Wednesday, October 30, 2013

025 - Siderophores Produced by Nitrogen-Fixing Azotobacter vinelandii OP in Iron-Limited Continuous Culture

One thing that's noteworthy about A. vinelandii is that when growing on agar plates or broth tubes of the medium commonly used for it (Burk's), after a certain time it produces this neon green pigment that diffuses through the medium. It's neon green under white light, but under ultraviolet light it fluoresces light blue!

This colorful feature is due to a molecule called a siderophore, which the bacteria secrete to scavenge (i.e. chelate) insoluble oxidized iron in the medium and bring it into the cell. Iron is an important element for A. vinelandii, especially for fixing nitrogen; all of its nitrogenases require it.

In the present study, the researchers wanted to find out how A. vinelandii responds to insufficient iron in its environment, and the function of proteins and compounds it produces. They used continuous culture to do this, analyzing steady states at different concentrations of iron.

The scientists grew A. vinelandii strain OP (aka CA) not with Burk's but with a medium called B6, which also works I guess. Obviously this didn't always include the same amount of iron that it normally contained. They actually cleaned their medium vessels and reactor with EDTA (a chelator that binds tightly to metals) to get rid of all traces of iron.

So they grew OP at four different concentrations of iron, and at steady state for each concentration, measured the concentration of cells (dry weight), quantities of different potential chemical siderophores (extraction and separation by electrophoresis), and quantity of yellow-green fluorescent protein (fluorescence measurement). They also measured the total amount of chelated (bound) metals from all the siderophores.

What they saw was not surprising: at higher levels of iron, there was a higher concentration of cells in the culture and a lower amount of chelation going on. This makes sense because the more iron there is, the less need there is for the bacteria to produce special chelators to scavenge it. The numbers they got were very consistent, differing only about 5% even between different chemostat runs.

Regarding the levels of specific siderophores, one was clearly the predominant one compared to the others. This one went down dramatically as iron increased, dropping all the way to zero when iron was sufficient; the others dropped also but still had low levels even in iron-sufficient conditions.

A few other things to note is that, according to the authors, the lack of fixed nitrogen in the medium prevented contamination of the chemostat, and since the reactor was made of Teflon, no bacterial growth built up on the walls (which presumably would happen on a different material).

So this study shows pretty well which siderophore is important for iron scavenging in A. vinelandii, and that iron deficiency (in nitrogen-fixing conditions at least) impairs its growth.

Citation: Fekete, F. A., Spence, J. T. & Emery, T. Siderophores Produced by Nitrogen-Fixing Azotobacter vinelandii OP in Iron-Limited Continuous Culture. Appl. Environ. Microbiol. 46, 1297–1300 (1983).

Tuesday, October 22, 2013

023 - Genome Sequence of Azotobacter vinelandii, an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes

One good way to learn a lot about a bacterial species, or at least to get a lot of hints about what it might do or be capable of doing, is to sequence its genome. So that's what a bunch of people decided to do with Azotobacter vinelandii. It makes sense, since this organism is one of the better-studied ones and has interesting capabilities, such as nitrogen fixation.

The strain they chose was called DJ, a variant of the wild-type strain CA. DJ is supposed to be easier to manipulate genetically than its parent. So they sequenced its entire genome, but focused mainly on the surprising amount of oxygen-sensitive enzymes they found in an obligately aerobic organism.

The method of sequencing, for those who care, was plain shotgun Sanger dye-terminator sequencing after generating a clone library. (Apparently, for some reason, Monsanto did a lot of the work.) I guess this was before the next-generation sequencing technologies were available or affordable. And teams of undergrads did much of the work labeling genes and such.

Overall, the genome is pretty similar to that of pseudomonads, especially Pseudomonas stutzeri (another nitrogen-fixing soil microbe). Some of their genes have been rearranged compared to each other, though, and A. vinelandii has almost 1,000 more genes.

In terms of energy-generating systems, A. vinelandii's genome has all the genes needed for aerobic metabolism but seems to lack any complete system for anaerobic respiration or fermentation. It is well-equipped for aerobic respiration though, which it seems to use to consume large amounts of oxygen that would otherwise damage its nitrogenase and other enzymes. The other mechanism it has to protect its nitrogenase is called the FeSII or Shethna protein, which can temporarily deactivate the nitrogenase when oxygen is too high, protecting it from damage.

The sequence showed the precise location of each set of nitrogenase genes relative to each other. They're somewhat spread out. It also located the mod genes for molybdenum (Mo) transport and the hox genes of the uptake hydrogenase (which are pretty close together). Though it turns out there is a second set of genes similar to the original mod operon elsewhere in the genome, that may be a second Mo transport system. Possibly even a third set right next to the first, but it's not certain what it does.

Somewhat interesting is a set of genes that are similar to something called carbon monoxide dehydrogenase (CODH) that is present in some anaerobic organisms. This can convert CO to CO2 and H2, effectively using it as an energy source instead of something toxic. But it's not certain whether this is functional in A. vinelandii at all. It may be related to some genes that seem to be related to soluble hydrogenases in other organisms, but their function isn't clear either.

A. vinelandii, some strains of it at least, is well-known for producing certain polymers: polyhydroxybutyrate (PHB), which can be used to make a kind of bioplastic; and alginate, and kind of mucusy stuff that has various uses too. The strains that make alginate are rather slimy and hard to work with, and supposedly this provides a further barrier against oxygen poisoning, but strains CA and DJ don't make it, and this sequence revealed why: a transposon inserted itself in the middle of a regulatory gene, inactivating it. That's all it takes.
The genes for PHB synthesis seem to be intact though.

Knowing the sequence of an organism is very helpful; if you want to check for new capabilities, you can just check the genome. So this is a good study.

Citation: Setubal, J. C. et al. Genome Sequence of Azotobacter vinelandii, an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes. J. Bacteriol. 191, 4534–4545 (2009).

Tuesday, October 15, 2013

020 - Comparative characterization of H2 production by the conventional Mo nitrogenase and the alternative "iron-only" nitrogenase of Rhodobacter capsulatus hup- mutants

As I've mentioned before, hydrogen gas is a byproduct of nitrogen fixation (which makes sense; nitrogenase adds protons and electrons to N2 to form NH3, and in the process some protons and electrons stick to each other, forming H2. And the different kinds of nitrogenase have different efficiencies—that is, different proportions of protons and electrons that come off as hydrogen instead of actually useful stuff. The molybdenum-containing nitrogenase is most efficient, with one H2 per N2 fixed, and the others have higher ratios.

So this study intended to compare different nitrogenases to find out how much hydrogen they produced. It was done with Rhodobacter capsulatus, not Azotobacter, but the enzymes are similar. R. capsulatus is a type of phototrophic bacterium that possesses the primary Mo nitrogenase and also the iron-only alternative, as well as an uptake hydrogenase.

In order to get around the confounding effects of an uptake hydrogenase, which would significantly reduce the amount of measurable hydrogen given off by all nitrogenases, the scientists used a hydrogenase-negative strain they had generated using a transposon (jumping gene). Besides this strain and its parent, they had a strain with the Mo nitrogenase and Mo transport genes deleted (so it could use only the iron nitrogenase), and a hydrogenase-negative mutant of this strain.

The strains were each grown in broth and then exposed to an atmosphere of argon or nitrogen, sometimes mixed with acetylene or oxygen. The purpose of argon is that, when the nitrogenase enzyme lacks any other substrate (nitrogen, acetylene, etc), it will still work but just devote all its protons and electrons to producing hydrogen gas, producing a lot more than in any other condition. Adding acetylene measured the enzyme activity converting acetylene to ethylene (and a little ethane too, in the case of the iron nitrogenase), and adding oxygen measured its effect on the enzymes. Then after some time for the reaction to occur, concentrations of ethylene, ethane, and hydrogen in the headspace were measured.

In an argon atmosphere with nothing else, the hydrogenase-negative Mo nitrogenase strain produced the most hydrogen. In the parent strain that was hydrogenase-positive, the hydrogenase consumed about 1/4 the hydrogen produced. The hydrogenase-negative strain using the iron nitrogenase produced about 1/2 the hydrogen of the top producer, and when present, hydrogenase consumed about 1/2 its hydrogen, resulting in 1/4 the amount of the top producer. So like this:

Under argon:
  • nif+ hup-: 100%
  • nif+ hup+: 75%
  • nif- hup-: 50%
  • nif- hup+: 25%
This may seem odd because the iron nitrogenase is supposed to produce more hydrogen (relative to other substrates), but that is not the only difference between the enzymes; the Mo nitrogenase's rate of production (productivity) is also higher, such that it produces more of any product in a given time. Since these reactions were measured after 1 hour, the Mo nitrogenase was able to produce more hydrogen in that time than the alternative, though they might have produced the same amount (or the iron version more) if allowed to consume all their substrate.

The story of hydrogen production is somewhat different in a nitrogen gas atmosphere:
  • nif+ hup-: 62%
  • nif+ hup+: 5%
  • nif- hup-: 100%
  • nif- hup+: 3%
Not surprisingly, when nitrogen is present for the enzymes to fix, the alternative nitrogenase produces a lot more hydrogen than the Mo nitrogenase. However, even in the hydrogenase-negative iron nitrogenase strain's case, the hydrogen produced is a bit more than 1/4 of that produced by the top producer under argon. When hydrogenase is present, it is able to consume most of the hydrogen. Similar results are obtained when acetylene is added to an argon atmosphere.

When acetylene was added, the hydrocarbon results were as expected also. Presence or absence of hydrogenase didn't make much difference regarding ethylene and ethane produced. Mo nitrogenase produced much more ethylene than iron nitrogenase, and hardly any ethane; while the iron nitrogenase produced about 17 times more ethane than the Mo nitrogenase, and 11 times less ethylene. Total products for Mo nitrogenase were also about 11 times more than for iron nitrogenase. More efficient, I say.

In terms of protons and electrons, these results suggest that 80% of Mo nitrogenase's electrons go toward nitrogen fixation (that is, 1 hydrogen for every nitrogen fixed, as I said), but only 45% of the alternative's electrons (so, it makes 3-4 hydrogens for each nitrogen).

Oxygen had different effects on the different enzymes also. In the hydrogenase-negative strains, higher levels of oxygen inhibited each, but the alternative nitrogenase's activity dropped to below 40% with very small increases in oxygen levels, whereas the decrease in Mo nitrogenase activity was almost linear with increasing O2, retaining as much activity as the alternative at more than five times the level of oxygen. So the iron nitrogenase seems to be about 4 times more sensitive to oxygen. Understandably alternative.

So, at least in this species, the iron nitrogenase is 3-4 times less efficient in terms of electrons going to hydrogen, about 11 times slower, and 4 times more sensitive to oxygen than the Mo nitrogenase.

Citation: Krahn, E., Schneider, K. & Müller, A. Comparative characterization of H2 production by the conventional Mo nitrogenase and the alternative ‘iron-only’ nitrogenase of Rhodobacter capsulatus hup- mutants. Appl. Microbiol. Biotechnol. 46, 285–290 (1996).

Monday, October 14, 2013

019 - The Effect of Nutrient Limitation on the Competition between an H2-uptake Hydrogenase Positive (Hup+) Recombinant Strain of Azotobacter chroococcum and the Hup- Mutant Parent in Mixed Populations

Since Azotobacter has this uptake hydrogenase enzyme that seems to recover the energy lost as hydrogen gas from nitrogen fixation, it is worth testing to see if this hydrogenase actually does provide a benefit to the bacteria. This study was done in A. chroococcum, not A. vinelandii, but they're related enough that we should be able to generalize the data gathered, with caution.

The hydrogenase works by taking H2's electrons and passing them through the electron transport chain to oxygen to generate a membrane potential (like voltage) that provides energy for the production of ATP (the cell's energy currency, that it uses to power many of its reactions). This can be an important process for crop production, since some crops (legumes) are colonized with bacteria that fix nitrogen for them, but at the time it was unclear whether the uptake hydrogenase in this system was actually helpful for the crop at all. So the authors decided to study the question in an easier system: free-living Azotobacter.

In this study, the strains under investigation were a mutant strain, offspring of the wild-type, that lacked hydrogenase, called MCD103; and another strain derived from MCD103, called MCD503, that did have a hydrogenase because they crossed MCD103 with a plasmid containing wild-type hydrogenase genes, resulting in MCD503, which was the same as MCD103 in every way except the hydrogenase (presumably).

These strains were grown together in continuous culture/chemostats, fixing nitrogen. If one had a growth advantage over the other, it would come to dominate the culture in time. They measured proportions of the strains in two ways: first, by plating them out on agar and using a technique called "scrying" (which seemed to consist of exposing the colonies to H2 in a sealed box with an indicator present, such that those that had hydrogenase would stay white and those that didn't would turn blue-black) and then counting the colonies of each kind. Second, by plating and transferring individual colonies to a 96-well plate, then exposing all of them to radioactive hydrogen (tritium, 3H2) and measuring which ones retained radioactivity (indicative of consuming the hydrogen with their hydrogenase). These sound like rather painful and burdensome procedures; nowadays people would probably just do gene sequencing or transcript analysis to see how many copies of hydrogenase genes/transcripts were present over time. I suppose the techniques in this paper may give a more direct measure though.

So anyway, about what they found. When sucrose (sugar) was the limiting nutrient (that is, when the cells were consuming all the sugar they were given and could've consumed even more), the hydrogenase-positive strain MCD503 came to dominate the culture over time, regardless of the proportions of the strains at the beginning of the experiment. Even when initially there were 99 hydrogenase-negative cells for every one hydrogenase-positive cell, before too long they saw the amount of hydrogen produced falling quickly as hydrogenase activity increased. The domination happened faster at higher dilution rates, which makes sense because faster-growing cells would be able to tolerate these better. When fixed nitrogen (ammonium) was added to the cultures, neither strain dominated the other consistently. So it seems that hydrogenase is important when fixing nitrogen.

When nutrients other than sucrose were limiting, though, the situation was not always the same. When phosphate was limiting, MCD503 still dominated, though not as well as with sucrose limitation. However, when oxygen was limiting, the strain missing its hydrogenase (MCD103) was dominant! Even when MCD503 started as 78% of the cells present, it fell to less than 20% before stabilizing. So it seems that hydrogenase-negative strains can deal with low oxygen better.

When sulfate was limiting, MCD503 declined slowly, but it declined more quickly when iron was limiting (makes some sense because the hydrogenase requires iron in its cofactor).

It was interesting to note, also, that MCD503 (hydrogenase-positive) consumed its own hydrogen but also that produced by MCD103 (hydrogenase-negative). Might've contributed to its faster growth in some conditions, and this is consistent with how the domination slows down as more and more of the population is MCD503 (thus there is less hydrogen produced by its competitor to steal).

Speculations about explanations for the findings, as far as I understand them: carbon-limiting results make sense because H2 oxidation adds to the energy recovery and makes up for some of the limitation.
Phosphate-limiting results make sense because hydrogen could help increase consumption of oxygen to protect nitrogenase and hydrogenase, which are sensitive to it.
Oxygen-limiting results make sense because hydrogen oxidation might take precedence over other kinds in the electron transport chain due to greater affinity.
And sulfate- and iron-limiting results make sense because hydrogenase requires sulfur in addition to iron, so the cells would devote some of their nutrients to this enzyme instead of other, more important ones; while hydrogenase-negative strains wouldn't have this disadvantage.

So it seems that the hydrogenase is helpful in some circumstances and harmful in others. Interesting results.

Thursday, October 10, 2013

017 - Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum

Everyone now knows that Azotobacter vinelandii has three nitrogenases, and that the Mo-containing one is the best. The iron-only nitrogenase is the least efficient but requires the fewest different metals (only iron, obviously, compared to iron plus another for the other two). But does A. vinelandii ever encounter situations in nature where it can't find molybdenum? It seems likely.

In this study, the scientists tested the difference in nitrogen fixation between cells growing on agar plates and cells growing in liquid broth. This is important because in liquid, nutrients are constantly being mixed and cells can all experience the same concentration of them, pretty much; whereas on agar, nutrients don't move around much (they're trapped in the gel) so the concentration around the bacteria decreases as cells use them up.

They tried growing two strains, wild-type CA and strain CA70 which lacks the genes for the iron-only nitrogenase, to see if one or the other grew more quickly on each type of medium. It turned out that, with the same total concentration of Mo in each, on agar CA outgrew CA70 more and more over time, while in liquid the numbers of cells of each strain remained about the same. And when they tested the strains on agar with different concentrations of Mo, CA outgrew CA70 more and more the lower the concentration of Mo was.

To confirm this, they used a strain (CA73) that had a fusion between anfH (one of the iron nitrogenase genes) and lacZ (makes an enzyme that can break down a compound into color, used to determine amount of a gene produced). They tested this on agar or in liquid with different concentrations of Mo, and found that expression of anfH was much higher on agar than in liquid for mid-range concentrations of Mo (on the low end, A. vinelandii expressed anfH in both conditions, while on the high end, it didn't need to express anfH in either).

Finally, they used a 2-D gel to confirm the presence of iron nitrogenase subunits.

So it seems that, when A. vinelandii is growing on agar, it can sometimes deplete the Mo present in the agar to the point that it needs to switch over to use the iron nitrogenase. In the soil, where A. vinelandii is found naturally, it is probably a common occurrence to encounter areas of low Mo and have to switch.

Citation: Maynard, R. H., Premakumar, R. & Bishop, P. E. Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum. J. Bacteriol. 176, 5583–5586 (1994).

Wednesday, October 9, 2013

015 - Characterization of genes involved in molybdenum transport in Azotobacter vinelandii

It is known that molybdenum (Mo) represses A. vinelandii's alternative nitrogenases, at least in the wild-type. What is not exactly known is the mechanism of this repression. Concentrations of Mo as low as 10 μM repress the alternatives, but some strains (such as CA6) produce the alternatives even with high concentrations of Mo. The question is, is this because Mo is not being transported into the cell, or because the protein that represses the alternatives is not functioning properly somehow?

So in the current study, they took a strain of A. vinelandii that can't make the primary nitrogenase but still represses the alternatives when Mo is present, and created mutants using a transposon, Tn5, then grew it on medium with Mo but without nitrogen, so that only those that had a mutation in a relevant gene could grow. And they found two Tn5-induced mutants, and also some that had mutated spontaneously to be able to grow in such conditions.

They named the two transposon mutants FL2 and FL4, focusing on them because the transposon insertion allowed them to locate and study the genes of interest. The mutants could grow pretty much just as fast with Mo present as the parent strain could grow with Mo absent (though these rates were all somewhat slow because they all needed to use the alternative nitrogenases, which are less efficient). Actually FL4 grew a bit faster than the parent.

They isolated and sequenced the section of the genome that the transposons had inserted themselves into, and it turned out that it was the mod operon that I've discussed before (012). FL2 had an insertion in modE, the regulatory gene, and FL4 had an insertion near the end of modB. So it seems like these genes are important for Mo-induced repression of alternative nitrogenases.

Citation: Luque, F., Mitchenall, L. A., Chapman, M., Christine, R. & Pau, R. N. Characterization of genes involved in molybdenum transport in Azotobacter vinelandii. Mol. Microbiol. 7, 447–459 (1993).

Monday, October 7, 2013

014 - Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii

Since the primary nitrogenase of Azotobacter vinelandii (and other nitrogen-fixing organisms) is so dependent on molybdenum (Mo), such that they turn off expression of the nitrogenase in the absence of Mo, I thought it worthwhile to read about A. vinelandii's system for storing Mo. Klebsiella pneumoniae, another well-studied nitrogen-fixing soil organism (given its intimidating name because it is an opportunistic pathogen, I think), is used as a comparison.

Previous research had shown that A. vinelandii takes up and stores Mo continuously when it is present, whether or not the bacteria need to fix nitrogen. This is a different strategy from other diazotrophs (nitrogen-fixing organisms). In this study, bacteria were starved of Mo but given fixed nitrogen, and then put into media with varying amounts of Mo with or without fixed nitrogen. When fixed nitrogen was absent, nitrogenase activity maxed out at low concentrations of Mo, but the more Mo present, the more accumulated in the cells, much more than needed to make nitrogenase. When fixed nitrogen was present, the cells showed the same pattern of increasing Mo accumulation, though the levels seemed lower.

In contrast, K. pneumoniae seemed not to accumulate any Mo when fixed nitrogen was available, and while it did store Mo when fixing nitrogen, it was more than 10x less than the levels in A. vinelandii in any condition.

The researchers also measured accumulation of Mo over time. A. vinelandii took up 100% of the Mo provided within 1 hour (it was only a low concentration though), but K. pneumoniae didn't even start uptake until 2 hours in, and then took 2 hours to reach only 25% accumulation. Clearly the two organisms have different strategies; Klebsiella's uptake pattern correlated with its nitrogen-fixation pattern.

They tested the effect of oxygen on Mo uptake; oxygen is toxic to nitrogenase, such that K. pneumoniae doesn't fix nitrogen in its presence, and A. vinelandii takes steps to protect its nitrogenase. But A. vinelandii is an obligate aerobe, so it must still fix nitrogen and accumulate Mo in the presence of oxygen. Klebsiella, on the other hand, shuts off its nitrogenase and Mo uptake both when oxygen is present.

Chloramphenicol, an antibiotic that inhibits protein synthesis in bacteria, inhibited K. pneumoniae's Mo uptake too, but surprisingly not A. vinelandii's; the latter actually had more Mo per cell with chloramphenicol than without, because it was unable to multiply, so there was the same amount of Mo divided among fewer cells.

So there must be some protein in A. vinelandii that is able to store lots of molybdenum. Indeed, when transferred to Mo-free medium after accumulating Mo, the bacteria could fix nitrogen at the same rate as when growing in Mo-containing medium, at least for a while. Trying to purify the Mo-containing proteins from each organism, the scientists found that pretty much all the Mo in Klebsiella was contained in the nitrogenase, whereas (depending on the amount available) that only accounted for a fraction of Mo in Azotobacter. There is another protein that is able to store large amounts of the metal, about 14-15 atoms per molecule of storage protein.

As a bonus, they tested A. vinelandii's ability to store tungsten, and found that it was accumulated in the same way as Mo. It's a sneaky element, apparently.

Citation: Pienkos, P. T. & Brill, W. J. Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii. J. Bacteriol. 145, 743–751 (1981).

Wednesday, October 2, 2013

012 - Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii

So Azotobacter vinelandii CA6 has impaired molybdenum (Mo) uptake (003). This paper studies the Mo transport system of A. vinelandii, encoded by the mod genes, modEABC. It seems like ModA is a protein that binds Mo outside the cell, ModB brings them inside across the membrane, and ModC powers this process. ModE's role is unknown at this point.

The scientists generated a number of mutant strains of A. vinelandii, knocking out a given gene while also fusing it with lacZ to quantify its expression. They also discovered another mod gene, modG, adjacent to the others but in the opposite strand direction. It looks similar to half of modE, so the protein may have a similar function.

Another enzyme in A. vinelandii that requires Mo is nitrate reductase; the authors measured activity of this enzyme as a proxy for Mo transport activity. The wild-type's activity rises quickly as concentration of Mo in the environment increases, levels off, then rises quickly again at higher concentrations (supporting the idea of two different Mo transport systems). With mutants of modA, modB, and modC, the pattern was always the same: no activity until the concentration reached a certain point (the same point when the wild-type's activity started rising quickly the second time).

Other results were more puzzling: when modE was knocked out in a way that didn't inhibit expression of the other mod genes, it seemed to have good transport activity at lower Mo concentrations but not at higher; and the opposite when its knockout inhibited the other genes.
Strain CA11.6, which genetically combined the lack of Mo nitrogenase in CA11 (002) with the tungsten-tolerant phenotype of CA6 (003), showed good Mo uptake at low concentrations but not at higher. When the modB gene was specifically knocked out of CA11.6, there was hardly any uptake activity at any concentration. Considering my own research, it's difficult to say what is going on genetically in these cases.

When modG was targeted for knockout, it looked pretty much the same as wild-type activity, except when both modG and modE were deleted, in which case it showed activity at much lower concentrations even than wild-type. Explain that, science!

They also directly measured uptake of a radioactive isotope of molybdenum (99Mo) in the wild-type and modA or modB mutants. The rate of transport in the wild-type and modB mutant were pretty much constant, though the latter was slower than the former. In the modA mutant though, there was very little transport. They tried adding nonradioactive compounds (Mo, vanadium, sulfate, or tungsten) to compete with transport of radioactive Mo, and found that only Mo and tungsten inhibited radioactive Mo transport by competition. Evidence that the mod genes transport tungsten in addition to Mo.

Lastly, the scientists tested the nitrogen-fixing abilities of mod mutants. With Mo present, nitrogen-fixing growth of modE and G mutants was similar to wild-type. When it was absent, modG knockout grew more slowly and modE more quickly. A double mutant didn't grow hardly at all in either condition, in normal aerobic conditions, but with lower levels of oxygen it grew as well as the wild-type (both very slowly). It could also grow using vanadium (V) and the V-containing alternative nitrogenase.

The conclusions, I suppose, are that modABC are all important for Mo transport, especially at low concentrations. modE's role is not exactly clear, but it may regulate which Mo transporter system is working at a given time (possibly by repressing one and activating the other at low concentrations, and vice versa at high). modG's role is even less clear.

Citation: Mouncey, N. J., Mitchenall, L. A. & Pau, R. N. Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii. J. Bacteriol. 177, 5294–5302 (1995).

Tuesday, October 1, 2013

003 - Phenotypic characterization of a tungsten-tolerant mutant of Azotobacter vinelandii

One tungsten-tolerant strain from 001 in particular caught the attention of the researchers. Azotobacter vinelandii strain CA6 just happened to mutate spontaneously to be able to fix nitrogen in the presence of tungsten (W).

Later research showed that A. vinelandii possesses three nitrogenase system, actually: the primary, molybdenum-containing one, and two alternatives: one with vanadium instead of molybdenum, and a third with iron. The third is least efficient, but iron is most likely to be available, so it is the most versatile.

But CA6 was still interesting, because somehow it was able to overcome the repressive effect that molybdenum (Mo) and W have on the alternative nitrogenases. So in order to study it, among other things, the scientists made a number of recombinant strains of A. vinelandii, to test the functions of different nitrogenase genes.

They tried growing wild-type strain CA and mutant strain CA6 with different concentrations of W. All tested concentrations of W inhibited CA, and above 1 μM (0.184mg W per liter) all concentrations inhibited it the same amount. With CA6, however, no amount of W seemed to affect its growth. However, when Mo was present (and no W), CA grew about twice as fast as CA6.

To figure out why, they deleted the genes for the alternative nitrogenases to create strain CA6.1.71 (sounds like software versions, heh). Obviously this couldn't fix nitrogen or grow without Mo present for its primary nitrogenase. But when Mo was present, it could grow just as fast as the wild-type, showing that the difference in growth rate is probably because CA6 wastes its energy producing less efficient nitrogenases instead of focusing on the efficient primary one.

They also made some genetic fusions of nitrogenase genes with a gene called lacZ, which codes for an enzyme that breaks the bond between the two sugar molecules of lactose, resulting in one molecule of glucose and one galactose. The purpose of this is that this enzyme also breaks the bond in a molecule called o-nitrophenyl-β-galactoside (ONPG), which releases a molecule of galactose but also o-nitrophenyl, which is a bright yellow color. So when you add ONPG to liquid containing the enzyme, you can tell how much enzyme is present by how yellow the liquid becomes. And by fusing lacZ to other genes, you can get an idea of how much those other genes are expressed in the cell.

So this way, they found that, in the wild-type strain CA, Mo-nitrogenase genes are expressed when Mo or W are present (not surprisingly), and alternative nitrogenase genes are only expressed when Mo or W is absent. In CA6, the iron-nitrogenase is produced with or without Mo or W; only vanadium represses it. And the vanadium nitrogenase in both is expressed only when vanadium is present. They confirmed these results with 2-D gels (described in 001).
(Side note: vnfH, vanadium dinitrogenase reductase, is expressed in CA whenever Mo or W is absent, whether or not V is present; in CA6, it is always expressed regardless of the metals in question.)

One possible reason for the difference between CA and CA6 is the latter's ability to take Mo into its cells; if its uptake of Mo is impaired, that could result in the observed phenotype. So the scientists tested that. They found that, not only was CA6's Mo uptake slower than CA's, but it ceased to take up more above a certain concentration, whereas for CA, the more that was available, the more CA took up. It seemed like there were two separate Mo-uptake systems, one that worked better in low concentrations and one in higher, and CA6 lacked the latter. However, there was still enough Mo present in CA6 that it should have repressed the alternative nitrogenases, so this explanation didn't quite work; there must be something else. These observations just add to the mystery of A. vinelandii CA6.

Citation: Premakumar, R., Jacobitz, S., Ricke, S. C. & Bishop, P. E. Phenotypic characterization of a tungsten-tolerant mutant of Azotobacter vinelandii. J. Bacteriol. 178, 691–696 (1996).