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).

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).

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).

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

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

What They Wanted to Know

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

What They Did

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

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

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

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

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

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

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

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

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

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

207 - Transformation of Azotobacter vinelandii with plasmid DNA

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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