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).