159 - Effect of chelating agents on hydrogenase in Azotobacter chroococcum: Evidence that nickel is required for hydrogenase synthesis

This study used chelating (metal-binding) compounds to study the cofactor of hydrogenase in Azotobacter chroococcum.

What They Saw
All the chelators they added (NTA, EDTA, etc.) decreased the hydrogenase activity in batch cultures, though to different extents. NTA was much stronger than EDTA. The effect was not inhibition of already-formed enzyme (since adding chelators to resting cells or extracts didn't affect activity), so it must be from preventing formation of additional enzyme.

They tried adding trace metal salts along with the chelators to see if pure excess of whatever was missing could restore activity. Copper, zinc, and manganese didn't really do anything. Cobalt helped if it was added in fairly large amounts, but the most helpful was nickel. Adding extra iron boosted this effect even more.

Monitoring nickel uptake by adding radioactive nickel, they saw that cyanide completely wiped out uptake (possibly by binding the nickel), but juglone and 2,4-dinitrophenol enhanced it (despite inhibiting respiration). Sodium azide didn't really affect either. The chelators they tested earlier generally seemed to inhibit nickel uptake too, generally in the same proportions as they had inhibited hydrogenase activity.

What This Means
Chelators seem to inhibit hydrogenase, but rather than acting on the enzyme directly, it seems to be by inhibiting its synthesis, and even this mechanism seems to be by inhibiting nickel uptake in most cases, rather than something more direct. Nickel is important for synthesizing the enzyme; it's a part of its essential cofactor. It seems like cobalt might be able to substitute for nickel somewhat though. I wonder if palladium would work too, since it has similar orbital arrangements. But apparently cobalt doesn't help in the absence of chelators or contaminating trace metals, so maybe it only helped here because it distracted the chelators away from nickel (so to speak).

Reference:

Partridge, C. D. & Yates, M. G. Effect of chelating agents on hydrogenase in Azotobacter chroococcum: Evidence that nickel is required for hydrogenase synthesis. Biochem. J. 204, 339–344 (1982).

071 - Two open reading frames (ORFs) identified near the hydrogenase structural genes in Azotobacter vinelandii, the first ORF may encode for a polypeptide similar to rubredoxins

This study looked at the genetics of the two open reading frames (ORFs) near the hydrogenase structural genes (hoxZM).

What They Saw
They sequenced the ORFs and compared to known genes. hoxZ seemed to have most homology with genes encoding proteins called rubredoxin from other species. These are typically small proteins that play roles in electron transport, which makes sense. And that's all.

Reference:

Chen, J. & Mortenson, L. Two open reading frames (ORFs) identified near the hydrogenase structural genes in Azotobacter vinelandii, the first ORF may encode for a polypeptide similar to rubredoxins. Biochim Biophys Acta 1131, 122–124 (1992).

070 - The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase

Here they wanted to look into the hoxZ gene more closely. Previous studies suggested that the product might be involved in electron transport for the hydrogenase.

What They Saw
They grew Azotobacter vinelandii DJ (an easy-to-transform strain) and knocked out hoxZ and hoxKG by transformation and screening for hydrogen production.

Comparing the hoxZ mutant to DJ (positive control) and the hoxKG mutant (negative control), they observed an intermediate rate of hydrogen oxidation, so there seemed to still be some activity. DJ consumed nearly all the hydrogen, and hoxKG consumed very little (the graph showed a decrease but it was apparently because gas leaked out of the vial, so it's a good thing they had good controls!).

Then they tried measuring short-term hydrogen oxidation with different electron acceptors: oxygen or methylene blue. DJ quickly oxidized all the hydrogen while reducing oxygen or methylene blue, as expected. Both mutants didn't show activity with either acceptor at first, despite the difference in the previous assay. But then they added sodium dithionite (a powerful reducer of oxygen) and more methylene blue, and the hoxZ mutant showed up to 80% of the activity of DJ. As far as I can tell, the hoxKG didn't show the same effect when they gave it the same treatment, but they don't say that explicitly. But it seems like the hydrogenase needs to be activated somehow, as by dithionite.

These results were confirmed by observing methylene blue color change too; DJ quickly started oxidizing hydrogen, but the hoxZ mutant did too after a longer lag period.

When they isolated membrane-bound hydrogenase from cells (still embedded in membranes), even DJ needed activation with dithionite. hoxKG mutants had no activity in any case, of course. But hoxZ mutant had more activity in the soluble supernatant portion than DJ did, at least when membranes were isolated aerobically; it seemed like lack of hoxZ led to more soluble enzyme. But it had low activity in general so this conclusion was uncertain. Though membrane-bound activity in general was higher when isolated anaerobically, and they didn't measure soluble activity in that case for some reason. So HoxZ may help stabilize hydrogenase in the presence of oxygen.

The increased presence of detached hydrogenase in the mutant was not confirmed by Western blot, so it seems like an artifact.

What This Means
HoxZ seems to have a role in shuttling electrons between hydrogenase and oxygen, though there may be other components involved in this path. It's possible that when HoxZ is missing, another acceptor can take the electrons, but isn't as good at it.

It also may be involved in activating the enzyme (which requires removing oxygen and providing reduction); somehow hydrogen is not enough for this. And it may help stabilize the hydrogenase to keep oxygen from inactivating it, maybe also using its role as electron transporter.

Reference:

Sayavedra-Soto, L. A. & Arp, D. J. The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase. J. Bacteriol. 174, 5295–5301 (1992).

067 - Analysis of a gene region required for dihydrogen oxidation in Azotobacter vinelandii

This study looks more closely into the genes discovered in previous studies (518,050,051,052) to be required for hydrogenase in Azotobacter vinelandii.

What They Saw
They knocked out different genes (or the whole operon) in the hyp operons of A. vinelandii CA by inserting resistance + lacZ cassettes, then tested these strains for hydrogen oxidation and expression of the genes (via beta-galactosidase activity). The medium they used had a lot more trace elements than typical Burk medium, including nickel.

They found that when lacZ was inserted in the same direction as the gene, they saw expression in all cases, whether fixing nitrogen or not, but if it was inserted in the opposite direction, they didn't. So apparently the genes are expressed to some extent even when not fixing nitrogen.

When they measured hydrogen oxidation, there was about 6x more when fixing nitrogen though, in the wild-type, and none in the mutants. The growth rates of the mutants were similar to the wild-type though, or so they claim without reporting any details.

Finally they tried growing the strains with extra added nickel, because the hydrogenase is a nickel-containing enzyme. This didn't have much effect on the wild-type, but the hypB mutant actually showed some hydrogenase activity in nitrogen-fixing conditions with the extra nickel, and activity was higher with more nickel added.

What This Means
It seems like extra added nickel can substitute for the lack of HypB, so maybe the enzyme has a role in nickel cofactor processing somehow. This makes sense considering the multiple histidines it contains. It's interesting that nickel didn't help activity in non-fixing conditions; maybe there are multiple hydrogenases, active in different conditions, and hypB is required for all but nickel doesn't help some of them.

Reference:

Chen, J. C., Mortenson, L. E. & Seefeldt, L. C. Analysis of a gene region required for dihydrogen oxidation in Azotobacter vinelandii. Curr. Microbiol. 30, 351–355 (1995).

056 - Properties of Hydrogenase from Azotobacter vinelandii

Another study tries to purify the uptake hydrogenase from Azotobacter vinelandii.

What They Saw
They were unable to get pure, soluble enzyme; they couldn't separate it from the membrane, so it remained in insoluble particles.

They observed that hydrogenase activity of these particles didn't decrease after 18 days of exposure to air, unlike particles from other species. They did lose activity within a few weeks though.

They also observed that the enzyme reduces cytochrome C, possibly as part of its electron transport role. They found that carbon monoxide and cyanide inhibit the enzyme. Azide does not, at least not at pH 8.

What This Means
Some of the observations here may be due to the presence of other membrane-bound proteins, not just hydrogenase.

Reference:

Hyndman, L. A., Burris, R. H. & Wilson, P. W. Properties of Hydrogenase from Azotobacter vinelandii. J. Bacteriol. 65, 522–531 (1953).

054 - Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium

This study investigates how the alternative (vanadium) nitrogenase interacts with tungsten.

What They Saw
They grew Azotobacter vinelandii UW (aka CA) or a tungsten-tolerant mutant with tungsten and Mo or V, then extracted the nitrogenase.

This mutant was derived by growing UW with W and ammonium over several passages to try to remove all the Mo the cells might be storing, and then plating the cells on plates without ammonium, so they would have to fix nitrogen in the presence of W and absence of Mo to survive. Stuff that grew was tungsten-tolerant: strain LM2.

Then to get nitrogenase, they grew LM2 with tungsten and UW with tungsten and ammonium (not sure why it would produce nitrogenase in that condition, though I guess it might when it ran out of fixed nitrogen).

Both UW and LM2 could grow on medium with Mo, though LM2 was sometimes slower. With W, LM2 grew faster or slower depending on amount of W, while UW did not grow.

They used something called rocket immunoelectrophoresis to measure how much MoFe nitrogenase components each strain produced with W. Each produced about the same amount of dinitrogenase reductase in all conditions, but UW with 1mM W produced about 56% the amount of dinitrogenase as it produced with Mo, and LM2 with 10mM W produced about 7% of the amount that UW produced with Mo. I wonder how accurate this technique is. But they also note that the specific acetylene reduction activity of crude extracts was about 6-8% for each strain grown in W compared to UW in Mo. Does this include alternative nitrogenase activity? They say no. But overall, it seems that both strains produce less Mo nitrogenase with W, LM2 less than UW, but LM2's is relatively more active.

I think what happened next was that they couldn't isolate nitrogenase well from LM2, so they studied it from UW. They had two kinds: typical Mo nitrogenase, and Mo/W nitrogenase that had one FeW-cofactor and one FeMo-cofactor. This latter showed less activity in every way: hydrogen under argon, nitrogen, or acetylene atmospheres, nitrogen reduction, and acetylene reduction. This fits with at least one previous study (045). There is still activity though, even with nitrogen reduction, so it's not clear why the cells can't grow in W; maybe its interaction with other important proteins?

What This Means
It's possible that instead of each molecule of Mo/W protein containing one W and one Mo, half the protein could have all Mo and the other half all W; this would give the same results, but seems a less likely explanation. Of course, both possibilities are pretty weird and confusing.

According to some chemistry stuff they did, it seems like the enzyme can't reduce FeW-cofactors, which could reduce the possible electron flux by half, I think. There are a lot of mysteries here.

Reference:

Hales, B. J. & Case, E. E. Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium. J. Biol. Chem. 262, 16205–16211 (1987).

045 - Tungsten incorporation into Azotobacter vinelandii nitrogenase

Tungsten is known to cause problems for molybdenum-containing enzymes. This report looks into its effect on Azotobacter vinelandii's Mo nitrogenase.

What They Saw
They grew A. vinelandii OP (aka CA) in Burk without Mo, with added ammonium phosphate. Because it's really hard to get rid of every little bit of Mo, they added lots of tungsten (W) to make sure that they could see it if it got incorporated into enzymes. Some of the W was radioactive.

W didn't inhibit growth when ammonia was present, which makes sense. But it did inhibit it, about the same, with N2, nitrate, or urea. The enzymes that use these N sources all need Mo. When Mo was about 0.1 μM, it took 20 μM W to inhibit growth 50%; when Mo was 10 μM, it took 4 mM W. When just a little ammonia was added, it took about 5000 times more W than Mo to stop growth.

When they purified nitrogenase from these W-grown cells, they actually did see acetylene reduction activity, though not nearly as much as with normal Mo nitrogenase. The W content of extracts was very high, though it seemed to be easily removable. Specifically purifying Fe-W protein and comparing to the Fe-Mo version, all activities seemed relatively low: acetylene reduction, hydrogen production, and ATP hydrolysis.

What This Means
A. vinelandii might treat W the same as it treats Mo: taking up as much as it can and storing what it doesn't incorporate. But it does seem to incorporate some into the Mo nitrogenase. This seems to result in a poorly functional enzyme, but is that enough to stop cells from growing entirely? Maybe W's effects on other enzymes cause some problems too.

Reference:

Benemann, J. R., Smith, G. M., Kostel, P. J. & McKenna, C. E. Tungsten incorporation into Azotobacter vinelandii nitrogenase. FEBS Lett 29, 219–221 (1973).

053 - Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing αβ dimer

This study purified and characterized the uptake hydrogenase from Azotobacter vinelandii.

What They Saw
As a single unit, not denatured, the hydrogenase ran as a single band on a gel. On a denaturing gel it was two bands though, 31 and 67 kDa, indicating that it had two subunits, a larger and a smaller (HoxG and HoxK). The whole thing is about 98.6 kDa. This contrasts with previous reports that it was a single subunit of 60 kDa (049), but it's not really clear why there's this discrepancy.

They also measured metal contents, and found 6.6 mol iron and 0.68 mol nickel per mol hydrogenase, so they rounded up to 1 mol Ni and 10 mol Fe, 1:10 ratio.

They also contradicted the previous result that the hydrogenase couldn't donate to acceptors with negative potential. But they confirmed the possibility that hydrogenase produces hydrogen when paired with a very reduced donor, except this peaked at a higher pH (6 to 8.5).

Reference:

Seefeldt, L. C. & Arp, D. J. Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing αβ dimer. Biochimie 68, 25–34 (1986).

518 - Cloning, sequencing and characterization of the [NiFe]hydrogenase-encoding structural genes (hoxK and hoxG) from Azotobacter vinelandii

To continue our journey back in time through the discovery of the uptake hydrogenase genetics in Azotobacter vinelandii, this study describes the sequencing of the hydrogenase structural genes, hoxKG.

What They Saw
They probed A. vinelandii DNA with a chunk from A. chroococcum with its hydrogenase genes, and sequenced a fragment they found. This fragment contained three full open reading frames (ORFs) and possibly the beginning of a fourth. The first two encode the structural subunits, HoxK and HoxG (for hydrogen oxidation), based on comparison to sequences in other organisms.

HoxK seems to undergo some processing, since the final protein is smaller than that predicted by the gene sequence; there might be a signal sequence that localizes the protein to the membrane or something. The structural ORFs overlap each other a little, indicating that they're probably transcribed as a unit (in the same operon) which helps the proportions come out right.

The third ORF, ORF3 (which we know now is hoxZ) was also similar to other organisms, and seemed to be a membrane protein of some sort. Possibly part of the hydrogenase electron transport chain.

Reference:

Menon, A. L., Stults, L. W., Robson, R. L. & Mortenson, L. E. Cloning, sequencing and characterization of the [NiFe]hydrogenase-encoding structural genes (hoxK and hoxG) from Azotobacter vinelandii. Gene 96, 67–74 (1990).

052 - Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii

This study looks at the hydrogenase-related genes in Azotobacter vinelandii in between the structural genes and the hyp genes.

What They Saw
They already knew about the structural genes, hoxKG. So they got some overlapping fragments containing those and sequences downstream of them, and sequenced them. They found 5 new open reading frames (ORFs), ORF3-7. They named these hoxZMLOQ. There was also part of an eighth at the end. All of them were homologous to hydrogenase genes in other organisms.

Then they knocked out each gene by inserting kanamycin or kan+lacZ cassettes, though probably some of these insertions affected other genes (polar effects) so it wasn't possible to study each gene individually. Each insertion abolished hydrogen oxidation. All 5 new ORFs seem to be involved in subunit processing, as the unprocessed form was present in higher proportion (or alone) in their knockouts.

The knockouts didn't seem to be impaired in growth, even when fixing nitrogen, though they might show more effect in certain conditions (like carbon limitation).

HoxZ seems to be a membrane protein, possibly a cytochrome, so part of the electron transport chain. HoxL seems like it might be involved in metal-binding somehow.

What This Means
Knowing more of the sequence of the operon helps to study it. The whole thing, hox and hyp, is pretty big, kinda surprising for an enzyme that doesn't seem to help the organism in some conditions. Probably in nature, the likelihood of encountering carbon-limited circumstances is much higher, so having an uptake hydrogenase is important.

Reference:

Menon, A. L., Mortenson, L. E. & Robson, R. L. Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii. J Bacteriol 174, 4549–4557 (1992).

051 - The hypE Gene Completes the Gene Cluster for H2-oxidation in Azotobacter vinelandii

As a followup to 050, this study completed the hyp operon by sequencing hypE. They suspected its existence because other sequenced operons always had a hypE after the hypD, so they purified a fragment from Azotobacter vinelandii that hybridized hypD and contained some downstream region.

What They Saw
The open reading frame (ORF) in the downstream region had good homology to hypE genes in other organisms. A few bases of hypD and hypE genes overlap, suggesting that they're translated together so there should be equal amounts present so they can work together equally.

They knocked out hypE by inserting a lacZ and kanamycin resistance cassette, which also let them detect expression (the product of lacZ can break down certain compounds to make color, so color intensity can be measured as a proxy for enzyme activity). It seemed like transcription of all the hox and hyp genes was in the same direction, and knocking out hypE meant that A. vinelandii couldn't oxidize hydrogen anymore.

They also noticed that there was a higher proportion of unprocessed hydrogenase structural component, so HypE might be involved in processing; this seems true in other organisms. It also affects the localization of hydrogenase, soluble or membrane-bound. It may affect pre-protein folding.

What This Means
This study completes the discovery of the whole uptake hydrogenase gene set in A. vinelandii. It's pretty similar to the gene sets in other organisms, including A. chroococcum, and sequencing A. vinelandii's genome confirmed the results.

Reference:

Garg, R. P., Menon, A. L., Jacobs, K., Robson, R. M. & Robson, R. L. The hypE Gene Completes the Gene Cluster for H₂-oxidation in Azotobacter vinelandii. J. Mol. Biol. 236, 390–396 (1994).

050 - Identification of six open reading frames from a region of the Azotobacter vinelandii genome likely involved in dihydrogen metabolism

This study looked into which genes in Azotobacter vinelandii are required for its uptake hydrogenase other than the structural genes, hoxKG.

What They Saw
They sequenced almost 6 kilobases somewhat downstream of the structural genes and found 6 open reading frames (ORFs), the last incomplete, so they sequenced some more to complete it. All 8 seemed to be a single operon. These genes are now called hoxV and hypABFCD (based on homology to E. coli genes).

They predicted that the middle 3 and last ORF (now called hypABF and hypD) would produce proteins good at binding Fe-S clusters. HypB has a histidine-rich region that might be good for binding nickel, and HypF has a zinc finger-like region. They have good homology to genes related to hydrogen metabolism in other organisms. They could be related to hydrogenase regulation or assembly, especially related to Ni and Fe; some (HypBF) have homology to other Ni-related proteins, such as urease.

Reference:

Chen, J. C. & Mortenson, L. E. Identification of six open reading frames from a region of the Azotobacter vinelandii genome likely involved in dihydrogen metabolism. Biochim. Biophys. Acta 1131, 199–202 (1992).

049 - Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii

Azotobacter has a hydrogenase. Some organisms have hydrogenases that produce hydrogen, or sometimes produce and sometimes break down (reversible). Azotobacter's had not been observed to break down hydrogen (so was considered unidirectional). This study purified the membrane-bound uptake hydrogenase from A. vinelandii and tested its properties.

What They Saw
They measured activity of purified enzyme with an electrode measuring hydrogen oxidation in the presence of methylene blue dye (an electron acceptor).

They measured stability of the enzyme in the presence of oxygen, and found that crude extracts were very stable (and could go for weeks without losing activity), but the more pure the preparation, the less oxygen-tolerant it was: the most pure lost half its activity in 20 minutes at 20% oxygen. This inactivation was irreversible.

With a very good electron donor (methyl viologen), the hydrogenase could produce hydrogen. The highest rate they saw was 3.4 μmol hydrogen per minute per mg protein, which peaked at a fairly low pH (around 4). The rate was almost 0 closer to neutral. Also, the presence of hydrogen in the environment inhibits its production by the enzyme.

Even when electron acceptors were not present, the enzyme could combine one deuterium from D₂ with one hydrogen from water to make HD.

The enzyme seemed to be good at donating electrons to acceptors with positive mid-point potentials but not to negative ones, so it seems to have a higher potential than reversible hydrogenases.

Reference:

Kow, Y. W. & Burris, R. H. Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii. J Bacteriol 159, 564–569 (1984).

256 - Construction of a recF deletion mutant of Azotobacter vinelandii and its characterization

So we saw that Azotobacter vinelandii seems to have multiple copies of its chromosome in each cell (253,255), but we also seemed to see that it doesn't exhibit the behavior expected of a polyploid organism, at least sometimes (445). Other times it might. One proposed explanation is that it has some mechanism by which it homogenizes its genotype, a type of homologous recombination or "homogenotization."

A possible mechanism for this is the RecF system found in E. coli, which is involved in repairing the genome after recombination. A. vinelandii has a homologous system, so this study investigated the phenotype of cells with it knocked out.

What They Saw
They knocked out recF, recA, or both from A. vinelandii UW (aka CA), by inserting tetracycline resistance cassettes. This was confirmed by Southern blotting. Actually they already had a recA knockout, so they just made that a double-knockout. That was somewhat tricky, since the recombination frequency is much lower than in the wild-type; that's not very surprising, since RecA is probably pretty important for recombination.

They found that knocking out recF seemed to impair recombination too (also not surprising), though not as much as lack of recA. With both gone, the recombination proficiency was even lower. They saw similar results with UV sensitivity (and thus ability to repair DNA damage).

Finally, the question of homogenotization: to test this, they knocked out the nifLA genes (which regulate/activate nitrogenase) by inserting a kanamycin resistance cassette, expecting that if RecA or RecF were involved in homogenotization, the lack of them would mean that it would be easy to isolate cells with kan resistance in some chromosome copies and yet capable of nitrogen fixation (because wild-type nifLA is still present in other copies to turn on the nitrogenase). This approach, again, seems questionable to me due to the presence of multiple nitrogenases. I'm not sure if NifLA are necessary to regulate all three versions, or just the Mo one.

However, they didn't find any transformants from any of the strains (wild-type or recA or recF or double-knockouts) that were both kan-resistant and nitrogen-fixing. So they conclude that these deleted genes aren't involved in so-called homogenotization, since their absence didn't make a difference. Is it possible that the homogenotization happened when cells divided and only those with kan resistance survived? I'm not sure that question was addressed. It doesn't seem known if progeny get just one copy and then make more, or if copies are equally partitioned. I suppose the latter makes more sense, since it is binary fission, as far as we know.

Also, they did say that antibiotics weren't necessary to prevent nitrogen fixation phenotypes, so I guess that addresses my question. And they did more Southern blotting to show that there was a cassette inserted in the nifLA locus in all the transformants, with no wild-type alleles visible.

I'm not convinced that homogenotization is a real thing, but I'm not yet sure how to explain these data. Somehow it seems like A. vinelandii has many copies of its genome, but it's just as possible to transform all of them the same way as to transform just some. Guess I'll keep reading.

Reference:

Badran, H., Sohoni, R., Venkatesh, T. V. & Das, H. K. Construction of a recF deletion mutant of Azotobacter vinelandii and its characterization. FEMS Microbiology Letters 174, 363–369 (1999).

255 - Segregation characteristics of multiple chromosomes of Azotobacter vinelandii

Again on the subject of multiple chromosome copies in Azotobacter vinelandii, they wanted to see if the larger amount of DNA in the cells compared to E. coli actually meant that many more copies of each gene.

What They Saw

They mutagenized A. vinelandii UW (aka CA) with transposons and tried to see if these insertions were present in as many copies as they had observed for other genes (30-40). They tried to find auxotroph mutants by conjugating with E. coli carrying a transposon. They didn't find any A. vinelandii that failed to grow on plates lacking certain amino acids, but they did find some that didn't grow very well unless the amino acids were present. This poor growth got less poor over time in successive generations though, but the cells were still resistant to the selective marker (ampicillin). They interpret this as being related to the proportion of genome copies with an insertion vs. without.

They also saw that mutagenized cells mostly couldn't grow with ampicillin when plated directly, though they could grow on antibiotic-free Burk medium. Then when cells from each of these conditions were transferred again to plates with antibiotic, only some from selective plates grew, while all from non-selective plates grew. The idea is that growing on selective plates, the ability to grow without added amino acids would be lost (since the transposon knocked out that ability in some copies, and those copies would be higher in proportion because of the selection). They should've tried growing cells from selective medium on non-selective medium to see if they got the same result.

And gaining the ability to resist the antibiotic? Shouldn't they have had that from the beginning? This whole set of experiments is unclear.

They also mutagenized cells with a different transposon that conferred tetracycline resistance, isolated DNA from them after growing on different amounts of tet, and probed with radioactive probes, correlating radioactivity to number of copies. They observed more radioactivity in cells grown with greater selective pressure, implying that there were multiple alleles and selection increased the proportion of resistant allele in a population.

I'm still somewhat dubious, but it seems like the data might be fairly solid.

Reference:

Phadnis, S. H., Dimri, G. P. & Das, H. K. Segregation characteristics of multiple chromosomes of Azotobacter vinelandii. J. Genet. 67, 37–42 (1988).

445 - Segregation pattern of kanamycin resistance marker in Azotobacter vinelandii did not show the constraints expected in a polyploid bacterium

This is a paper similar to the last one, by the same authors (254), investigating Azotobacter vinelandii's seeming ability to possess multiple copies of its chromosome, and thus exhibit polyploidy (multiple genotypes in the same locus).

What They Saw
They used A. vinelandii ATCC12837 and knocked out the nifY gene (whose product makes the central Mo cofactor) with a kanamycin resistance insertion, then grew it with or without nitrogen or kanamycin. It shouldn't be able to fix nitrogen with Mo present, though maybe with Mo absent.

Looking at the methods, I couldn't find one of the restriction sites they claim to have used, but it's possible that it was present in their strain and not in mine. All the others seem to be there.

Actually, they transformed cells, plated them out to obtain single colonies on non-selective plates, and then screened these colonies for kanamycin resistance. The idea was that if each cell had multiple copies of its chromosome, only some of the offspring of a resistant cell would have kan resistance, because some would inherit the resistant version of the genome and others wouldn't.

But when they grew more colonies from originally resistant colonies and tested their resistance, all of them were resistant that came from resistant colonies. So once the kan-resistant phenotype was present in a cell, it got passed to all offspring; no evidence of polyploidy.

This study is lacking in some ways: I would've liked to see confirmation of the locus of insertion by sequencing, to make sure it's in the right place. And it would be interesting to see what happened if they selected for nitrogen fixation, as in 254. So the question isn't quite settled.

Reference:

Pulakat, L., Efuet, E. T. & Gavini, N. Segregation pattern of kanamycin resistance marker in Azotobacter vinelandii did not show the constraints expected in a polyploid bacterium. FEMS Microbiology Letters 160, 247–252 (1998).

254 - Isolation and characterization of nifDK::kanamycin and nitrogen fixation proficient Azotobacter vinelandii strain, and its implication on the status of multiple chromosomes in Azotobacter

Others seemed to find that Azotobacter vinelandii had many copies of its chromosome (253). This was done partly by comparison to E. coli, but it seems that A. vinelandii's genome size is similar to E. coli's. And probing for specific genes seemed to show high copy numbers. A. vinelandii cells seem larger (about 12.5x), but is this enough to accommodate 40-80x more DNA? Also, it's possible to knock out genes from the species, which would be difficult if they had many copies of a gene that could substitute for each other. This study investigated.

What They Saw
They grew A. vinelandii OP (aka CA) and did genetic transformations with it, knocking out the nifDK genes (which encode the Mo dinitrogenase) by inserting a kanamycin resistance cassette by homologous recombination.

They plated transformant colonies on plate with or without kanamycin and with or without fixed nitrogen (so four different kinds of plate). The wild-type of course could grow on either plate without kanamycin but neither plate with it. They also saw two kinds of mutant phenotype: one that could grow with fixed nitrogen either with or without kanamycin, and another that could grow on all plates, with or without antibiotic or fixed nitrogen.

They tried to confirm this using PCR across the insertion, and restriction digestions followed by electrophoresis. The primers they report seem to be appropriate for getting the Mo nitrogenase genes.

Trying to figure out what they did, this paper seems to have lots of problems with reporting exactly what kind of digestions/cloning they did... sites they claim to have used don't seem to exist in my copy of the genome, or aren't in the right place, or are in too many places. So who knows what's actually going on genetically with this strain they isolated.

The gel they show from the PCR shows two bands in the kan-resistant, N-fixing mutant, and the bands seem to be the right size to correspond to sequences with and without the resistance cassette insertion. They claim to have sequenced the region but don't report the sequence in the paper.

What This Means
The authors conclude that A. vinelandii is exhibiting behavior suggesting multiple copies of a single gene locus (both with kan resistance and with nitrogen fixation). While it does seem to exhibit both phenotypes (assuming no contamination with multiple strains), I'm not sure the interpretation is clear. A. vinelandii has multiple different enzymes capable of fixing nitrogen, so a mutation in their regulation could explain that phenotype, though it wouldn't explain the bands on the gel. Still, it would be good to see this study replicated; unfortunately, the methods are explained poorly.

Reference:

Suh, M.-H., Pulakat, L. & Gavini, N. Isolation and characterization of nifDK::kanamycin and nitrogen fixation proficient Azotobacter vinelandii strain, and its implication on the status of multiple chromosomes in Azotobacter. Genetica 110, 101–107 (2000).

253 - Multiple chromosomes of Azotobacter vinelandii

Previous studies and some preliminary data supposedly showed that Azotobacter vinelandii cells had up to 40 times as much DNA material as Escherichia coli cells, so this study looks at the form that this excess of DNA takes. Is it all one molecule or are there multiple chromosomes or copies of the same chromosome?

What They Saw
They extracted DNA from A. vinelandii and E. coli and determined the amount per cell (by counting number of cells), finding that E. coli had about 3.4 femtograms per cell (3.4 * 10^-15 g) and A. vinelandii had 135 femtograms. Assuming E. coli's genome is about 4 megabases (actually 4.6, though it depends on the strain) and it has only one copy per cell, A. vinelandii should have about 160 megabases-worth of DNA per cell. A. vinelandii's genome is only about 5.4 megabases, so that's about 30 copies of the genome per cell.

However, they made an artificial mixture of known numbers of copies of a certain gene in A. vinelandii and measured the intensity of radioactivity for the correct band of a Southern blot when probed with a probe labeled with radioactive phosphorus. The amount of radioactivity for DNA extracted from cells was about twice the amount seen with 40 copies of the gene, so they concluded there must be about 80 copies of the gene present. Does this mean there are 80 copies of the chromosome per cell? Not necessarily; there could be multiple copies of the gene per chromosome. However, I don't think that is the case.

They also tried with nitrogenase nifDK genes and got similar amounts of radioactivity. nifH gave multiple bands (presumably because of the alternative nitrogenases), but the main band gave a similar brightness.

To distinguish between a giant chromosome with 80 copies of each gene, and 80 copies of a smaller chromosome (or something in between), they integrated a resistance marker in a particular place in the genome. If it were a single large genome, the marker would probably only integrate once or a few times, whereas after several generations, the cell would make copies of a smaller genome with an integrated marker such that the amount of marker would increase over time. They observed the latter result, suggesting single copies of genes on a chromosome with many copies.

What This Means
The copy number of Azotobacter chromosomes was about 30-40 compared to E. coli but about 80 in terms of specific gene copies. A possible way to reconcile this is that the E. coli cells might actually have had more than one copy too. In any case, this many copies of the genome might make it difficult to stably transform the organism.

Reference:

Nagpal, P., Jafri, S., Reddy, M. A. & Das, H. K. Multiple chromosomes of Azotobacter vinelandii. J. Bacteriol. 171, 3133–3138 (1989).

225 - O2-repression of nitrogenase synthesis in Azotobacter chroococcum

Klebsiella pneumoniae only fixes nitrogen when oxygen levels are very low (or absent), so oxygen represses nitrogenase synthesis. This study looks at whether the same is true in Azotobacter chroococcum; it seems unlikely, since this organism is known for its very aerobic nitrogen fixation; however, it is possible to stress this organism with oxygen such that it may shut down nitrogenase, at least temporarily.

What Robson Saw
He grew A. chroococcum in chemostats with Burk medium with mannitol, with or without ammonium for nitrogen, and stressed them with oxygen either by moving ammonium-grown cells to ammonium-free medium or by suddenly increasing aeration in nitrogen-fixing cells. He looked at levels of nitrogenase proteins (by labeling with radioactive sulfur isotopes) and nitrogenase activity.

At initial low oxygen levels, activity increased and radioisotype-labeled protein levels were relatively high (indicating high levels of nitrogenase protein synthesis). Upon oxygen shock, activity went to zero and protein synthesis levels dropped a lot. When the stress was relieved, both measures increased again.

With ammonium removed and then re-added, things were similar: nitrogenase activity went up and synthesis gradually increased to a plateau, but decreased when more ammonium was added. It did take a relatively longer time for nitrogenase activity to pick up after ammonium was removed, about 80 minutes.

Along with nitrogenase, flavodoxin and the small protein that protects nitrogenase from oxygen by temporarily inactivating it both showed up in radiolabeling. The former matched nitrogenase synthesis patterns, but the latter was fairly constant.

What This Means
It seems that oxygen stress can repress synthesis of nitrogenase even in Azotobacter, though radiolabeling might not be the best method for studying this question. This adds another layer to Azotobacter's protection of its enzymes from oxygen.

Reference:

Robson, R. L. O₂-repression of nitrogenase synthesis in Azotobacter chroococcum. FEMS Microbiology Letters 5, 259–262 (1979).

216 - Effect of Dissolved Oxygen on Growth Yield and Aldolase Activity in Chemostat Culture of Azotobacter vinelandii

This study looked at carbon- or oxygen-limited cultures of Azotobacter vinelandii and the effects on enzymes of the TCA cycle: aldolase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase, and isocitrate lyase.

What They Saw
They grew A. vinelandii in chemostats with Burk medium with glucose at different agitation speeds and different dilution rates.

At the second-lowest agitation, dissolved oxygen was low and a bit of glucose was residual in the outflow (about 0.44 g/L); this increased as D increased. Growth yield and carbon dioxide production didn't change much as D changed. Things were similar in the lowest agitation, except there was less biomass and presumably more residual sugar.

At the second-highest agitation, residual glucose was very low up to D = 0.2, then went up, so the culture seemed to switch from glucose-limited to oxygen-limited at that point. Biomass increased up to that point too, and then decreased. Growth yield and CO₂ production changed inversely, with yield increasing up to a certain D before 0.2 and then leveling off. Once leveled off, values were similar to those from lower aerations.

So overall for all aerations, as dissolved oxygen increased, growth yield from glucose decreased; growth became less efficient. Carbon dioxide production increased though, indicating that the carbon was being consumed but going toward that gas, complete oxidation.

In terms of enzyme activities, of the four tested enzymes, only aldolase increased as oxygen increased. Below is their model of what effect this has:

Fig 4, Nagai et al. 1971

The rise in aldolase activity meant that carbon was cycling through the pentose cycle more instead of moving on to the TCA cycle (which could lead to greater ATP generation and anabolism), so that explains the increase in CO₂ production and decreased growth yield.

Reference:

Nagai, S., Nishizawa, Y., Onodera, M. & Aiba, S. Effect of Dissolved Oxygen on Growth Yield and Aldolase Activity in Chemostat Culture of Azotobacter vinelandii. J Gen Microbiol 66, 197–203 (1971).

215 - Respiratory protection of nitrogenase in Azotobacter vinelandii

This study is pretty similar to 214, on how oxygen levels influence the respiratory chain of Azotobacter vinelandii.

What They Saw
They grew cells in batch at low aeration, and then increased the aeration to expose cells to excess oxygen.

The results were similar to 214: when aeration increased, cells stopped growing until respiration had ramped up and leveled off. Despite the large increase in respiration, cellular ATP levels dropped 40%. P/O ratios decreased as respiration increased to accommodate the increased oxygen, at least for NADH dehydrogenase.

They tried adding chloramphenicol again, since last time it didn't affect the growth lag, but this time observed that it lessened the increase in respiration about half, and prevented cytochrome a₂ and other dehydrogenase increases.

What This Means
The M/N ratio, meaning maintenance M (moles ATP consumed per weight of cells over time) over phosphorylating efficiency N (P/O ratio x 2), explains doubling of respiratory activity upon increase in oxygen only if M increases or N decreases. It doesn't seem like M does increase, so N seems to be decreasing, because respiration uncouples from phosphorylation.

A. vinelandii seems to have different branches in its respiratory chain, which allows it to tolerate different and suddenly changing levels of oxygen.

Reference:

Jones, C. W., Brice, J. M., Wright, V. & Ackrell, B. A. C. Respiratory protection of nitrogenase in Azotobacter vinelandii. FEBS Letters 29, 77–81 (1973).

214 - The Respiratory System of Azotobacter vinelandii 2. Oxygen Effects

This study looks at how oxygen levels influence the respiratory chain in Azotobacter vinelandii.

What They Saw
They grew cells with high or low aeration (based on volume of culture in the same size flask), then isolated respiratory membranes and measured P/O ratios.

The cells grew and quickly used up all the dissolved oxygen. With high aeration, they grew much faster and leveled off once the oxygen was gone, and with low aeration the growth was slower (not even really logarithmic) but continued long after the oxygen was gone. Respiratory activities were 2-5x higher when cells were growing logarithmically with excess oxygen than when oxygen was limited.

If they suddenly increased the aeration when oxygen had run out, respiratory activity increased back up to high levels (1000 μl/h/mg dry weight). Cells didn't start growing again until it had leveled off. This lag was the same when chloramphenicol (which inhibits protein synthesis) was present, suggesting that it wasn't due to the synthesis of new enzymes for respiration.

When oxygen was being consumed, cytochrome and oxidase levels were pretty constant (except cyt o oxidase, which increased), but when oxygen ran out, levels of c₄, c₅, and b₁ increased quickly and a₂ more slowly. Cytochrome o oxidase and a₂ oxidase also increased a lot, a₁ less so. The increase of o was fastest and greatest.

P/O ratios were similar to those seen before at maximum, but they didn't reach this maximum until oxygen was mostly used up late in the logarithmic growth phase, at least for NADH dehydrogenase.

With low aeration, cytochrome levels were pretty constant (since oxygen ran out almost immediately), increasing just a little, except for cyt a₂ oxidase which went up pretty constantly. P/O ratios were pretty level too, and fairly high, similar to when oxygen ran out in high aeration.

What This Means
This kind of pattern fits in well with those seen in other obligate aerobes. Cytochromes probably increase during oxygen limitation to try to compensate for the limitation. But the low P/O ratios at high oxygen makes sense in light of respiratory protection; respiration is uncoupled from energy generation.

Reference:

Ackrell, B. A. C. & Jones, C. W. The Respiratory System of Azotobacter vinelandii 2. Oxygen Effects. Eur. J. Biochem. 20, 29–35 (1971).

352 - The Respiratory System of Azotobacter vinelandii: 1. Properties of Phosphorylating Respiratory Membranes

Similar to 161, this study looks at elements of Azotobacter vinelandii's respiratory chain and P/O ratios thereof.

What They Saw
They isolated respiratory membranes (basically pieces of cell with the respiratory chain intact but not much else, I think) and looked at oxygen reduction with different electron donors.

They saw P/O levels up to 1.1 with NADH and about 0.7 with malate, higher than in 161.

Reference:

Ackrell, B. A. C. & Jones, C. W. The Respiratory System of Azotobacter vinelandii: 1. Properties of Phosphorylating Respiratory Membranes. Eur. J. Biochem. 20, 22–28 (1971).

213 - Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum

When respiratory protection fails in Azotobacter, it can temporarily inactivate its nitrogenase to protect it, by association with another protein, called FeSII or Shethna. This study purifies this whole complex (nitrogenase and FeSII) and investigates its characteristics in A. chroococcum.

What They Saw
The more pure the nitrogenase, the less protection from oxygen inactivation they observed. But while crude extract had the most protection, more pure forms were pretty similar until the protective FeSII protein was absent, in which case the nitrogenase was rapidly inactivated. Magnesium ions (or possibly other divalent ions) were also necessary for this stabilization.

This protective protein was 14 kDa, orange in color, and had 2 Fe and 2 S atoms, so a 2Fe-2S center (thus the name). This version seems smaller than the A. vinelandii version though, which is 23 kDa. In stable complexes, the three components (dinitrogenase, dinitrogenase reductase, and FeSII) were present in about 1:1:1 ratios.

What This Means
This FeSII (with Mg ions) appears to be sufficient to protect the nitrogenase complex from oxygen, stabilizing it even outside of the cellular environment. This stability is perhaps not complete though, since crude extracts did show more activity after exposure to oxygen.

Reference:

Robson, R. L. Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum. Biochem J 181, 569–575 (1979).

198 - Oxidation of nitrogenase iron protein by dioxygen without inactivation could contribute to high respiration rates of Azotobacter species and facilitate nitrogen fixation in other aerobic environments

This study looks at oxygen interactions with dinitrogenase reductase (DNR) in Azotobacter chroococcum (and Klebsiella pneumoniae) to see if it's possible that the enzyme can be oxidized without being totally inactivated.

When the DNR was present in high enough levels relative to the amount of oxygen (4-fold molar excess), it was protected from inactivation by oxygen: it seemed to reduce superoxides to peroxide (and then water if catalase doesn't get to it first) before the reactive oxygen species could harm it. Superoxide seems to be the harmful form of oxygen for DNR. They suggest this helps with high respiration rates (i.e. consumption of oxygen). They calculated that if 10% of the cell's protein were DNR, it could account for pretty much all of the oxygen consumption. This is unlikely, but it could still be a significant part of respiratory protection. They call this "autoprotection."

Reference:

Thorneley, R. N. & Ashby, G. A. Oxidation of nitrogenase iron protein by dioxygen without inactivation could contribute to high respiration rates of Azotobacter species and facilitate nitrogen fixation in other aerobic environments. Biochem J 261, 181–187 (1989).

197 - Superoxide dismutase and catalase in Azotobacter vinelandii grown in continuous culture at different dissolved oxygen concentrations

Considering how oxygen-sensitive nitrogenase is, it might be expected that enzymes specifically involved in oxygen detoxification (such as catalase and superoxide dismutase (SOD)) might be involved in protecting such oxygen-sensitive enzymes, especially since ramping up respiration in response to increased oxygen might also ramp up the production of reactive oxygen species. This study investigates the activity of SOD and catalase in Azotobacter vinelandii at different oxygen levels.

What They Saw
They grew A. vinelandii OP (aka CA) in chemostats with different levels of oxygen, with 3 or 15 g/L sucrose. They extracted enzymes from samples and assayed them for SOD or catalase activity, and also by electrophoresis.

As oxygen saturation increased from 1% to 90%, SOD activity increased linearly (when standardized to total protein); the increase was slightly faster at lower oxygen when standardized by number of cells (probably because cell size increases as oxygen increases, 098). The sucrose concentration didn't affect things. When cells were given ammonia, SOD activity was about 2x lower.

Based on electrophoresis, they concluded that the SOD is iron-containing, rather than manganese. They tried adding manganese but still didn't see any Mn-SOD.

It didn't seem like catalase activity increased with increasing oxygen, standardized by protein. The increase standardized by cells was much more apparent.

What This Means
It seems like SOD at least might contribute to A. vinelandii's protection of its nitrogenase enzyme from oxygen.

Reference:

Dingler, C. & Oelze, J. Superoxide dismutase and catalase in Azotobacter vinelandii grown in continuous culture at different dissolved oxygen concentrations. Arch. Microbiol. 147, 291–294 (1987).

195 - Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations

Obviously oxygen levels have a big effect on nitrogen-fixing Azotobacter. This study looked into specific effects on levels of different proteins related to nitrogen fixation, at different oxygen levels.

What They Saw
They grew A. vinelandii OP (aka CA) in chemostats with 3 g/L sucrose at different oxygen levels (or dilution rates). They measured nitrogenase activity and purified nitrogenase components as well as flavodoxin and FeSII protein.

Unlike in previous studies (111, 165, 183), increasing the oxygen levels didn't seem to reduce nitrogenase activity (in steady state), at least not in the range they tried (except maybe a little at low levels). At all different levels, nitrogenase activity (i.e. acetylene reduction) correlated only with dilution rate.

With Western blots, they found that levels of different nitrogenase proteins (and others) didn't really vary much across different oxygen levels; they were always about 10% of total protein. And if the activity doesn't vary, this means the proportion of active enzyme is constant too. The enzyme activity does match previous numbers though (106).

Trying to grow cells in ammonia, the two components of nitrogenase disappeared, flavodoxin decreased, but FeSII remained constant.

They tried measuring nitrogenase levels at different dilution rates and oxygen levels. At the lowest D, nitrogenase increased as oxygen increased, but it remained pretty constant at higher rates (as shown before). In contrast to the earlier data though, levels didn't seem to increase consistently with increasing D; it could be that the same quantity of enzyme is less active at lower D, probably related to the flow of electrons to the enzymes.

Then they tried inhibiting protein synthesis with chloramphenicol, at either low or high oxygen. The culture started to wash out, of course. Levels of the four proteins didn't change much with oxygen or with time passed after addition of the antibiotic, but nitrogenase activity decreased greatly over time (while respiratory activity didn't change much). This wasn't due to damage to the nitrogenase components; nitrogenase extracts had just as much activity as cells grown without chloramphenicol. Somehow the activity is inhibited.

What This Means
As suggested before, it seems like the absolute presence/concentration of oxygen doesn't determine its toxicity so much as the ratio of oxygen to availability of energy and reducing equivalents. So if there's enough energy and electrons available to nitrogenase, it can keep going up to high levels of oxygen.

Reference:

Dingler, C., Kuhla, J., Wassink, H. & Oelze, J. Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations. J Bacteriol 170, 2148–2152 (1988).

099 - Oxygen and Hydrogen in Biological Nitrogen Fixation

Oxygen is pretty toxic to nitrogen fixation enzymes, so organisms or the people studying them need to take steps to protect them. They lose more than half their activity within minutes exposed to air. The dinitrogenase reductase is more sensitive than the dinitrogenase itself, at least the Mo version. Some can retain some activity even up to an hour in air. But the FeMo-cofactor, when extracted, is even more sensitive than the dinitrogenase reductase. Overall, it seems that the metal-sulfur centers are the most sensitive parts.

And yet, there are nitrogen-fixing species that are obligate aerobes, or even oxygenic. How do they do it?

Azotobacter has been shown to increase its respiration while its growth efficiency decreases as oxygen increases, seeming to waste the oxygen: this has been called "respiratory protection." The mechanism for this is not simple though; it involves carefully regulated shifts in respiratory components throughout the whole catabolic system.

Azotobacter also has the ability to reversibly inactivate its nitrogenase if respiratory protection is not possible (such as in carbon-limited conditions, or upon a sudden increase in oxygen). This seems to depend on FeSII protein (aka Shethna), though it is suggested that there may be other mechanisms.

When oxygen is too high and cells' supply of fixed nitrogen runs out, production of nitrogenase may be regulated (no sense making an enzyme when it can't function). This regulation may be done by the products of nifAL genes.

Azotobacter also produces gummy alginate which might have a role in protection from oxygen, but non-gummy strains (such as CA) have been isolated that don't seem especially oxygen-sensitive. I wonder if they have higher rates of respiration though, or if they might be more sensitive in carbon-limited conditions.

Nitrogenase also produces hydrogen gas, whether or not it's reducing anything else. This reaction seems separate from the nitrogen fixation reaction, since some things can inhibit the latter without inhibiting the former. Acetylene seems to inhibit hydrogen production though. Nitrogen can't compete with hydrogen for electrons completely, even with pure pressurized nitrogen; the enzyme always produces at least 1 mol hydrogen for each mol nitrogen gas fixed.

Of course, this hydrogen usually doesn't just escape; Azotobacter and other diazotrophs recapture it with their uptake hydrogenase. The exact purpose this serves is not clear though.

Reference:

Robson, R. L. & Postgate, J. R. Oxygen and Hydrogen in Biological Nitrogen Fixation. Ann Rev Microbiol 34, 183–207 (1980).

374 - The Azotobacteriaceae

This was a very interesting review of the Azotobacter family from more than fifty years ago. It was interesting to see observations in this paper that I had made myself in my own research.

It discusses the taxonomy of Azotobacter somewhat: the genus includes A. chroococcum, A. beijerinckii, and A. vinelandii of course, and A. agile (which I'm not sure is considered a real separate species now); other alleged species (A. indicum for example) seemed like they should be separated into another genus, Beijerinckia. A. chroococcum was the first, discovered by Martius Beijerinck in 1901.

Characteristic features of Azotobacter are their large size, short thick rod-shaped cells (often found in pairs), nitrogen fixation (despite being obligate aerobes), and poor growth on digestions of meat extracts and such, like LB (something I've noticed myself). The cells change shape depending on their conditions though, which can be confusing. Worse, they can be difficult to isolate from contaminating strains. They also form resistant, dormant cysts in some conditions, though I don't think I've observed this personally. They also can form storage granules of different kinds which are observable under a microscope.

They're pretty versatile in their ability to use different carbon compounds. They can use alcohols (ethanol, propanol, butanol, etc), organic acids (acetate, citrate, butyrate, etc), and saccharides (glucose, fructose, galactose, sucrose, etc.). This depends on the species and strain somewhat; some seem to be able to use lactose, others not. Some can use starch and some other polysaccharides. Some can even use cyclic compounds (benzoic acid, phenol, salicylic acid) which are generally toxic. There are some things they can't use, such as xylose, methanol, and formic acid. Their respiration rate can be very high, the highest observed in nature (at that time, at least).

Their versatility regarding nitrogen compounds seems to be lower though. They can fix nitrogen, of course, and use basic inorganic forms (ammonia, nitrate) and some common organic forms (urea, glutamate, asparagine), but otherwise are limited. So they don't grow well on complex forms such as protein digestions (peptone, tryptone).

Otherwise, as represented in Burk medium, they need phosphorus, sulfur, potassium, calcium, magnesium, iron (amount depending on whether they were fixing nitrogen), and of course molybdenum or vanadium helped when fixing nitrogen too. Other trace elements or vitamins seem unnecessary, at least in many conditions.

The review suggests that azotobacters can produce compounds that stimulate or inhibit plant roots; I wonder if that is true.

The organisms are obligate aerobes, of course, capable of tolerating very high levels of oxygen, especially when not fixing nitrogen. They're mesophiles, preferring around 30ºC. Preferred pH depends on the strain, but around 6-8 is typical.

Some have actually reported that the weather can affect their growth, especially high-pressure areas, but this hasn't been confirmed.

Many have noticed that azotobacters seem to mutate fairly frequently; this is probably due to transposons and natural competence.

How much nitrogen do azotobacters actually fix in soils? It's hard to tell, of course, because it depends on many things and it's hard to measure the contribution of a single genus in such a complex environment, so it couldn't be said.

Jensen, 1954

Reference:

Jensen, H. L. The Azotobacteriaceae. Bacteriol. Rev. 18, 195–214 (1954).

183 - Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii

Later investigators criticized the previous study (182) as too simplistic, not explaining the total potential of the nitrogenase system. Azotobacter seems to make three flavodoxins, not just azotoflavin; flavodoxin II seems like the important one but it wasn't known how it got reduced; and ferredoxin might not be involved at all.

So this study grew A. vinelandii with and without ammonium, then observed the differences in its redox systems.

What They Saw
They grew cells in a chemostat with ammonium, and then removed samples and washed with nitrogen-free medium to remove the fixed nitrogen. They measured nitrogenase activity of these samples and labeled newly formed proteins with radioactive sulfur compounds.

They observed that nitrogenase activity correlated well with rate of respiration in different conditions, so they wondered if the two might be linked.

After they removed the fixed nitrogen from cells that had been growing with it, they observed nitrogenase activity within 20 minutes. Then the activity increased linearly over time for at least 40 minutes in this condition. On protein gels, they observed the nitrogenase proteins produced quickly, within 5 minutes, and flavodoxin II showed up some time later. There are some others of uncertain identity, and some interesting ones showing up only in the membrane protein fraction.

What This Means
The linear relationship between respiration and nitrogenase activity has a number of possible explanations. Extra respiration could mean higher membrane potential or ATP levels, so more energy for nitrogenase, or there could be more enzymes (or enzymes that are more active) to transport electrons to nitrogenase. The former seems unlikely, since Azotobacter uncouples respiration from energy generation at higher oxygen levels, so extra respiration doesn't necessarily mean more energy. And extra enzymes seems unlikely too, based on the protein results.

So it seems like increased nitrogenase activity might be due to increased transport of electrons to the enzyme, though it's not clear how that happens.

Reference:

Klugkist, J., Haaker, H. & Veeger, C. Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii. European Journal of Biochemistry 155, 41–46 (1986).

182 - The electron transport system in nitrogen fixation by Azotobacter. III. Requirements for NADPH-supported nitrogenase activity

Nitrogenase requires a steady flow of reductant to reduce nitrogen gas to ammonia. This study looks at how electrons move from catabolism to the nitrogenase in Azotobacter vinelandii. Previous studies seemed to isolate two components of the transport chain to nitrogenase: azotoflavin and azotobacter ferredoxin. But it was still unknown how these compounds themselves were reduced, since A. vinelandii can't reduce them directly from pyruvate as others can (such as Clostridium pasteurianum).

What They Saw
They extracted cell components and fractionated them, separating the carriers mentioned above from nitrogenase. Then they tested the potential of NADH and NADPH to reduce these carriers, hypothesizing that these reductants are used for many other redox reactions.

This reaction seems energetically unfavorable because the redox potential of NAD(P)H is higher than that of ferredoxins, but it has been shown to happen before, especially when the NAD(P)H/NAD(P)⁺ ratio is high. So it's plausible.

They found that NADPH seemed to be able to reduce azotoflavin, and could support nitrogenase activity, at least in vitro. NADH did not seem to have the same activity. Adding extra azotoflavin or ferredoxin increased activity too.

There was one more factor that they didn't identify or include in their assays, one that they replaced with spinach ferredoxin-NADP⁺ reductase. Presumably it is some reductase in A. vinelandii that they didn't purify right. They did figure out which fraction contained it though, and it restored most activity. It could be denatured by mild heating, apparently.

Of other substrates that might support nitrogenase activity, only those linked to NADP⁺ seemed to be useful in vitro: malate, glucose-6-phosphate, alpha-ketoglutarate, and isocitrate. And apparently NADP⁺-linked isocitrate dehydrogenase is pretty common in A. vinelandii cells.

So it seems like NADPH is the donor that starts the electron transport branch to nitrogenase.

Reference:

Benemann, J. R., Yoch, D. C., Valentine, R. C. & Arnon, D. I. The electron transport system in nitrogen fixation by Azotobacter. III. Requirements for NADPH-supported nitrogenase activity. Biochim. Biophys. Acta 226, 205–212 (1971).

178 - Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii

This study looks at what it takes for oxygen stress to induce Azotobacter vinelandii to shut off its nitrogenase.

What They Saw
They grew A. vinelandii OP (aka CA) in chemostats, fixing nitrogen, with limited carbon (3 g/L of sucrose, acetate, or citrate). They stressed the culture with oxygen by increasing the aeration for 6-minute periods. They measured acetylene reduction, and nitrogen fixation directly (by fixed nitrogen increase). They also measured oxygen levels going in and coming out, to determine consumption.

As usual, with sucrose, they observed that respiration rates increased as oxygen levels rose. At a given oxygen level, respiration also increased as the dilution rate D increased. The amount of respiration increase wasn't the same at different dilution rates though, even with the same change in oxygen. The oxygen maintenance requirement increases as oxygen increases, but not linearly (the rate of increase goes down).

With acetate or citrate, the oxygen maintenance coefficient (and respiration at a given oxygen level) was much lower than with sucrose. Also as D increased, respiration with citrate increased linearly, but with acetate it leveled off at some point.

The rate of nitrogen fixation depended only on D, and increased linearly with D. The carbon source didn't affect it.

When they did the oxygen challenges, they found that up to D = 0.15 h^-1, increasing the oxygen shut off nitrogenase completely. Above that D, the shut-off was less severe. With a less severe challenge, there was less shut-off at the same D too, as expected. Substrate didn't seems to matter.

They couldn't measure a change in respiration from oxygen stress directly, because it was too short, but they knew that cultures grown in acetate or citrate couldn't increase their respiration because they had already consumed all the substrate. There was still residual sucrose though, but the amount didn't seem to change with oxygen challenge, so they concluded that respiration didn't suddenly increase.

Finally they tried controlling nitrogenase activity by giving cells small amounts of ammonium, not enough to repress nitrogen fixation, just reduce it. So giving cells 1 mM ammonium when D = 0.16 resulted in the same nitrogenase activity as when D = 0.06 with no ammonium. And they found that an equivalent oxygen challenge led to the same amount of nitrogenase shut-off.

What This Means
So the rate of oxygen consumption doesn't affect how severe the nitrogenase shut-off is, only the rates of substrate feeding and nitrogenase activity, and more so the latter.

This is kinda weird, because if respiration is how the cells protect nitrogenase from oxygen by removing it (respiratory protection), then higher respiration should correlate with higher nitrogenase activity, but it doesn't seem to here. Also, oxygen level and oxygen consumption should correlate linearly, but they don't, especially considering different carbon substrates.

The authors propose that, instead of respiratory protection, the cells' redox state is what matters: nitrogenase requires a reduced state to function, and more oxygen leads to a more oxidized state. Reduction is made possible by the carbon substrate, which provides energy and electrons; at higher dilution rates, more reduction is possible, so the nitrogenase activity can be higher. That also explains why at higher D, the same oxygen challenge leads to less nitrogenase shut-off, because the change in the redox state is less severe. That way, the cells don't need to create an anaerobic environment in their cytoplasm, just maintain a low redox potential and good flow of electrons.

Respiratory protection as a concept is still useful, since it is still true that the cells' respiration increases as oxygen increases when fixing nitrogen, to allow nitrogenase to function; it's just the details that have been challenged here.

Reference:

Kuhla, J. & Oelze, J. Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii. J. Bacteriol. 170, 5325–5329 (1988).

156 - Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii

This study was intended to study carbon metabolism in Azotobacter and the formation of PHB.

What They Saw
The enzymes they studied (glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, 6-phosphogluconate dehydratase, 3-deoxy-2-oxo-6-phosphogluconate aldolase, citrate synthase, and isocitrate dehydrogenase) all seemed to be inhibited by NADH and/or NADPH.

What This Means
PHB synthesis consumes reduced nucleotides such as NADH, which could reduce their inhibitory effects when oxygen is limited.

Reference:

Senior, P. J. & Dawes, E. A. Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii. Biochem. J. 125, 55–66 (1971).

175 - Regulation of the Tricarboxylic Acid Cycle and Poly-β-hydroxybutyrate Metabolism in Azotobacter beijerinckii Grown under Nitrogen or Oxygen Limitation

When Azotobacter grows in oxygen-limited conditions, one expects that reducing equivalents such as NADH and NADPH would accumulate (since the electrons have no good acceptor). This could inhibit the TCA cycle. This study looked at different catabolic enzymes and their behavior under different nutrient limitations.

What They Saw
They grew A. beijerinckii in a chemostat with oxygen or (alleged) nitrogen limitations. As seen before, low oxygen induced PHB formation to store the carbon and as an electron sink. Adding extra oxygen caused a drop in PHB and dry weight, and then removing it reversed the effect. Enzymes for PHB synthesis showed a similar pattern.

NADH oxidase and enzymes involved in the TCA cycle of carbon catabolism (2-oxoglutarate dehydrogenase and isocitrate dehydrogenase) tended to increase activity as oxygen increased, and vice versa. NADH oxidase (part of the electron transport chain), though, fell after the initial increase and then rose again at the second increase. This enzyme seems like an important part of the genus's respiratory protection.

Some other TCA enzymes, citrate synthase and pyruvate dehydrogenase, didn't change much with oxygen changes.

They saw again that the lower the oxygen, the higher the growth yield (amount of biomass produced per unit sucrose), though probably some of this was due to PHB formation increasing the dry weight. The amount of sucrose consumed increased as oxygen increased, indicating less efficient growth.

With NADH/NAD+, when oxygen limitation was first imposed, the ratio rose greatly but then went down again quickly, and remained mostly steady with some fluctuations.

What This Means
The recovery of NADH/NAD+ ratio is likely due to the formation of PHB acting as an electron sink.

Azotobacter is kind of an interesting mix of aerobic and facultative organisms; they need oxygen, but not too much, and excess is harmful, so their respiratory systems are tightly regulated to deal with multiple different levels. The ability to fix nitrogen puts them in kind of a different category.

Reference:

Jackson, F. A. & Dawes, E. A. Regulation of the Tricarboxylic Acid Cycle and Poly-β-hydroxybutyrate Metabolism in Azotobacter beijerinckii Grown under Nitrogen or Oxygen Limitation. J. Gen. Microbiol. 97, 303–312 (1976).

165 - The Behaviour of Azotobacter chroococcum in Oxygen- and Phosphate-limited Chemostat Culture

This study looked at Azotobacter chroococcum behavior when limited in oxygen or phosphate.

What They Saw
They grew A. chroococcum in chemostats, similar to previous studies (163). In their allegedly oxygen-limited culture, biomass levels fell as dilution rate increased, while carbon dioxide production fell and then rose again for some reason; they claim it's just random, and that CO₂ was actually fairly constant (or blame it on consistency of electricity in Britain). They also observed that as dilution rate decreased, the amount of PHB in the cells rose. They could tell the cells were oxygen-limited because when more oxygen was added, it disappeared pretty quickly.

When cells were phosphate-limited, extra oxygen was pretty toxic; the cells couldn't handle it. Plating cells out after this challenge revealed that there was very little viability, if any. In contrast, cells that had been oxygen-limited didn't seem to mind extra oxygen much, especially when they were grown on medium with fixed nitrogen.

What This Means
It fits pretty well with previous results. PHB seems to be an electron sink, useful when cells don't have enough oxygen to receive all their electrons.

The fact that oxygen didn't reduce cells' viability when plated on medium with fixed nitrogen suggests that the cells' problem with extra oxygen is lack of ability to fix nitrogen immediately, rather than loss of viability. Then the cells ramp up respiration to consume the extra oxygen—respiratory protection.

Reference:

Lees, H. & Postgate, J. R. The Behaviour of Azotobacter chroococcum in Oxygen- and Phosphate-limited Chemostat Culture. J Gen Microbiol 75, 161–166 (1973).

164 - Reassessment of Maintenance and Energy Uncoupling in the Growth of Azotobacter vinelandii

Maintenance energy reflects a cell's inefficiency, the energy it needs but that does not go toward growth; it may go toward maintaining cellular components somehow, or just reflect waste. These possibilities aren't easily distinguishable.

Previous results looked weird for A. vinelandii when its glucose feed was suddenly changed, so Nagai and Aiba wanted to clarify their understanding of this bacterium's maintenance and yield values.

They grew A. vinelandii ATCC9046 in chemostats with 5 or 8 g/L glucose, 0.05 g/L sodium citrate, and other things in Burk medium, limited either in glucose or oxygen.

The important equations are:

1: v = m + μ_x/Y_G

and

2: Q_O2 = m_o + μ_x/Y_GO

which, being interpreted, mean that 1) the specific rate of glucose use (mmol glucose/mg bacteria/h) = maintenance (same units) + biomass growth rate/true yield (g bacteria/mol glucose). Which makes sense: maintenance takes glucose, and growth takes more, so adding them together with the growth rate you get the total glucose use;

and 2) the specific rate of respiration (mmol oxygen/mg bacteria/h) = respiration maintenance (same units) + biomass growth rate/true oxygen yield (mg bacteria/mmol oxygen). Which is parallel to the first, except with oxygen instead of glucose.

So with these equations in mind, they measured specific glucose use and specific respiration rate over a series of dilution rates (equivalent to growth rates):

Positive slopes indicate oxygen-limited points, negative slopes glucose-limited points, at different agitation speeds.
Nagai and Aiba, 1972

So from these graphs and the equations, v and Q_O2 can be the y in the equation of a line (y = mx + b), while D is the x, and so 1/Y_GO or 1/Y_G become the slopes, and the y-intercept is m, maintenance.

What's weird is with the glucose-limited points, the slope of the line (and thus the growth yield) is negative (so the amount of biomass should decrease as the glucose or oxygen increases); are glucose and oxygen toxic in this case? The other thing is m, which is within a reasonable range when oxygen-limited (around 0.8 mmol glucose/g bacteria/h, comparable to other organisms), gets super-high when carbon-limited: between about 19.5 and 26 mmol glucose/g bacteria/h, depending on the agitation speed and glucose concentration. The explanation for this is likely energy-uncoupled growth: when cells increase their use of substrates without increasing their growth rate (like with respiratory protection, and oxygen-wasting system).

The maintenance requirement of oxygen, or oxygen wasting, increases almost linearly as the amount of oxygen present increases. So the reason that the yields were negative when carbon-limited was that the cells receive more carbon per hour as D increases, so they produce more cells, but the rate of consumption remains the same. Therefore a lower proportion is being wasted per cell, but the same amount overall. So increasing the amount of substrate doesn't decrease the amount of biomass, but rather the 1/Y_GO term encompasses both actual yield and substrate-wasting values, and the latter is negative because it goes down proportional to the number of cells as D increases, and the negative outweighs the positive in this case. So the true growth yield is still positive.


Reference:

Nagai, S. & Aiba, S. Reassessment of Maintenance and Energy Uncoupling in the Growth of Azotobacter vinelandii. J Gen Microbiol 73, 531–538 (1972).

097 - Aeration in fermentations

Providing oxygen in culture conditions can be tricky. It should be done homogeneously across time and space in the reactor, so all cells experience the same levels all the time. But homogenizing aeration adequately and measuring real-time dissolved oxygen levels are difficult tasks, because oxygen is not very soluble in aqueous solutions.

Azotobacter vinelandii, for example, is an obligate aerobe, so it needs some level of oxygen, but too much can be harmful when the cells are fixing nitrogen (or, at least, reduces the efficiency of the cells' product formation). So optimizing A. vinelandii culture requires determining the optimal oxygen level and how to reach that level.

What They Did
Phillips and Johnson had a reactor with pH and dissolved oxygen electrodes, aerated by agitation and sparging, and sensors of oxygen and CO₂ in the exhaust gas. So a pretty nice setup.

They grew various organisms, including A. vinelandii strain O, in media with 5% glucose and measured their oxygen demands. Sometimes they added ammonia as fixed nitrogen, sometimes they didn't.

It seemed like A. vinelandii's oxygen utilization rate was the lowest, compared to E. coli, Aspergillus, and Penicillium; all of these remained constant as oxygen tension increased. It seems odd though.

In the reactor, when fixing nitrogen, A. vinelandii seemed to use excess sugar just to get rid of oxygen; its oxygen demand was much higher than expected for the growth, yield, and number of cells observed. When they added ammonia so the cells didn't fix nitrogen, they claim the same effect was observed, but I don't really understand how they reached that conclusion. Their graphs seem kind of messed up, not fitting their descriptions very well.

What This Means
Overall, the conclusion was that cells don't need more oxygen than just enough to keep the level above a critical threshold. Measuring dissolved oxygen is good for keeping above this threshold, but it doesn't help determine how much extra oxygen is needed if levels drop below measurable amounts. But when oxygen demand and uptake rates are measured, the oxygen deficit can be measured, except maybe with A. vinelandii which is more complicated.

Reference:

Phillips, D. H. & Johnson, M. J. Aeration in fermentations. Journal of Biochemical and Microbiological Technology and Engineering 3, 277–309 (1961).

161 - On the Efficiency of Oxidative Phosphorylation in Membrane Vesicles of Azotobacter vinelandii and of Rhizobium leguminosarum Bacteroids

The authors wanted to study oxidative phosphorylation (generating ATP through respiration) in Azotobacter vinelandii and another related to oxygen, nitrogen fixation, and hydrogen oxidation by hydrogenase.

What They Saw
They grew A. vinelandii strain OP (aka CA) in chemostats, at D = 0.1 h^-1, limited in oxygen or nitrogen, then broke up the cells and isolated membrane vesicles anaerobically.

The P/O ratio is how much ATP is produced by moving 2 electrons through the electron transport chain to reduce one oxygen atom. The pattern of P/O over a range of oxygen levels is similar for different electron donors (NADH, malate, hydrogen, and NADH + hydrogen): it goes up to a peak, then falls as dissolved oxygen levels rise above the limit of detection. ATP production mostly levels off near that peak too (except with hydrogen, where it declines). The peak occurs at higher oxygen levels with NADH and malate than with hydrogen though. The height of the peak is 0.7 (ATPs per O reduced) for NADH and hydrogen, but only 0.5 with malate.

They tried adding acetylene up to 20%, but didn't see any indication that hydrogenase was inhibited. Previous studies showed that 40% acetylene was required to show inhibition, so it's not surprising. They also found that the hydrogen branch seemed to be very efficiently coupled to phosphorylation, and doesn't seem to involve flavoproteins.

What This Means
The lower values from hydrogen at high oxygen is probably due to inactivation of the hydrogenase, which is sensitive to oxygen.

The fall in P/O ratio is likely due to excess oxygen going through a different cytochrome branch, with cytochrome b to cytochrome d, which isn't involved in ATP production, so respiration and ATP are decoupled.

It also appears that the electrons from hydrogen oxidation don't travel through the same branch of the electron transport chain as electrons from carbon sources; hydrogenase has its own branch.

Reference:

Laane, C., Haaker, H. & Veeger, C. On the Efficiency of Oxidative Phosphorylation in Membrane Vesicles of Azotobacter vinelandii and of Rhizobium leguminosarum Bacteroids. European Journal of Biochemistry 97, 369–377 (1979).

153 - The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii

This is a study on what factors initiate production of large amounts of PHB in Azotobacter beijerinckii.

What They Saw
In batch with 5 g/L glucose, bacteria started producing PHB near the end of the exponential phase. They stopped when they had consumed all the glucose, and then consumed the PHB, but this didn't help them increase in density, possibly because the PHB itself had been a proportion of the bacterial density; it seemed to permit the increase in actual biomass.

When glucose was 20 g/L, the cells continued producing PHB and the bacterial dry weight kept increasing long after exponential phase ended; the polymer got up to 74% of the dry weight.

Using an oxygen electrode, they observed that PHB production didn't start until dissolved oxygen reached 0%, at which point exponential phase was over. So they thought it might be oxygen limitation that induced the production, unlike in other organisms where nitrogen limitation is the inducer. But to be sure that it was really oxygen and not nitrogen, they turned to chemostats.

They found that nitrogen limitation didn't induce PHB formation at any dilution rate, though growth yield increased as D increased. In contrast, in oxygen-limited conditions, the PHB content (as a proportion of dry weight) and yield seemed to decreased as D increased, starting around 45% and going down to 20%, while growth yield seemed to peak at mid-range and then fall. Glucose limitation didn't induce PHB either, though some was produced at lower dilution rates.

The sudden imposition of oxygen limitation on nitrogen-limited cultures immediately induced PHB formation, and content increased over at least 10 hours. Dry weight and OD initially went up but then back down below what it had been, possibly reflecting more efficient growth until oxygen was completely depleted. They don't show it, but claim that PHB went back down from 45% to 20% after 32 hours.

What This Means
The authors speculate that the limitation of low oxygen comes in at the level of the TCA cycle; acetyl-CoA stops being oxidized as much as before, so it starts going toward PHB synthesis.

So limited oxygen might be the best condition for these microbes: they have some but not too much, their growth is more efficient, and they produce a nice storage polymer to save food for harder times.

Reference:

Senior, P. J., Beech, G. A., Ritchie, G. A. F. & Dawes, E. A. The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii. Biochem. J. 128, 1193–1201 (1972).