374 - The Azotobacteriaceae

This was a very interesting review of the Azotobacter family from more than fifty years ago. It was interesting to see observations in this paper that I had made myself in my own research.

It discusses the taxonomy of Azotobacter somewhat: the genus includes A. chroococcum, A. beijerinckii, and A. vinelandii of course, and A. agile (which I'm not sure is considered a real separate species now); other alleged species (A. indicum for example) seemed like they should be separated into another genus, Beijerinckia. A. chroococcum was the first, discovered by Martius Beijerinck in 1901.

Characteristic features of Azotobacter are their large size, short thick rod-shaped cells (often found in pairs), nitrogen fixation (despite being obligate aerobes), and poor growth on digestions of meat extracts and such, like LB (something I've noticed myself). The cells change shape depending on their conditions though, which can be confusing. Worse, they can be difficult to isolate from contaminating strains. They also form resistant, dormant cysts in some conditions, though I don't think I've observed this personally. They also can form storage granules of different kinds which are observable under a microscope.

They're pretty versatile in their ability to use different carbon compounds. They can use alcohols (ethanol, propanol, butanol, etc), organic acids (acetate, citrate, butyrate, etc), and saccharides (glucose, fructose, galactose, sucrose, etc.). This depends on the species and strain somewhat; some seem to be able to use lactose, others not. Some can use starch and some other polysaccharides. Some can even use cyclic compounds (benzoic acid, phenol, salicylic acid) which are generally toxic. There are some things they can't use, such as xylose, methanol, and formic acid. Their respiration rate can be very high, the highest observed in nature (at that time, at least).

Their versatility regarding nitrogen compounds seems to be lower though. They can fix nitrogen, of course, and use basic inorganic forms (ammonia, nitrate) and some common organic forms (urea, glutamate, asparagine), but otherwise are limited. So they don't grow well on complex forms such as protein digestions (peptone, tryptone).

Otherwise, as represented in Burk medium, they need phosphorus, sulfur, potassium, calcium, magnesium, iron (amount depending on whether they were fixing nitrogen), and of course molybdenum or vanadium helped when fixing nitrogen too. Other trace elements or vitamins seem unnecessary, at least in many conditions.

The review suggests that azotobacters can produce compounds that stimulate or inhibit plant roots; I wonder if that is true.

The organisms are obligate aerobes, of course, capable of tolerating very high levels of oxygen, especially when not fixing nitrogen. They're mesophiles, preferring around 30ºC. Preferred pH depends on the strain, but around 6-8 is typical.

Some have actually reported that the weather can affect their growth, especially high-pressure areas, but this hasn't been confirmed.

Many have noticed that azotobacters seem to mutate fairly frequently; this is probably due to transposons and natural competence.

How much nitrogen do azotobacters actually fix in soils? It's hard to tell, of course, because it depends on many things and it's hard to measure the contribution of a single genus in such a complex environment, so it couldn't be said.

Jensen, 1954

Reference:

Jensen, H. L. The Azotobacteriaceae. Bacteriol. Rev. 18, 195–214 (1954).

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).