compound?

Is there a reason why high-molecular-weight PAHs aren’t studied as much?

How do they know if they have the right metabolite?

In the supplemental text, they found a lot of genes but I’m having a hard time relating them back to the initial goal of the experiment. Can you explain?

Here they are identifying the PAH ring-hydroxylating genes.

Oddly, these genes are in the same place. It hasn’t been this way with many of the other genes in the papers we read before. This is a guess, but I suppose different genus’s store their DNA differently than others.

Is there a reason for why their levels vary?

Does this mean new genes for the degradation of the PAH were found?

In comparison to the other studies, it seems like the information found in this part didn’t go as far as the other papers. In other papers this sort of information would be used to physically test if the conclusion was true or not.

Is computational genetics a common way of discovering the function of genes? Did the information used in the other papers rely on this kind of work to point the researchers in the right direction?

I want to know what separates these bacteria from other bacteria. What allows them to be able to degrade naphalene while others cannot.

This is a good point. The paper states that the pollutants are conversed to biomass, carbon dioxide and water. Which I guess either way you look at it, the remaining end products are less toxic or non toxic in comparison to the original pollutant.

I was thinking the same thing. Could these Naphthalene-degrading bacteria be used for clean-up of the BP oil spill in the Gulf?

Did they want to slow down the activity of their samples to make sure that the results they got were due strictly to experimental variables, and not the environmental changes while being transported?

Why did they choose to get sediment samples from 1-12 cm? Is this the depth where most of the oil had settled in the sediment? And why are the seawater samples taken from 15 cm? How big of a difference does it make in the results if these samples were chosen from different depths?

I was curious about this too. You would think they would be closer in range. Thank you for the clarification. It is true that we are all similar, but all so uniquely different.

I was interested in the GC-FID method, because I wasn’t sure what this was, so I researched it. I read that it was developed for analysis of oleic acid and fatty acids. What do these have in relation to nephthalene-degradation?

I am interested to in how the gram-negative can tolerate PAH better than gram-positive. I’m assuming it has to do with the peptidoglycan layers of the gram-negative.

Is the isolation of the naphthalene-degrading bacteria process similar to that which we performed in lab. Just test using different agars that they may possibly grow in until they get pure cultures?

What is the difference between substitutes and unsubstituted PAH’s?

Since this paper is saying that marine bacteria may be more significant in degrading PAH’s than terrestrial bacteria…. does this mean terrestrial degraders are limited only to land and vice versa, or are there some bacterial PAH degraders that are present in both marine and terrestrial environments?

The genomic library is used to represent all of the genomic DNA from an organism. This is done through DNA extraction in which the DNA is then cut to certain sizes. They are then inserted into vectors which are taken up by hosts. These hosts can then be used to amplify. The genomic library is used for sequencing genomes.

I looked up what agarose gel electrophoresis was, and I read that once a DNA molecule is digested, this method can then be used to see the fragments, because the electrical current moves the molecules across the agar.

Why are homologs for oxygenase subunits located in pairs usually? And what do you think the reasoning for the separation/absence is here in phnA1b?

A biotransformation is just a change or alteration, correct? What is the goal for biotransforming the substrates?

** My second comment is in supposed to be posted on paragraph 18 **

Today in lab, when you drew the picture and explained how the clones can be an “overlap” of a region really helped clarify the idea of having the same 10.5-kb Sau3AI fragment.

I think the significance of not finding the gene clusters for PAH degradation would be that the organisms lacking the gene cluster could not degrade PAH’s.

What is the importance of the major cluster? I get that it shows divergence of a-subunit sequences and similarity of amino acid sequences, but what exactly is meant by the major cluster?

I think that it would be better conservation to have one pair that can partner with many different dioxygenases.

I was researching this topic and read that arsenate is toxic because it can uncouple glycolysis, is this true?

I was wondering the same thing, especially since they included “surprisingly”. I mean they did have aerobic As(III) oxidation activity, so why would there be no authentic PCR products?

Why did they choose sites with differences in temperatures?

Aren’t cyanobateria a type of photosynthetic bacteria? If that is the case are only photosynthetic bacteria the only type of bacteria of interest when it comes to arsenic use?

I researched this after reading your question. DNA libraries are made from fragments of DNA. Where cloned libraries are made from cloned, reverse transcriptase of mRNA. So cloned libraries do not actually consist of DNA sequences.

Going through this figure today, it really helped see what was actually going on in the figure. While the Arsenite is in light it is being oxidized, then when it is in the dark, it is being reduced.

Even though we went over this figure in class, I still don’t understand why they used the three different variables of 100% H2, 2mM sulfide, and no added electron donors. What are each of these three variables offering us? I understand that they are driving forces for reduction, so is it just offering us information on which is the greater of the three for Arsenate reduction?

I was wondering the same thing, because here it doesn’t really help us to get a clear conclusion on specific microbes. But I guess that could allow for a follow up experiment using the techniques we will discuss on Tuesday.

In response to Rhyann’s comment… Is it because photoheterotrophy was responsible for the oxidation, and acetate was the electron donor for photosynthesis?

In my opinion this is the most interesting paper we have done this semester. I enjoy reading about the links made between our anoxic Earth vs. oxic. And it now makes sense that more recent biological communities would share relation to As(III) oxidation and As(V) reduction.

In reference to the deoxygenation and monooxygenation in this paragraph, is this what you were showing us an example of last Friday?

They can determine that the ring hydroxylating dioxygenase is the initiator by doing the same processes we are doing in class this semester?

So what is the point it “knocking out” the PAH degradation gene? It is just to make sure you are pin-pointing the correct genes involved in PAH degradation?

That sounds correct Daniel, because in the last sentence it says that the metabolites were determined using the GC-MS.

These are very extensive figures. How do you even begin reading maps like these? Or are they strictly read by computers?

I didn’t even initially think about genes being horizontally transferred to increase PAH degradation.

This is an important finding since it correlates with aromatic compound uptake.

The COG numbers ranged from 134-3. When it mentions poorly characterized, which is it referring to?

Is there normally a correlation between multiple antibiotic resistance regulators and PAH degradation pathways. I don’t remember reading this in our papers from last semester, but then I also wasn’t sure if I over read it.

They found 6 ring hydroxylating dioxygenase genes. Just because these code for this, does that mean they all actually assist in PAH degradation?

What kind of further studies could they do to study the functions and mechanisms of the P73T strain?

What is meta cleavage? and what is protocatechuate?

In PAH degraders, is it more common for the PAH catabolic genes to be arranged in discrete or dispersed throughout the genome? I’ve never really thought about which arrangement is more common/likely.

Is the topic of interest here, why PAH dioxygenases can oxidize fluorine, but not use it as a sole carbon source? Or will we discover that along with determining locations of PAH degrading genes?

Thank you for sharing this Molly. Background information is always a plus. Dr. Ni Chadhain, what is GSL and LPS? I remember hearing you mention LPS in class today or Monday, but I can’t remember what you were referring to.

The southern blotting made much more sense when you went over it today in class. I did not quite understand about wanting two different sticky ends while also containing the RHD probe, but you clarified this concept.

So, from what you were saying in class today, this part of the experiment is basically just to tell us yes you can force the E. coli to make the protein, or no you cannot?

Is the answer that cells use pyruvate during glycolysis to produce acetyl CoA?

This is what you were discussing with us in class today, that sphingomonas have some genes from gram positives.

What is meant by angular attack mean? It is pertaining to stereospecifity?

The southern blot technique made more sense after you explained it in class, as well as the “walking” done in designed primers.

Are there different types of dioxygenase genes? Like does each PAH degrader have different types of dioxygenase genes or are all dioxygenase genes the same because they do the same job?

So are you saying that even though the RHD is present, it doesn’t mean that it will utilize the PAH as an energy source? Does this mean that the degrader will use whatever route is most catabolically favorable?

When they mention the band extraction are they referring to agarose gel electrophoresis or southern hybridization?

The glass powder technique is pretty interesting.

How do you choose which strain of E.coli you want to perform a transformation with? Is it just based on availability or are there certain strains that work better than others?

Thank you for the information on the shotgun cloning Bentley. As I was reading that was my first question.

Homework 4 helped with understanding the information in Table 1. I understand what the table is actually talking about after performing our own BLASTs and listing our ORFs.

What is the importance of retention time in gas chromatography in comparison to expression of dioxygenase activity?

Can you better explain this sentence please? I’m not understanding how they can serve as substrates, but cannot grow on them. “Although strain A4 is not able to grow on naphthalene, phenanthrene, anthracene, and fluoranthene, these substrates could serve as substrates for the ring-hydroxylating dioxygenase system from this strain, since their corresponding cis-dihydrodiol products were detected in the resting cell reaction experiments.”

I’m surprised that monitoring PAH-dioxygenases in sphingomonads isn’t possible, because I feel like the species came up a lot in the BLASTs we did for homework 4, so it seems to be a well studied species.

I know that xenobiotic are something that is foreign to the body or environment. What is an example of a bodily xenobiotic? Are xenobiotics hard to degrade? I’m assuming degrading capabilities are based on what the foreign substance is.

When finding gene clusters of PAH  degrading genes, how does this work if they are scattered out? Do they all have the same sequences, so when using certain primers, they all show up? Or does the DNA have to be cut multiple times in order to find every cluster throughout the genome?

I would think so, because if you find the genes involved first, you can then focus on the pathways they carryout.

I tried to look at the primers they used listed in Table 2, but it sent me to an online journal site, and I could not see the table because it only shows a preview of the article.

I think that transposon mutagenesis is really interesting, and it is probably my favorite part of what we have learned all semester. And I was really interested in our plasposon mutagenesis experiment in class, even though it did not work like we hoped.

Why did they have double crossover recombinants? And what exactly does that mean?

Why do some strains produce little indigo formations such as strain A4-PCM1(pBBadA13), and other like A4-PCM1(pBBadA14) produce it significantly?  What causes this difference? are they metabolically utilizing other things available in the agar first?

I’m glad that were are getting to perform RT-PCR in our own class experiments. I read on the ThermoFisher website that RT-PCR is so powerful that it can “enable quantitation of RNA from a single cell”. 

Why are they saying that the loose operon structure seems inefficient?

How can they find the genes responsible for further degradation of acenaphthene if they are dispersed throughout the genome? How do you even determine which step is next in the pathway?

This could be a stupid question, but it has me wondering. Since this is concerning oil in the Persian Gulf, it makes me curious after the oil spill in the Gulf of Mexico. Could these Naphthalene-degrading bacteria be found in in the Gulf of Mexico?

Why did the researchers choose this particular oil spill? Is it a special type of crude oil, or did the environment in which it is found play a role in the selection?

Why did the researchers decide not to add an IR Spectrum to the analytical chemistry? It would help further narrow down any isomers that could be found in the tested substance.

Is the purpose of this research to identify PAH degraders or is the purpose to explore PAH degraders and their effectiveness in bioremediation of the surrounding environment?

Could PAH degraders be under-researched because of the lack of equipment or resources available for such exploration? 2000m is a long way down and we definitely cant send a human down there.

What measures did they take to ensure that the extremely low temperature did not kill the bacteria that was isolated?

Later in the document E. Coli is used as a standard for unmarked DNA, does the first sample site act as the standard for the PAH degraders that are pursued in the second sample, or does this experiment assume PAH degraders are found dispersed throughout the sea floor?

Where does this Se(VI) and Se(IV) at high valence states come from?  Is it waste from natural processes or is it pollution related?

Is there an aerobic component to E. cloacae? Is this bacteria an anaerobe due to high competition of resources found in the ocean?

I believe that a transcriptomic/proteomic approach would be useful, assuming that the researcher already knows the sequence that they will be looking for. On the off chance that their bacteria has a new sequence that has not been found, then the screen would surpass the gene and mis identify the bacterium. 

With the fnr gene knocked out of E. Coli would this cause a completely anaerobic metabolism for E. Coli causing it to only use the Se as a source of carbon?

There is no further mention of the ogt gene in the rest of the results section so is it safe to assume that this gene played little to no role in the reduction of Se(VI)?

Based on this section E. Coli appears to naturally reduce Se(VI), but at a lower or less efficient rate than E. cloacae. Would this be the reason they did not try to incorporate the fnr gene from E. cloacae to E. Coli to prove that the fnr gene is responsible for the reduction of Se(VI)?

For E. cloacae to have a real world use in the reduction of Se(VI) that could reach toxic levels in an aquatic community, would there need to be a mutation of the fnr gene where Se(VI) is reduced in the presence and absence of O2 or would the rate of reduction be significant enough with out it?

Would these proteins be a way to make E. cloacae a suitable candidate for real world applications where a Se(VI) reduction capable bacteria would be needed for biosynthesis? Could these proteins be transplanted into the genome horizontally or would they have to be transplanted virally?

I am interested in the amount of arsenic V that was reduced to arsenic III. 

Wouldn’t this be a good opportunity for the authors to employ transcriptomics? What would be the benefits of employing the experimentation in the lab versus out in the field.

It is quite amazing how versatile rhodococci is and how it is able to degrade environmental pollutants that are too difficult to degrade for other organisms. While the rhodococci can metabolize xenobiotic, and halogenated compounds, which can be found in various herbicides. Is it possible for the rhodococci to metabolize other sorts of controlled substances such as pesticides or possibly fertilizers?

It is very interesting to learn that during degradation Rhodococcus uses aromatic rings that catalyze the initial reaction of bacterial degradation. During this degradation it uses two subunits, one of which is the alpha subunit that aids in electron transfer to the dioxygen molecule. But the other subunit, the small beta, was not explained further. Does this subunit have a purpose similar to the alpha unit? Or does it perform it’s own necessary task in the degradation process?

I think it is really interesting that the sample chosen was from an oil-contaminated sample in Japan. The fact that the oil could impact the growth of the culture compared to if the soil from that area was not contaminated. It makes me wonder what kind of oil was present in the soil and if it was contaminated with a different kind how would the affects differ. 

I believe that OD600 is an abbreviation for absorbance or more specifically optical density. In this instance the sample was measured at a wavelength of 600 nm. This method helps in estimating the concentration of bacterial cells in the liquid. 

I think it is really cool to be able to visualize the relation between the different mycobacterium and where Rhodococcus falls in the phylogenetic tree. It makes me wonder how other closely related would react under the same experiment or even their common ancestor.

I was thinking the same thing Aubrianna! It’s really cool to think that I am able to understand the paper better after every lecture and lab day. A lot of these terms and processes I had no idea what they were about but now I can formulate a general idea of what is happening during this experiment.

Since the Rhodococcus only exhibited an amplifies 100-bp band compared to its similar counterparts exhibiting a 78 bp band, would it not be expected that the Rhodococcus to perform the way it did? What is so different between these genes that allows Rhodococcus to do what it does?

It’s really amazing that a new naphthalene dioxygenase was reported in Rhodococcus during this experiment. Since it only shows a distant relation to the pseudomonas enzyme I hope to read more papers about this dioxygenase and its capabilities and be able to see what it is capable of in other situations. 

It is really interesting that these PAH’s can be brought up the food chain and affect humans. I wonder what kind of effects they  have on humans and what some of the symptoms would be to in order to diagnose  a patient with being affected by PAH’s. 

While it suggests that obligate marine  bacteria may have more significant PaH degraders, could the physical parameters of their ecosystem be helping them? Terrestrial organisms have a restricted form of movement through their ecosystems where substances can only be moved via wind, water, or other organisms, but in marine ecosystems the movement of substances is much faster and less restricted possibly exposing the bacteria to more PAH’s. 

I understand that the Marmur method allows DNA to be isolated from microorganisms and keeps the needed material active, and polymerized while keeping the majority of proteins and RNA away, but is there anything else about this method that makes it a necessity for this experiment? 

So in this case, the important thing to remember is the fact that the converted products were being identified and measured as well as their purification that was produced in the culture supernatant, correct?

In the flanking region of phnA1b it is said that there is no present small-subunit gene in the oxygenase pair. Was this subunit lost in transformation? Also how does this hinder the genes functionality throughout the cell?

Tara, I also looked up P. stutzeri and found out about the spinal fluid and it’s similarity to the NADH ferredoxin. It’s really interesting that they are similar and it would be cool to be able to learn more about the history of the relationship between the two, just like you said. 

I also have the same question Jesse. Is there anyway to access the Figures they are referring to in the text? I believe that if I was able of view them I might have a better understanding of what they are trying to say about their trees and how that relates to them falling out of the cluster. 

Jennifer, I think it would make it more efficient. Since the PhnC is involved in both aerobic and anaerobic pathways, not only is it degrading the compounds fast but it is also able to degrade them when there is no oxygen is available. 

Hey Alexandria, there are a bunch of other relationships between cyanobacteria and chemolithotrophs, and even some pretty large eukaryotes too. In some cases the cyanobacteria will provide its host with fixed nitrogen and the roles can even be switched around where the cyanobacteria is being provided for. So it sldo makes me think the same thing about whether or not they might be breaking down the arsenic to help each other out. 

I was also thinking the same thing Louie. I can’t wait to continue reading this paper, it seems like a lot of the information will tie into some information from the Biology of Algae class that I took and how cyanobacteria could have nitrogen fixing activity during daylight hours if induced by low ammonium. I wonder if that sort of reaction will be shown in this different type of environment. 

I agree Tim,  identifying the protein produced would definitely help in figuring out which genes are responsible for the arsenic cycling or resistance quality since some of the PCR primers were not giving us enough information.  

Caleb, I was also thinking about the environmental parameters of these anaerobics. Since the temperature range is so broad, it makes me wonder if they hold any sort of extremophile qualities, where their tolerance can withstand very cold or hot temperatures. It’ll be interesting to see the phylogeny tree and find out if they are related to any temperature tolerant organisms. 

I wonder what kind of pigments these arsenic cyclers have, considering that they undergo anoxygenic photosynthesis it can be assumed that they use bacteriochlorophyll, but determining which kind might help with understanding the limits of their photosynthesis.

It would be interesting, considering the technology available now, if they redid the experiment with a omics approach to annotate the gene that oxidizes AS(III).

I was confused when I first read this paper as well. Thank you for your definition! This concept seems interesting, and I appreciated the explanations in the text to both co-metabolism and the use of consortium strains. These definitions helped me understand the biological aspects of bioremediation as it relates to microbiology and how it can be efficiently used to preserve the environment. It shows that there are natural ways that we can effectively target toxic oil spills instead of introducing harsh methods that could potentially devastate the environments even more. 

I wondered the same thing, especially since there has been no further study for PAH degrading bacterial strains from the Nakheel region. Were there other factors contributing to the unique degradation patterns besides the low nutrients and high hydrocarbon pollution?

It does not specify that a control was collected. Is that already assumed, or is there even a need for a control in this type of experiment?

The paper does not specify that a control was collected. Is that to be assumed, or is there even a need for a control in this type of experiment?

Why would conductivity of the sediment samples affect the PAH-degraders? Do conductivity and pH affect each other, or were they measured together for the sake of efficiency?

Does it matter how much PAH they end up with? It seems as if they would not have a large number of PAH left after this rigorous isolation process if they began with 1% PAH. It could be assumed that it would be a big inconvenience for them to repeat that process if they did not end up with a desired amount of PAH.

Why did they construct the phylogenic trees? Was this to organize and assess the complete variation of the bacteria present? Or did this help them in some way analyze which bacteria would degrade the toxic oils more efficiently?

What would the difference between the gram-negative and gram-positive bacteria have on the effectiveness of the PAH degradation? Why would that make a difference?

I was wondering the same thing. It would seem that LB would be more useful, but that conclusion cannot always be applied in such seemingly obvious situations. 

Was this step in the experiment the reason why the phylogenic tree created earlier on in the methods portion of the paper? Does this make identifying effective bacteria and related bacteria easier? It seems that this could help find possibly both cheaper and mire effective cousin bacteria.

I too thought of your first question, if related bacteria are just as effective at PAH degradation as their cousins.

Since P. aeruginosa is possibly unsafe to test due to human pathogenic qualities, would there be any instance where that could be tested in a safe environment using technology? It begs the question of if it would be ethical or worth exploring other testing methods if it means finding the most effective PAH degrading solution, though it would be ethically irresponsible. 

I had the same question, Cody. Does this mean that the other isolates can be defined as the bacteria that they are so similar to, or are the 1% and 2% differences significant enough for them to be labeled as something else?

It’s interesting to note the drastic difference of the 3 isolates on degrading pyrene vs phenanthrene. They all performed similarly on pyrene, but there are varying levels of effectiveness on the phenanthrene. In both cases, most of the isolates seem to follow the general pattern of the control and follow its fluctuations, just at a much higher degradation % rate. 

That was also my first thought upon reading this section. After learning what turbidity and optical density are/consist of, it also made more sense that the solutions would be clearer if there were larger microbes present. 

What is the purpose of the bacterial mat? It is mentioned here and in the next paragraph. Does it influence the temperature of the microbes? And would the absence of the microbial mats allow for more effective extreme cooling as apposed to them being present?

What is the purpose of using the different carbon isotopes 12, 13, and 14? I see that all 3 are used throughout the experiment. Do they relate to the bacteria being degraded, or do they relate to the methodology to extract the strains?

I agree, Kelsey. It seems as if the researchers obtained different results then they originally hypothesized. Once they identified the most efficient mineralization technique, they were able to continue experimentation to see which compounds would be degraded best by which means. It is clear in the graph in paragraph 2 that the 4567-24 has a much higher percentage of initial CO2 concentration captured. 

It seems as if there are drastic differences in both the 14C-labeled PHE and the HPLC regarding the live cultures vs the acid-inhibited controls. Their effects are opposite of one another. 
The PAH performed better with live cultures, but poorly in the control group.
The HPLC performed better in the control group and more poorly in the live cultures.
Was this effect to be expected since the PAH was removed in the HPLC?

How would the oxygen levels in the cell in the medium show selenate reduction? Would the less oxygen show more reduction?

What would the importance of determining the shape and size of the selenium particles formed be? Is this to determine how much of the selenium is degraded?

I had the same question. There didn’t seem to be a clear reason as to why one cosmic clone was chosen over the other or that any evidence of that would have been indicated later in the next paragraph or the rest of the paper. 

How was the fragment pEC223 chosen from the 4 sub-clones? Does that have any significance as to which genes were isolated for selenate reduction? 

If the FNR gene operates best in anaerobic conditions, how would one be able to monitor this in a natural environment?

Was the strain K-12 introduced to show that there are other means that E.coli can use to reduce selenate without the presence of the FNR gene? 
Is this paragraph saying that the FNR gene was needed for the S17-1 strain and the YgfK,M,N gene was needed for the K-12 strain? 

I would presume that the hydrocarbon degraders would degrade the oil differently, even if the oil in the surface waters is the same that is in the deep sea. The extreme environment of the deep sea would cause these bacteria to rely on other energy sources, possibly chemicals such as sulfur, etc. How much of a difference would this make on the effects of the hydrocarbon degraders?

I also made that observation, the extreme living conditions cause me to believe that these deep sea bacteria are a different strand than the coastal bacteria.

How would the phylogenetic identity of am organism determine its function, in this scenario with PAH degrading bacteria? I understand the concept of creating a phylogenetic tree for the purpose of identifying related and efficient PAH degrading bacteria, as conducted in the last paper. How would one determine the function of a certain phenotype or genotype of a PAH degrading bacteria? 

How different are the microbial reduction of the selenate and selenite vs the abiotic reduction? Are they equally effective, or would one be much more effective than the other?

Would the bacterium being gram-negative be significant in its metabolic functions, or is that just a way to further identify the bacteria?

Does the process of Degradation depends upon Number and kind of micro organisms or the Chemical property of the PAH which needs to be degraded or both?
 

As Microbial degradation of PAH is quite interesting topic, I also read few other short articles about this, and I would like to share some information here. Prolonged exposure to chemical toxicants can cause adaptations in microbial populations that results in greater resistance to toxicity or enhanced ability to utilize toxicants as substrates for metabolism ( source of energy). Actual mechanisms for these adaptations are still unknown. 

Does this biodegradation takes place in the absence of Oxygen also? I mean in anaerobic conditions ?

I am excited to learn more about PAH degrading bacteria and bioremediation. I hope many of my questions will be answered in the upcoming sections.

I wonder what if gene insertion(from other PAH degrading bacteria) is done, will be the affect of degradation of PAH increase or decrease or remains same!. 

Does C . baekdonensis B30 strain degrade high molecular weight PAHs?  

In this context , I think there are referring to the intermediate metabolites in fluoranthene degradation only.

I just want to know how the mutant work, what will be the results? I hope I will find my answers in coming sections.

As far as I remember it is around 1%-2%.

Why is there a differentiation in 48tRNA gene represent 19 amino acids and a tRNA for other amino acid? why not they considered 49tRNA and 20 amino acids? 

Does COGs transfer between the generations or are they differ among the generations of same species?

I was thinking the same! I’d like to find out if there was potentially any type of symbiosis between the two!

I feel like the dark reductions occurred without a light source, which might cause it to have fluctuating temperatures as the experiment wore on from day to day. However, in the light controlled experiments, I think that the ability to control the temperature was easier as light was a component of the experiment!

I think the same! I wonder if the microbial populations were dependent vs. independent on light and if mutations had occurred in order to select for light oxidation and dark reduction. 

It is definitely cool to see that hydrogen sulfide stimulated both the anoxygenic photosynthesis and in the dark incorporation of acetate. I think this makes sense because both reactions use sulfide as the beginning electron inorganic donor. 

Since the bacterium rhodococci are capable of biodegradation of environmental pollutants, I wonder if it is capable of a mutualistic relationship with other organisms? Its ability to metabolize xenobiotic compounds that are not naturally occurring is quite unique and poses the question of whether it can exist symbiotically in different environments. 

I was wondering the same? What is the specific function of the small beta unit? To my understanding, the alpha unit is involved in electron transfer at the iron catalytic domain. Does the beta unit function in reduction as well? 

It is very interesting to note that the two ringed and three ringed PAHs are more readily degraded than the four or five ringed PAHs. I wonder if this has anything to do with the chemistry of the PAHs. Also, how can the enrichment be better modified to target four and five ringed PAHs?

I definitely noticed that too. I wonder if there was not continual enrichment and subculturing of the Rhodococcus on Fla would have resulted in different outcomes? What if the cells were instead subcultured with Nap or Phe?

It is very interesting that the attempt to enhance Nap, Phe, and Fla degradation actually resulted in their inhibition or had insignificant results. This can prove to be a beneficial discovery for producing a napthalene-inhibiting substance. 

Since there was no homology between the tested rhodococci ARHDs in this study to any prior studies, does this quantify a new type of dioxygenases unique to this species of rhodococci? Are there any additional tests to confirm this?

I agree with this. It’s very encouraging to see education in real-world application. This studies shows that there are methods to inhibit degradation of certain compounds like Nap and methods to increase the biodegradation of Fla.

PAHs seem to be a by-product of factory and industry activities. Is there a way to regulate these processes or regulate the release of PAHs before it enters the natural environment? 

Since Cycloclasticus operates in a marine environment, this bacterium must be salt-tolerant. Although I had no growth on the mannitol salt agar plate in lab 5, I wonder if I isolated an NP-degrader from a water sample if I would have growth on the mannitol agar and if it could possibly be a strain of Cycloclasticus?

Can PAHs also be transferred to humans through respiration or contact? Does it have to be ingested?

Were the dioxygenase proteins tested for the presence of the substrates or were its activity actually measured? How was it measured and quantified?

I was wondering the same! Perhaps it may not have an effect at all? Maybe the presence or absence of this gene just serves as a comparison point for the two? I wish there was more information about the small subunit!

What does the data mean by “agreed well”? Does this conclude that, yes, the phnA1A2A3A4 genes actually did code for the holoenzyme of the aromatic dioxygenase? Is there an explanation for why the data may be a little off from the predicted sizes of Phn?

I think so too! Is there a common ancestor that we can compare some of this data too since the basic sequence of features are conserved?

Since these proteins may be multipurpose electron transport proteins, would there be high regulation of these proteins since its functionality is enhanced?

I’m wondering if there is a specific reason as to why the activities of the fused, unfused, and sing-ring compounds were not different in these situations. Does it have to do with hydrogen bonding?

I’m wondering if these bacteria can use arsenate and arsenite as an electron donor in major pathways like glycolysis or the electron transport chain due to its high potential?

From the last sentence of this paragraph, are they suggesting that there are certain bacteria that can undergo “arsenification” and “dearsenification”? Also, what would be the ecological importance of forming arsenate and arsenite if they are toxic compounds?

Okay, so reading further, I do indeed see the the suggested coupling of arsenic metabolism. I wonder if one part of the couple is to recycle arsenic under anoxic conditions, will this also be tested under oxic conditions? Also, will the recycled products from this arsenic metabolism still have toxic effects on the environment or will they be neutralized?

I think that this is very cool that they took samples from rocks and cobbles that we would see on everyday hikes. This just reminds us of how much microbes rule the world! But on a more serious note, why was the slurry stored in the dark? Were the biofilms found in shady/dark areas?

I wonder why there is a distinction between the light incubated and dark incubated bacteria samples. Do they exhibit phototropism?
 

I was wondering the same! Does it have something to do with the photosystems and the light dependent and light independent reactions?

I feel like they mean that out of all the clones they did, there was only one clone that matched the arsenic oxyanion translocation pump gene. 

I, too, agree that it is interesting that the sample was collected from a city in Japan. Why was Japan the site of sample collection? I wonder if there would be a difference if the sample was collected from an oil field in the Middle East. What type of soil and oil is optimal for the cultivation of PAH degraders?

I wonder why gas chromatograph was used as the method of analyzation. Do the bacterial strains give off a certain molecule as it degrades the aromatic hydrocarbons? Could a spectrophotometer also been used as a qualitative measure of analysis for bacterial growth?

In paragraph 9 of the introduction, it was mentioned that present study and isolation of Rhodococcus does not have homology with catalytic domain of any prior ARHD Rhodococcus species. In this study, it is reported that the isolated bacteria exhibited 99% homology with the Rhodococcus species in the 16S rRNA gene, which is a highly conserved gene region that is used to identify microbes in complex environments. I wonder, if this bacterial strain exhibited any homology with the Rieske center of ARHD’s as well. Does it also have homology with the catalytic domains of prior ARHD’s of Rhodococcus species?

It appears that Fla degradation followed a logistic rate according figure 2. I wonder how the degradation rates of Nap and Phe compares to Fla on a regression curve. I wonder what the difference in molecular structure led to the high rate of degradation of Fla in comparison to Nap and Phe. Knowing this, is there any way to select for the degradation of Nap and Phe better?

Is there a particular reason why the ferredoxin and ferredoxin reductase genes were amplified through PCR rather than another genetic set? Is this code unique to the bacteria?

I wonder how the results would turn out if PAH samples like from the previous paper and from our lab experiments were used instead. How would the collection of a variety of PAH samples have an effect on the results?

Is the degradation process of high-molecular-weight PAHs unclear because there is a lack of research in that aspect, or have researchers tried to study it but were unsuccessful? 

If the P73T strain is the first fluoranthene-degrading bacterium to be found within the Rhodobacteraceae family, would that suggest that there may be other bacteria in the family that could degrade PAHs that we haven’t discovered or haven’t studied enough yet? Also, could the PAH-degrading genes be moved from P73T to other bacteria within the family using horizontal gene transfer?

What makes fluoranthene different from other high-molecular-weight PAHs that it is a good model? The previous paragraph mentioned that the degradation of high-molecular-weight PAHs wasn’t fully understood, so I’m curious as to why fluoranthene is the model of choice. I’m also intrigued as to why fewer fluoranthene degraders have been isolated from the marine environment. Is it because there is a lower fluoranthene concentration in those environments, or because bacteria marine environments aren’t the preferred research specimens?

I’m also wondering about this. Especially since the degradation of the high-molecular-weight PAHs is not fully understood. Some bacteria can degrade high-molecular-weight PAHs and some can, so is it the PAHs themselves that cause this, the bacteria doing the degrading, or both?

Me too. The fact that only a small percentage (like 10% I’m pretty sure) of the Earth’s oceans have been mapped and researched always has me curious about what else will be found in the ocean as time goes on. There’s so much to be found and researched from and about the oceans.

The mutant strain was generated to analyze and compare to the wild strain during experimentation. I’m curious to see if they will only try to examine the differences between the mutated strain and the wild strain, or if they will be trying to  amplify the PAH-degrading genes in both the mutant and wild strains to see how the mutation will affect the function of the gene.

If fluoranthene is a PAH and is the only source of carbon and energy for the bacteria, then the mutated strain should starve and die since it is unable to degrade the fluoranthene. I’d like to see if this will be the case, or if the mutated strain will have an alternative PAH-degrading gene that will allow it to survive. 

The coding density of the human genome is about 3% from what I know.

I thought it was really interesting that so many horizontally transferred genes were found. The P73T strain using HGT to adapt its PAH degradation is not something I expected, as I didn’t ever consider bacteria able to quickly adapt like that.

This answers a previous question of mine. I was curious to know if the bacteria with the mutated gene would be unable to use the fluoranthene, or if they would have an alternate PAH-degradation gene that would be used. 

This is the same thing I was wondering! I looked up a few things, but there are mixed answers from what I have seen so far. I would like to know a for sure answer as well.

What makes PAHs become resistant to biodegradation?

So if non-sporulating aerobic rhodococci are commonly found as biodegrading environmental than could they be used more prevalently in the world to break down manmade pollutants?

I find that it is interesting how different types of bacteria take different types of stains. It is important that we can use stains to distinguish different types of bacteria.

I wonder how much Fla would have been degraded if the samples had been allowed to incubate for longer amounts of time. For instance, how long would it have taken for Fla to completely degrade.

I wonder why the Rhodococcus sp. was able to degrade only that much of the ILCO in two weeks. How long would it take for the Rhodococcus sp. to degrade more of the ILCO.

I find it interesting how the writer chose to focus on how PAHs are present in the marine environment instead of the terrestrial environment. Perhaps this paper will focus more on how PAHs affect marine organisms.

I would be interested in learning more about how this genus of bacteria, Cycloclasticus, can degrade petroleum PAHs. That seems like it could have important real-world applications if used in the right way.

If less pure chemicals had been used I wonder what the consequences would have been to the results.

I wonder why the Luria-Bertani agar plates were chosen for the plasmids and to why there were added antibiotics. Also, why was that concentration of antibiotics chosen for the plasmids?

I may have missed this but what is the flanking region?

It is interesting that the researchers were able to show that PhnC is involved in the upper and lower pathways of degrading naphthalene, phenanthrene, and biphenyl. Since it’s involved in the upper and lower degrading pathways does it mean that these are more efficient in degrading these molecules? 

It’s cool that the PhnC is able to use both the upper and lower pathways to degrade naphthalene, phenanthrene, and biphenyl. Since PhnC uses both pathways does that make it more efficient in degrading naphthalene?

I like that this paper is different from the other two papers that we have done. It is cool that there are bacteria that are able to use arsenic to survive when it is so toxic to a lot of other organisms. It is even more interesting that the bacteria are able to use the Arsenate and Arsenite to create energy for themselves.

I am also interested in learning why their PCR failed. Perhaps it was because the primers were not right for the genome. I wonder how it would be different if the researchers had taken samples from a different biofilm that had similar bacteria. I am interested in learning about how these different bacteria work to get their energy since it is an anaerobic system that is light-driven.

I wonder if the results would change if they took the bacteria samples from a different hot spring with the same conditions?

Maybe the slurry did not need to be intact because it was going to be used for growing the biofilm on media so they needed it in a liquid state. But that just a guess.

I think it was interesting that there were only two main types of bacteria that were found in these biofilms. It makes sense that several types of Archea were found in the biofilm since the biofilm was taken from a harsher environment.

I found it interesting how the As(V)  and  As (III) had nearly opposite reactions when introduced to the light and dark regimens. I don’t think I was expecting that to happen.

It is interesting that Rhodococcus has the potential for such varied uses. I would be interested in learning about what kinds of applications it has in environmental remediation.

I agree with you that Rhodococcus could have great benefits for the environment. It would be great for the many uses of Rhodococcus to be recognized and well developed in the coming years.

I also found it interesting how the addition of the YE resulted in the inhibition of Nap while did not really decrease the degradation of Phe and Fla.

Why did the YE initiate the early degradation of Fla? Looking at the rest of the paragraph it certainly sped up the degradation of Fla during the incubation period significantly.

It is interesting how the increase of Fla concentrations could be inversely proportional to is degradation as the paragraph does not make it very clear. But reading the other comments does help to clear it up a bit with how Fla could be toxic in high doses.

I find it quite interesting that Rhodococcus what able to degrade aromatic fraction of Arabian light crude oil and the aromatics and alkanes of motor oil. But in the present study why was only the aliphatic fraction of ILCO able to be degraded by the Rhodococcus?

I also find it interesting that a new naphthalene dioxygenase was found in Rhodococcus sp. NCIMB12038 

It is interesting how a new naphthalene dioxygenase was discovered during this experiment in the Rhodococcus sp. This is cool as it only showed a distant sequence relationship with the Pseudomonas enzyme. 

I am curious to see how the Rhodococcus could be used in the future for biodegradation of different hydrocarbons. This is a very interesting product but I believe needs further research to help develop the methods to effectively use it as a biodegradation tool.

I am also curious as to the age of the paper and what kind of implications that this paper has had and/or will have to the study of Rhodococcus.

Are there be any benefit to not converting some of the molecules to their cis-dihydrodiol forms or to having the other molecules stay in their monohydroxylated forms? From the reading it says that the second set of molecules mentioned stayed in their monohydroxylated form due to structural instability in the cis-dihyfrodoils. But could there be any other reasons for keeping or changing the molecules one way or the other?

How many of these kinds of degraders where found to have the ability to oxidize indole to produce indigo? Do the ones in our experiments use that since the colonies will appear blue.

I would like to know what makes filter mating better for this study rather than other mating protocols.

I was wondering the same thing as Hampton. Is this like a standard dilution or is it based off what they need for the experiment.

I was wondering the same thing while reading this. Is this a standard dilatation or does it go by the amount of cDNA solution?

Why would they not show the data for the Southern Hybridization? I feel like it that confirm the loss of the region that you would want to show that data?

This is because of the size. Plasmids are only approximately 10kb, while cosmids are for approximately 40kb. Now they could have opted for a Phage instead possibly because the phages are used for the same size, but plasmids are going to be too small.

So since they are studying a new strain here, I am assuming that they will be incorporating the techniques in the previous papers here to identify the genes and pathways?

Is there a reason why there is not much known on marine-sourced degraders? Are they harder to study or just not as common in studies?

So GIs are the areas involved in horizontal gene transfer correct? Would they not run into problems with this later if they are only studying these areas?

This is because they have the genomic islands which give them ideas of where things are located, if I am understand the method correctly.

I find it interesting that it was this was found to be the only pathway, in comparison to those who have two or more.

So for studying the function of the catabolic gene, would they just move on to mutagenesis?

Is bioremediation a process of introducing foreign microbes to the environment to breakdown the pollution? Is this considered a natural technique?

Why would they not isolate and analyze the bacteria from that location? Was it because of the lack of enough nutrients and the excessive amount of pollution? 

Was the purpose of taking the temperature of the water to compare it with the abundance of bacteria found at each location?

Are the nutrients’ analyses vital to the decision of the bioremediation microbes selected to attempt to naturally correct the issues? Is that how biologists know from the get go if bioremediation is an option?

Why is the potassium in location B & C so high compared to location A?

I think gram-negative bacteria is only pink when performing “Gram Stain” tests, which stain the gram-negative bacteria pinkish red.

It’s interesting that the rod bacteria are favored over coccoid bacteria. I researched on the internet and a website said that the large surface area’s don’t necessarily mean a large adhesive force.

I believe that heme is a co-factor (prosthetic) that is required in hemoglobin and myoglobin for the proteins to function. 

I believe the purpose of the study was to identify any other possible genes that could reduce selenium.

Knowing the morphology of the particles would tell us how the bacteria reacted to the transformation?

The rate of selenate reduction was measured because of the direct relation to the changes of concentrations, correct?

Did the colonies turn red because they were unable to reduce nitrate?

So, the exterior cell membrane of the bacteria would determine the morphology of the Se particles? 

My takeaway is that information from each experiment helped them come to that conclusion after their own study results. 

It appears that they are saying that FNR regulation isn’t necessary if certain proteins are present. So yes, I think they are saying there are possibly multiple ways to reduce Se(VI).

I was wondering this same thing. If we are exposed to PAH’s through digestion do you think that the way certain foods are processed could increase the harmful effects? Also, regardless of whether the food we are digesting is processed or not, it could still have been contaminated from the environment. While it is important in protecting workers who could potentially be exposed to PAH’s via respiratory uptake, I feel it is just as important for people to be informed that these are in foods we are ingesting.  

I also find it interesting that these can be identified using the same techniques that we are going to have the opportunity to use in the lab. If we were to do this testing in an area with greater exposure to PAH’s do you think it would correlate with the rates of cancer in that particular area? This is something that I was interested in reading a little further into. This lead me to asking myself if these areas that have greater exposure to PAH’s are well informed of this and are aware of the effects they can have on the human body.  

Is there a chance that a person can be contaminated with PAHs such as ingestion without you realizing it? I’m wondering if the effects are immediate or are they something that can sometimes be over looked, such as shortness of breath and dry cough. It is hard for me to believe that I have never heard of PAHs yet they are potentially everywhere around us and even in what we eat or drink. I see that it says prolonged exposure causes more serious side effects but once you are exposed to these do they stay in our bodies or does our body eliminate them?

I was also thinking this same thing. In our lab I’m interested to see what the difference in growth is between our diluted cultures and our more concentrated cultures. Along with this I am wondering the difference in R2A plates and MSM plates. Will they differ in growth, color, etc ?

I was also thinking about the amount of PAHs in the environment. It it alarming that Dr. Ni Chadhain found this just simply in her neighbors yard. Is the public aware of things like this? I feel that there should be more awareness of the harmful effects PAH’s have and also more awareness of their abundance in the environment. 

After reviewing the results I was wondering the same thing. I feel as though this is a question we will be able to answer after completing this course. After finding out what differs in their molecular structure I feel that we would be able to identify a better way to detect for the degradation and not only these but others as well.

It is interesting that I can immediately relate this back to what we are doing in our lab.  Although, we are only a few weeks in to our course I already can identify what the difference is in growing a medium in an MSM plate vs an R2A plate. Even before we began this weeks lab we were told to look for a blue/black colony on our plates.  

I did not initially expect the results with the added YE to completely inhibit the degradation of Nap. This makes me wonder if there are any studies about different strains that could possibly be tested on Nap. While this study was successful in regards to the degradation of Fla, I feel that there should be more experiments done to see what strains could be successful with degrading things like ILCO. Obviously there is going to be a lot more to the composition of something like ILCO, but I think it would be worth the extra work to help protect our environment.  

I am curious what exactly (with the addition of YE) caused it to be so successful in degrading Fla, yet completely inhibiting the degradation of Nap. The Fla degrader seems to be more complex as they stated that another study found there to be two clusters depending on their PAH utilization capabilities. Do we know which cluster the one used in this experiment belonged to? Which also makes me wonder would a strain from the other cluster have been as successful in this experiment? 

I guess I expected there to be a direct relationship between the concentration and the amount of degradation. Also, in this section of the paper it doesn’t state what the time frame was for the 750 and 1000 mg1-1 degradation but assuming that it would also be 8 days, would a longer time frame allow this to have a more successful rate? Or does it reach a type of thresh hold

I am interested to compare and contrast between these and the Rhodococcus that we read about in paper 1. The first question that comes to mind is wondering if one of these are generally more affective than the other? I am also interested to see the difference in the results of their degrading ability on different PAHs. If these are used in costal marine environments is there a chance that they have been used around our area for any type of bioremediation?  

If marine bacteria is potentially more successful at PAH degradation this makes me think that Cycloclasticus will be the more successful one when comparing the degrades from this paper and the last paper. Also, it seems that the success of their degradation might be effected by what type of environment they are in, and not only which strain etc. Would we be able to conclude that one environment supports PAH degraders better than another? 

This is the overall question I have had since the beginning of reading these papers. In just a short time we have learned the multiple routes and the extent to which PAHs can effect humans, animals and the environment… yet before this course I had never heard of “PAHs”. This raises the question of how many people are aware of the extent to which we are exposed to them and also the extent to which they effect everything in the environment surrounding us. 

I was also wondering this. I think it is realistic to assume that depending on the situation one may be more effective than the other. But when they describe either one as effective do the byproducts play a role? Furthermore, does one give off more toxic by products than the other? I feel as though this is definitely something that they would consider when using either Fla or Cycloclasticus in bioremediation. 

Will we get to learn more about the specific endonuclease that they are using to do this experiment? This also makes me wonder over what range can endonucleases be used or if they have to be very specific to the DNA sections that they are cleaving. Also, what was significant about the size range of the fragments that they recovered? 

When I think of centrifuging I always think of chemistry 131 lab. I never expected further education to reveal the importance of this and that we can actually separate cells by simply centrifuging. After reviewing our pre-lab it’s so cool that we are going to get to do this Wednesday! The idea of us digesting the RNA and being left with DNA from our own culture is exciting and it’s also interesting to relate what we our doing in our lab directly to these papers.   

I was also wondering the same thing after reading this paragraph! I do feel as though it is saying that there would be a specific DNA sequence that gives microorganisms the ability to degrade PAH, but I could be misinterpreting the wording. So does the order of the genes have any effect on their ability to degrade PAHs? Or does it matter that the order of the genes were different from the previously reported genes? 

I was also thinking this! I’m wondering what the effects of the opposite direction of ORF7 is in the homology   since later in the paper it states that the order of the genes (that exhibited high degrees of similarity with the polypeptide sequence) is different than the order from that previously reported. 

Since they are located on separate transcriptional units does that completely determine that the electron transport proteins are shared with other redox systems or is it just assumed that they are shared as the reason for why they are on separate units? 

When I was reading this paragraph I also was wondering about what degree of significance evolution would have on this expression mechanisms. Since it is so highly organized I feel that this is something that would be very interesting to read on from an evolutionary perspective. Could scientist alter the gene expressions to see what increases and also decreases the expression of the specific genes we see here? 

I am interested to know if the conditions of Antarctica and its wildlife have changed in the past year, with less travel/tourism and possibly less exploitation of natural resources that leads to spills; and if any change has been for the better for the organisms that inhabit it.

Why and How is Diesel oil the most commonly used fuel in Antarctica? I have always known the continent to be a “cold and snowy desert” with nothing and no one around.

I wonder why they chose to only take 4 samples from each site (exposed and not exposed to diesel fuel). I would think they would want to collect more samples from the sites to have a larger sample size.

Why did they choose 168 h to grow the cultures? and Why was E. coli used as a control in this experiment?

I find it interesting that S. xenophagum did not show a chemotactic response toward any stimuli used. Is this due to it not having the proper mechanisms? Does this microbe have chemoreceptors that can detect cues? Does it have flagellum that could help it move toward a cue? I tried to Google this strain to see what the cells looked like but was unlucky.

Wow! I think it is very cool that Salicylic Acid is produced in this process. This is a chemical that I am very familiar with in my day to day life (in skincare) and I know that many people are familiar with it’s close relative- Acetylsalicylic Acid aka Aspirin.

Can these bacterial isolates work outside of extreme environments? Would this screening process work in our daily environments? 

I think it is interesting that other strains have been isolated in other locations that have cold environments and it makes me wonder if there are strains that work similarly in hot to very hot environments.

How is Marine Oil Snow formed? Is it just an accumulation of oil particles that float back down from the surface of the spill? If so, is it possible to prevent this from forming by cleaning up the surface spill quicker?

Does the mineralization of hydrocarbon contaminants have any lasting effects on these environments? Is the product of this process not considered to be a contaminant or is it turned into a useful product for the environment? 

I think it is referring to the controls that were killed with acid. But I do wonder why acid-killed controls were used rather than an E.coli strain as used in the last paper?

This was the comment I was looking for! The Meselson and Stahl experiments that we learned about in genetics were exactly what popped into my head when I read about heavy versus light DNA. 

“Clearing”… is this similar to the Casein Hydrolysis test we did in Lab 5. Does this show the presence of caseinase exoenzyme production or something that has the ability to clear agar in the same way?

To add onto your question, I would also like to know if we have found this species or similar species in other ecosystems (like on land or in extreme conditions) and if they have the same roles in these areas or if maybe PAH degrading is something only the ocean-stricken species has adapted to?

How common is it for a microbial habitat to shift between an oxic and anoxic state in a 24 hour period? Is this similar to the day and night changes of plant photosynthesis and respiration?

I think it is interesting that this process has been found or studied in multiple very different sources: periphyton, which is located the vegetation found on the bottom of freshwater systems; the soil in Japan (assuming this is not from the sediment of a water system); and hot spring biofilms.

Would this anoxygenic photosynthetic As oxidation be common during algal blooms? Has this been studied?

I think this idea comes from haloarchaea being around for a long time, and the process of anoxygenic photosynthesis comes from early earth being anoxic.

I would be interested in further understanding the process of endpoint determination using liquid scintillation and why this was used to measure mineralization.

As with the last paper and although I do not fully understand these processes, I enjoy reading about real-life experiments that correlate with what we are doing in our own lab – I recognize those bacterial primers!

Does the color of these spring correlate to the abundance of each bacteria present?

What was the purpose of collecting samples months apart like this?

Yes, I think it may be dominated by Ectothiorhodospira because of the dominance of it’s ability to cycle arsenic, it may overpower any other organisms ability to cycle the arsenic.

Would the temperature range increases at night have anything to do with the respiration processes of other organisms in the environment?

I am just wondering why these various and specific locations for the soil, water, or sediment samples. I understand that the samples that were collected have various levels of salinity, because the bacteria needs to thrive in hypersaline environments as said in the introduction, but is there another property that the samples need to have in order to be used for their experiment?

The Lowry method sounded familiar, but I could not recall what exactly it was. So I googled it and found that the Lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques. This now makes sense as to why they used this method while using the UV spectrophotometer.
 

The fact that these archaea have been found to degrade both aliphatic and aromatic hydrocarbons in high-salinity environments leaves me to wonder if they can also degrade beneficial compounds to these type of environments? Or is it just exclusively degrading the hydrocarbons? What would be the cons to exposing these fragile environments to more bacteria and archaea?

I find the topic of bioremediation very interesting. The use of naturally occurring or introduced microorganisms to consume or break down environmental pollutants. This should be the first step when cleaning up pollutants instead of introducing more harmful chemicals in order to clean up other harmful hydrocarbons. This is especially important for the Gulf of Mexico. The Gulf has a relatively high number of oil rigs, while also being the home of many estuaries. This can be a dangerous combination.

[Approximately 40 to 60% of the added [14C]benzene was converted to 14CO2 in a period of 3 weeks (data not shown), further suggesting utilization of benzene as the carbon source.]
This is good news in the sense that over half of the added benzene was converted to CO2 in a period of three weeks. Which is relatively fast working in the world of science. I just need clarification on this part, as the strain Seminole degrades benzene, it is stated that benzene is converted into CO2. Does this mean as Seminole uptakes benzene, it releases CO2? Correct me if I am wrong, but could that just add more problems to these estuarine environments?

It is said that the pob and pca genes are found clustered together in a contiguous pattern or scattered over several portions of the genome. I wonder is it better for these genes to be clustered or scattered? Or does it make no difference to the expression of these genes? I happened to google it, but could not find any reliable information to go on

I am curious as to why the organism was not able to grow on catechol if it was predicted to be an intermediate in the benzene and toluene degradation pathway. I understand they didn’t find a conclusion for this, but is there a specific test to find out why? Or could it possibly be a mistake on their part when making predictions about the organism’s genome?

I would be interested in finding more out about the genes that they predicted would show during the degradation process, but didn’t. I am assuming once all the genes are well known, that is when we can start to build a plan towards degrading the compounds that are harmful to marine environments.

I am interested in learning more about fluorene degradation. Being in marine sciences, the main focus is usually carbon dioxide, methane, nitrogen, and other major greenhouse gases. People rarely think about other compounds that come into play as well. Then to learn about microbes that can degrade these toxic compounds and the science behind it is super interesting, 

I looked up the bacterial species as well and found that there are 8 halophilic and 10 halotolerant different strains for this particular bacteria. Im curious to see any similarities with the last degrading halotolerant bacteria we discussed for paper 1

I was wondering why they mentioned that they used phosphate buffer instead of Tris buffer. So I looked up the difference of the two that might lead as to why they choose one over another. Tris buffer is used as a running buffer in agarose gel electrophoresis to identify a species by looking at the base pairs. On the other hand, phosphate buffer is used in isolating cells from tissue to maintain pH and keep cells alive. I also saw where phosphate is more expensive, but I see why it was needed instead of the Tris buffer for this experiment.

I like how detailed these authors are with their experiment. It makes it easier to read and understand when they explain each step and what the product resulted after doing each step and not just assuming the reader knows how they got to a specific result.
Coming from a now second time reader of scientific papers.

Looking at figure 2, does this mean that the Terrabacter sp. DBF63 is the species found to degrade fluorene more successfully compared to the others?

I too would like more explanation on this. I am also confused on the importance of the functional expression of FlnA1 and FlnA2

I think it is interesting how we are now on our third paper about degradation of contaminants in the marine environment by naturally occurring or introduced organisms, and I have never heard anything about it beforehand. Introducing these organisms to polluted areas seems like such a good idea. I wonder if this is actually taking affect anywhere around the world

Since fluorene had three different degradation pathways, I wonder if fluoranthene will have similar pathways

I didn’t know about the cre-lox recombination method so I did a little research. Cre-lox recombination involves the targeting of a specific sequence of DNA and splicing it with the help of an enzyme called cre recombinase. Cre-lox recombination is commonly used t circumvent embryonic lethality caused by systematic inactivation of many genes.

I think it is interesting that they took samples from the Indian Ocean sediment, but then grew the strain on artificial sea water. If this is to work in the marine environment, wouldn’t it be more beneficial to take ocean water samples as well instead of something man made?

Could you further explain this figure? There is so much small information, I am not sure what to take out of the circular map. I am also confused to what the inner circle is showing

This is the most genes involved in the degradation of PAHs in a single strain that we have read about. Does this mean that the P73 is more capable of degradation of hydrocarbons than any of the other strains that we have learned about in the previous papers?

Why don’t we know about the degradation of PAHs with higher molecular weights? Is it due to the large size of those PAHs?

How does naphthalene hinder mitochondrial resiration?

The variety and number of tests performed should show relationships among naphthalene degrading bacteria. Are there any other tests that would help with the study? Is there any particular test that is more beneficial or conclusive in determining naphthalene degrading bacteria?

ONR7a medium was used since it contains naphthalene as the only carbon source. Are there other media that have similar characteristics?

Since strains N2, N7, N9, and N10 are all phylogenetically related (from figure 2), one would assume their growth rates would be nearly the same; however, the growth rates range from 0.345 to 0.928 (from table 1). Why is their so much variation?

Why is it that 400 ppm was the optimum concentration of naphthalene? Is it more important simply to understand that 400 ppm is the optimum concentration of naphthalene?

I find it interesting that in paragraph 4 gram positive bacteria were isolated and found to play a role in naphthalene degradation, but in this paragraph, gram negative bacteria were favored for naphthalene degradation. Does the difference have to do with the lipopolysaccharides in the gram negative bacteria’s outer membrane?

Since bacteria were isolated from an oil polluted area in the Persian Gulf, could bacteria be isolated from the Gulf of Mexico which was polluted during the 2010 BP oil spill? I think it would be interesting to isolate bacteria from a waterway that most people in this region have encountered.

What is the largest aromatic compound ring number that bacteria are able to degrade? Is this dependent on the type/complexity of the bacteria?

I know some of the main points of the study were to isolate and classify naphthalene degrading bacteria, but unless I missed it, the paper didn’t discuss how to insert these degrading bacteria into oil polluted environments for controlled bioremediation processes. Is this being tested?

Are there any preventative measures to see if the seafood we are consuming has toxic PAHs in it?

Where are these Cycloclasticus bacteria located? Are they in any marine environment polluted by petroleum?

Various chromatography techniques were performed in the method of this lab. Chromatography is useful in separation by size, charge, and affinity each of which is useful in identifying a pure isolate.

Various chromatography techniques were performed in the method of this lab. Chromatography is useful in separation by size, charge, and affinity each of which is useful in characterizing a pure isolate.

Since naphthalene is a two ringed structure and phenanthrene is a three ringed structure, is it important to know which bacteria can use which chemical? Or is the fact that they have the ability to degrade either and/or both of  the compounds the topic of interest?

Why are E. coli containing pPhnA able to transform certain molecules but not others?

All of the aromatic substrates were hydroxylated in the figure. Is this useful for them to be degraded?

The Sau3AI fragment of DNA is the gene which allows pH1a and pH1b to oxidize certain aromatic compounds. Is this inerpretation of the paragraph correct?

What is the significance of not finding the gene clusters for PAH degradation on the plasmid, and why doesn’t the strain have a plasmid at all? This is a unique characteristic as plasmids often contain genes for antibiotic resistance, degradation properties, etc.

Monooxygenases are enzymes that transport one oxygen atom from the air to the substrate, and dioxygenases are enzymes that transport both oxygen atoms from the air to the substrate. I think this comparison is useful if, like me, you have trouble keeping these definitions straight.

If other dioxygenase genes in Cycloclasticus were examined, what would those results conclude? Since this paper is several years old, I assume additional research has been done.

Is there another method for obtaining the aresenite oxidase gene since the PCR for the genes was unable to be obtained?

How are some organisms able to utilize arsenic, but for others arsenic is toxic? I understand that if an organism can utilize arsenic it does so by using it as an electron donor or acceptor, but what determines whether or not an organism can use arsenic?

Alkalithermophiles are extremophiles that are grow optimally at high pH (around pH of 10). The rod-shaped bacteria, Anaerobranca californiensis, is an example of an alkalithermophile. From further research, I found that this bacteria grows best in the presence of elemental sulfur, polysulfide, or thiosulfate which are found in the spring waters.

A radioassay tests a radioactive sample to determine the intensity of its radiation. Incubation is a process that maintains a favorable temperature and other conditions promoting development. In this experiment, is it correct to deduce that radioassay incubation was used to “grow” the bacteria?

Assimilative reduction is where an element is consumed and incorporated into new cell material. In light-incubated slurries, assimilation was not stimulated by the presence of arsenic oxyanions. Why is that?

Are there further tests that could be performed to prove that the clones were of the genus Ectothiorhodospira, or are the results obtained sufficient?

Senescence or biological aging is the gradual deterioration of function characteristic of most complex lifeforms.  Essentially the third sentence is saying growth rates decrease because the microbes are aging and toxins are accumulating.

Is there any way to make a guess on which microbes reduced arsenate?

Sulfide and hydrogen increase the rates of arsenate reduction because they are at the top of the standard reduction potential chart. This means they give off a lot of energy.

Where do the microbes get the bicarbonate?

Fluoranthene is a fusion of naphthalene and benzene by a 5 member ring. An alternate PAH is one where the pi centers are not adjacent to another pi center. In a non-alternate PAH, somewhere on the ring there are pi centers that are adjacent to one another.

From further reading of the paper we will find the answer to the following question, but for now, could it be guessed that the fluoranthene degradation pathway of Celeribacter indicus is initiated by dioxygenation as said in paragraph 2?

One of the goals of this experiment was to isolate the genes needed for PAH degradation. Should genome sequencing help to determine the fluoranthene degradation pathway?

I had never heard of the term silylation, so after a quick google search, I found that this is replacing a proton with a trialkylsilyl group. The purpose of this is for analysis using GC and mass spec. How is this helpful in our understanding of the fluoranthene degradation pathway?

If glucose-6-phosphatase is missing, how does gluconeogenesis proceed? Were these genes not transferred during horizontal gene transfer?

I’m having a difficult time understanding why strain B30 and strain P73T were compared. Are the two strains more or less the same other than B30 lacks most of the RHD genes found in P73T?

Are there other genes throughout the genome that could also be responsible for the integration of foreign sequences needed for HGT? In this paragraph it talks about region B, but what about regions A, C, and D?

Knocking out a gene is a good way to determine if a gene has a suspected function. In this approach, gene function is studied by examining gene loss. What other methods can one use to determine a gene’s function? Genomic DNA analysis?

Why is aromatic ring hydroxylation the most difficult step in fluoranthene degradation? Is it a difficult reaction to get started, does it make unnecessary side products/reactions, or is it something completely different?

Horizontal gene transfer is a good way for new genes with new functions to be inserted into a genome, but can HGT be harmful to an organism? Can it insert a gene that inhibits a necessary gene? I assume such organisms wouldn’t survive in nature since this would decrease the organisms’ fitness.

Sphingomonas sp. strain LB126 is unique from other microbes we’ve looked at since it was isolated from PAH-contaminated soil. We usually look at microbes from PAH-contaminated marine environments. Is there any difference among microbes found in soil vs in a marine environment?

Why is there more research on fluorene degradation of gram-positive bacteria than gram-negative bacteria?

I did some googling on degenerate primers. I found that they’re useful when isolating the same gene from several organisms, as the genes are most likely similar but not identical to one another. Degenerate primers are also used when primer design is based on protein sequence.

From my biochemistry course last semester, I learned that SDS-PAGE is used when separating proteins by electrophoresis.

From what I found, sonication involves applying sound energy to agitate particles in a sample. Some of the purposes include: to break intermolecular interactions, for the production of nanoparticles, to disrupt or deactivate a biological material, to evenly disperse nanoparticles in liquids, initiate crystillization, loosening particles adhering to surfaces, etc.

For this experiment, is sonication used to break up the pellet into the liquid?

Is this because they wanted to obtain a clone with a site for one enzyme on one end and a site for another enzyme on the other end?

I’m trying to make sense of the information found in paragraphs 6-8 and in Table 2. From what I understand, this is to compare what substrates FlnA1-FlnA2 can use to which substrates two common dioxygenases, DFDO and CARDO can use. Are FlnA1-FlnA2 genes that were found in Sphingomonas’ genome that function in fluorene degradation? From these 3 paragraphs and table 2, what exactly should we find to be noteworthy? 

I think it is very interesting that a gene usually only found in gram positive bacteria is found in Sphingomonas, a gram negative bacteria.

Additionally, I think this paragraph helps to clear up some of the questions I had about FlnA1-FlnA2.

Cells use glucose in glycolysis to produce pyruvate. Pyruvate can then be broken down to lactate in lactic acid fermentation, ethanol in ethanol fermentation, or acetyl CoA in the citric acid cycle.

Shouldn’t we expect that fluorene is not the best carbon source since smaller PAHs such as naphthalene are easier to degrade? Or am I confusing this with the fact that smaller PAHs are more widely studied as they’re model PAHs?

Is it unique that Sphingomonas sp. strain A4 is only able to grow on acenaphthene & acenaphthylene and not lower molecular weight PAHs?

I looked up the 16 priority PAHs. From what I found, these PAHs are of high priority because of their potential toxicity in humans & other organisms and their prevalence & persistence in the environment.

Is this describing targeted mutagenesis like we went over in class today?

From what I found on google glass power is used for purifying fragments from agarose gel electrophoresis, is that correct? What is the purpose of using glass powder?

Blunting of sticky ends is done to allow non-compatible ends to be joined.

Shotgun cloning is different than shotgun sequencing. Shotgun cloning is used to duplicate gDNA by fragmenting DNA with a restriction enzyme. These fragments are then cloned into a vector. In shotgun sequencing, DNA is randomly fragmented with a restriction enzyme, but then, the fragments are sequenced. These sequences are reassembled on the basis of their overlapping regions.

In this paragraph, they used targeted mutagenesis which relies on homologous recombination. I’m going to attempt to explain it, so please let me know where I explain it incorrectly. In this type of mutagenesis, the arhA1 gene was cloned into a suicide vector. The antibiotic resistance gene (Gm) is inserted into the arhA1 gene (in the vector) which disrupts its function of PAH degradation. Homologous recombination occurs, so now the mutated arhA1 gene is inserted. This allows the gene to show antibiotic resistance, but not PAH degradation. You know you’ve successfully mutated the gene when the original function is inhibited.

Why was the phylogenetic tree constructed like this? Is this advantageous to the trees we’ve made?

Have the additional studies mentioned been done to show other genes needed in the degradation of acenaphthene and acenapthylene?

So in this experiment, genes encoding for ferredoxin & ferredoxin reductase were on one of the plasmids that was transformed in E. coli. I know ferredoxin works in electron transfer, but what exactly is happening with it in this experiment? What is the purpose of having a plasmid with genes encoding for ferredoxin and ferredoxin reductase?

Transposon mutagenesis allows genes to be moved to the chromosome of your organism of interest. This causes mutation because the gene is inserted in the middle of a functioning gene. In this experiment, they’ll mutate the genes involved with acenaphthene degradation.

In paper 3, the authors found the genes involved in the initial steps of acenaphthene degradation, oxygenation. In this paper, the authors hope to find genes involved in the next steps of acenaphthene degradation and the genes involved with acenaphthene degradation regulation. Would sequencing the organism’s genome give you this information?

I tried to do some googling on what a xenobiotic is. From what I found, they’re substances which are foreign to the organism. Xenobiotics include drugs, industrial chemicals, naturally occuring poison, and environmental pollutants. This website might help to answer some of your questions.

http://www.ilocis.org/documents/chpt33e.htm

I know that sometimes the order of events in the papers is not always written in the way they were carried out. Is that the case here after the DNA manipulations paragraph?

I’m assuming arhR is a gene involved in acenaphthene degradation. Disrupting the gene will give us insight into the function of the arhR gene. From this paragraph, are we able to determine what disrupting the gene caused to happen, or would this information be found in the results section?

Does the location of the insert tell us that acenaphthene degradation is dependent on these genes?

I was unfamiliar with what a primer extension analysis was. From my google search I found the following:

Primer extension allows the 5′ ends of RNA to be mapped. It can be used to determine the start site of transcription by using a primer which is radiolabeled.

A transposase gene is often a sign that horizontal gene transfer occurred, right? Is that the case here?

I’m not sure how old this paper is, but has further investigation of the degradative genes and regulatory mechanisms been done to help clarify the acenaphthene degradation pathway?

Is there a way to place an insert into a particular spot in an organism’s genome? If not, is that why there were 10,000 strains with inserts?

After the process of bioremediation, what type of effect does the end product of microbial biomass have on the environment? Does it show an effect at all?

What are the costs and effects of using different strains of bacteria for biodegradation? What difference would you expect to see when deciding between bacteria based on their biodegradation ability?

What is the purpose of transporting the samples on ice? Is there a certain temperature that is required to maintain?

What is the CTAB method and how does it work?

What is the GC-FID method and how does it differ from spectrometry?

What factor would determine the amount of growth of strains after 400 ppm concentration?

Would Gram-negative naphthalene bacteria be able to play as great a role as Gram-positive in an oil-contaminated area? Or is that possible?

Is it possible for Gram positive bacteria to be found in an area of high pollution of heavy crude oil with Gram negative bacteria?

What other gene sequences besides C23O are most useful in identifying phylogeny of a strain?

By what measurement determines which bacterial strain is best suited for metabolizing PAHs?

I read that sonication can cause cell lysis. But in this case, is it used to just agitate the cells?

I looked up SalI, but I am still unsure of exactly what it is and what it does?

Looking at the data table, I am unsure of exactly what the numbers corresponding to each carbon substrate represent? I understand the table shows the utilization of each carbon substrate but I am unsure of how the numbers are calculated.

Is the basic 7.0 pH level usually the ideal when it comes to the strain degrading PAHs?

How would evolutionary pressures cause species of Pseudomonas putida to be scattered to different clusters and those from Sphingomonas to be in the same group?

How does the structure of the  meta-pathway operon change the way S. paucimobilis degrades PAHs?

What would cause an inability to obtain PCR products for axoB genes?

Would bacteria using arsenate as a respiratory electron acceptor benefit more than bacteria using arsenite as an electron donor?

What is the significance in the colorization of the different bacteria? Is green always associated with cyanobacteria?

Is there a particular reason all aoxB clones failed to show any similarity to the database?

Why would the re-introduction of light cause a diminished rate of As (III) oxidation?

Since the promotion of O2 exchange didn’t affect the rates, what can be concluded?

Would there be a method to more directly determine which biofilm microbes were associated with these arrA amplicons involved with As(V) reduction in dark incubations?

My guess to your question based off the section is that V. fischeri ES114 contained something that the others did not which inhibited the growth specifically to that culture, because it was stated that the other culture fluids from the other tested strains did not inhibit V. harveyi growth.

I also noticed how well of a specimen vibrio seemed for the experiment, knowing this information makes me think the results of this study will be great in terms how much data they will be able to collect. 

Reading the paragraph right above this one, siderophores have a high affinity for Ferric or Fe^3+ iron which is the abundant form of iron in at neural pH in the presence of oxygen, so I came to the conclusion that if it is not that specific affinity for iron then it would not actually bind. In conclusion not all siderophore producing microbes would not have the same affinity to iron.

Oops! This comment was supposed to be in response to Ryan. 

I actually knew that nitrification played a role in marine life, but I was not aware of the significant role that it played until I just read the data. So, I also agree that this was a pretty interesting finding. 

After reading your comment, I was curious and decided to research your question. From the looks of it, as if right now, they are not able to give too much more information other than that diversity is negatively impacted. So, I am guessing some life exists but not a lot. 

Based on your definition and the paper’s definition of what a steady flux is, it seems to a constant considering it is distinguished by the amount of atoms crossing. 

I think they were determined or at least thought to have made an appearance in the step above where it says “Anammox rates were measured by the means of isotope-pairing” and were then verified using the CARD-FISH. 

 “Hydrocarbon contamination in Antarctica has profound effects that have been shown to reshape the structure of microbial communities as well as affecting the abundance of small invertebrate organisms”. How exactly are the microbial communities reshaped? What is to be expected of the communities when they are encountered? Also, does that mean there is a reduced amount of small invertebrate organisms or more of a diversity when taking into account their amount?

In response to your question regarding limiting the amount of contamination, we would have to figure out what might be one of the biggest contributions in causing the contamination in order to reduce it. Because the causes of oil contamination could easily very. If that was done, then I feel confident that we could play a role in the reduction of contamination. 

Finding your question interesting as to why biofilm may not occur in this experiment, I researched it. Based on my research, the formation of biofilm has to do with antibiotics, biocides, and ion coating. Those can interfere with the attachment stage in biofilm which will cause the other stages not to necessarily be applied. 

I also wondered what the change in temperature would do to the buffer. Personally, I think it would make the 90 minute incubation drop, but what will the changes in temperature do to the results of the experiment? what direction will it go? 

[ To this end, we assessed the ability of culture supernatants to emulsify diesel (E24 index) as well as the ability of supernatants to generate water droplet collapse by altering surface tension (Figure 3G). In both tests, D43FB supernatants lacked emulsifying abilities, and exhibited similar behaviors to negative controls, suggesting D43FB does not secrete biosurfactants to aid in its degradation of phenanthrene.]
While reading, I was not necessarily sure what “emulsify” or “emulsifying” meant. I researched the terms and they basically mean to have two liquids in a colloid and not completely mixed together. So, in other words, it now makes sense as to why emulsification had nothing to do with the degradation of phenanthrene due to the lack of emulsifying abilities.

I agree with you as far as not truly believing the D43FB will thrive when put into the conditions of Antartica. It seems as if they are more so of hoping for the results will turn out good solely because it had the best results when it came to the others, but the analyzation doesn’t seem too solid. 

In addition to what you’re saying Ryan, I would also love to compare how S. xenophagum D43FB would respond in the situation in which the temperatures change drastically either much colder or warmer. Does the weather play a major role in how the isolate degrades and replicate? 

I was not aware until reading the article that it was forbidden to bring foreign organisms into Antartica, so I was caught by surprise as well. Also, I feel like this should be enforced in a lot more other places as well because according to the data, strict laws such as this are what is making this environment such a great place. There are so many places that could benefit from the narrowing down of decontamination techniques/strategies.

My initial thoughts after reading was what necessarily makes iron a great source of competition as opposed to any other source and what lead them into making the decision as to why they should use it? 

I would assume that there would be a larger possibility to make a mistake considering how broad the approach is rather than specific. 

In response to your question, I do not necessarily think it shows evidence of that because of it stating the V. fischeri ES114 does not produce aerobactin and/or sufficient iron. In other words, it doesn’t clearly state if low iron is the sole reason. 

I think it’s very interesting to see the drastic change when the oxygen has nicely depicted lines decreasing into the suboxic area then travels backwards increasing back into the lower oxic area. Then the H2S starts off at a plateu or a completely flat rate then gradually increases as O2 completely runs out. 

I am a little confused in Figure 2D because what I am interpreting based on data is that the NOx- decreases in some areas of the lower oxic area while it slightly increases then reaches a rate where it plateaus and does nothing at all like it was at the beginning where there was no oxygen? So, is this saying that the oxygen ran out? 

I definitely agree with your comment, without the genes being transferred in that manner, the experiment would not be as effective. I wasn’t aware of the horizontal transfer being across multiple lineages either until reading this section of the article. 

Yes, this last sentence gave a great/more clear summary of what was actually concluded at the end of the experiment. Before reading it, I did have some confusion of what they were suggesting. I found this experiment interesting because I have never seen this type of interaction “occur in nature”. 
 

I honestly don’t think it would be considering the genes were rarely seen to begin with, so I think it just happened to be abundant in that particular location based on the conditions. The only way I feel like would be observed is if they had similar conditions. 

I would actually like to know the answer to that question as well, because I thought they initially chose the Black Sea due to its great source of microaerobic nitrification. If I had to guess though I would say that it would not be as directly coupled which it what would make the Black Sea so unique to begin with. 

I found bioaugmentation very interesting. I found a study where they actually used bioaugmentation and biostimulation hand in hand and discovered that together these two processes were optimal for oil degrading. Interestingly, there are Antarctic bacteria that are capable of hydrocarbon degradation at extremely low temperatures and they concluded that the use of bioaugmentation in those situations could enhance the rate of bioremediation.   

I found myself pondering similar things during this reading. My initial thought was that it would be doing more harm than good as far as bringing those pollutants over as well as any non-native species of microbes and things that are on or in the icebergs. I typically tend to believe that disrupting the natural order of things means it shouldn’t happen. However, with what I’ve learned about serious drought issues that people are facing I feel this is a fairly logical route to supply the people. The captain who conceived the plan seems to have an ecological, well- thought out plan to make it work. I think the pollutants would be in such minimal amounts that purifying that would become the easiest part of the process.

I don’t necessarily believe the experiment would have been hindered if another media was chosen that suppressed certain bacterial growth as long as there were runs made with a control media. However, this is obviously very dependent on which media is used. 
When pondering which media (if any) I though would have been more successful, I found myself thinking that R2A is typically used with slower growing bacteria and can be often suppressed by faster growing colonies. This made me wonder if they came across this issue in the field. Unfortunately, I never really found the answer for which agar would be better than R2A. I would lve to hear others thoughts.

 After reading about biofilm production as well as this paragraph, I was curious as to which of the 350 colonies produced were the ones producing the biofilm and if there is a method to differentiate between those working in biofilm production and the colonies who didn’t. If it wasn’t all of them. 

I believe the answer is yes! When undergoing transformation, the cell lyses and releases those intracellular components into the environment. That includes DNA fragments which a bacterium could take up to gain resistance. 

I was also confused. From what I understood when looking it up, I found that while most siderophores are strong enough to pull iron from host-binding proteins so maybe since the microorganisms producing them are in iron deficient areas its just another case of survival of the fittest we observe in countless environments. There are siderophores who outcompete others based on better genetics. 

From what I looked into I understand it to be a more specific type of PCR (DOP-PCR). It allows the employment of olignucleotides of partially degenerate sequences specific for genome mapping. Olignucleotides primary use seem to be primers for PCR so your assumption is correct. As for specific reasoning, I found they have the ability to amplify a small amount of DNA as well as being designed for length and G/C content. They flank the target reason so it seems just to be a wonderful primer for this use. 

I read that this type of assay seems to be the most accurate as compared to counting-based methods for plotting growth curves. Is this because the fluorescence- based method isn’t dependent on determination of total cell numbers, but more so assessing fluorescence of the given sample of single cell from the population at a given time? I feel that contributes to lower variation rates among the plated samples which in turn only increases reliability especially when assessing the lag, log, and stationary phases. 

I understood it to be both. They kind of go hand in hand as you can predict antibiotics resistance based off of mutations. Mutant construction of essential genes aid in resistance. Or am I missing some context? 

Specifically CAS because of its ability to detect various types of siderophores. I found a paper that was saying in comparison of solid versus liquid that the liquid reaction-rate was an positive over the solid in all species tested. So maybe it was easier to quantify this way?

[The inability to grow was not specific to V. harveyi: V. fischeri ES114 culture fluids also prevented growth of Photobacterium angustum S14 and Vibrio cholerae C6706, while no diminishment of growth yield of Vibrio parahaemolyticus BB22OP and Vibrio vulnificus ATCC 29306 occurred (Fig. 1B). ]
comparing this to figure 1B made for much easier understanding. However, I found it a bit confusing that the growth inhibition was specific to V fischeri ES114 while others still had the inability to grow. If someone could offer some clarification. 

[In communities, public goods can be exploited by cheaters who acquire advantages through use of the good but who do not pay the energetic cost of goods production. In the context of siderophores, a cheater need only possess genes required for recognition and uptake of the siderophore-iron (Fe3+) complex. ]
This comparison helped my understanding greatly. I found it very interesting that the cluster of siderophore biosynthetic genes encode for the ancillary functions of reception and transport for the V. fischeri.

I know that this just saying that V. fischeri ES114 outcompetes the others by uptaking all the iron which in turn also deprives the other of iron. Would the cheaters not steal the siderophores to outcompete V.fisceri ES114 since it wouldn’t be paying the metabolic cost to produce/secrete?

[ This co-culture system, in which either or both species can be genetically manipulated, provides a route to the quantitative investigation of both competitive and cooperative interspecies interactions that occur in nature.]
The identification of aerobactin acts as an inhibitor or the V. harveyi while working towards eliminating production in the V. fischeri ES114 thus creating this co-culture. I found this so interesting and somehow it helped the whole discussion click easier for me. It’s cool that this experiment led to quantitative data for the competitive and cooperative interspecies interactions. 

[ nonthermophilic MGI Crenarchaeota constitute a significant portion of oceanic picoplankton (up to 30%) (21, 22) and a considerable fraction are likely autotrophic (23, 24), it is speculated that these MGI Crenarchaeota could be more important nitrifiers in the oceans than the usually less abundant AOB (18, 19). ]
As a student who has been studying marine science, I found this particularly interesting specifically for its higher abundance. Nitrification in oceans is important to the nitrogen cycle. For several reasons, including the microorganisms that use it for nutrients. I am eager to read where this study went and infer the impact it will have on things I’ve learned this far. 

I think nitrate levels increase with depth because there’s no producers consuming it as well as regeneration due to decomposition of organics and the hydrothermal vent systems.  

 I looked up what a steady state flux is and from what I read its a diffusion process distinguished by amount of atoms crossing a unit of area perpendicular to a given direction per unit of time.  So basically it is a constant concentration gradient? 

I noticed they got their water samples and examined physical properties. Chemical analyses, dark carbon fixation, and Anammox rates were measured. Presumably, this data could be used to provide evidence of the amoA activities in the water column from which the samples were taken. This also provides comparative data for the coupling between the nitrification process and anammox along side the chemical profiling. 

In Fig. 1, I would say these data points would be expected. In part a it shows nitrogen levels tend to be higher/largely distributed in greater depths due to lack of light and producers as well as regeneration through decomposition of organics by bacterial colonies. 
Part b: Oxygen is most abundant in surface waters and decreases with depth. Sulfide would increase with depth as we get deposits of decomposing matter settling down. 
Part c: light transmission is obviously one that is higher in surface waters and dissipates with depth as the light is scattered or absorbed by particles floating in the water. The particles increase with depth and in return decrease light penetration.  

Part d: Anammox bacterial cells are more abundant with depth as they love abundant ammonium and nitrite as well as dark, anaerobic conditions (which matches the graphs before). The NH4 spikes where there arent more dense colonies and decreases when bacterial densities increase.  

Fig. 4 shows that the most efficient production of 14N15N and 15N15N are converted from 15NH4 (a) where both numbers steadily increase. 
(b) From 15NO2, we get equal 14N15N, however, 15N15N was not produced. 
(c)Finally, with 15NH4 + 14NO2, we see 15NO2- produced to equal amounts, but drop off. And 14N15N had steady production, but the numbers weren’t quite there. Also showing no production of 15N15N. 

[In accord with previous findings, dissolved oxygen in the central Black Sea (43°14.9′N, 34°00.0′E) [supporting information (SI) Fig. 5] decreased from fully oxic to <5 μM at 85 m (σt = 15.83, for comparison with studies in other parts of the basin) (Fig. 1). The suboxic zone extended from this depth to 112 m (σt = 16.15) below which sulfide started to accumulate. ]
I was expecting a similar discussion to this based off of just the graphs, however, I was not as well spoken. Although I falsely assumed the graph down to an anoxic level instead of just a suboxic zone. The sulfide was predicted to increase in the suboxic zone because of the sulfur-reducing bacteria and their tendency to thrive in a lower  oxygen environment. 

[ The total NOx − production in the oxic zone (55.5 μmol of N m−2 day−1) also could not match this NH4 + loss because of anammox, which consumes 1 mol of NO2 − per mol of NH4 + oxidized. Therefore, an additional local source of NO2 − and/or an additional loss of NH4 + must be present to reconcile the difference. ¶ 16 Leave a comment on paragraph 16 0 The best candidate to explain this phenomenon is microaerobic or anaerobic nitrification, whose direct coupling with anammox, the so-called completely autotrophic nitrogen removal over nitrite (CANON), has been demonstrated in bioreactors (35, 36). At 100 m, nitrification was evidenced by the production of 15NO2 − (12.9 nM day−1) in incubations with 15NH4 + + 14NO2 − and no measurable oxygen (Fig. 4).]
The coupling of anammox and microaerobic or anaerobic nitrification was apparent when analyzing anammox rate measurements and the isotope pairings in Fig. 4. I just found this part of the experiment particularly interesting because it gave me more insight into whether NO2- in marine water columns comes from the nitrification process, nitrate reduction, or both. Directly ties into my studies in physical oceanography. 

Good question Justin, I was thinking the same thing. I think that it does generally occur when smoke enters the air, rather it be burning firewood or smoking a cigarette. PAHs  are found in a various amount of things that we tend to burn, therefore I feel a common way they get exposed to our environment is through smoke in the air.

I agree with you Dawson! I find it intriguing myself how much science and new technology have advanced allowing us to isolate and identify one bacterium from another. I find it interesting that we can isolate these enzymes and further manipulate them to see what they can do.

Why is it that more information is available on bacterial biodegradation of lower molecular weigh than of high molecular weight? What factors play a role in performing microbial degradation? Are these processes also harmful to the environment?

Is the Persian Gulf still suffering from being polluted in 1990? How does the PAH bioremediation process converge the pollutant material into a useable substance that is not harmful to the environment? Can the marine environment actually benefit from these strategies?

Why did the samples have to be incubated for a seven day period? How could they actually determine the amount of growth if the samples weren’t  being directly measured?

What does the action ” agitating in the vortex” mean?  How does this process of using gas chromatography work? How long does it take? Is this the most accurate way to calculate the culture medium?

What caused the strains show a dramatic decrease in growth after 400 ppm concentration? Why did the Salegentibacter sp. strain N7 have the maximum growth.

What is the process on how the activity and adhesion were examined? What was different about strain N1 that allowed it to have the highest values of emulsification activity and values of cell surface hydrophobicity?

What is a catechol? How would they be able catalyze? Why is catechol 2,3- dioxygenase the most important member of the meta-cleavage pathways?

What does the 16S rDNA sequence control? Why was it necessary for them to clone the catechol meta-pathway operon genes? What characterizations did the strain contain that was necessary for the conclusion that it is a good model organism?

What is the difference between the BHY plate from the plates we use in lab such as MSM and R2A? What type of plate is the BHY plate?

Why was it necessary for them to disrupt the cells? What would happen if the cells were not disrupted? How does the centrifuge remove the cell debris?

What other organisms belong to the genus Sphingomonas? Did all of the results show that the strain should be apart of this genus or were there specific traits that made them think this?

Why did the strain ZX4 oxidize only 55 out of the 95 carbon sources? What does the paucimobilis describe?

Is there any relationship with the distance of spacing between encoding genes? When it says the arrangements are similar to others, does it mean they function the same?

What is the ideal or preferred structure of the meta-pathway operon? How do the differences in structure alternate the understanding of metabolic capacities?

Are there specific advantages in being oxic, anoic, or shifting between the two?

Why weren’t they able to obtain authentic PCR products for the arsenite oxidase genes? Was there an error? Would they normally have been able to obtain this information?

Why did they take more samples and focus most of their attention on the samples that were taken from the red springs instead of the green colored ones?

Why did they use a toothbrush to remove the biofilm first? Then used a spatula to remove the biofilm for the DNA analysis?

When the light was reintroduced, why was the rate of oxidation lower than when it was introduced the first time.

Did they make intentionally want the temperatures to range, or was that one of their findings?

Are they saying that since the biofilms have already been exposed to variations in the environment and have already been introduced to sulfide and hydrogen, that they did not respond drastically to the addition of sulfide and hydrogen that they added?

Was there a specific reason that they decided to use sulfide and hydrogen in their study? Why didn’t they use any of the other elements?

What is the function of a benzene ring? So the more rings it has the harder it is to break them down?

Why does the Persian Gulf have such a high annual oil contamination? Has this increased or decreased recently? Are there any other places that have higher numbers of oil contamination?

Since the bacteria have different decomposing enzymes, is it possible to combine multiple bioremediation methods to degrade PAHs with higher molecular weights? If these methods were combined, is it possible that they could have an inverse effect & end up doing more harm than good?

Since PAH bioremediation is effective and environmentally benign, are they being done on large scales? Are the residents of the area accepting of the bioremediation, or are they doubtful and afraid of the science behind the treatment?

Is there a reason why the culture was incubated just for 7 days?

Were the phenotypically different colonies placed on the fresh  ONR7a agar together or were they separated according to different size requirements?

Is there a reason why the strains were incubated just for 15 days? Could they have endured a longer incubation period?

These numbers have a lot of variation. Could it be due to some of the strains dying off and some of them regenerating?

Were the seawater samples taken in the same area? Could differences of salinity of the seawater samples effect the number of naphthalene-degrading bacteria strains collected?

Although this form of bioremediation is helpful for marine ecosystems in the Persian Gulf, can it be helpful in more rural marine areas as well? Could the rural environment alter the type and number of naphthalene-degrading bacteria?

Why is the 16S rDNA sequence being used instead of other DNA markers?

Are there trade-offs for being able to degrade PAHs rapidly?

Bushnell-Haas minimal salt medium is ideal for studying the microbial degradation of hydrocarbons and examining microbial contamination for fuels.

Luria-Bertani medium is ideal for bacterial growth because it’s rich in nutrition.

Both of these mediums seem like good choices to produce phenanthrene cultures.

I found that Glutathione S-tranferase are detoxifying enzymes, which may help with the biodegradation of phenanthrene.

Since the non-inoculated media are used as references, are they also considered the negative control?

I found that trichloromethane can be used for oil extractions and the purification of antibiotics.

Although, Sphingomonas is a naturally metabolic bacteria that is very versatile and works well in bioremediation, some of its species can be dangerous for some organisms. It seems that its versatility could be a trade-off for safety.

Although 16S rDNA sequences have wide ranges of information about bacteria, could other genetic markers be used as well?

Although Sphingomonos is versatile and biodegrades PAHs well, it is corrosive to copper pipes. This could undo the progress of the bioremediation and possibly effect neighboring water supplies.

GSTs can be used to detoxify xenobiotic cells, which is good in some organisms, but they have the chance of causing resistance. They can resistance in chemotherapy. Is it possible that they can form resistance in the biodegradation of phenanthrene and other PAHs as well?

Periphyton contains a mixture of autotrophic and heterotrophic aquatic microbes, including cyanobacteria.

Halobacteriacaea are able to survive extreme environments such as the hot islands of Paoha Island.

Ectothiorhodospira are purple sulfur bacteria that can be found in anaerobic, marine ecosystems.

HPLC works fast, yet it is able to separate small particles efficiently.

If there were environmental variables, such as air pollution, would that need to be accounted for in the artificial medium as well?

Photosynthesis occurs during the light-incubation, using arsenite as the electron donor and arsenite as the electron acceptor.

As(III) and As (V) have inverse relationships due to being incubated in the light or in darkness. Therefore, as one increases, the other decreases.

Photosynthesis drives the light reaction, which decreases As(III). The chemolithotrophic process drives the dark reaction, which increases As(V).

Since sulfide was the more readily available electron donor than H2 in the oxidizing step, the H2 levels dropped.

While As(V) reduced to As(III), H2 donated as many electrons as it could and sulfide rates increases.

Would a weaker terminal substrate work better than acetate?

I found it interesting that both the Ectothiorhodospira and the uncultured both had the highest identity percentages, although the Archean seems to be unidentified.

I can understand how PAH exposure through respiratory uptake can cause harmful effects. I’m curious whether or not the dermal uptake is present in the same situations where respiratory uptake is occurring. I would guess higher concentrations of PAH would be required for dermal uptake.

Are inorganic nutrients able to provide energy in order to power the metabolic pathways used to degrade PAH? This could be be accomplished by harvesting energy from redox reactions or possibly promoting an excess of reactants forcing the reaction to move forward.

It would be interesting to see if the bacteria of interest in this study could be supplemented by certain nutrients to target Phe instead of Fla. After reading the entire study, it seems like this could be a possibility.

I wonder if pH 7 is the optimal growing condition for most of the cultures used throughout the experiment or if it was selected for simplicity purposes. Keeping it consistent throughout the experiment is a good standard but I wonder how it affects the growth of various bacterias.

Extractions were commonly done throughout organic lab. It’s interesting to learn about an example in which this lab technique is relevant to microbiology!

This experiment seemed to be a little bit of a side track. Was the purpose of this experiment just to compare how this bacteria degrades ILCO as opposed to other strains that had already been studied. 

It’s very interesting how the rate of degradation is not linear. I would have expected a linear line or even a line demonstrating exponential decay. There’s also a fairly large standard deviation on the R. erythropolis in MSM for a few values that may distort the line.

It’s very cool in this experiment that two methods of identification were used – one chemical and one biological. Certain functional groups such as an aromatic were identified through the degradation of ILCO and then the genes of the bacteria of interest were sequenced to find similarities to known genes. 

It is quite interesting how the expected outcome and observed outcome of this particular experiment differed. The method on this section seemed to place a focus on intending to observe the degradation of aromatics in the Arabian light crude oil, although the results explained that only aliphatic components of the oil were degraded.

I agree that more research should be done before implementing Rhodococcus sp. CMGCZ as an environmental PAH degrader. It seemed as though some of the observed results from this experiment did not match the expected results, which may mean there is more to be considered in regards to PAHs and degraders.

I wonder where there obligate marine bacteria reside mainly. It would be interesting to know their exact locations – such as being coastal or in deep waters.

I would be interesting to know how the abundance of Cycloclasticus either increased, decreased, or stayed the same following the oil spill in the Gulf. This bacterium seems very relevant to this area.

Is it possible that PAHs are more plentiful relative to other carbon energy sources in marine environments so bacterial strains have adapted to utilizing this resource more? 

Is 50 micrograms/ mL a fairly common antibiotic dosage? In other words, I’m curious as to why this concentration was chosen.

This paragraph provides another great example of how much overlap there is between organic chemistry lab and microbiology research. 

It’s interesting how orf7 in A is read in the opposite direction from every other open reading form.

orf7 was the open reading frame that was read in a different directions from the other orfs. Is it possible that this is an anomaly considering no function or obvious homolog was found?

I think it’s really neat that these genes can be introduced into E. coli as a way to better study the importance of the gene. I’m sure E. coli has been studied so extensively that it’s quite easy to observe differences.

I also found this very interesting! Only 44% similarity in amino acid sequence seems to leave a lot of room for different protein folding.

Is the PAH dioxygenase system a newer evolutionary product seeing as it shares many electron transport proteins with other redox systems? 

I’m very interested to read about why they could not find the PCR products for arsenite oxidase genes even though this specific oxidation activity was observed.

It’s pretty interesting how manipulating the oxygen conditions can influence the oxidation and reduction of different elemental forms of As.

I think this is a very cool experiment, tagging specific elemental oxidation states. I did not know this experiment type would allow for different oxidation states to be known.

I wonder if the anaerobic in the dark has a broader temperature range because on environmental adaptation. In dark locations, it could be very cold or extremely hot if near a thermal vent. I would expect light conditions near the surface to be much more consistent in temperature.

I also thought this finding was interesting. It seems to me that at 50 C some sort of denaturation occurs and prevents further activity from occuring.

From this paragraph it seems that the researchers concluded that acetate is utilized as an electron donor as a result of photoheterotrophy. 

Acetate was not utilized in the reduction of As (V) because that reaction occurs in the dark and results from chemoheterotrophy. The difference in energy sources results in different molecules being utilized in the respective redox pathways.

I think it’s pretty neat that although acetate itself couldn’t be utilized in the reduction of As (V) its byproduct, CO2, could be so radio labels from acetate are visible in As (V).

You made a very good point. Would the bioremediation of Rhodocuccus for PAH degradation purposes have unintended or potentially harmful side effects on different aspects of the environment? I did not think about that while reading this experiment, but it seems necessary to answer your question before proceeding to introduce degraders into the environment.

I’m curious about how the researchers figured out the composition of the hot spring water in order to mimic the environment. It seems like the chemical composition could possibly shift due to certain environmental effects and not be entirely consistent.

Jesse,
I also thought this part of the materials and methods was interesting. I’m curious if the researches dismissed these two clones because they suspected issues or if there is any possibility that studying these two clones could lead to possible new discoveries.

I know you can do mutagenesis to enhance the compatability for transformation and expression using E. coli. They may have found some problems with all but ArhA1A2.

In paragraph 4 it mentions that multiple clusters can be found within the genome of some sphingomonads. They seem to wanting to find out how these clusters work if the genes aren’t being regulated within the same operon.

I remember that mutagenesis can be done to aid transformation and expression in a host. ArhA1A2 may be easily transformed while the others need to be mutated first to be accepted by E. coli

By paragraph 4, It seems as if the approach is to find out how the clusters come together to work on the same pathway when the genes are not coordinated through the same operons.

Would they be using this knockout to observe  it’s effect, or lack thereof, to determine it’s link to the genes that have already been studied?

Could they have also done mutants without ArhA1A2, and also take out ArhR to see if its effects can be seen on the A4 electron transport proteins? The same could be done without the ArhA3A4 genes instead of A1A2 to assess which genes it possibly inhibits or induces.

I know that cosmids are able to utilize the cos site as sticky ends like a phage vector, but the extra phage genes are gone. Transduction happens really easily with this type of vector so maybe that plays a part.

Would this be an important find because all of the genes studied are downstream of ArhA3 and upstream of ORF6? Would this suggest that these genes are all required to function as a cluster and not be located seperately?

It seems as if AG3-69 doesnt grow on acenaphthene or its intermediate 1,8,-NA suggesting that the mutagenesis carried out removed the strains ability to oxygenate acenaphthene.

The marine environment is a huge dump for pollutants, I would assume that by now these organisms probably have many acquired genes for degradation of PAHs and would prove to be an asset to studying mechanism pathways of multiple degrading genes.

Are the bacteria being studied from the family Rhodobacteraceae too? I’m unsure if they are or if they were just pointing out another organism who displays flouranthene degradation.

Would the genes being located close by have anything to do with how many genes they are finding? It seems they have located way more genes than the previous papers.

Are these metabolites produced during degradation of a specific PAH or are they just providing a list of metabolites produced as a whole, rather than for flouranthene degradarion alone?

Could they have also checked for the lipoproteins and glycolipid’s mentioned in paragraph 15? They could have assessed whether they are constitutive or based on response to fluoranthene.

Do we know if B30 can degrade fluoranthene? If the genes which aren’t shared with B30 are important for degradation, could they just enhance the ability of the B30 genes found in P73?

I think a microarray could be a convenient way to assess which of the many genes they have found are produced under an environment containing solely fluoranthene as a carbon and energy source.

What is the significance of this gene being in the toulene/biphenyl family?

When compared to the other studies it seems this one gathered more information with the methods they used. The way this organisms PAH degradation genes were more closely organized seems to have benefitted the results of this paper.

“However because siderophores are released from cells, they are considered public goods and are susceptible to exploitation by non-producing cells”
Vibrios bacterial species utilize cooperative behavior. An environment with limited resources affects cooperative behavior by increasing costs for the producing cell, because resources are released away from growth towards cooperative functions as public goods. Siderophore production qualifies as a cooperative benefit. Many species are siderophore non-producers but have the genes to bind and utilize it. So these “cheaters” benefit from the cooperative actions of others without suffering the cost and energy themselves.

A gene knockout will cause the DNA to mutate in such a way which prevents the expression of a specific gene. Here, Vibro chromosomal mutants are generated by introducing plasmids incorporating the V. Fisheri genes iutA with fhuCDB. Assumingly, these genes enable V. fischeri to prevent the growth of other vibrio species including V. harveyi.

I researched the co-culture competition between V. fischeri and V. harveyi, under  iron-depleted conditions. Aerobactin, a bacterial iron chelating agent production allows V. fischeri to competitively exclude V. harveyi— which does not have aerobactin production and uptake genes. In contrast, V. fischeri mutants incapable of aerobactin production lose in competition with V. harveyi. 
And introduction of these genes are sufficient to convert V. harveyi into an aerobactin cheater.

Once the microorganisms completed their purpose, would they continue to be benign? If not, would measures be taken to eliminate them?

What is the next step if no naphthalene degrading bacteria can thrive in a highly selective environment? Would alternate bioremediation strategies need to be considered?

Was this step performed in order to confirm that these microbes were NP degrading bacteria, or was it performed in order to further characterize them?

How does this fact translate logistically for biodegradation of oil and pollutants? Would strain N7 be the best and/or most efficient biodegrading bacteria?

When choosing a bacterial strain to use for biodegradation, is emulsification activity or BATH a more important indicator?

How does the knowledge of optimum concentration translate to the practical use of bioremediation?

Is one of these two types of NP-degrading bacteria (gram-negative or gram-positive) more eco-friendly than the other?

Since the gene coding for the most important member of meta-cleavage pathways is located in plasmid, how difficult would it be to bio-engineer non-PAH-degrading bacteria to be PAH-degrading?

Was this strain anticipated to be a good model organism because of the characterization of the catechol meta-pathway operon genes and their neighboring glutathione-S-transferase gene?

Why is identifying the different degrading bacteria strains and their efficiency important? Based on which bacteria is the most efficient, would environmentalists introduce those strains into environments that need more bioremediation than what was naturally taking place?

I looked it up, and the way I understand it is Biolog-GN tests are a way to determine bacterial strains’ ability to grow with different carbon sources. Is this right?

Is the slant medium referenced in this paragraph similar to the slant medium we used in the previous lab?

I don’t remember reading it, and I looked back and didn’t see anything in the Materials and Methods. Is it mentioned in the paper how they determined the fatty acid composition? Would gas chromatography be used?

I read online that GST was a class of molecules best known for detoxification. Does this mean that GST is directly responsible for PAH-degrading bacteria’s ability to be used for bioremediation?

I looked back at paper 1. At 200 ppm naphthalene, the optimal strain had 89.94% naphthalene degradation, and at 100 mg l-1 phenanthrene, this strain had 98.74% phenanthrene degradation.

The fact that they mentioned this strain could degrade phenanthrene “thoroughly” made me wonder if it is possible that other degrading bacteria only partially degrade phenanthrene.

Does this meta-pathway arrangement account for this strain’s efficiency at degrading phenanthrene?

Why would they have not been able to obtain authentic PCR products for arsenite oxidase genes? Were they not able to design a primer suitable to these genes?

For respiratory As(V) reduction, is As(V) the final electron acceptor? What is the electron donor?

This question was answered this week in lab. When this paper was written, the GenBank only had a few sequences to design a primer. The primer designed was probably not suitable for these particular genes.

What does this mean that these clones failed to show significant similarity to anything in the GenBank database? Does this mean that no one has annotated these genes yet?

If the arsenic speciation and acetate concentration were measured by HPLC, was the purpose of radioactively labeling them to make them detectable?

Why did the reduction/oxidation rates diminish each time?

Why was the sulfide condition not tested for acetate assimilation?

The percentage is above the baseline number given with no additions; therefore, assimilation using acetate was stimulated by the addition of As(V) and As(V)+H2. It was not stimulated by the addition of H2,killed. The number for the addition of H2 is too close to the baseline number to give a definite yes or no.

So in the light, As(III) acts as the electron donor (what water would do in aerobic photosynthesis) in anoxygenic photosynthesis, and in the dark, As(V) acts as the terminal electron acceptor in anaerobic respiration (how oxygen would act in aerobic respiration)?

I think you answered this question in lab today, but I cannot remember what you said, and I didn’t write it down. In figure 4, why was there a slight reduction of As(V) when no electron donor was added?

Why is it that this gap in knowledge exists for marine-sourced degraders? Are these harder to isolate and/or sequence?

What determines which intermediate the PAH will be transformed into?

I Googled paired-end sequencing; is it just sequencing the DNA segment from both ends? Why would they choose to sequence the DNA this way?

Are suicide vectors always used when performing recombination, or is this particular to the cre-lox method?

Why would this strain possess genes for chemotaxis, yet be non-motile/not possess genes for flagellar proteins? Why have they not lost the genes for chemotaxis?

Only one pathway is referenced in this paragraph; however, would the strain utilize one of multiple pathways depending on the intermediate?

When ring hydroxylation is labeled “the most difficult catalytic step”, is this meaning it’s the most energetically demanding?

How do PAHs compare to other compounds as a carbon source?

I don’t understand the significance of this gene being of the toluene/biphenyl family?

To further study the functions of the catabolic genes. would they perform more gene knockouts? What else would they do?

Why does the presence of heavy metals inhibit the enzymes? Is it a form of noncompetitive inhibition where the metals are interacting with the enzyme?

Why is information on this topic limited? Has little research been conducted, or has research been conducted and not yielded a lot of information?

So for this experiment, the amount of PHE degraded should decrease as the concentration of Cu(II) is increased because the copper hinders the activity of the degradation enzymes. Also, will they test each initial concentration of PHE with each concentration of Cu(II)?

For the blank control, would PAH-RHD and C230 genes likely not be transcribed at all?

From what I understand, they’re interested in the effect of copper because the PHE needs to be degraded because it is a pollutant; however, heavy metals (like copper) can hinder the activity of the enzymes necessary.

Why would the biodegradation rate of PHE in PAHs-contaminated soils improve in the presence of cadmium and arsenic?

From what I understand, was the only unexpected result that the transcription of PAHs-degrading genes was promoted by high level of Cu(II)?

I googled a different comparison of cluster structure, and in that one, the length of the arrows corresponded with the length of the ORFs and the direction corresponded with the direction in which it was read.

Are the PAH-degradation genes frequently dispersed because they’re often acquired through horizontal gene transfer?

The way I’m understanding this paragraph, degradation enzymes have been located and described for a fluorene-degrader; however, it’s gram-positive. Fluorene-oxidizing enzymes have been located and described for gram-negative degrader; however, the hosts doesn’t grow when fluorene is the sole carbon/energy source. This paper will be the first that locates and describes fluorene-degrading enzymes for gram-negative degrader that can grow when fluorene is the sole carbon/energy source.

So for this overexpression experiment, the dioxygenase genes (flnA1-flnA2) were cloned into the overexpression plasmids from paragraph 6, allowed to grow, and then the products from that were extracted and measured using GC as in paragraph 9?

A follow-up to my last question: is the SDS-PAGE also a step in this experiment? Do they perform SDS-PAGE before GC? I get a little confused with the methods not being in order of the experiments.

So if we were to do expression cloning for our project, how would we test if the protein was made? Would we be able to do GC or SDS-PAGE?

The differences in expected and observed sizes mentioned here – are these significant? I can’t tell from the paragraph if this is important.

Should we focus of substrate range? Because I found this paragraph very confusing.

They performed RT-PCR of flnA1 and flnA2 using cloning vectors to see if they were transcribed more in strain LB126 when grown on fluoranthene and not when grown on glucose. This was the case. They then used expression vectors to see the enzymatic activity of these same genes.

Phenanthrene is one of the preferred substrates; however, it cannot be used as a sole source of carbon and energy by strain LB126. Could this be because, like you mentioned in one of your comments in the introduction, the initial enzyme has the relaxed substrate specificity to accommodate phenanthrene, but the later enzymes in the pathway do not?

Looking at the product concentrations in Table 3, it seems as though fluorine and phenanthrene were drastically the preferred substrates.

This paper is looking at the degradation of carbofuran even though several metabolites cause effects on the reproductive system in female rats. Is this because biodegradation is the only way to eliminate carbofuran even if there are negative repercussions?

Sphingomonad strains seem to appear in papers for the degradation of many different substrates. Is this because Sphingomonas is just more capable (than other bacteria) of degrading a wide variety or is it because this is just what is isolated when researchers collect samples?

So for this experiment they performed random mutagenesis on the strain KN65.2 to look for the loss of function (degradation of Carbofuran)?

The gene that when interrupted resulted in the loss of Carbofuran degradation was identified, and then sequenced to design primers in order to perform RT-PCR to confirm function?

I would think this result would be a surprise. All of the degradative pathways we’ve looked at so far in these papers have been inducible if I’m remembering right. Usually glucose would be the preferred C source and then the alternate pathway would be induced when glucose was used up.

The mutants for the most part exhibited reduced or slower mineralization rather than the abolition of mineralization. Is this because the pathway was shown to be constitutive?

So they selected mutants and assigned them to five groups based on whether how they grew, degraded, and/or mineralized carbofuran; they then selected ten of those mutants and verified whether carbofuran phenol (what they think is the first metabolite of degradation) is detected during the process?

How rare is this deviation from the expected regulation of catabolic genes?

So this paragraph is saying that although they used plasposon mutagenesis to identify genes responsible for carbofuran degradation, they had to be careful assigning loss of phenotype to specific genes because the loss of phenotype could be a result of polar effects instead?

Group I: C1 metabolism genes affected -> C1 metabolism is predicted to be related to conversion of methylamine -> hydrolytic release of methylamine moiety is first step in carbofuran-degradation

So for group I, the delayed/decreased growth rate on carbofuran is explained by C1 metabolism genes being affected, which in turn affects the first step of degradation, or am I misunderstanding?

Does the ONR7a medium select for NP degrading bacteria more efficiently than the MSM we used in lab?

I’m very interested to see specifically how they collected the bacteria and recreated an environment similar to their habitat. If the bacteria proves to be successful, how long will it take before they can actually start using this bacteria in the wild?

I figured that the oil eventually floats to the bottom of the sea floor, but I always wondered how it impacted the organisms down there since it’s already a pretty extreme environment. If bacteria is used for bioremediation here, would the goal be to remove all of the oil or just decrease the concentration?

These methods are super dense, but I love that I can recognize the primers and semi-understand the processes. Why did they use “near complete 16S rRNA gene sequences,” I feel like it would make more sense to use fully complete sequences?

What is the special characterization of ONR7a agar plates and why was it selected? 

I’m confused. I thought only Eukaryotes have mitochondrial respiration and Bacteria has respiration through cell membrane. If Naphthalene hinders mitochondrial respiration, it would not do anything for the bacteria, right?

If we were to use one of the common naphthalene degrading bacteria against a PAH compound that has not been well characterized and its not able to compete with the bacteria that’s polluting the water and ecosystem, will the selective pressure make the bacteria that we are trying to fight worse and even harder to get rid of?

Since the N1 and N2 strains has the most growth, does this mean those are the two stains that are causing the most pollution in the Persian Gulf? If so, would bioremediation of these pollutants significantly reduce the pollution in the water?

I was looking at the phylogenetic tree tables, and I was wondering if the originator of all the stains was targeted with the bioremediation approach would all the strains that followed be eliminated as well?

Since the different genera produce biosurfactant and bioemulsifiers that can uptake and dissolve the component of the crude oil that is contaminating the Persian Gulf already, is there a need to anything else? It seems as if the ecosystem is fixing itself.

Would it be better to have more N1 and N7 strains in the Persian Gulf so that more of the oil contamination can be dissolved?

It seems like they should focus on the obligate marine bacteria in coastal marine environment versus terrestrial habitats since this bacteria has only partially been studied. They are studying something that has already been extensively characterized.

I don’t feel like the author really told us the importance of their study as opposed to all the other studies on this topic. What will this paper tell us that the others didn’t ?

What makes ampicillin and kanamycin the best antibiotics to add to the media in order to select only the bacteria with recombinant genes?

Why was the library replica plated onto LB agar plates with kanamycin and not ampicillin also?

If the large subunits do not have small subunits to pair with, what will happen?

Since they did not have any small subunits to make their dendrograms, did they just use the previously published amino acid sequences based of the large subunits found in their experiment?

Is there any benefit to having gene clusters responsible for PAH degradation on the plasmids rather than the chromosomes and vice versa?

What does it mean for a gene product to fall outside the major cluster?

Why does the rate of oxidation and reduction of the samples get slower each time the light and dark regimen is shifted?

Is the reason why the killed controls neither oxidize or reduce because of their proteins being denatured by heat?

How did they come to the conclusion that As (III) was oxidized due to anoxygenic photosynthesis? They should not have been able to conclude this just from this one figure. Couldn’t the  oxidation be due to chemolithtrophy since it can occur in the light or darkness?

So does this mean that As(III) will continue to reduce due to the microbes losing their ability to oxidize and not due to the microbes being incubated in the dark?

Why do the 16s genes have to go through PCR amplification in order to be identified? If all PCR amplification does is make more copies of the gene, why can’t the single gene be identified?

Why did the samples include both contaminated sea water and marine sediment ? If the sea water and the sediment was collected at the same location wouldn’t it have the same type of bacteria? Why the need for both?

Why were they unable to obtain authentic PCR products for the gene even though they observed aerobic oxidation activity? Will they elaborate more later on why they were unable to obtain PCR products?

Since the Ectothiorhodospira bacteria is dominant in the biofilm, does this mean this bacteria has the best resistance to arsenic opposed to other bacteria ?

Does arsenic support anaerobic respiration since the medium they used was oxygen free ?

What is the signicance of the shaking of the incubated tubes ? Does this have something to do with the electrons being donated ?

What does PAHs stand for? Do these compounds referred to in this paragraph still pollute the environment badly in the U.S. because of sewage waste water and home sewage?

How much cleanup has bioremediation done since the oil pollution during the Persian Gulf War? How affective is PAH bioremediation really since there is already so much damage?

When did researchers realize that cyclic aromatic compounds are harmful to mammals? How long has this fact been known or in literature?

I know that in lab we pour agar. Do you turn solid agar into liquid for GC purposes? How would you turn solid agar into a liquid to extract the different compounds mixed in it?

What is starch hydrolysis? What would be the significance of starch hydrolysis in this experiment?

Why were 18 out of 54 strains isolated? What happened to the other strains that made them not have an adequate growth rate? Could some of these strains be cross-contaminated?

What is a shaker incubator? Why wasn’t the microbial growth and naphthalene biodegradation monitored every day instead of just 15 days after? What made the experimenters choose 15 days of growth and then monitoring?

It would be interesting to read about why researchers think that Gram-positive bacteria might play a role in naphthalene degradation in the highly variable environment of oil-contaminated sediments. What brought them to these conclusions?

Are there any other regions that have a high diversity of naphthalene-degrading bacteria besides the Persian Gulf?

In this study, did the researchers choose to study Gram-negative bacteria instead of Gram-positive or was that just a coincidence?

How do researchers know which bacteria types are best for bioremediation? What tests and check-marks were used to come upon such assumptions?

Was phenanthrene-degrading bacteria chosen to study because is an aromatic compound? Are we going to discover/read if naphthalene-degrading bacteria are better or worse than phenanthrene-degrading bacteria?

What were the identifications and characteristics of distinct colony morphology? What were the researchers looking for?

Why did the amplification have to go through 29 cycles of denaturation? Was is the “extension” process?

Was there only one isolate that was found that would be able to degrade phenanthrene? This is different from the last article because those researchers found several strains of bacteria capable of degrading naphthalene. Does this possibly mean that phenanthrene is harder to degrade because there was only one isolate found?

There are quite a few aromatic compounds that Strain ZX4 can use for carbon and energy sources.

There are quite a few aromatic compounds that Strain ZX4 is able to use as carbon and energy sources.

Out of the 95 carbon sources, what were “wrong” with 40 of the sources to where Strain ZX4 could not oxidize them?

I am interested to know what was the two percent difference between strain ZX4 and strain UT26, since they had a 98% similarity.

In this paragraph, it says that strain ZX4 could be a potential strain used for bioremediation. What other types of tests and research would be need to know if this strain could be used for bioremediation rather than just having the potential to be used?

Should the initial step involving meta-cleavage of catechol have been catalyzed by PhnI, but it was done by PhnH instead? I am a little confused on the first statement.

What is the salicylate pathway?

Why did the researchers not focus on the cycling of arsenic under oxic conditions too other than just focusing on anoxic conditions?

Why were the researchers unable to obtain authentic PCR products for arsenide oxidase genes? Will this be discussed more in the study?

Why were the researchers unable to obtain authentic PCR products for arsenite oxidase genes? Will this be discussed later in the paper?

Why were the slurries stored in the dark before being used in experiments?

Why were only groups containing two or more clones sequenced?

Why did the rates not change after constant shaking of the samples? I would think that after they shook the samples to increase oxygen exchange then the rates would favor oxic conditions.

What is a hypersaline environment?

So with increasing ever 100h of incubation, does the rate of As(V) reduction further diminish every time?

Is acetate a key terminal substrate in aerobic process or is it just one in anaerobic processes?

When this paragraph says, ” We recently proposed that the respiratory arsenate reductase of strain PHS-1, and presumably the very similar sequence found in the biofilms, functions in vivo as a de facto arsenite oxidase”, what does “in vivo as a defacto arsenite oxidase” mean?

Will the researchers continue to search out this unexpected result? Will they research out about why there was an observed aerobic As(III) oxidation carried out by a novel mechanism? Will they see what this “novel mechanism” is?

About how long does the process of bioremediation take in order to efficiently eliminate all of the environmental pollutants in the area?

I am curious to know the effect of more complex PAH’s on certain bacteria. Would they be able to efficiently eliminate environmental pollutants or would the by-products be malignant to the environment and people?

Why is it necessary to transport the seawater on ice when the samples were gathered from just 15cm below the surface? I feel as though the ice would bring the temperature above normal conditions therefore having effects on the organisms that live within it.

How did they know what temperatures and times to hold the PCR at during the 35 cycles?

Why does the increment pattern cease at 400ppm concentration?

I am wondering the same thing. Is the GC-FID method some sort of gas chromatography method?

What factors play a role in the reason for the high diversity of naphthalene-degrading bacteria in the Persian Gulf besides the pollutant. If this is the only reason, could the same experiment be done in a different part of the world that has a similar pollutant?

I am curious to know what the optimum concentration for naphthalene-degradation would be for isolates found in different areas besides this specific area in the Persian Gulf.

What characteristics of the strain made the researchers anticipate that it would be a good model organism for their experiment?

What do they mean when they say the catechol 2,3-dioxygenase mirrors the taxonomic grouping of the host bacteria? Does this mean that it possesses similar characteristics of the host bacteria?

What is the purpose of amplifying the 16S rDNA? I see that you are basically breaking the strain down, building it back up again, and I think making it longer? But what purpose does that serve?

Why use the supernatant for enzyme assay rather than disposing of the supernatant and using a different enzyme? Does this result in different enzymes being assayed?

What are BHY plates? And what are some benefits of using these types of plates over others (i.e. R2A, MSM, etc.)?

I found that the Sphingomonas paucimobilis strain is commonly found in hospital equipment such as vents and can cause disease. I think it is interesting that a strain with these characteristics also possesses positive characteristics in the sense that it is able to degrade phenanthrene which is found in cigarette smoke.

Is the pH range for this strain considered a wide range or no? I would assume that a 2.5 difference would be considered a wide range. This should allow for a greater variety of conditions that this strain is able to grow and survive in compared to other strains.

I think its extremely interesting that this ZX4 strain offers an environmentally friendly alternative to creating indigo instead of having to harvest it from plants. Another positive capability of this strain is its ability to be used in bioremediation. Two environmentally friendly uses.

How would the researchers go about obtaining information on the structure of the meta-pathway?

I looked up what xenobiotic compounds were and have concluded that the ability for the ZX4 strain to degrade these compounds is yet another environmentally friendly characteristic that this strain possess. What are some environments that xenobiotic compounds can be found in? How can this strain be introduced in those environments in order to rid the environments of those pollutants and how long will it take for them to be completely eliminated?

Did the researchers decide to focus on examination of the cycling of arsenic under anoxic conditions because of how the coculture responded in the manipulated laboratory conditions?

What makes the biofilms used in this experiment a good model system for this oxidation/reduction reaction?

I was wondering the same thing, but also what made the green-colored springs and small ponds so rare compared to the red springs?

How did they keep track of the tubes that were shaken before they were inoculated and the ones that were not? Is there any specific reason for shaken some of them and not shaking others. I am curious to see the results of the tubes that were not shaken and and how they may differ from the tubes that were.

I believe they did not include single clones in the sequence analysis because it would not be financially worth it.

So, since the rates did not change can it be said that the conditions of the environment, whether its oxic or anoxic, does not play a major role?

How could they test to determine what the functional gene is since it is not aoxB even though it shares similarities?

How could the authors know for sure whether the capacity for aerobic Arsenite oxidation is due to the shallowness of the pond or if it is another reason?

What are some ways that the researchers could determine which biofilm microbes that were associated with the arrA amplicons?

What is a novel mechanism?

Do all regions have unique PAH-degradation patterns or is only the Nakheel region unique from the others? I would assume that if each region has a unique pattern, then removing PAH’s completely would become an extremely difficult task.

They mention how the concentration of the PAH is important for removal. Are the co-substrates like yeast used for this purpose, or how do they go about lowering the concentration of the PAH?

I am a bit confused by this paragraph as to why they are testing the sediment samples ability to withstand the stress. What does it mean about the sample if it is not able to do so? 

What is the advantage of constructing the phylogenetic trees? Could they use information from them to determine similar degradation methods that have already been determined, or would this not be possible with the unique PAH samples they have found?

If bio-emulsifiers “act like surfactants” and the water solubility of LMW PAHs is improved when surfactant-production is present, could these bio-emulsifiers ever be modified to be useful for both LMW and HMW PAHs?

I know they mention that there is no significant difference among the 3 sites, but table 1 shows that there is a slight difference in temperature at all 3 sites. Does this have any impact on the concentrations of the different nutrients found at each site? 

I think it is pretty interesting how the table shows that none of the isolates showed ‘excellent growth’ when it came to a PAH substrate. They were all ‘good’ or ‘poor.’ Could that mean there are much better strains to use for bioremediation? 

If all of the isolates were either at 100% similarity or very close, then why would they not look at different sequences aside from the partial ones they did? I guess I am wondering how they are able to draw any unique conclusions here if all the results are almost identical. 

My question is a bit related to both papers, so if hydrocarbon contaminants can be toxic in the waters, wouldn’t evolution have created better mechanisms within microorganisms for treating them? Or has not enough time passed yet because most of these pollutants are more recent and man-made? 

Does the temperature of the environment have any effect on the hydrocarbons that are present? 

It says that the cores were stored for subsequent use within 2 weeks. Does the storage time have any effect on how the following experiments on the core go? Or would the results be the same as one day later or two weeks later?

16s rRNA sequencing has been in both of the papers we have read. Is is just a common thing in general for sequencing or just for use in studying types of microorganisms/PAHs in these types of marine environments?

All of the genera in this paper are different from the last paper. I just find it kind of interesting how there seems to be a lot of species with potential of being PAH-degraders but it is still a huge problem in the environment. 

I am a bit confused on obligate/non-obligate. I know what those mean in general but does it mean here that the obligate PAHs only responsibility is to degrade them while non-obligates have other uses in addition to degrading?

How did they select these PAHs as the ones to test the incubations on? 

What exactly is a singleton sequence and why did it mean that those two weren’t further analyzed? I tried to google it but all the answers were a bit confusing. 

They say that Se-reducing bacteria is ubiquitous so is the high concentrations of toxic forms of selenium natural or caused by something?

I am a bit confused because the previous paragraph says only two reductase genes have been characterized but this paragraph talks about a separate one, or am I reading this incorrectly? 

How do putative knockout mutants differ from other mutants aside from the fact that they didn’t grow on the LB agar?

I am a bit confused here. So they are trying to screen the E. cloacae library and they need a strain that doesn’t have selenate reductase activity? Why is that?

So were FM-1,2 and 3 all just separate isolates of the mutated SLD1a-1?

So the pECL1e clone was chosen for further characterization because t he pLAFR3 showed no ability to reduce SE(VI)?

So just to clarify, the fnr gene was inserted into the S17-1 and that is responsible for oxygen-sensing and therefore responsible for reducing Se(VI)?

So the Se reducing ability is not related to presence of oxygen? The presence of oxygen only applies to the fnr gene? 

After reading the introductory paragraph, it’s clear that individuals exposed to PAHs mainly suffer from symptoms relating to the respiratory system. My question would be which type of exposure yields worse symptoms, dermal uptake or respiratory uptake? And what are the various types of preventive measures that have been put in place to avoid exposure to PAHs?

The article briefly mentions how both LMW and HMW microorganisms are used as a means of bioremediation against PAHs. However, does one prove to be more efficient than the other? Is the reason LMW microorganisms are more commonly used in studies because they’re more easily accessible or because they’re the better option of the two?

In paragraph 2.3, the procedures detail how they let colonies grow on MSM plates for approximately two weeks before the isolating and sequencing the cultured colonies. However, in paragraph 2.4 the procedures mention how they only let the naphthalene degrading bacterium grow on MSM plates for 8 days. Why weren’t the plates given 2 weeks to grow? Did they not need as much time to grow?

What difference did using the YMSM plates use for the experiment? What was the advantage of using YMSM over regular MSM plates?

For the MSM plates, why were the percentage degraded for Nap and Phe so close compared to the Fla? Are the chemical structures for Nap and Phe somewhat similar? And if that’s the case, shouldn’t they also have similar percentages degraded when placed in the YMSM plates? 

Given that the degradation percentage of crude oil was so low, would it make sense to search for an alternate degrading bacteria? Or are these results typical to what you’d expect when attempting to degrade crude oil via bioremediation over such a short period of time?

It’s definitely interesting that CMGCZ was better able to degrade Fla more efficiently than a structure like Napthalene. Especially considering Napthalene is only a two ring PAH while Fla has four rings.

It sounds like the addition of YE in a medium didn’t lead to a significant change of results. Why was YE chosen to be used in the first place?

Given that we’ve already read about the bacteria Fla, I wonder which one (Fla or Cycloclasticus) is a more effective means of bioremediation

I accidentally compared Fla and Cycloclasticus.. when Fla is a PAH and Cycloclasticus is a degrading bacterium. So my original post should’ve asked if Cycloclasticus is a better bacterium at degrading Fla than the original bacterium we studied, Rhodocuccus.

Of the PAHs being studied, I wonder which ones are of a low molecular weight and which are of a high molecular weight. Which will be easier for the Cycloclasticus strain to degrade? Does it have anything to do with the marine environment?

The LB agar plates are the same used from the first experiment we read about for Blog Summary 1. To me it seems to be a standard growth plate used in labs everywhere.

After reading the introduction of the paper, I thought that the mission of the experiment was similar to the first blog we read; therefore leading to similar experimental methods. However, the further I read into this paper, the more complex this experiment seems. I’m interested to see how the results vary between the two experiments.

I wonder what the specific location of the 10.5-kb SauAI fragment was. And if it was in identical locations in both the pH1a and pH1b constructs. In Experimental Designs with Kroetz, we learned that the location of a specific sequence was key in determining its characteristics/influence on the DNA.

Does this paragraph hint at the idea that there’s a specific sequence of DNA that gives microorganisms the ability to degrade PAHs? It would seem to be the case considered there were 7 cases of PAH-degrading constructs found with similar if not the same sequence.

Why were the chemical structures from Fig 4.4 converted to a different form? What was the purpose, especially considering the form they were transformed to were stated to be more structurally unstable.

So does this hint at the chance that having a shared dioxygenase system is a contributing factor of the increased ability of a strain to degrade a PAH?

This may have went over my head but what is an ISP and what processes does it typically carryout?

Does the ability to degrade PAHs go up as more dioxygenase subunit genes are found in a strain? That’s what this paragraph suggests, yet you would think that the attachment of additional DNA would have the opposite effect.

I wonder if a bacteria’s ability to metabolize substances that are normally considered toxic to organisms means that its categorized as an extremophile. This would also make sense because the paper discusses how some bacteria with this ability live in extreme environments such as hot springs.

I wonder if the inability to obtain PCR products for arsenite genes has anything to do with the specimen being collected from an extreme condition (i.e. a hotspring). Why weren’t samples from somewhere else used and would they have likely yielded the same results? Interested to finding out what the next step in their experimentation was.

I honestly was wondering the same thing Justin. I wonder if the details given about the location were significant  in a way that isn’t obvious to a reader. Because to me, the details seemed pretty generic.

Why was there no type of pattern in the intervals that the samples were taken? It seems odd to me that they retrieved 2 samples in 2008 only 2 months apart and waited 6 months to retrieve another one in 2009.

It’s interesting reading about how exactly the researchers set up and prepared their experiment. It seems as if they’re was plenty of room for human error. Which may explain the inability to produce authentic PCR products for the arsenite oxidase gene later on in the experiment. I wonder if there is any correlation.
 

I wonder if there are any major similarities between the bacteria strain compared to the archaean strains. Because to me it seems like the bacterial strain would have to have some of the same properties in order to maintain survival in an environment typically known to be home to extremophiles (aka archaea).

It’s cool seeing how the slurries were able to go back and forth between oxidizing and reducing arsenic based on their environmental conditions. I wonder if the amount of exposure to either light or darkness affected the speed of oxidation/reduction.

This paragraph answers my question that I wrote under paragraph 5. I’m curious as to why the temperature range for reduction is so much bigger than that of oxidation.

How can it be determined that acetate was used as an electron donor when in the paragraph above it’s stated that there wasn’t any observation of As(V) reduction with acetate?

Would the next step be to try to make new and more specific primers in order to better detect arsenite oxidase genes?

This paragraph is important if the reader has no background knowledge of what PAH is or the dangers of it to the environment. It not only breaks down the toxicity of PAH, but also discusses why the chemical is so hard to remove. 

This paragraph highlights the overall purpose/goal of the experiment. In the previous paragraph, it is noted that there are bacterial strains capable of surviving in the toxic environment of the PAH stricken Arabian Gulf. The goal of this study is to isolate the bacterial strains growing in the PAH environment and further study their functionalities. A better understanding of these bacteria would help future bioremediation efforts across the world. 

I noticed that the researchers used 16S rRNA for constructuing the evolutionary relationships of these bacteria. My question for this portion is; is sequencing the 16S rRNA more effective than the 23S and the 5S rRNA segments? 

What makes the temperature of a particular sample site important? Was this to ensure that the sample area could sustain the PAH degrading microorganisms or is it something more technical? 

Does the fact that Isolate LC having 100% similarity to P. aeruginosa imply that it is definitely the same species?

What is the reasoning behind the difference in degradation abilities of the same organism on different PAH compounds? Are the compounds so unlike one another that certain bacteria will specialize into a particular PAH compound, rather than PAH’s as a whole?

The PAH isolating bacteria isolated from soil on the surface would have little to no effect on oil degradation at such low depths, could this class of organism be understudied due to the expenses involved in isolating it?

Since these areas are ridden with hydrothermal plumes, could one expect to also search and isolate archaea PAH degrading organisms, as their extremophilic nature would make them a dominating organism in the ecosystem?

Could removing cores from their pressured environment have changed the ecosystem too drastically for the organisms to preform at the same compacity?

Would an PAH degrading archaeon that could sustain higher temperatures preform better the closer one got to the hydrothermal center? Could a selection of two microorganisms capable of degrading PAH, one being a dominant PAH degrading bacteria and another being a more niche archaeon work together since they would occupy separate spaces along the seafloor.  

Could Cycloclasticus have an impact on other deep sea enviroments with abundant sediment surface hydrocarbons? Would Cycloclasticus preform better if it was deposited alongside Halomonas, Thalassospira, and Lutibacterium in a new enviroment?

Is this “rusty-yellowish” coloration able to be observed at the site of extraction? It would be interesting to compare the coloration and presence of oxidized intermediates in the original location and the studied organisms. 

The prosthetic heme group acts as an Oxygen binding site in hemoglobin of animal cells. Why would an organism utilizing anerobic metabolism of Se require the capture of Oxygen?

Will the selenium metabolic mechanism always produce a similar mineral product, or will a selenium metabolic mechanism from a different organism produce a different mineral product?

Genome sequencing was clearly important in order to identify the presence of known selenium metabolic genes, but would a transcriptomic/proteomic approach be useful in this experiment when searching if an organism contains rna/enzymes capable of metabolizing selenium? 

What would be the proper procedural steps to take if the organisms were capable of selenium metabolism, but none of the three strains contained the gene that they were searching for?

When looking at the graph, it is notable that pLAFR3 expresses a relatively constant Se (VI) Reduction, whereas the reduction rate of pECL1e gradually decreases over time.

This experiment was preformed to confirm that the fnr gene is essential for the reduction of Se (VI) to Se(0) in E. cloacae SLD1a-1. The results from this experiment have shown the importance of the fnr gene. 

What causes this nirate inhibition on selenate reductase activity. What can be done to further study this complication?

The FNR gene is essential in the detection of Oxygen concentrations, but is also essential to initiation the degradation of Se(VI). Without FNR, no Se(0) precipitation will occur.
If there was a way to mutate FNR to make it sense another molecule OTHER than oxygen (i.e. mutating the gene so that it detected the presence of nitrogen), could you make a pathway that was dependent on the presence of another gas? Are there other similar pathways in nature that are dependent on such gas concentrations. 
Apologizing for all the questions in advance, this is just a fascinating concept. 

Many questions arose when I first started reading this article in Paragraph 2. For starters, it states that a main concern to Antarctica’s natural region is being damaged by the increase in tourist and other people visiting the continent. When I googled how many people are annually visiting Antarctica I was shocked by the numbers. It states that when Antarctica first allowed for people to visit in the 1950’s only a couple hundred a year visited. Recently, more than 56,000 tourists visited Antarctica during the 2018-2019 season. That is a huge increase in only 60 years for a continent that is mainly an ice wasteland. I can completely see why native Antarctician’s are becoming more afraid of their climate due to the increase in visitors. My guess is that more people will result in warmer areas and increasing the use of various fossil fuels to bring people on and off the continent. 

We probably can all agree that various Oil spills and burning of fossil fuels has in fact caused the Earth’s atmosphere to become warmer and also resulting in Antarctica losing areas of ice each year. [ Oil contamination can generate detrimental changes in soil properties, including modifications in maximum surface temperature, pH, and carbon and nitrogen levels (Aislabie et al., 2004).] From this quote in the article, I was shocked to see so many different types of Proteobacteria being affected from oil contamination and my overall response to this is, well that cannot be good, and its not. From my understanding of paragraph 2, Antarctica are in real trouble when it comes to oil and other fossil fuel contaminations and if it is not improved, many of the bacteria essential for Antarctica’s nature could no longer exist and the entire world will feel its effects. 
 

[53 cultures that metabolized phenanthrene were selected, and the concentration of PAH quantified as described below. The three highest metabolizing strains were then selected for further studies.]
It is very interesting to me to see how these scientists are creating 53 different cultures that will ultimately metabolize phenanthrene and their PAH concentration and pick upon the nest three with the highest metabolizing rate. I believe out of the 53 some might even be rather similar, and if you have multiple that are very similar, it must be quite challenging to select to best options to experiment on?

[Briefly, non-polar compounds were extracted from culture media using two volumes of hexane and vigorous mixing for 60s]
From the Phenanthrene quantification section in this article, i wondered why it was so important to use two volumes of hexane and vigorous mixing for 60s in order to extract the non-polar compounds. So when I browsed the internet trying to find an answer to my question, I quickly realized that the main reason behind it is to just clarify the non-polar compound you are trying to extract, while it also helps purify the substances being extracted. This is also why the procedure is repeated for a total of three times in order to make sure the non-polar compounds were fully and properly extracted. 

The main part of paragraph 3 that stood out to me was the similarity in how the glucose in both the unexposed and the diesel-fuel exposed were realitivly the same. On another note, the other 2 aspects of the graph showed increases in CFU for the control group as well as the phenanthrene. To me this shows that an increase in phenanthrene is present in the diesel fuel sample.  

[Analysis of optimal growth temperature revealed that all isolates exhibit maximum growth rate at 28°C (Figure 2C), indicating that these strains are psychrotolerant rather than psychrophylic, a behavior we have seen in previously isolated Antarctic strains]
The main question that I derived from paragraph 4 was that why were all of the samples experience maximum growth at 28 degrees Celsius? It is just strange to me to think that the variety of different samples all hit their maximum growth rate at the same temperature rate. As stated in the paragraph as well, this means that the strains are psychrotolerant from our previous examples from the Antarctica stains. 

One thing that stood out to me in this paragraph was the fact that Antarctica is a certain region where bringing over foreign organisms is forbidden in the continent. Like the article continues to say, this increases the use of native bacteria samples and also will help narrow down decontamination techniques and strategies. This is something I never knew was true and can fully understand and see where Antarctica are coming from in the fact that they don’t want to cause anymore unwanted bacteria to grow. 

To respond to your question Rachel, I agree with you on the point that they were just trying to achieve their main goal, but I also believe because there are so many different varieties of bacteria in the area they are testing, it is more likely that they will not have a completely isolated degrader. 

After reading this paragraph, the statement about the variety of competitive strategies intrigued me the most. From raid culture of limiting resources, to contact-dependent delivery of other toxic, can ultimately effect competitive cells to change. 

I was confused on the part when the writer explains how species can produce siderophores that will have a competitive advantage to other cells, but what eventually caught my eye was how when these cells are released they are public goods and are exploited by non-producing cells. I found this very interesting and believe to be very important for the upcoming experiment. 

There is so much going on in this paragraph it is difficult to grasp what the author is trying to present. Other than all the equipment and type of bacteria’s/ antibiotics. What was the point of so many different samples that had to be added? What was the main advantage for doing so?

I found it interesting that the scientists did not filter the remaining mutant just for simplification purposes, but also how come ploymyxin B was added to prevent any more growth. I’m curious to understand how this actually works. 

[Finally, the presence of V. fischeri ES114 culture fluids prevented the growth but did not kill V. harveyi. While below the level of detection by OD600 measurements, a small increase in V. harveyi cell density could be detected by counting colony forming units (CFUs) (Fig. S2D)]
I found this sentence to be very interesting to me and caused me to form a question in my head. I was wondering why does this culture prevent the growth, but still does not kill the V. harveyi. Usually I would assume that if a process or anything is stopped growing by an outside force, majority of the time that product will eventually die off. 

[It was curious that growth inhibition of V. harveyi occurred only when culture fluids were obtained from V. fischeri ES114 grown in minimal marine medium but not in rich medium (Fig. S2B). ]
I found this interesting as well, to the point that the inhibitions were only obtained from V. fischeri ES114 when grown in medium but not in rich. I believe this is because the medium was the minimal required for the product to actually grow, and too much medium like in rich, the product might not like and abundance of medium and could harm or damage the process. 

[Alternatively, the genes encoding IutA and FhuCDB could be acquired by horizontal gene transfer. Horizontal transfer is known to have distributed aerobactin genes across multiple vibrio phylogenetic lineages ]
This was a statement that caught my eye and I thought was quite interesting. I did not realize that horizontal transfer is known to distribute aerobactin genes across multiple lineages. I believe that this is actually necessary for the given experiment, and without them, it could be hard to encode these genomes, like with siderophores for an example. 

[It could be beneficial to repress iron uptake under oxidative stress because ferrous (Fe2+) iron reacts with hydrogen peroxide in the Fenton reaction to generate harmful hydroxyl radicals that damage DNA (Imlay et al., 1988).]
Another statement in this section that I found interesting was the fact that more iron uptake under oxidative stress can be better beneficial. This is very interesting in the fact that they could have been using this technique for a long time in the experiment, and also helps understand how some things are changed and processed in this experiment, that could eventually harm and damage the DNA they are sequencing. 

The fact that nitrogen recycling is important for marine life and having a nitrate reservoir, is something that I did not know and found very intriguing. It is interesting to see how nitrification can play a role in promoting marine and nitrogen loss. 

[Since then, similar sequences of crenarchaeal AMO gene subunit A (amoA) (69–99% amino acid homology) have been detected in various marine water columns and sediments, including the Black Sea (20)]
I wondered why are these sequences so similar? And also the black sea caught my eye as well and also wondered if this body of water had some extra resources containing nitrogen than other seas. Since there was in fact various water columns and sediments in the black sea. 

I was interested in what the CTD looked like and how it performed, so after looking at the link Dr. Ni Chadhain, I had a more understanding of how the equipment worked. Overall, the picture helped me better understand what was going on in this paragraph, because I gained an idea of how the machine works. 

I was interested in how a FISH actually worked and why the CARD-FISH was better to use than the other. After reading some more information about it and following the link attached above, I can see why they used this system because it does indeed work well will samples that have low abundance. The CARD-FISH is shown overall as an improvement when compared with the normal FISH mainly in aquatic environments. 

In figure 2, after observing the charts, I had a hard understanding of them but after todays class where we had an example of this figure, I have a better understanding. Although Figure 2d was some what confusing but can be seen to show the relationship between a,b, and c. 

I feel like this figure was easier to understand than the Figure 2, mainly because each color has a different label. From this, I believe that NO2- is probably the best source of vertical distribution in water, since its lines are so close to the edge of the chart. It is also good to know that O2 levels are very high near the surface, and of course very low as the water level drops. 

From this graph it was similar to the others that we discussed. Since the oxygen gets lower as the water depth drops, this can help apply the theory that sulfur reducing bacteria will successfully live in. Nitrogen can also be shown  in the water levels as well. 

Its good to know that this was a vertical distribution of the amoA expression with the crenarchaea. The crenarchaea levels seem to be the greatest as the water level drops. Also this can be seen in the graph in figure 2 where it is better represented than betaAOB and gammaAOB. 

This part of the introduction provides background information to the reader. It explains what PAHs are and how they’re harmful as well as a summary of how and which microorganisms break them down. However, the authors caveat by saying that there is much to PAH degradation that we still don’t understand, especially with regard to high MW variants.

It seems according to this paragraph that while we’re capable of sequencing the genomes of PAH-degrading bacteria, without a better understanding of the metabolic pathways that actually serve in breaking down PAHs, the knowledge of these bacterias’ genomes is of limited use to us in understanding the breakdown of PAHs.

I am somewhat curious as to how the sediment for C. indicus and C. baekdonensis were extracted from the ocean, as well as what depth they came from (even though it’s not exactly relevant to the article). I also tried looking up Ausubel et al. DNA extraction, but most of what I found were similar citations to it.

I looked more into GC-MS and it was pretty interesting. Apparently the SCAN mode, if I looked at the description correctly, is useful in determining unknown compounds and scanning ranges of mass fragments. I’m not entirely sure what it’s being used to determine in this paper, but I read that GC-MS can be used to determine metabolic activity, which I suppose could be useful in trying to isolate a pathway for PAH degradation.

So to see if I understand this figure correctly, this figure (along with paragraph 11) is showing the predicted phylogenetic tree containing P73 and its 5 plasmids present, and that the trees are based off comparing the protein sequences of the parA gene (for figure 2A) and the rep gene (for figure 2B) with other available genomes, and that the closest related bacterial species to those plasmids is where they might have originated from?

Interesting that the P73T strain has the genes for flagella assembly, motility, and chemotaxis and yet phenotypically demonstrates none of those traits. Does simply having those genes assist in increase bioavailability of PAHs, or is P73T perhaps relatively closely related to a strain that is both motile and capable of breaking down PAHs, and this strain for whatever reason lost the ability to assemble flagella or become motile?

So the way GC-MS can be used to determine metabolic pathways is that it can detect potential products and byproducts of degradation, like it did here for fluoranthene degradation?

So this pathway corresponds to the top pathway illustrated in Figure 5, the numbers above the arrows being the genes involved. I’m curious as to what ways you could experiment to see if you could confirm steps in the process, or perhaps that’s not necessary so long as the end result is fluoranthene degradation.

The last sentence about P73T being potentially useful in marine oil spill bioremediation is a pretty cool possibility. I would imagine many more experiments and studies involving it would have to be carried out before it come be used practically, I imagine.

So while the researchers found these 138 genes that may be involved in aromatic metabolism, it’s likely not explicit that all of these genes are used in PAH degradation or fluoranthene metabolism. Could some of these genes hae been acquired via lateral gene transfer but otherwise not utilized (or utilized in other aromatic metabolic pathways not related to this study)?

Considering the age of this paper, have other significant groups of microorganisms capable of degrading aromatic hydrocarbons been discovered or have been the subject of greater focus in recent years? Are sphingomonads still the intense subject for researching PAH-degradation that they were when this paper was written?

So strain LB126 is especially significant because not only have the researchers identified the genes for fluorene degredation in a gram-negative bacteria, but in one that can sustain itself only on fluorine? And that makes it more significant than the pCAR3 plasmid from strain KA1?

So in order to test the expression of the dioxygenase-coding gene, they essentially constructed a plasmid by isolating the gene using PCR, subcloned it into the expression vector, and inserted that vector into the BL21(DE3) strain of E. coli?

I would guess that the purpose of dioxygenase overexpression is to grow colonies of E. coli with the vector containing the gene that codes for dioxygenase that would be able to express the ability oxidize PAHs, which would demonstrate that gene’s role in the process. Could you use gene knockout of that gene to reach a similar conclusion, like as was done in the previous paper?

Since they had to incubate the E. coli at 42 degrees C rather than the initial 37, are the proteins coded by FlnA1-FlnA2 in the E. coli different from those coded by the same genes in the LB126 strain of Sphingomonas because they have different activation temperatures.

I’m not entirely sure what the significance of the truncated orf2 encoding for a transposase. It apparently suggests horizontal gene transfer, but in the context of the whole experiment, it’s unclear to me why that’s important (or is it just an interesting observation?).

If both FlnA1-FlnA2 and CARDO can make 1-hydro-1,1a-dihydroxy-9-fluorenone from from 9-hydroxyfluorine, why is it that FlnA1-FlnA2 can make it from fluorine and not CARDO? Does this imply FlnA1-FlnA2 has a greater substrate range pertaining to fluorine degradation?

I suppose a closer reading of this paragraph actually answers my question on paragraph 3 about the importance of the truncated transposase gene. Is it the transposase’s proximity that leads them to the conclusion that LB126 acquired its genes for fluorine degradation through lateral gene transfer, as the paper only says it was ‘upstream’ (are they likely in the same cluster or group or something like that)?

Is it common for strains of Sphingomonads or other genera of microorganisms to use only a select few carbon and energy sources? Even if not, I imagine strains like A4 and the strain from the previous paper are novel since you only have one or two sets of metabolic processes to make observations about and detect enzymes and genes for.

Is the lack of reporting for the identification of dioxygenase genes for these sphingomonads due to just the lack of interest in identifying them, or is there some sort of significant difficulty in identifying them. If it were the latter, would reports exist that mention the challenges of identifying those genes (and the lack of said reports would indicate that difficulty is not the reason for the lack of identification)?

I guess this was their negative control to see if disrupting the arhA1 gene in A4 would diminish its ability to utilize acenapththene and acenaphthylene. By “blunting”, does that refer to removing the overhangs from the sticky ends of the DNA?

Is this preparation similar to what we did in lab where we used the oxidation of indole to determine which colonies did not have our inserts? I would guess it would serve the same purpose in this experiment.

Seems like the process of disrupting the arhA1 gene was effective. What is the putative arhA1 homologue mentioned referring to though? Is it referring to the gene they used in homologous recombination with arhA1?

While the arhA1 homologue seems to still be intact in the mutant, according to the results of introducing the mutant strain to the acenaphthene environment the strain couldn’t degrade it. So it’s interesting to consider what this homologue is. I’m curious as to what follow up research could be done to better characterize it.

So is the interest in the intrinsic electron-transport protein for ArhA1A2 sort of looking forward to the next step in PAH degradation, or is it another part of the initial process that hasn’t been well studied at the time?

In terms of bioremediation, does the Sphingomonad genus’s ability to degrade xenobiotics function similarly to its ability to degrade contaminants in the environment. Are ingested PAHs considered xenobiotics?

Primer extension is apparently useful in mapping the 5′ ends of RNA sequences. I’m curious to see what purpose this serves in the paper, since we never did primer extension (and I don’t recall if or how much we touched on it during lectures).

I imagine it’s common to use this many strains and plasmids pertaining to a particular species of bacteria in these studies. It feels like in comparison the number of strains me and my group have worked with pales (3 initial strains, plasmid vectors, and a few others). Of course, this paper probably took at least months of studies to complete, so if anything this would be what our research project would look more like were time not a limiting factor.

It’s a little sad that we never had time to do anything with Southern hybridization beyond discussing how it works, seeing how ubiquitous it seems to be as a method in these papers.

AG2-45, 2-48. and 3-15 lost the ability to produce indigo, but also maintained their ability to grow on 1,8-NA. Is that expected, or something not relevant to the conclusions suggested by these observations.

So it would seem the ArhA3 and ArhA4 genes were what the researchers were looking for in terms of the electron-transport system. It seems that incubation increased in cells expressing A1A2A3 without A4 (although not as great as with all four), so they’re complimentary, but are not both required for increasing acenaphthene oxygenase activity.

The arhR gene, interestingly, seems like a sort of “pre-step” for acenaphthene degradation, as its expression seems to affect the transcription of the arhA genes, and its disruption was shown to negatively affect acenaphthene degradation. It also seems to be active separate from the arhA genes.

So does this loose operon structure indicate an area for further research, or is it not relevant to PAH degradation since it doesn’t seem to have anything to do with acenaphthene degradation? Could it have been a result of mutation or gene transfer potentially?

I remember one of the papers (the first, I think?) involved theorizing a potential metabolic pathway in the breakdown of certain PAHs. They could potential do the same for future experiments. I agree that it seems difficult to accomplish with how dispersed they are, but I imagine there are ways we’re not aware of.

So since the arhA1 homologue’s function wasn’t identified, I imagine that finding its purpose would be a good jumping-off point for future research. Could you make preliminary assumptions of its function due to its homology to arhA1?

It seems so odd to me that despite the importance of Sphingomonas in PAH degradation we still know so little about their deoxygenation genes. It’s still somewhat unclear to me how this study increases our body of knowledge on the subject, but so long as it serves as a base point for further research, I suppose it doesn’t have to that much.

I remember during the oil spill, how the oil being released in to the deep ocean was causing big blooms in the bacterial populations that consumed the oil. this made big areas of hypoxias water as the oxygen was used up. and how this was one of the most damaging secondary events from the spill. 

Its good how Antarctica is for the most part being protected from possible invasive species so we can keep it’s research opportunities available. It is interesting to see how these local environments are providing solutions for the problems we have created and how we have to be very inventive with finding these solutions since external fixes aren’t allowed.

I can see how our labs mimic this in our isolation of our naphthalene degraders. as our bacteria is isolated to single colonies to obtain strains for farther study.

PCR can be a highly powerful tool in the study of genomes as it can let us obtain as many copies of a piece of DNA as needed. most of the current Covid-19 tests are run through PCR to get the viral Genome large enough that it can be easily detected. which is also why it can take a while as PCR has to cycle through temperature ranges to successfully anneal and replicate the genomic material.

PCR and its readily availability has allowed for all the growth in genetic research over the last few decades as you can now easily get your hands on the equipment, proteins, and primers for replicating almost any DNA strain you want. I has need to all this testing with recombinant DNA and Plasmids as they are now cheap to produce once your initial sample is synthesized.

I probably wont be the three same strains but other bacteria in those areas can probably degrade oil. Oil and produces made from it are now everywhere thanks to humans,  but oil will naturally enter different environments as it seeps up through the ground.

Life is a truly amazingly resilient thing. you can find some form of life at almost every point on earth. they have found microbes in salt water pockets at the bottom of mineshafts in Johannesburg. we have even done studies of exposing bacteria to space onboard the international space station, and found that several survived with minimal damage after an extended exposure. 

Its interesting how they are using a naturally occurring environment with a significant amount of hydrocarbons as a base examination to compare to the areas effected by the oil spill. 

Do these mats refer to biofilms or just large bacterial communities living off the oil on the ocean floor?

I see this group is also using 16S to identify and match what species of bacteria these are. but I don’t quite understand why they are using carbon 13 for this. is it just a way to mark the bacteria they could culture from the samples?

Its interesting that phenanthrene is the PAH that is mineralized the most. Especially with PHE being the PAH that was used in the last paper. Is there some reason, maybe with the origin of the genes that allow for the use of PAH, that make PHE a globally common PAH that can be broken down? maybe the proteins that allow for the breakdown were originally specialized to a different energy source that PHE manages to be the most similar to.

Its interesting that there are these specific genera that have fulfilled their niche so strongly that they are only able to get their energy from the breakdown of PAHs.

It will be interesting to see how they will test to find a coculture of bacteria. along with this how they verified if it is a symbiotic relationship, and how the bacteria handle these reactions

It is interesting that they couldn’t isolate the arsenite oxidase genes. is it possible that there is some temporary form the arsenite is being made into before it self oxidizes? 

Since they all ready have primers for the genes dealing with the uses of arsenate and arsenite. I assume they are working off research that was already conducted before that had isolated the genes.

It is interesting the amount of sterility they are using to conduct these experiments. They are acting really carefully to prevent oxygen and sources of lab contaminates. The prevention of oxygen makes sense as the bacteria are supposed to be growing in a natural condition that lacks oxygen to use as the end electron receptor and are thus forced to use the arsenic.

Its interesting how few bacteria they found found in the samples and how only one close group photosynthetically oxidize arsenite. but this makes sense when you look at the graphs that the arsenite is being oxidized by phototrophic bacteria. 

With the large rate differences between the oxidation and the reduction of As(III) and As(V). would it be safe to assume that in natural conditions, that the soil and surfaces in which the biofilm where collected from would primarily contain As(V) with very low amounts of As(III)

I wonder why there is such a focus on the salinities and the comparison to oil spills and leaks, is it that the two are linked or just that the risk is high in these regions of high salinity because of concentration of extraction sources or if equipment disrepair and misuse is the reason.

I wonder what they used as the initial cultivation media. Because the bacteria was found to degrade all of these hydrocarbons but you need a pretty specific initial media as to isolate the bacteria in the first place as too many added hydrocarbons and the media would just act as a general purpose media grabbing multiple different types of bacteria.

Okay, this answered my question from the first page, they used benzene as the carbon source in their selective media. As benzene is the primary structure for the different compounds in the BTEX group. It would make sense for what ever proteins the bacteria uses can also bind to the other compounds and break those down as an energy source as well.

Looks like they did the pretty standard practice of using Carbon 14 enriched versions of compounds you are trying to see the bacteria degrade. The amount of released Carbon 14 CO2 released can show the rate in which the bacteria is degrading the benzene.

Its interesting that the bacteria ceased to degrade benzene at 4 M NaCl, although 4 M NaCl is 233.8 g of NaCl in a liter of liquid. A this level of salt it is safe to assume that the bacteria began to shutdown its intake mechanism to help lower internal salt concentrations. 

It is interesting that they didn’t manage to find all of the enzymes that would be used to breakdown these compounds. They believe that the enzymes should be known ones and not the possibly of unknown enzymes for the breakdown of these different compounds.  

Its cool that they actually go through and show the actual breakdown steps for the benzene, toluene, and other hydrocarbons. It takes these different compounds and converts them to acetyl-CoA which it can then use in most metabolic functions. 

This is looking a bit bit closer at the biochemical properties of the enzymes, they talk about the bacterial strain’s protein structure. The proteins are actively more soluble due to their abundance of negative charges. This helps the bacteria continue to function in high saline environments. 

Fluorene is a know to be highly toxic to aquatic life. I found a document by the Minnesota department of health that talked about the exposure risk from fossil fuel contaminated areas and cigarettes’ smoke. They haven’t preformed wide scale testing but they believe it can build up in lake sediments. 

This paper brings up that the fluorene oxidizing genes are on plasmids, this differs from the last paper where the genes were part of the bacterial chromosome. 

Protein electrophoresis is harder than DNA electrophoresis as you need SDS in order to smooth over the charges to negative so they can be attracted to a single electrode so you can actually get coordinated movement of all the proteins in the same directions and at speeds equivalent to their size and not their charge.

Its interesting how they have the exact primers for these genes although this was one of the first times they were supposed to have been isolated for testing. There is no degenerate nucleotides in these sequences.

Its interesting how in figure 2, terrabacter sp. DBF63 had a regulatory gene in the reverse direction surrounded by Fluorene degrading genes in the forward dirrection..

Reverse transcriptase is such a powerful tool in our tool belt of techniques and enzymes for genetic research. We took an important part of one of humanities most infamous viruses and made it into a tool to try and improve peoples lives through the research avenues it has opened.

Its interesting that the Proteins produced, if they were FLnA1 and FLnA2, were not fully translated in their initial conditions, but when they were produced at 42C they got full sized proteins. I wonder what the physical/mechanistic reason for the translation to have stopped is? 

I find it interesting that FlnA1 and FlnA2 could be a relatively novel way to break down fluorenone and other cyclic hydrocarbons, it would cool to see if the evolution of these genes could be determined to figure out how it came to be  and function in this way. 

Its interesting how this is a soil sample from deep sea sediment, this could mean that the ability to degrade these hydrocarbons comes from oil seep exposure. 

That’s interesting how this bacterium is believed to have acquired its genes for the breakdown from 4 different sources.
 

cre-lox recombination is a very interesting way to inactivate genes. Although it is semi limited in its use as you need the recognized sequence for the enzyme to work.

I see that they used the now standard method of using mass spec and chromatography to detect the breakdown compounds made from the bacteria.

Its interesting that this paper actually when and tested all the different sugars the bacteria could use. It looks like they are doing a full examination of this bacterial genes/

Its cool how these little left over fragments of DNA can give an idea at how these different genes were obtained by this bacteria. 

The inability of the knockout to use fluoranthene and naphthalene and the defectivity of the other hydrocarbons suggests that this was in fact the gene responsible.

I too have an interest in the differences between the inocula, or rather, what qualifies them as “very similar”. I can only infer that the differences and level of effectiveness will be further discussed in the remainder of the paper, for anyone reading would also pose the questions you did.