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

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

What They Wanted to Know

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

What They Did

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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