062 - Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability

This study looked at superoxide dismutase in Azotobacter vinelandii, an Fe-SOD encoded by sodB, and its importance.

What They Saw
Running proteins on a gel testing for SOD activity, they saw two bands: one was Fe-SOD and the other CuZnSOD (which sits in the periplasm). They tried knocking out sodB from A. vinelandii by introducing a kanamycin resistance cassette, and isolated a kan-resistant strain, but it appeared to have two copies of sodB (only one of which was knocked out). They tried increasing the concentration of kanamycin (presumably to force the strain to have multiple copies of the resistance gene), and got one that grew slowly at 100x more kanamycin than I use, but they couldn't get rid of the SOD. Seems like it's essential.

This also supports the idea that A. vinelandii can have multiple copies of its chromosome, since they saw multiple PCR products from the same locus, with and without the resistance marker. The genome only has one copy of sodB, so there must be multiple genome copies.

Reference:

Qurollo, B. A., Bishop, P. E. & Hassan, H. M. Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability. Can. J. Microbiol. 47, 63–71 (2001).

523 - Carboxyl-terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii

Based on amino acid prediction from gene sequence, the HoxG alpha subunit of the uptake hydrogenase should be about 66.6 kDa, but in the wild-type it appears smaller. In some mutants with accessory genes knocked out, the size matches this number. So this study tried to figure out if post-translational processing was involved in producing active enzyme. N-terminal modification was already ruled out, as that sequence matches the prediction.

What They Saw
They grew Azotobacter vinelandii CA and purified its hydrogenase, then studied its subunits with mass spectrometry.

They observed that the actual size of the larger subunit was 64.9 kDa, smaller than the 66.6 predicted size. The N-terminal was still the same as predicted, so they concluded that about 15 amino acids had been removed from the C-terminal of the subunit. This appears to be necessary for it to function.

Reference:

Gollin, D. J., Mortenson, L. E. & Robson, R. L. Carboxyl-terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii. FEBS Letters 309, 371–375 (1992).

362 - Mutants of Azotobacter chroococcum Defective in Hydrogenase Activity

This study isolated some hydrogenase-negative mutants of Azotobacter chroococcum by chemical mutagenesis and looked at how they behaved.

What They Saw
Almost all of the 16 mutants had almost no hydrogenase activity, as expected. Some had a little, <2% of wild-type. Some more had a little hydrogen-producing activity in the right conditions, usually less than 7% of the wild-type, but one had 40% of wild-type. That one also seemed to have a relatively active soluble hydrogenase (possibly the uptake hydrogenase in soluble form). All of them seemed able to take up nickel.

The one weirdest mutant, MCD-124, showed max activity at a different pH (5.5 instead of 8) and was weird in other ways.

Also, the authors were surprised by the frequency with which they could get hydrogenase mutants. They wondered whether the relevant genes were just more susceptible, or if the growth medium was more favorable to mutants somehow, or if there were just that many necessary genes. But judging from the genome sequence, this isn't quite a sufficient explanation.

Overall, it's hard to know exactly what's going on in this study.

Reference:

Yates, M. G. & Robson, R. L. Mutants of Azotobacter chroococcum Defective in Hydrogenase Activity. J Gen Microbiol 131, 1459–1466 (1985).

297 - The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H2-uptake hydrogenase of Azotobacter chroococcum

And finally, seeming to complete our journey back in time through the discovery of hydrogenase genetics in Azotobacter chroococcum, this study looks at the structural genes, hupSL.

What They Saw
The sequences were similar to A. vinelandii's hoxKG structural genes. They tried knocking each out, then measuring hydrogen oxidation (with methylene blue) and hydrogen production (with methyl viologen). As expected, hydrogen oxidation in mutants was no higher than negative controls. Surprisingly, they did see hydrogen production in the some of the different mutants with a strong electron donor, but it was less than in the wild-type. Only the mutant with an insertion very close to the start of the hupS gene had no hydrogen production.

What This Means
It seems like a fragment of HupS is sufficient to produce hydrogen with a strong electron donor, but not as much as with both HupS and HupL completely intact.

Reference:

Ford, C. M. et al. The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H₂-uptake hydrogenase of Azotobacter chroococcum. Mol Microbiol 4, 999–1008 (1990).

296 - The Azotobacter chroococcum hydrogenase gene cluster: sequences and genetic analysis of four accessory genes, hup A, hupB, hupY and hupC

So at this point, hupSL (hydrogenase structural genes) and hupDE (accessory genes at the end of the operon) had already been identified. This study found a few more upstream of hupDE.

What They Saw
They sequenced the DNA upstream of hupDE and found four open reading frames, which they called hupABYC. The AB and C were similar to E. coli genes, but the Y wasn't, so they called it Y (for Ynknown, I guess). These were all homologous to A. vinelandii genes though, and in the same order.

Then they tried knocking out each of these. Each knockout was unable to oxidize hydrogen, even in the presence of methylene blue as an electron acceptor.

They also made a fusion of HupL (the structural subunit) and beta-galactosidase, then knocked out hupY or hupB to see if this changed the expression of hupL. Beta-galactosidase activity rose a little bit, like 25% in each, but it didn't seem either was an important regulator.

Reference:

Tibelius, K. H., Du, L., Tito, D. & Stejskal, F. The Azotobacter chroococcum hydrogenase gene cluster: sequences and genetic analysis of four accessory genes, hup A, hupB, hupY and hupC. Gene 127, 53–61 (1993).

295 - Sequences, organization and analysis of the hupZMNOQRTV genes from the Azotobacter chroococcum hydrogenase gene cluster

This study focuses on the hydrogenase genes in Azotobacter chroococcum. They had already found hupSL encoding the structural genes (equivalent to A. vinelandii's hoxKG I guess), and then the accessory genes hupABYCDE further downstream (homologous to A. vinelandii's hypABFCDE). Now, in between, are the hupZMNOQRTV genes, completing the 16-gene operon.

What They Saw
This set of genes seems to be homologous to A. vinelandii's hoxZMLOQRTV string, making the whole operon very similar in both organisms.

As in previous studies, HupZ (and its analog, HoxZ) seems to be an electron carrier in the membrane, possibly a cytochrome. When they knocked it out, they observed similar results to 070: hydrogenase could oxidize hydrogen with methylene blue as an electron acceptor, but not with oxygen, so HupZ seems to be part of the transport chain to oxygen.

Based on comparison with homologs in other organisms, HupM may help the hydrogenase attach to the membrane, or with processing the subunits. It's unclear. The other genes may be involved in processing or metal stuff. HupR may be another electron-carrying protein. Further study is required.

Reference:

Du, L., Tibelius, K. H., Souza, E. M., Garg, R. P. & Yates, M. G. Sequences, organization and analysis of the hupZMNOQRTV genes from the Azotobacter chroococcum hydrogenase gene cluster. Journal of Molecular Biology 243, 549–557 (1994).

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).

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).

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).

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).

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).