203 - Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-β-hydroxybutyrate synthesis

As mentioned before, Azotobacter species can make some useful polymers, such as poly-β-hydroxybutyrate (PHB), a kind of bioplastic. A. vinelandii also makes some other potentially useful polymers: alginate, a kind of slimy polysaccharide; and compounds called alkylresorcinols. 

This last is involved in encystment, when the cells change into a more resistant, dormant state, called a cyst. Alginate is involved in that too, actually, but alkylresorcinols are lipids that replace phospholipids in the membrane. And PHB accumulates in granules in the cyst, possibly as food storage for when the cyst germinates.

What They Wanted to Know
Considering that PHB seems important for the encystment process, or at least shows up in cysts, Segura and colleagues wondered if mutating the phb genes in A. vinelandii might affect the cells' encystment.

What They Did
The scientists sequenced the region of genome containing the phb operon, and compared the open reading frames they found to known sequences. Of the genes they found, they made strains of A. vinelandii with two different genes knocked out, phbC (which makes PHB synthase) and phbB (acetoacetyl-CoA reductase), by inserting stuff into the genes; and characterized these mutants, testing their PHB production, aklylresorcinol production, and encystment. Measuring the PHB followed the usual methods, with boiling chloroform and concentrated sulfuric acid; sounds like tons of fun.

What They Observed
In the sequence they got, there were six open reading frames (ORFs). By comparing the sequences to other known PHB-related genes (such as from 174), they identified the ORFs from A. vinelandii as the PHB-producing operon phbBAC, along with the regulator-producing gene phbR. Another of the six was like phbP from Ralstonia eutropha, making a granule-associated protein, and next to that an ORF similar to phbF in R. eutropha, seemingly a putative regulator for PhbP.

Then they knocked out phbB and phbC, though not in the same strain. Neither of these mutants produced detectable levels of PHB. The phbB knockout had over 90% reduction in acetoacetyl-CoA reductase activity (makes sense) and also much less activity from PhbA or PhbC; it seemed like the mutation had polar effects on the operon. The phbC mutant only had much reduction in PHB synthase, about 95%, which makes sense, though the other enzymes were affected a little too (~40%), maybe because of unstable mRNA.

Then they induced encystment, apparently with n-butanol. Neither mutant seemed impaired; phbB knockout actually seemed to encyst more. And with a different induction method, they saw the same results, even in regular Burk medium. Obviously they didn't contain PHB granules, but this didn't seem to be a problem: their viability was the same or even higher than wild-type cysts.

Regarding alkylresorcinol production, A. vinelandii produces them when PHB or n-butanol replace glucose as a carbon source. But the authors tested the mutant strains first in regular Burk, since they apparently could form cysts in that; turns out they also were able to produce alkylresorcinols, unlike the wild-type, especially the phbB knockout, which also had greater alginate production (possibly contributing to its higher viability).

Under an electron microscope, the mutants' cysts didn't have PHB granules (of course), and in the phbB knockout strain, the exine of the cysts seemed thicker than other strains', probably due to extra alginate and alkylresorcinols.

What This Means
The phb operon is the one involved in PHB production in A. vinelandii too. Knocking out phbC seems to produce a cleaner phenotype, with less effect on the cells other than lack of PHB production. But it seems like lack of PHB channels more carbon through the lipid metabolism pathway. It doesn't seem to affect encystment much, at least not negatively, but this may only be because of the unnatural lab environment in which the cells are growing.

All this extra production of alkylresorcinol and alginate may be due to accumulation of acetyl-CoA that would normally go toward PHB. The mutant lacking PHB synthase may accumulate hydroxybutyrate instead; it's not clear what effects that might have.

Reference: Segura, D., Cruz, T. & Espín, G. Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-β-hydroxybutyrate synthesis. Arch Microbiol 179, 437–443 (2003).

248 - NAD-, NMN-, and NADP-dependent modification of dinitrogenase reductases from Rhodospirillum rubrum and Azotobacter vinelandii

What They Knew
Nitrogen fixation is a demanding process, using a lot of energy, so bacteria regulate it tightly, shutting it off whenever fixed nitrogen is available already. One diazotroph, Rhodospirillum rubrum, regulates its nitrogenase by ADP-ribosylating the dinitrogenase reductase component, using a protein called Dinitrogenase Reductase ADP-ribosyl Transferase, or DRAT. This takes ADP-ribose from NAD when ammonium is present. Another protein, Dinitrogenase Reductase-Activating Glycohydrolase (DRAG), reverses the process.

What They Wanted to Know
Ponnuraj and colleagues studied the nitrogenases of R. rubrum and Azotobacter vinelandii to see how specifically each interacted with the DRAT/DRAG system and various ADP-ribosyl donors.

What They Did
They took different ADP-ribosyl containing molecules (NAD, NADP, NADH, NMN), combined each with A. vinelandii's dinitrogenase reductase (DNR) and R. rubrum's DRAT, then ran them with SDS-PAGE along with samples lacking the molecules, to see which molecules could be used to donate ADP-ribosyl. They also exposed some of each sample to DRAG and ran that alongside to see if it could remove the modification.
Results from this were confirmed with another test, seeing if modified or de-modified DNR could function with dinitrogenase to reduce acetylene.

To see how small a modification works to inactivate the system, they removed a phosphate from phosphoribosylated DNR and tested it again.

In addition to testing A. vinelandii's DNR, they tested R. rubrum's too.

To see more specifically what was going on with A. vinelandii's DNR, they used MALDI-TOF mass spectroscopy.

What They Observed
Based on gels and activity assays, NAD, NMN, and NADP all seemed pretty good at donating to DRAT to inactivate A. vinelandii's DNR. NAAD not so much, or anything else they tried. DRAG seemed able to re-activate DNR with all of them too.

Surprisingly, with R. rubrum's DNR, only NAD seemed to be a good donor. Mass spec results confirmed their expectations about what was going on biochemically.

What This Means
R. rubrum uses DRAT and DRAG to regulate its nitrogenase activity based on whether fixed nitrogen is available already and whether its environment is illuminated or not. This helps save energy, so it doesn't have to break down the whole system and reconstruct it with every little environmental change.

It's not clear how relevant it is for A. vinelandii, though, because that organism doesn't appear to have the genes to produce DRAT/DRAG proteins. It's somewhat interesting that R. rubrum's proteins are able to modify A. vinelandii's nitrogenase, arguably even better than they can with R. rubrum's, but this actually makes some sense, that R. rubrum would have tighter control over its nitrogenase regulation system. Though apparently some in vivo studies suggest it might not be as tight as it seemed here.

This doesn't necessarily mean that A. vinelandii doesn't have a system for post-translational regulation, just that it isn't exactly this one. I haven't found what it is yet, if there is one. And other studies seem to imply that A. vinelandii might not have such tight control (010). I wonder why.

Reference:

Ponnuraj, R. K., Rubio, L. M., Grunwald, S. K. & Ludden, P. W. NAD-, NMN-, and NADP-dependent modification of dinitrogenase reductases from Rhodospirillum rubrum and Azotobacter vinelandii. FEBS Letters 579, 5751–5758 (2005).

174 - Poly(3-Hydroxybutyrate) Synthesis Genes in Azotobacter sp. Strain FA8

Azotobacter is a genus that makes polyhydroxyalkanoates, especially polyhydroxybutyrate (PHB). This is a carbon-storage polymer, so the cells can store extra carbon in their environment they can't use right away, and it forms intracellular granules that can be consumed later. Also it's basically a kind of plastic, but biodegradable, so it's of some economic and biotech interest.

What They Wanted to Know

Pettinari and colleagues had a species of Azotobacter called FA8 that makes PHB, but at the time they didn't know what genes were involved in the PHB synthesis process in Azotobacter, so they wanted to figure that out. The genes are more well-studied in other species, but might not all be the same.

What They Did

They cut up genome DNA with a restriction enzyme and put it into an Escherichia coli library, then screened the strains they got for one gene in particular: phbC, which encodes PHB synthase, the final enzyme in the PHB production pathway (at least in other species). This screening was done using a PHB synthase-deficient strain of another well-known PHB-producing species, Ralstonia eutropha. If they put a plasmid into this R. eutropha and suddenly it can produce PHB again, it must have the phbC gene.

When they found an transformant of R. eutropha that produced polymer, they extracted it from the cells to analyze the monomeric units: lyophilize the cells, extract the polymer with hot chloroform, precipitate with ethanol, then analyze methyl ester derivatives with a gas chromatograph.

Then they sequenced the gene they had found, responsible for this PHB synthesis, and compared it with other known synthases, from Pseudomonas, Ralstonia, and Burkholderia species; they also looked upstream and downstream from this gene for other relevant genes, and cloned and sequenced those that they found, comparing them to other species as before.

So then to make sure the phbC gene they found actually deserved its name (and was doing what they expected), they knocked it out in Azotobacter FA8 by inserting an antibiotic resistance marker in the middle of it. First they amplified a 578-bp section of gene, inserted that product into the pGEM-T Easy vector. Then they cut this piece open with PstI, inserted a kanamycin cassette from pUC4K, and transferred this whole construct into pAT18, which can replicate in E. coli but not Azotobacter. Finally they used conjugation to transfer the plasmid from E. coli to Azotobacter, where the latter recombined with it to transfer the kan cassette into its genome, thus becoming able to resist the kanamycin used to select for transconjugants. They confirmed the PHB-lacking phenotype using the GC method described above. To make extra-sure, they made another construct in pRK404 with an intact phbC gene, transferred that to Azotobacter, and observed that it restored the PHB-producing phenotype.

Finally they wanted to see if Azotobacter FA8 could make more kinds of polymers than just PHB. There are different kinds of PHA synthases that can incorporate different monomers with different carbon-chain lengths; some prefer short chains, some longer. So they grew the bacteria on media with different carbon sources, specifically glucose and/or octanoate or hexanoate. They also tried putting genes from Pseudomonas aeruginosa in the phbC-knockout strain, which they thought might help metabolize the longer chains better. And they tried putting Azotobacter genes in R. eutropha to see what polymers they could get from there.

What They Observed
Analyzing the polymer that R. eutropha produced with the Azotobacter gene, they found it was a homopolymer composed entirely of 3-hydroxybutyrate, so, basic PHB. The gene was closely related to PHB synthase genes from other related species, as mentioned, so they named it phbC in Azotobacter. Knocking the gene out and then restoring it by complementation confirmed that it was correctly named.

There was nothing interesting downstream of phbC, but upstream they found a couple other genes that were also very similar to PHB-related genes: phbA, that encodes β-ketoacyl-CoA thiolase, and phbB, which makes acetoacetyl-CoA reductase. Together these form the phb operon, apparently in Azotobacter as well as other species. They also found some consensus regions for σ⁷⁰-dependent promoters upstream of phbB.
In their experiments with polymer production, Azotobacter could only grow when glucose was present, regardless of other genes or carbon sources. And when it produced polymer at all, which was only when it had its own phbC gene, it produced only PHB. There were similar results in R. eutropha possessing Azotobacter's phbC.

What This Means
Pettinari and colleagues seem to have identified the PHA synthase-encoding gene in Azotobacter FA8 and identified the product as a short-chain specific synthase, that doesn't do much longer than four carbons per monomer. So that's good to know.

Apparently around the time this paper was in publication, someone else published A. vinelandii phbB and phbC gene sequences; comparing them to the ones in this paper, they're over 90% similar, which makes sense. So it should be possible to use knowledge of one when studying the other.

Citation: Pettinari, M. J. et al. Poly(3-Hydroxybutyrate) Synthesis Genes in Azotobacter sp. Strain FA8. Appl. Environ. Microbiol. 67, 5331–5334 (2001).

013 - Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor

What They Wanted to Know
Pau et al. knew that Azotobacter vinelandii had three versions of nitrogenase, including one with no heterometal (Mo or V), only iron. All of these had similar requirements for energy and conditions. They're all similar in structure too, except that the alternatives both have an extra subunit.

So Pau and colleagues wanted to purify the iron-only dinitrogenase from A. vinelandii and analyze its structure and such.

What They Did
They used a strain of A. vinelandii with the genes for Mo and V nitrogenases deleted, so the only one it could produce was the iron-only one. Since this strain couldn't fix nitrogen in the presence of Mo (it represses the alternatives), they selected for a mutant that didn't have this limitation: RP306. They grew large amounts of this strain (in a 400-L fermenter) and purified the nitrogenase from it. Then they analyzed the enzymatic activity and chemical structure of protein and metallic cofactor.

What They Saw
The parent of strain RP306 couldn't grow by fixing nitrogen when molybdenum (Mo) was higher than 5nM in the medium, but RP306 actually grew better as Mo increased, up to 20nM.

Since the V nitrogenase has an extra subunit (δ) encoded by the vnfG gene, and the Fe nitrogenase has a homologous gene, anfG, Pau et al. thought that it might encode a δ subunit also. So they analyzed the subunits of the dinitrogenase with SDS-PAGE, and did indeed see a third small subunit as expected, whose amino acid sequence corresponds to the sequence of the anfG gene.

In terms of metal content, the dinitrogenase seemed to have about 24 atoms of iron and 18 of sulfur, which corresponded well to previous work. Not surprisingly, it had negligible V, but surprisingly it had 1 atom of Mo. So they analyzed it with electron paramagnetic resonance or EPR spectroscopy, which gives different curves depending on the chemical composition, and it seemed like the iron-only nitrogenase actually had a Mo-containing cofactor! Though it seemed like only one of the two cofactors in the dinitrogenase contained Mo. They were able to extract this cofactor, observed that it had a Mo-to-Fe ratio of 1:4.3, and could insert into a cofactor-less Mo nitrogenase from Klebsiella pneumoniae and make it active.

This Fe dinitrogenase with a Mo cofactor could reduce acetylene, but only to ethylene, not to ethane like regular V and Fe nitrogenases could produce. This activity, or any other, was only present when the enzyme was paired with the iron-only version of dinitrogenase reductase, not with the other versions.

With other substrates (N₂) or no substrate (just argon), this Fe nitrogenase didn't perform as well as the Mo nitrogenase. With argon, it produced 350 nmol hydrogen per minute per mg of enzyme, compared to 2220 from the Mo version; with nitrogen, it produced about 100 times less ammonia than the Mo version, but twice as much hydrogen as ammonia. This is about 4 times as much as expected from the Mo version, which produces one hydrogen per nitrogen fixed. So about 57% or 4/7ths of its electron flux goes to hydrogen, compared to 25% of the Mo nitrogenase's. They also saw some ethane produced from acetylene somehow, especially when the ratio of dinitrogenase reductase to dinitrogenase was higher; at least half the electron flux went to ethane.

What This Means
Apparently the allegedly iron-only nitrogenase can incorporate Mo-containing cofactor, at least partially, and this affects its activity. I wouldn't expect this to happen much in nature, since in the presence of Mo the Fe nitrogenase wouldn't be produced, so it's not clear what this really means in terms of enzyme activity. It seems important to exclude Mo from the medium when studying the real activity of the Fe nitrogenase though.

From other results, it seems like the cofactors, despite their differences in metal content, can substitute for each other in the holoenzymes, though the resulting activity changes (not surprisingly). The cells rely on regulation of genes that produce the proteins and cofactors to keep things running the way they should be, rather than specificity of cofactor for protein. But it's probably usually not disastrous if there are a few mix-ups. The activity is best with the right match, but it still works somewhat with some mismatches.

Citation: Pau, R. N., Eldridge, M. E., Lowe, D. J., Mitchenall, L. A. & Eady, R. R. Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor. Biochem. J. 293, 101–107 (1993).

010 - Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii

Apparently, it had previously been shown that ammonium repressed nitrogen fixation in Azotobacter vinelandii, and even when fixing nitrogen, cells would immediately take up ammonium when it was given, but would not immediately start fixing nitrogen if they ran out of ammonium. They wanted to look at this lag period more closely.

What They Did

They grew A. vinelandii OP (aka CA) in Burk's nitrogen-free medium, and actually this is the paper most people later cited as the best recipe for Burk's, the standard medium for growing A. vinelandii.

So they grew the cells, sometimes with ammonium acetate or potassium nitrate as fixed nitrogen sources, sometimes with chloramphenicol to prevent protein synthesis. They also did enzyme activity assays with nitrogenase, using ¹⁵N₂. And determined protein content of cells.

What They Observed

The first figure, taken from Strandberg's master's thesis, shows that when A. vinelandii is grown in a nitrogen-free atmosphere with ammonium, growth eventually levels off; if N₂ is then added, cells start growing again after a short lag, 30-60 minutes. But if ammonium is added instead, there's no lag; the cells start growing again immediately. If N₂ was present the whole time, the cells switch to nitrogen-fixing pretty quickly when ammonium runs out, with a small decrease in growth rate.

A better demonstration for this lag was nitrogenase activity assays: it showed right when nitrogen fixation activity started, about 1 1/4 hours after ammonium ran out. Though oxygen levels and temperature possibly weren't ideal. It could be as little as 45 minutes later.

Another interesting observation was that when ammonium ran out and cells were in an environment of 40% oxygen (with the rest 60% helium or hydrogen), they didn't start producing nitrogenase, but they did start when oxygen was only 20%. The hydrogen level didn't seem to matter.

One problem they encountered was that there were small amounts of nitrogen in their gas tanks of oxygen, helium, and hydrogen, which could've been enough to affect the results. They tried to make purer oxygen by electrolysis (splitting water), though there was still a bit of nitrogen; still, it wasn't clear whether nitrogenase production was induced by the presence of nitrogen or merely repressed by ammonium. My guess would be the latter, since cells wouldn't normally encounter N₂-free environments in nature. But regulation can be complicated.

They noticed a slight rise in turbidity even after ammonium ran out, but speculated it could be due to color change that cells go through (from reddish brown to dark brown) when fixing nitrogen. The small amount of nitrogen in the gas flow was enough to get cells to produce nitrogenase, but not enough for them to use it. But cell-free extracts didn't show different absorbance for the two kinds of cells, despite the visible difference.

When they added chloramphenicol, an antibiotic that inhibits protein synthesis, obviously this inhibited nitrogenase formation. If the enzyme was already present, in vitro, the antibiotic didn't inhibit it. But it did inhibit it in cells, possibly because ammonium built up with no way to use it, repressing nitrogenase.

They tried adding 150 mg N (as ammonium) per liter to a culture of nitrogen-fixing cells, and saw that nitrogenase activity dropped off within about 3 hours. Not as fast as I would expect. They interpreted this to mean that the enzyme is not inhibited immediately, just diluted out as the cells stop producing it while continuing to multiply; but it seems to be inactivated faster than just by dilution, so there might be some inactivation or degradation going on.

Citation: Strandberg, G. W. & Wilson, P. W. Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii. Can. J. Microbiol. 14, 25–31 (1968).

009 - A Non-Gummy Chromogenic Strain of Azotobacter vinelandii

A popular wild-type Azotobacter vinelandii strain at the time was Wisconsin strain O, which was sometimes difficult to work with because it became "gummy," that is, kind of slimy and mucusy. This was due to alginate production, which helped production nitrogenase from oxygen.

So Bush and Wilson were trying to isolate a stable, non-gummy strain that would be easier to study. Eventually they found one that made dense, slime-free colonies on Burk agar plates, and once sure they had a pure culture, they called it A. vinelandii OP (now also called CA). This strain still produced its nice, yellow-green azotobactin pigment for iron-gathering.

Even in liquid, OP didn't become gummy, like other strains did. So they had found something useful. Later studies, especially sequencing the genome (023), revealed that a transposon had knocked out an alginate regulatory protein in OP.

Citation: Bush, J. A. & Wilson, P. W. A Non-Gummy Chromogenic Strain of Azotobacter vinelandii. Nature 184, 381–381 (1959).

004 - Bacterial Iron Transport: Coordination Properties of Azotobactin, the Highly Fluorescent Siderophore of Azotobacter vinelandii

In order to fix nitrogen, Azotobacter vinelandii needs a lot of iron for its nitrogenase enzyme. But in environments with oxygen, iron is usually found in insoluble, oxidized forms, so A. vinelandii secretes compounds called siderophores that bind/chelate the iron and make it easier to bring into the cell.

One of these in particular is a very pretty yellow-green compound that also can fluoresce blue under ultraviolet light, called azotobactin. The authors here wanted to study azotobactin properties, so they purified it, basically by filtering out bacterial cells and running the filtrate through columns.

They found that azotobactin has five sites that are involved in binding iron. This doesn't mean it binds five iron atoms though. Actually it seems only to bind one. It does seem to bind more strongly than another siderophore called aerobactin that Escherichia coli produces.

Once the cell takes iron-bound azotobactin back inside, it seems to reduce the complex, convert Fe(III) to Fe(II) and thus release the iron for other purposes (such as in nitrogenase).

The last interesting thing is that azotobactin is most fluorescent when it is not binding iron. Only one of the conformational states it goes through when bound to iron is slightly fluorescent.

Citation: Palanché, T., Blanc, S., Hennard, C., Abdallah, M. A. & Albrecht-Gary, A.-M. Bacterial Iron Transport: Coordination Properties of Azotobactin, the Highly Fluorescent Siderophore of Azotobacter vinelandii. Inorg. Chem. 43, 1137–1152 (2004).

106 - Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii

What They Wanted to Know

As mentioned last time (105), as the carbon-to-nitrogen ratio of culture medium increases (and the carbon becomes a lot more available than fixed nitrogen), Azotobacter vinelandii biomass stays fairly level for a time, and then starts increasing; it's like two different phases. This depends on the oxygen exposure somewhat (at low oxygen, biomass increases more linearly; at high oxygen, it doesn't increase much at all, at least at the C/N ratios tested), but is a definite phenomenon at some levels.

The hypothesis is that, when there's not much more carbon than fixed nitrogen, there's not enough nitrogen to produce much more biomass (nitrogen is limiting), and there's not enough carbon to make the cells start fixing nitrogen (because that takes a lot of energy; so carbon is also limiting). But as carbon increases, the cells start up their nitrogenase, and nitrogen stops being limiting, so biomass increases.

In this paper, Bühler, Oelze, and colleagues wanted to see if this was actually what was happening in the cells, by testing nitrogenase activity directly.

What They Did

As before, they grew A. vinelandii CA in a chemostat, but this time they measured nitrogenase activity by acetylene reduction (nitrogenase can reduce acetylene/ethyne to ethylene/ethene, which is easy to measure). They also measured total nitrogen and protein contents of the culture, corrected for added ammonium. And to make triple-sure, they did Western blots on samples of culture, using antibodies targeting nitrogen-fixing proteins.

What They Observed

As in 105, protein/nitrogen content remained fairly constant at a mid-range oxygen level up to a point as sucrose increased, and beyond that point, it increased proportionally along with dry weight.

For nitrogenase activity, they saw that the higher the oxygen, the higher the C/N ratio had to be before the cells had detectable nitrogenase activity (and the lower the peak activity at the highest carbon level). After nitrogenase started, it increased up to a certain C/N level, then leveled off. 

They give a formula for how to calculate the C/N ratio when nitrogenase starts working. And based on that, they figured out that cells started fixing nitrogen when the ammonium they were given was not enough for production of biomass from the sucrose they were given. Which makes sense. That happens at about 14 mmol ammonium per gram of protein.

Finally, they wanted to figure out whether nitrogenase proteins needed to be synthesized from scratch in ammonium-grown cells, or whether they were already present to some extent, just not active. So they used Western blots to look at nitrogenase proteins from cells at various C/N ratios. The lowest ratio showed no nitrogenase activity and no visible nitrogenase protein on the blot; mid-range showed slight activity and the faintest of bands; and the highest showed good activity and solid, visible bands. Flavodoxin proteins, related to electron transport to nitrogenase, showed up at all ratios, interestingly.

What This Means

It appears from this that cells have to produce nitrogenase proteins from scratch as C/N ratios increase, but I'm not sure it's clear that inactive versions of the proteins would show up on the blot. Maybe the antibodies they used to detect the active versions don't work well on inactive versions. It's possible.

The other possibility is that A. vinelandii does keep inactive nitrogenase around for short periods, but eventually breaks it down, and the cells in this study were kept too long in nitrogen-sufficient conditions, so they had to re-synthesize nitrogenase. This would make sense too.

In order to explain the nitrogenase regulation, the authors say it's tempting to say the need for respiratory protection is why cells don't fix nitrogen until C/N ratios are high enough, but clarify that another explanation could be that the cells just have enough fixed nitrogen until a certain point. A tricky conclusion to a tricky series of studies. Perhaps I will revisit later.

Citation: Bühler, T. et al. Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii. Archives of Microbiology 148, 247–251 (1987).

105 - Control of respiration and growth yield in ammonium-assimilating cultures of Azobacter vinelandii

What They Wanted to Know

As discussed in the previous posts, Bühler, Oelze, and others knew that Azotobacter vinelandii could fix nitrogen at high oxygen levels, but weren't sure how: the respiratory protection hypothesis, that the cells increased their respiration to consume all the oxygen, only seemed to make sense at relatively low levels of oxygen (below 30% saturation); respiratory activity doesn't increase very much beyond a certain point.

Also, since nitrogenase requires a large amount of energy, it's possible the respiration might just be increasing to provide for it. In 089, this lab showed that increasing the fixed nitrogen provided to A. vinelandii led to lower respiratory activity. So, similar to 090 which looked at substrate use efficiency in nitrogen-fixing conditions, here they wanted to see how the efficiency changed when fixed nitrogen was provided.

What They Did

As in previous, they grew A. vinelandii OP (aka CA) in chemostats. They added various levels of sucrose as a substrate and ammonium chloride as fixed nitrogen. They also added sodium citrate, 0.05g/L, to keep the iron they provided from precipitating.

They measured respiratory activities in different states based on oxygen consumption, and also cell dry weights and protein contents, as well as residual sucrose and ammonium.

What They Observed

At the lowest oxygen level (5%), biomass increased almost linearly with increasing sucrose. But as oxygen increased, biomass stayed constant at lower sucrose levels (up to 13-20 mM), and then rose linearly but not as steeply as at low oxygen. At high oxygen (60%), biomass barely rose at all. This was all with the same amount of ammonium added. So, at a given sucrose level, more oxygen meant less biomass. This is consistent with previous studies (090).

They also tried keeping the sucrose constant and varying the amount of ammonium, which affected the carbon-to-nitrogen ratio. They saw similar patterns of biomass vs. C/N ratio, with shifts in the biomass increase at higher ratios, as they had seen when varying the sucrose.

Residual sucrose and ammonium were always very low, so it was all being consumed, and thus limiting. Ratios of dry weight to protein contents were always constant, so there didn't seem to be any nutrient storage going on, even at high sucrose levels.

In nitrogen-limited states at low C/N ratios, yields of biomass were higher, though they decreased as sucrose increased. This is sorta the opposite of what was seen in purely nitrogen-fixing cultures (090), where yield increased as dilution rate (and thus, amount of sucrose) increased. They leveled off when cultures started fixing nitrogen. Even with ammonium though, higher oxygen meant lower yields.

Similarly, respiratory activity increased as C/N ratio increased, up until nitrogen-fixing started; the higher the oxygen, the higher the respiration. Though at a given C/N, higher oxygen always meant higher respiration, unlike in previous studies where it leveled off, though maybe the ranges of sucrose concentrations were different. Also, they measured both respiratory capacity and actual respiration, and cells always seemed to be using only about 50% of their capacity.

What This Means

The way to understand this is that at low sucrose levels, there's only enough ammonium to support a certain amount of biomass production, and not enough sucrose to make it worth turning on nitrogenase, but as the sucrose increases (or ammonium decreases), it becomes more worthwhile.

So C/N ratios seem to control respiratory capacity and activity. That kinda explains why respiration might level off at higher C/N ratios, when nitrogen-fixing activity has started: cells fix as much nitrogen as the carbon level permits, keeping the C/N ratio constant, so the respiration level is constant also. I guess. Look for more discussion about that in the next post.

One last cool thing about this paper: they give the ratios of the main components of A. vinelandii cells, based on the thesis of one H.W. van Verseveld in 1979. The composition is C₆H_10.8N_1.5O_2.9. Useful for calculating molar yields.

Given this, it appeared that the cells converted between 20-30% of the sucrose they consumed into biomass, getting rid of the rest of it, at the lowest oxygen level (5%). At 60% oxygen saturation, they only assimilated 5-10%. Overall, the results aren't really consistent with respiratory protection of nitrogenase, since these were cells grown with ammonium. Interesting.

Citation: Bühler, T. et al. Control of respiration and growth yield in ammonium-assimilating cultures of Azotobacter vinelandii. Arch. Microbiol. 148, 242–246 (1987).

090 - Dependency of growth yield, maintenance and Ks-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii

What They Wanted to Know

Considering Azotobacter vinelandii's high respiration rates at higher oxygen concentrations, it seemed like its energy metabolism was uncoupled from respiration, meaning that it was respiring without getting as much energy from it, proportionally. This meant that its maintenance energy, the amount of food it needs just to remain the same, should be pretty high in some conditions. So Kuhla and Oelze wanted to examine maintenance requirements and growth yields in more depth, at different levels of oxygen.

What They Did

Similar to previous posts, they grew A. vinelandii OP (aka CA) in a chemostat, limiting its carbon diet and controlling its oxygen exposure, under nitrogen-fixing conditions. They adjusted dilution rates (by adjusting the rate that fresh medium flowed into the reactor) and oxygen saturations.

They also tested different carbon substrates: grams per liter of sucrose, glucose, or acetate. They measured cell dry weights in different conditions, and protein content, nitrogen content, and residual substrates.

What They Observed

First they ruled out the possibility of nutrient storage as a confounding factor, by increasing the amount of food the bacteria received. This didn't really affect the nitrogen content of the cells much, so they didn't seem to be storing it for later. So protein content is a good proxy for total biomass.

Protein content increased as dilution rate increased, not surprising because it meant they were getting more food. At the lowest oxygen level, though, this increase leveled off before too long (around 0.1 h^-1), but even after leveling off the values were still much higher than at any other oxygen level, so they might've just run out of oxygen. Speaking of oxygen, as the saturation increased, the protein content decreased at a given dilution rate.

Growth yields followed almost exactly the same pattern as protein content. So the higher the oxygen, the less efficiently cells used the substrate.

They used a couple methods to calculate maintenance requirements in different conditions (including consumption vs. growth rate from 199 and 1/Y vs. 1/D from 065), but they gave similar results, at least for sucrose and glucose. As expected, maintenance increased as oxygen increased, at least for sucrose, but the maintenance on acetate at mid-range oxygen seemed relatively low and the yield relatively high, so it seems like acetate is used more efficiently.

What This Means

Oxygen above a certain level seems to place extra demands on a cell, preventing it from converting as much of the substrate it consumes into biomass. Exactly what these demands are is unclear though, but growing on acetate seems to reduce the demands somewhat, at least proportional to the amount of carbon consumed.

The authors calculated that, theoretically, A. vinelandii could convert up to 26.2% of the carbon it consumed into biomass, when fixing nitrogen. Presumably this would be significantly higher if not for the requirement to fix nitrogen.

Speaking of respiratory protection again, the expectation would be that maintenance requirements and oxygen levels would be linearly related, but this was only true at lower oxygen levels, suggesting that at higher oxygen, respiration isn't as good at removing oxygen.

Citation: Kuhla, J. & Oelze, J. Dependency of growth yield, maintenance and K_s-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii. Arch. Microbiol. 149, 509–514 (1988).

089 - Whole Cell Respiration and Nitrogenase Activities in Azotobacter vinelandii Growing in Oxygen Controlled Continuous Culture

This paper was basically a sequel to 098, by most of the same authors.

What They Wanted to Know

The question that Post, Kleiner, and Oelze wanted to answer in this paper was in regard to Azotobacter vinelandii's ability to protect its nitrogenases from the damaging effects of oxygen. Azotobacter is an obligate aerobe, so this is always an issue, but how it protects its sensitive enzymes was not clear.

The theory was that A. vinelandii employs respiratory protection, in which it consumes oxygen at so high a rate that oxygen cannot build up to toxic levels in the cell. If oxygen does build up too high, or there isn't enough substrate available to consume it all, A. vinelandii can reversibly change the conformation of its nitrogenase so it is protected, though it cannot fix nitrogen in this state, so the cell effectively goes dormant.

The idea of respiratory protection comes from the observation that A. vinelandii can only fix nitrogen aerobically when there is adequate substrate available to maintain high enough rates of respiration. If there's no substrate from which to get electrons to dump onto oxygen, the system doesn't work. It takes time to increase respiration rates, so this process doesn't cope well with sudden increases in oxygen. This is when conformational change helps. In theory.

What They Did

They grew A. vinelandii OP (aka CA) in a chemostat, limiting its carbon (at two different levels) and controlling oxygen exposure, either with nitrogen gas or ammonium as a source of N. Similar to 098. Oxygen was always kept higher than limiting, so they could measure exactly how much there was; I wonder if that was the best range to observe though, and it means that 0% wasn't really anaerobic.

They measured cell protein contents and nitrogenase and respiratory activities, as well as residual sucrose.

What They Observed

Cell protein levels were always higher in ammonium-grown cells, not surprisingly, and in both N conditions they rose a bit as oxygen rose to about 3% saturation, and then dropped, leveling off at around 30%. At higher carbon, N-fixing cells took a bit longer to level off, at about 50% oxygen.

Protein yield followed a similar pattern, dropping as oxygen increased up to 30%. Carbon level didn't affect N-fixing cells' yield, but ammonium-grown cells had higher yields with lower carbon levels.

The pattern of respiratory activity was similar to the above, but inverted: it rose between 1 and 30% oxygen saturation, then remained pretty constant. Nitrogen status didn't affect it much at the higher carbon level, and was always higher than the lower carbon values, but at the lower carbon it was about double when fixing nitrogen compared to when grown with ammonium.

Nitrogenase activity decreased quickly up to about 3% saturation, then gradually up to 100%. Carbon level didn't matter.

Then they tried holding the oxygen constant at 45% and increasing the dilution rate (how fast new medium flowed into the reactor, diluting out the contents). Respiration increased linearly with dilution rate, as did protein content and nitrogenase activities at first, but at a point (around D = 0.25 h^-1), the protein content dropped off and nitrogenase activity increased greatly.

Finally, instead of gradual increases in oxygen saturation, they adapted cells to one level and then suddenly changed it to a higher level for 7 minutes, then dropped it back. Regardless of the starting saturation or new peak of oxygen, the cells always switched off their nitrogenase activity when exposed to a larger amount of oxygen. They started it up again when the oxygen dropped back down, but not at the same level as before.

What This Means

Cell activity seemed to level off around 30% oxygen saturation, so either that's more than they can use, or their ability to deal with it has peaked and doesn't need to increase any more. However, a sudden large increase does cause them to suddenly shut down, even if they wouldn't have shut down with a gradual increase to the same level, so there's something else going on.

It's interesting to note the lower yields as oxygen increased, indicating that A. vinelandii was sorta wasting the carbon to deal with the oxygen. It wasn't just when fixing nitrogen though, so it might not be specifically to protect the nitrogenase. Hard to say from just this.

Inconsistent with the respiratory protection hypothesis is the large increase in nitrogenase activity at higher dilution rates without a simultaneous increase in respiration, while oxygen remained the same. Also the fairly constant rates of respiration and nitrogenase activity as oxygen increased above 30% to 100%; we would expect respiration to rise and nitrogenase to drop more severely.

So respiratory protection might be important at some levels of oxygen, but possibly not all.

Citation: Post, E., Kleiner, D. & Oelze, J. Whole Cell Respiration and Nitrogenase Activities in Azotobacter vinelandii Growing in Oxygen Controlled Continuous Culture. Arch. Microbiol. 134, 68–72 (1983).

144 - Experiments on the Transformation and Fixation of Nitrogen by Bacteria

This old and somewhat hard-to-find report (Update: apparently now it's on Google Books; see link below) by J.G. Lipman details the discovery of Azotobacter vinelandii, discovered in Vineland, New Jersey in the early 1900s.

Lipman's goal was to study the nitrogen-related behaviors of soil bacteria in general, and specifically to isolate nitrogen-fixing bacteria.

Behaviors of soil microbes in nitrogen-rich or nitrogen-poor media
When soil is added to nitrogen-rich medium, such as meat extract, many species grow. Some soluble nitrogen becomes insoluble (incorporated into bacterial cells/biomass), but more is broken into simpler forms, including nitrogen gas, so the total amount of nitrogen in the culture decreases.

But when there is a minimal medium of certain salts and organic compounds but not much fixed nitrogen, the amount of nitrogen in the medium increases when soil is added, because bacteria fix nitrogen gas.

So two results from the same soil in different media. This makes sense. Different bacteria grow to different levels in each.

Some of the flasks of these two kinds of media were inoculated with soil from Freehold, either pasteurized or not, or similar soils from Vineland. Others were kept sterile or inoculated with a single species (one B. pyocyaneus). These each differed in appearance after incubating for a number of days.

In high-nitrogen medium, the non-pasteurized soils reduced the total nitrogen the most, as should be expected.
In low-nitrogen medium, pasteurized Vineland soil actually remained sterile, but the others grew, and those increased the amount of nitrogen. Apparently the Freehold soil had more spore-forming organisms, which are more likely to survive pasteurization.

In the Freehold soil, nitrogen-fixers seemed to consist mostly of spore-forming organisms, while in the Vineland soil the main organism appeared as large, aerobic diplococci (paired roundish cells), which Lipman labeled Azotobacter (i.e. nitrogen bacteria).

Also noteworthy: non-pasteurized Vineland soil fixed more nitrogen than any of the others, by far.

Isolation of Azotobacter vinelandii
Trying to isolate Azotobacter from soil samples was somewhat difficult for Lipman, because small bacilli kept overgrowing his cultures, but eventually he found a good medium to select for the desired bacteria. Through certain tests (growth preferences, motility, shape of cells), Lipman distinguished his culture from Beijerinck's Azotobacter chroococcum isolate, naming it instead Azotobacter vinelandii (because it came from Vineland).

A. vinelandii Characteristics
Lipman noted A. vinelandii's growth characteristics in various media, and observed in some that the cells accumulated deposits of what he called fat, which looked like small globules, giving the body a granular appearance.

He also noted that the cells produce a bright yellow pigment that diffuses out from the cells, especially when well-aerated. He tried to figure out what the pigment is, but apparently a fire at the research station interfered with that goal.

A. vinelandii Fixing Nitrogen
Lipman did an experiment with his bacteria, inoculating several flasks of broth with them and testing how much nitrogen they fixed. He found that, for a given volume of culture, being in a bigger flask meant fixing more nitrogen; i.e. more surface area was helpful. Apparently oxygen is important.

Then he tried different forms of nitrogen (or none at all): potassium nitrate, peptone, or ammonium chloride, in two different quantities. After 18 days, all cultures had the same appearance, with growth and yellow pigment. The amount of fixed nitrogen in them was pretty similar for all. So it seemed like adding fixed nitrogen, at least in these low amounts, didn't affect nitrogen fixation much.

For some reason, Lipman also observed that adding filter paper to the culture more than doubled the amount of nitrogen fixed. Maybe extra carbon? Seems improbable. When they weighed the paper after removing it from the culture, it didn't seem to have decreased at all, so apparently it wasn't consumed.

A. vinelandii Carbon Substrates
Lipman tried growing the bacteria on a variety of carbon sources, to see which it could use: ethanol, glycerine, mannite (mannitol) + soil, propionate, succinate, and citrate. It seemed to grow on all of them to some extent; perhaps best on mannitol, then glycerine, then ethanol and propionate, then a bit on succinate, and very slightly on citrate. This is paralleled in the amounts of nitrogen fixed in each one.
They tested some other substrates, but didn't complete the experiments before the fire mentioned above. After 48 hours, A. vinelandii seemed to be growing on glucose, sucrose, and dextrin, but not lactose.

A. vinelandii and Bacillus 30
Apparently there was some mysterious bacterium, discovered around the same time as A. vinelandii, which, when grown together with A. vinelandii, enhanced the latter's nitrogen fixation for some reason, though by itself B. 30 couldn't fix very much, if any.
When grown together, A. vinelandii seemed to fix from 2-4 times more nitrogen. Considering that B. 30 seems to produce a large amount of hydrogen, this could be one way it helps nitrogen fixation, but I'm not sure exactly how that works.
None of the other soil microbes discovered around the same time seemed to help A. vinelandii fix nitrogen very much.

Adding pasteurized soil from different places also seemed to help A. vinelandii fix nitrogen, especially Vineland soil, but it wasn't clear why for that either.

Other Factors Influencing Nitrogen Fixation
Adding iron to the medium seemed to help increase nitrogen fixation a great deal.

There were some other experiments, but I didn't think them very important. Check it out if you're curious.

Citation: Lipman, J. G. Experiments on the Transformation and Fixation of Nitrogen by Bacteria. New Jersey State Agric. Exp. Sta. Ann. Rep. 24, 217-285 (1903).

098 - Morphological and Ultrastructural Variations in Azotobacter vinelandii Growing in Oxygen-Controlled Continous Culture

What They Wanted to Know
At the time of this paper, some had noticed that Azotobacter vinelandii seemed to produce extra cell membrane surface area protruding into its cytoplasm in some conditions. It seemed to happen when the cells were fixing nitrogen, to the extent that some thought it related only to nitrogen and not to oxygen/respiration at all, though others disagreed and thought it could happen with high enough cell densities or low aerations, regardless of nitrogen (implying that oxygen was the important factor).

So in this paper, Post, Golecki, and Oelze investigated conditions resulting in membrane formation very specifically, using chemostats with controlled levels of oxygen and nitrogen.

What They Did
They grew A. vinelandii strain OP (aka CA), either with ammonium acetate added as a fixed nitrogen source, or no fixed nitrogen (so it had to fix its own). They used an oxygen probe to measure and control the oxygen dissolved in the medium, as a percentage of saturation from 1-100%. The dilution rate was 0.15 h^-1 for 800mL of culture, stirred at 1000 rpm, so the doubling time for the bacteria was 4.6 hours.

They measured the vesicles/invaginations of cell membrane in each condition by transmission electron microscopy, and cell shrinkage with light microscopy, calculating average cell volume.

What They Found
What they found was that for all nitrogen conditions, cells got bigger as oxygen levels increased, up to about 1.6-fold. At all levels, nitrogen-fixing cells were about 10% larger than cells growing with fixed nitrogen. Length:width ratio remained the same for all though. The size increase happened mostly between 1% and 25% oxygen saturation.

The result of this increase meant less area on the surface of the cell, which could mean less penetration of oxygen inside, but also less area available for establishing a proton gradient for energy generation. However, in nitrogen-fixing cells, the number of membrane vesicles also increased with higher oxygen levels, which meant more membrane surface area for proton gradients. The two effects nearly balanced each other, such that membrane surface area per cell volume increased only 1.5x.

In ammonium-grown cells, by contrast, the amount of membrane increase was lower, so the ratio of membrane area to cell volume dropped a little.

Another contrast is that in nitrogen-fixing cells, the ratio of vesicle membrane area to cytoplasmic membrane area increased almost 3-fold, but in ammonium-grown cells it stayed pretty constant.

What It Means
This means that both nitrogen status and oxygen levels can affect cell size and area of membranes in the cells. Oxygen may affect whether or not vesicles are formed at all, while nitrogen affects the numbers and proportions at a given oxygen level, at least between oxygen saturations of 0 and 25%. But the amount of vesicle area increased as oxygen increased, rather than decreasing (as others had proposed).

It makes some sense that nitrogen-fixing cells would try to increase membrane area while decreasing cell surface area (by increasing volume): that would help increase potential respiration rates while decreasing oxygen penetration, so it'd be easier to protect oxygen-sensitive enzymes. This doesn't really explain why ammonium-grown cells also increased cell volume though. But it's an interesting result.

Citation: Post, E., Golecki, J. R. & Oelze, J. Morphological and Ultrastructural Variations in Azotobacter vinelandii Growing in Oxygen-Controlled Continous Culture. Arch. Microbiol. 133, 75–82 (1982).

207 - Transformation of Azotobacter vinelandii with plasmid DNA

One cool thing about Azotobacter vinelandii is that it is naturally competent—that is, in certain conditions it takes up DNA from its environment and sometimes incorporates it into its genome. This is a useful characteristic for a species to have when studied in the lab, because it means a researcher can modify its genes and such to see what they do.

But besides taking up straight pieces of DNA and incorporating them (recombination), it'd also be useful if A. vinelandii could take up and maintain plasmids, which are small circular pieces of DNA, usually with only a few genes on them (for example, a gene of interest and an antibiotic resistance gene as a selectable marker). Plasmids are useful for studying overexpression or complementation of genes, for example, when it's not necessary to incorporate anything into the genome.

So Glick, Brooks, and Pasternak attempted to transform A. vinelandii with several broad-host-range plasmids:

• pRK2501 (IncP-1 group, tetracycline and kanamycin resistance) 
• RSF1010 (IncQ group, sulfonamide and streptomycin resistance)
• pGSS15 (IncQ group, tetracycline and ampicillin resistance)

They selected for transformants using kanamycin, streptomycin, and tetracycline, respectively.

To transform A. vinelandii, it's necessary to use Transformation (TF) medium:

• 1.9718 g/L MgSO₄
• 0.0136 g/L CaSO₄
• 1.1 g/L ammonium acetate
• 10 g/L glucose
• 0.25 g/L KH₂PO₄
• 0.55 g/L K₂HPO₄
• For solid medium, 18 g/L agar

This low-iron, nitrogen-containing medium, developed by Page and von Tigerstrom, induces competence in A. vinelandii.

So Glick et al. picked a colony into TF medium, grew at 30ºC to an optical density (620nm) of less than 0.2, then transferred to fresh TF broth and grew some more. They tested transformation at a variety of optical densities at this point to see which is best, standardizing the density of cells transformed to 1.6 x 10⁸ cells per mL with TF broth.

50 µL of cells mixed with 300 µL fresh TF and 50 µL DNA (~22 µg/mL) sat at 30ºC for 30 minutes. These were spun down and resuspended in 400 µL fresh TF and incubated for another hour.

Then the cells were plated onto regular A. vinelandii agar plates, with or without antibiotics, and grown for 3 days at 30ºC. Plates without antibiotics revealed numbers of viable cells after transformation, and plates with antibiotics (compared to those without) indicated frequency of transformation.

From the results, it seemed like cells grown up to optical densities between 0 and 1 (which took 2-24 hours) could be transformed at very similar efficiencies; maybe a slight negative slope, but hardly noticeable. After the first 5 hours of growth, the culture should take on a yellow-green color as it becomes iron-limited.

Interestingly, transformed colonies on plates without antibiotics could be distinguished from non-transformed colonies; the transformed ones grew a lot bigger and more gooey.

The authors also found that, not surprisingly, the more DNA added to the transformation mix (from 0.1 up to 51 µg), the higher the frequency of transformation. At 51, 44% of the viable cells were transformed, which is not bad.

Also useful to note is that even without antibiotic pressure, transformed cells kept their plasmid around for at least 10 generations (not sure if that means 10 cell divisions or 10 transfers from one culture to another); and that the plasmid remained separate from the genome, rather than integrating or recombining or anything.

So this is useful for those who want to introduce genes into A. vinelandii and do some genetic modification; it's not required to integrate anything into the genome to express new proteins.

Citation: Glick, B. R., Brooks, H. E. & Pasternak, J. J. Transformation of Azotobacter vinelandii with plasmid DNA. J. Bacteriol. 162, 276–279 (1985).

199 - Maintenance energy: a general model for energy-limited and energy-sufficient growth

Even when a microbe is actively growing, it is not devoting all of the energy it gathers toward growth; some always goes toward maintaining basic functions. This is called "maintenance energy." When there's plenty of food around, this constant consumption (being relatively small) is minute and hard to perceive, but when food is scarce, it becomes more apparent.

In this paper, John Pirt (who apparently is one of my academic forebears) defined a number of ways to determine and use the maintenance energy in continuous culture experiments.

Through a series of equations are difficult to conceptualize except mathematically, Pirt derives a theory that only if maintenance energy were zero could growth rate and substrate utilization efficiency reach their theoretical maximum values. If maintenance is greater than zero, which it always is, the maximum growth rate is reduced some amount called a, which is the specific growth rate at which the actual growth yield is half of the maximum theoretical growth yield, so substrate utilization is not as efficient as it could be.

However, this assumes that the maintenance energy requirement stays constant over different rates of growth. But if maintenance decreases as growth rate increases, the formula is different. In this case, maintenance energy falls to zero as growth rate increases up to the reduced amount mentioned above. In this case, while growth rate never reaches its theoretical maximum, substrate utilization does.

Allow me to quote, since I don't think I could word this concept better:

"The specific maintenance rate (a) may be regarded as an endogenous metabolism rate which results in decrease in the biomass and expenditure of the corresponding amount of maintenance energy."

But this article goes beyond theory and tries to use data from live cells to test this concept. The data are take from this study.

The data show the behavior of bacteria, Klebsiella aerogenes, growing in conditions where either carbon, nitrogen, phosphorus, or sulfur is insufficient. In a graph with oxygen consumption on the y-axis and growth rate on the x-axis, a comparison between actual culture data and lines drawn on the graph based on theoretical patterns, the data fit the lines amazingly well. This is especially true for carbon, sulfur, and nitrogen; not so much with phosphorus, in which the oxygen consumption doesn't increase as much as expected at higher growth rates.

Overall, the maximum growth rate and oxygen consumption don't change between different limitations, either in theory or in real data (possibly excepting P). This is working under the assumption mentioned above, that maintenance energy decreases as growth rate increases. But at low growth rates, the different limitations have very different effects on the oxygen consumption: there is much lower consumption when carbon is limiting compared to the others, especially phosphorus.

To explain the divergence from theory in the P-limiting experiment, Pirt suggests that the cells may be storing excess nutrients in their cells, which the researchers that collected the data did not consider or control for. This is one thing about cells that can be tricky: they change their behavior in certain conditions. And this could be how the maintenance energy requirement changes.

And that's about it. So... I'm not sure I understand this completely, but probably better than before.

Citation: Pirt, S. J. Maintenance energy: a general model for energy-limited and energy-sufficient growth. Arch. Microbiol. 133, 300–302 (1982).

185 - Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii

Azotobacter vinelandii is known for its amazing ability to fix nitrogen, converting nitrogen gas into biological forms like protein even in the presence of oxygen. This is apparent even in its name—Azotobacter—which translates approximately to "nitrogen bacteria."

But the nitrogen-fixing process and machinery is pretty complicated and difficult to study in a reductionist fashion. Many components don't work the same outside of the context of the rest of them or outside of the cell itself.

So in this study, scientists (some of whom were involved in sequencing the first A. vinelandii genome) took a broad approach to the subject, by looking at the expression of all the genes in the organism, comparing their expression when the cells were fixing nitrogen compared to when they weren't.

In addition, they examined the expression of genes when the bacteria were grown with or without certain metals needed for the various nitrogenase versions: molybdenum, vanadium, etc. More specifically:

• To study non-nitrogen-fixing cells: they grew cells with ammonium
• Cells using the molybdenum nitrogenase: they grew cells with molybdenum and no ammonium
• Cells using the vanadium nitrogenase: they grew cells with vanadium and no Mo or ammonium
• Cells using the iron-only nitrogenase: they grew cells with iron and no other metals or ammonium

Simple enough.

Then, to measure levels of expression, they extracted RNA from the cells in each condition, converted the RNA sequences to DNA (called cDNA or complementary DNA, converted from RNA messengers), and then these chunks of DNA were sequenced using a high-throughput technology called SOLiD.

SOLiD (or Sequencing by Oligonucleotide Ligation and Detection) is one of the main kinds of next-generation sequencing, alongside Roche's 454 pyrosequencing and Illumina. I had to look it up. Apparently one machine these days can sequence 5 trillion bases per day (for reference, the human genome is about 3.2 billion bases long, so SOLiD could sequence more than 1500 human genomes per day). Of course, this is not cheap: that much sequence data would cost about $500,000.

The way it works is by cutting a piece of DNA into short sequences, binding them to tiny magnetic beads so there's one sequence per bead. Then the beads are mixed into an emulsion of oil so that on average, each bead is encased in a small bit of water in a sea of oil, along with reagents needed for polymerase chain reaction (PCR). This allows the DNA on each bead to be copied many times, all at once yet individually, so that many reactions can be done in the volume of liquid that would normally allow only one. Very cool. This is the same technology used to prepare samples for 454 pyrosequencing.

In pyrosequencing, the sequencing is done with DNA polymerase, which is what is normally used to copy DNA, and each base is added one at a time, so each bead will incorporate only the base that fits with the sequence bound to the bead. When a base is added, a tiny bit of light is given off, which a camera in the machine detects and registers it as the base that was added at that time.
However, SOLiD works a bit differently: instead of DNA polymerase, the enzyme is DNA ligase, which links together two strands of DNA. And instead of single bases, short DNA sequences called probes are added, with the two bases at one end known. When one of these matches the bead's sequence, ligase links it up. The probes are labeled with fluorescent molecules, so the next two bases in a sequence can be determined from the color of the fluorescent probe. Then this is cleaved off and another two bases are added. It's somewhat confusing; this site might help a little.
This system avoids some of the problems that pyrosequencing experiences, such as with accuracy, but has some problems of its own (especially price), so it's useful in some cases and not as much in others.

So what good is sequencing all the cDNA? The number of copies of one cDNA, relative to the copies of others, shows how much the cell is transcribing that gene, which can be an indicator of expression levels (transcribing more may mean that the gene is translated into protein more, so there may be more of that specific protein in the cell). It doesn't always work quite proportionally, since there are mechanisms other than transcription for regulating cellular protein levels, but it can usually provide some interesting data. So they sequenced all the cDNA in the cell and compared the number of copies for each gene to see which ones were present in higher or lower numbers in different growth conditions.

Then after analyzing all this data (a large undertaking in itself) and finding genes that seemed to be expressed at higher or lower levels in the different conditions, the scientists confirmed the most interesting findings using real-time quantitative PCR, which is a more sensitive way of measuring the same information. It works by doing PCR on a gene but adding some kind of fluorescent molecule to detect exactly how much of that sequence is present over time in the PCR. Ideally PCR should double the number of sequences in every round of the reaction, but this doesn't always work exactly, so the most accurate measure is to determine the point at which the fluorescence becomes bright enough that the PCR machine can detect it, and then extrapolate back to figure out how much of the sequence was present at the beginning. It's another way to compare transcription levels.

Ok, finally on to results. The authors found that almost 30% of A. vinelandii's genes were affected when fixing nitrogen compared to when not doing so. Many of these were affected regardless of which nitrogenase the cells were using. Mo nitrogenase growth affected the most genes on its own compared to the other two, but the two alternative nitrogenases (V and Fe) together affected more genes than any nitrogenase on its own. So overall, compared to non-fixing conditions, the using the alternatives affected many more genes than using the Mo version; but compared to each other, using the alternatives didn't change many genes. Apparently using the alternatives involves a large shift in the cell's gene regulation, compared to using the main Mo nitrogenase. I wonder why.

Genes Expressed When Using Mo Nitrogenase
The nif genes that make up the Mo nitrogenase are found in two clusters in the genome, one near the beginning (relative to the origin of replication) and one near the end. Some of these genes form the actual enzyme structure itself, some help to put it together with its metal-containing cofactor and such, and some (especially nifA) regulate the process.

Not surprisingly, the main structural genes increased their expression greatly when the cells switched to nitrogen-fixing mode, between 50 and 150 times higher. The primary dinitrogenase reductase, NifH, increased the most, which fits in with previous observations that a high ratio of this protein to the Mo-containing dinitrogenase allows higher nitrogenase activity.

More surprisingly, other nif genes in the major cluster only increased expression up to about 14 times more. This could be because not much of their proteins is needed, or possibly that they were already expressed at high levels and their regulation is mostly post-transcriptional, so not much change would be visible in transcript levels. In the minor cluster, some genes necessary for making the metal cofactor increased around 20-fold.

Other than these expected increases, lots of other genes changed as well; not surprising, considering that nitrogen fixation is essential for growth in low-nitrogen environments but is also very energy-intensive. The most significant changes were in type IV pilus genes. These pili, little hairlike projections from the cell, are involved in lots of things: motility, sensing the environment, attachment to surfaces, etc. It's not entirely clear what they're doing in this context, but apparently something.

Another important factor for nitrogen fixation is protecting the nitrogenase from oxygen. A. vinelandii seems to do this by consuming a lot of carbon in order to reduce whatever oxygen is present, transforming it to water. Its genome has many electron-transporting proteins such as oxidoreductases and terminal oxidases, some of which did appear to be somewhat upregulated in nitrogen-fixing conditions. This could also be useful for producing more energy to power the nitrogenase. The genes for the uptake hydrogenase, which recovers hydrogen produced by the nitrogenase and regenerates some energy from it, also showed increased expression.

There also seems to be a change in some genes associated with iron and sulfur organization, something else that is important for nitrogenase because it contains multiple atoms of these elements. Also, not very surprisingly, the genes related to molybdenum gathering increased also.

Genes Expressed When Using Alternative Nitrogenases
Obviously, the genes that encode the alternative nitrogenases themselves (vnf genes for the vanadium-containing nitrogenase and anf genes for the iron-only nitrogenase) are going to be upregulated when these are in use. The V (vanadium) nitrogenase is used when molybdenum is not present but vanadium is, and the Fe nitrogenase is used when neither of these metals is available.

But these alternative systems don't have alternative versions of all of the necessary enzymes for fixing nitrogen, only the main ones, so they share some of the proteins that the Mo nitrogenases uses. This is especially true of enzymes involved in assembling the nitrogenases and their cofactors, such as NifUSVMB.

When vanadium was present, vnf genes were upregulated, as I said, but in this case, the vnfH gene encoding the vanadium dinitrogenase reductase wasn't as high relative to the other V nitrogenase components as was the case with the molybdenum nitrogenase. Not sure why. There were some other differences, especially that vnf homologs of nif proteins involved in cofactor synthesis were expressed in different proportions, so the process of V-containing cofactor synthesis might be different somehow.

In the case of the iron-only nitrogenase, the nifH and other components' homologs (anfH, etc.) were upregulated in the same ratio as the nif genes, distinct from the vnf homologs: that is, anfH was expressed much higher than anfDK, around four- to five-fold higher.

Of the genes that don't have anf homologs, some nif genes were upregulated (nifUSVMG again), but in other cases the vnf versions were preferred (vnfENXY). vnfH was also upregulated, even though there is a separate anfH. This is in agreement with other previous studies (003), and may be because vnfH has some kind of role in regulating gene expression.

Genes Related to Electron Transport
All of the nitrogenases require electron transport machinery, since the nitrogenase functions by putting electrons (and protons) onto nitrogen gas (N₂) to make ammonia (NH₃). This takes at least eight electrons for each molecule of nitrogen: six for two molecules of ammonia, and two for one molecule of hydrogen as a byproduct. The alternative nitrogenases produce more molecules of hydrogen, so they need even more.

Some of the genes involved are nifF and vnfF, which encode proteins called flavodoxins that transport electrons. They may not be necessary to fix nitrogen, but presumably they're helpful. When the cells were using the Mo nitrogenase, nifF was upregulated, and both were higher when Mo was absent (though vnfF much more so).

Some other genes that seemed involved included rnf1 genes, whose products are membrane-bound and also help to transport electrons to nitrogenase; they also seem to be important for the iron-sulfur cofactor of dinitrogenase reductase. And fix genes also seem important for electron transport. All of these were expressed more when fixing nitrogen in all conditions, but when Mo was absent, fix genes were much higher than rnf1 genes.

Regulatory Genes
Clearly A. vinelandii's nitrogenase system has a lot of regulation going on, so regulatory genes are important. vnfA and anfA are necessary to use the alternative nitrogenases, as is nifA for the primary nitrogenase, and these regulatory genes increased whenever their respective isozyme was in use, though low levels of them were present constantly. Regulation of these genes is likely to be how the cells turn on and off the alternative nitrogenases.

There are a few other homologs of nifA and vnfA that show similar patterns, but may fine-tune the regulation somehow (how is not yet known).

Other Differences in Global Expression
As mentioned, the transcriptional profile when using the Mo nitrogenase is very different from when using V or Fe nitrogenases, probably because the latter are less efficient. The most apparent difference in this study was in the hutU gene, for urocatanase hydratase, which increased greatly when Mo was absent. This gene is necessary to degrade histidine, one of the 20 common amino acids, which makes sense because cells using a less efficient nitrogen-fixing enzyme might want to get nitrogen from other places too, like breaking down some less essential proteins. Similar results have been seen in other diazotrophs.

Also quite interesting, genes for a putative soluble hydrogenase discovered when the genome was sequenced were upregulated, especially when using the Fe nitrogenase. This may be a backup system for recycling the extra hydrogen molecules that these nitrogenases produce, to recover the valuable energy that would otherwise escape.

Some other genes increased also, but the function of their products is as yet unknown. Might be worth investigating.

Comparing expression when using the V nitrogenase vs. the Fe nitrogenase, there was at least one interesting point: there were a few genes near a vnf operon upregulated during V nitrogenase growth, seemingly related to a transporter system, so they're probably a vanadate transporter.

Evolution of Nitrogenase
There's some debate about which came first in history: the Mo nitrogenase or the alternatives. Since the alternatives are less efficient, it would make some sense if they came first and the Mo nitrogenase just improved on them, especially since Mo and possibly V were probably difficult to find before there was much oxygen in the atmosphere. But no one has discovered a species that has alternative nitrogenases and not the Mo nitrogenase, though there are plenty that have only the latter, and not many that have all three. And this study seems to show that the alternatives evolved from the Mo nitrogenase to allow cells to thrive in environments where Mo is absent, which also makes sense.

So these results are very interesting and potentially useful, and seem to tell us a lot about what's going on inside the cells in different conditions!


Citation: Hamilton, T. L. et al. Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii. J. Bacteriol. 193, 4477–4486 (2011).

205 - Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool

Saccharomyces cerevisiae is a well-known species of yeast, commonly used for making bread, beer, wine, and other such fermented products. It can convert sugar effectively into ethanol (useful in alcohol production) and carbon dioxide (useful for creating pockets of air that make bread dough rise).

This yeast's products are inextricably linked to its anaerobic fermentative metabolism—what happens when it consumes food without consuming oxygen. (Note: S. cerevisiae does have the ability to use oxygen, but it chooses not to when its food is abundant, or when oxygen is not present, obviously.)

And an essential part of this metabolism is nucleotide molecules that carry protons and electrons from one cellular chemical reaction to another, oxidizing and reducing various compounds in the cell. These are called NAD⁺/NADH and NADP⁺/NADPH (the + refers to the oxidized form with one fewer electron and proton, and the H refers to the reduced form), and are slightly different, both in function and chemical structure.

NAD is used more in breaking down food and shuttling the electrons from this process to the final reactions of the metabolic process. For example, electrons from glucose transfer to NAD⁺, forming NADH. Then in an aerobic condition, the electrons would be pushed across the membrane in the Electron Transport Chain to generate energy for the cell, ending up on oxygen to form water; in anaerobic metabolism, they end up being dumped to generate ethanol and CO₂ and regenerate NAD⁺ to repeat the cycle.

NADP is used more for synthesizing important compounds for cell growth, such as proteins and nucleotides.

There are some organisms that have enzymes that can transfer electrons between these two systems, usually from NADH to NADPH. These enzymes are called transhydrogenases, of which there are two kinds: those bound to the membrane, which are pretty common in mammals and bacteria; and those that are floating in the cytoplasm of the cell, found especially in bacteria such as E. coli, Pseudomonas, and Azotobacter vinelandii.

The authors suspected that if this kind of enzyme were present in S. cerevisiae, it might increase the amount of ethanol the yeast produces from the same amount of sugar, and since ethanol is a valuable product for biofuels (and for drinking), they thought it was worth a try.

But first they had to find the DNA sequence of the gene in A. vinelandii, since at this point the genome hadn't been published. To do this, they purified the transhydrogenase protein from the bacteria (detecting it by its chemical activity rather than its sequence) and determined the sequence of amino acids at one end of it, by chopping off one amino acid at a time, identifying it, then the next, up to the first four. Then, since a number of different DNA sequences can code for the same sequence of amino acids, they made primers that could bind to a number of similar but not identical sequences (called "degenerate primers") and used these for PCR to amplify the gene out of the bacteria. They named it sthA (for Soluble Transhydrogenase A).

Compared to the amino acid sequences from the other bacteria in which the enzyme is found, A. vinelandii's SthA protein is pretty similar. And the more closely related the species, the more similar the protein, as might be expected.

So they took the transhydrogenase gene from A. vinelandii, sthA, put it on a plasmid (a small circular piece of DNA) and put it into the yeast along with a gene that provides resistance to an anti-bacterial and anti-yeast compound, so they could be reasonably sure that any cells that could grow in this compound would also contain the gene of interest. The result was a strain of yeast called TN21.

When they tested extracts of this strain for STH activity, they found a decent amount, and none in strains TN1 and TN26 which lacked the gene. So the new protein seemed to be working in the yeast, and at higher levels than it is naturally in the bacteria. Producing the transhydrogenase also slowed down the growth rate of strain TN21, down to less than half of the parent strain, but did it work as they wanted?

They tested levels of an enzyme in a metabolic pathway called the Pentose Phosphate Pathway, which is a way cells break down sugar that is good for generating NADPH. Their hypothesis was that the SthA activity might reduce the activity of this pathway, since the cell would already have plenty of NADPH converted directly from NADH. So they tested the activity of one of the enzymes in this pathway, but found that it was just as active in the STH strain as in the others, almost exactly the same. So that hypothesis failed.

The other hypothesis was that SthA might increase the yield of ethanol (by simultaneously reducing the production of glycerol). In actuality, all three strains produced similar levels of these compounds, and ironically the strain with SthA produced a little less ethanol (11% less) and a little more glycerol, if these are even real differences. The authors attempted to explain this by pointing out that the cell biomass produced in strain TN21 was slightly lower also (13%), so it seemed that this strain was less efficient in general.

What they did notice, though, was that TN21 produced a lot of a compound called 2-oxoglutarate. About 23 times more than the other strains. This compound is normally consumed by oxidizing NADPH, so an overabundance of it indicates a lack of NADPH; the transhydrogenase may be converting all of it to NADH.

This is the opposite of what the authors wanted to happen, but such is the nature of science. They modeled what they thought was happening in the cells energy-wise, and found that there was so much more NADPH than NADH in the cells to begin with that it's no wonder which way the reaction went. I'm not sure why they expected anything different, considering that the same thing happens in bacteria. But in science, you gotta try! Good on them for publishing negative results.

Still, the transhydrogenase enzyme seems like it could be useful for some things, depending on the reaction you want to happen. I will have to investigate further.

Citation: Nissen, T. L., Anderlund, M., Nielsen, J., Villadsen, J. & Kielland-Brandt, M. C. Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool. Yeast 18, 19–32 (2001).