Friday, December 12, 2014

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).

Wednesday, December 10, 2014

248 - NAD-, NMN-, and NADP-dependent modification of dinitrogenase reductases from Rhodospirillum rubrum and Azotobacter vinelandii

What They Knew
Nitrogen fixation is a demanding process, using a lot of energy, so bacteria regulate it tightly, shutting it off whenever fixed nitrogen is available already. One diazotroph, Rhodospirillum rubrum, regulates its nitrogenase by ADP-ribosylating the dinitrogenase reductase component, using a protein called Dinitrogenase Reductase ADP-ribosyl Transferase, or DRAT. This takes ADP-ribose from NAD when ammonium is present. Another protein, Dinitrogenase Reductase-Activating Glycohydrolase (DRAG), reverses the process.

What They Wanted to Know
Ponnuraj and colleagues studied the nitrogenases of R. rubrum and Azotobacter vinelandii to see how specifically each interacted with the DRAT/DRAG system and various ADP-ribosyl donors.

What They Did
They took different ADP-ribosyl containing molecules (NAD, NADP, NADH, NMN), combined each with A. vinelandii's dinitrogenase reductase (DNR) and R. rubrum's DRAT, then ran them with SDS-PAGE along with samples lacking the molecules, to see which molecules could be used to donate ADP-ribosyl. They also exposed some of each sample to DRAG and ran that alongside to see if it could remove the modification.
Results from this were confirmed with another test, seeing if modified or de-modified DNR could function with dinitrogenase to reduce acetylene.

To see how small a modification works to inactivate the system, they removed a phosphate from phosphoribosylated DNR and tested it again.

In addition to testing A. vinelandii's DNR, they tested R. rubrum's too.

To see more specifically what was going on with A. vinelandii's DNR, they used MALDI-TOF mass spectroscopy.

What They Observed
Based on gels and activity assays, NAD, NMN, and NADP all seemed pretty good at donating to DRAT to inactivate A. vinelandii's DNR. NAAD not so much, or anything else they tried. DRAG seemed able to re-activate DNR with all of them too.

Surprisingly, with R. rubrum's DNR, only NAD seemed to be a good donor. Mass spec results confirmed their expectations about what was going on biochemically.

What This Means
R. rubrum uses DRAT and DRAG to regulate its nitrogenase activity based on whether fixed nitrogen is available already and whether its environment is illuminated or not. This helps save energy, so it doesn't have to break down the whole system and reconstruct it with every little environmental change.

It's not clear how relevant it is for A. vinelandii, though, because that organism doesn't appear to have the genes to produce DRAT/DRAG proteins. It's somewhat interesting that R. rubrum's proteins are able to modify A. vinelandii's nitrogenase, arguably even better than they can with R. rubrum's, but this actually makes some sense, that R. rubrum would have tighter control over its nitrogenase regulation system. Though apparently some in vivo studies suggest it might not be as tight as it seemed here.

This doesn't necessarily mean that A. vinelandii doesn't have a system for post-translational regulation, just that it isn't exactly this one. I haven't found what it is yet, if there is one. And other studies seem to imply that A. vinelandii might not have such tight control (010). I wonder why.

Reference:
Ponnuraj, R. K., Rubio, L. M., Grunwald, S. K. & Ludden, P. W. NAD-, NMN-, and NADP-dependent modification of dinitrogenase reductases from Rhodospirillum rubrum and Azotobacter vinelandii. FEBS Letters 579, 5751–5758 (2005).