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).

009 - A Non-Gummy Chromogenic Strain of Azotobacter vinelandii

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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