Propionic acid metabolism and poly-3-hydroxybutyrate-co-3-hydroxyvalerate production by a prpC mutant of Herbaspirillum seropedicae Z69.

Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters produced by a wide range of bacteria as carbon and energy reserves. PHA accumulation is typically increased under unbalanced growth conditions and with carbon source in excess. Although polyhydroxybutyrate (PHB) could be used for specific applications, it is brittle and not a useful alternative for plastics like polypropylene. Far more useful polypropylene-like PHAs, are copolymers composed of 3-hydroxybutyrate and 3-hydroxyvalerate, P(3HB-co-3HV). Propionic acid is one of the carbon sources that can be used to generate 3HV. A mutant derived from Herbaspirillum seropedicae Z69, a strain previously described as capable of producing P(3HB-co-3HV) from propionic acid, was constructed to increase 3HV biosynthetic efficiency. The strategy involved elimination of a catabolic route for propionyl-CoA by deficiency marker exchange of a selected gene. The mutant (Z69Prp) was constructed by elimination of the 2-methylcitrate synthase (PrpC) gene of the 2-methylcitrate cycle for propionate catabolism. Strain Z69Prp was unable to grow on sodium propionate, but in cultures with glucose-propionate accumulated 50% of its dry weight as copolymer. Z69Prp had 14.1 mol% 3HV; greater than that of strain Z69 (2.89 mol%). The 3HV yield from propionic acid (Y3HV/prop) was 0.80 g g-1, and below the maximum theoretical value (1.35 g g-1).

Phylotype diversity in a benthic cyanobacterial mat community on King George Island, maritime Antarctica.

Cyanobacterial 16S ribosomal RNA gene diversity was examined in a benthic mat on Fildes Peninsula of King George Island (62º09'54.4''S, 58º57'20.9''W), maritime Antarctica. Environmental DNA was isolated from the mat, a clone library of PCR-amplified 16S rRNA gene fragments was prepared, and amplified ribosomal DNA restriction analysis (ARDRA) was done to assign clones to seven groups. Low cyanobacterial diversity in the mat was suggested in that 83% of the clones were represented by one ARDRA group. DNA sequences from this group had high similarity with 16S rRNA genes of Tychonema bourrellyi and T. bornetii isolates, whose geographic origins were southern Norway and Northern Ireland. Cyanobacterial morphotypes corresponding to Tychonema have not been reported in Antarctica, however, this morphotype was previously found at Ward Hunt Lake (83ºN), and in western Europe (52ºN). DNA sequences of three of the ARDRA groups had highest similarity with 16S rDNA sequences of the Tychonema group accounting for 9.4% of the clones. Sequences of the remaining three groups (7.6%) had highest similarity with 16S rRNA genes of uncultured cyanobacteria clones from benthic mats of Lake Fryxell and fresh meltwater on the McMurdo Ice Shelf.

An alternative succinate (2-oxoglutarate) transport system in Rhizobium tropici is induced in nodules of Phaseolus vulgaris.

The rhizobial DctA permease is essential for the development of effective nitrogen-fixing bacteroids, which was correlated with its requirement for growth on C(4)-dicarboxylates. A previously described dctA mutant of Rhizobium tropici CIAT899, strain GA1 (dctA), however, was unexpectedly still able to grow on succinate as a sole carbon source but less efficiently than CIAT899. Like other rhizobial dctA mutants, GA1 was unable to grow on fumarate or malate as a carbon source and induced the formation of ineffective nodules. We report an alternative succinate uptake system identified by Tn5 mutagenesis of strain GA1 that was required for the remaining ability to transport and utilize succinate. The alternative uptake system required a three-gene cluster that is highly characteristic of a dctABD locus. The predicted permease-encoding gene had high sequence similarity with open reading frames encoding putative 2-oxoglutarate permeases (KgtP) of Ralstonia solanacearum and Agrobacterium tumefaciens. This analysis was in agreement with the requirement for this gene for optimal growth on and induction by 2-oxoglutarate. The permease-encoding gene of the alternative system was also designated kgtP in R. tropici. The dctBD-like genes in this cluster were found to be required for kgtP expression and were designated kgtSR. Analysis of a kgtP::lacZ transcriptional fusion indicated that a kgtSR-dependent promoter of kgtP was specifically induced by 2-oxoglutarate. The expression of kgtPp was found in bacteroids of nodules formed with either CIAT899 or GA1 on roots of Phaseolus vulgaris. Results suggested that 2-oxoglutarate might be transported or conceivably exported in nodules induced by R. tropici on roots of P. vulgaris.

Cellular and biochemical response to Cr(VI) in Stenotrophomonas sp.

Chromium (Cr)-resistant bacteria isolated from a soil with 6 g kg(-1) of Cr were identified based on 16S rRNA gene sequence analysis as a Stenotrophomonas, and designated as JD1. Growth of JD1 was accompanied by transformation of Cr(VI) to Cr(III) in liquid medium initially containing 300 mg L(-1) Cr(VI), the maximum concentration allowing growth. JD1 produced the highest levels of a Cr(VI)-binding exopolysaccharide when grown in medium with 100 mg L(-1) Cr(VI). The relative exopolysaccharide monosaccharide composition was analysed by HPLC, which showed that rhamnose+galactose was the major component, and that its relative level increased when cells were grown with Cr(VI). JD1 grew as a biofilm on various inert surfaces. Biofilm macromolecular composition analysis indicated that the relative levels of exopolysaccharide and protein were more abundant in biofilms grown in 100 mg L(-1) Cr(VI), whereas relative uronic acid levels remained constant. Biofilm cells exposed to Cr(VI) were elongated, grouped in clusters and exopolysaccharide obtained from the biofilm extracellular matrix had an enhanced capacity to bind Cr(VI). Exopolysaccharide production and composition, and biofilm growth are discussed as a mechanism of protection that allows survival during Cr(VI) stress.

Iron depletion affects nitrogenase activity and expression of nifH and nifA genes in Herbaspirillum seropedicae.

Herbaspirillum seropedicae Z67 is a nitrogen-fixing bacterium able to colonize the rhizosphere and the interior of several plants. As iron is a key element for nitrogen fixation, we examined the response of this microorganism to iron deficiency under nitrogen fixing conditions. We identified a H. seropedicae exbD gene that was induced in response to iron limitation and is involved in iron homeostasis. We found that an exbD mutant grown in iron-chelated medium is unable to fix nitrogen. Moreover, we provide evidence that expression of the nifH and nifA genes is iron dependent in a H. seropedicae genetic background.