Role of aniA in the regulation of Polyhydroxyalkanoate synthesis in Sinorhizobium meliloti

Sinorhizobium meliloti is a nitrogen-fixing soil bacterium that can accumulate poly-3-hydroxybutyrate (PHB), poly-3(hydroxybutyrate-co-hydroxyvalerate), glycogen and produce considerable amounts of exopolysaccharides . PHB in rhizobia and other bacteria is thought to serve as a carbon and/or energy reserve to be utilized under suboptimal growth conditions. To control the production and accumulation of these different polymers a fine regulation must be operating, possibly involving a group of genes. Recently, we studied a gene referred to as aniA (1). The occurrence of AniA orthologs (described in some cases as PhaR) and organization of the respective genes were described in detail in at least 29 different bacteria (Ref. ??). Based on sequence similarity and on the presence of a DNA-binding domain we have proposed that AniA functions in S. meliloti as a transcriptional regulator (1). The product of the aniA ortholog PhaR of P. denitrificans has been shown to bind to the intergenic region phaC-phaP and to repress the in vivo expression of the granule-associated phasin PhaP (2). Recently, similar results were shown in R. eutropha (3, 4). Other putative ortholog includes a steroid-inducible gene (stdC) in Comamonas testosteroni (5). An aniA-defective mutant of S. meliloti produced larger amounts of exopolysaccharides (EPS), especially after induction under low oxygen concentration, were expression of aniA is induced. This mutant accumulates less PHB and glycogen than the parental strain under conditions of polymer production (Ref. 1??). In this study we present new data regarding physiological and genetic characterization of aniA in S. meliloti. Recently, aniA was studied in Rhizobium etli and was also shown to have a role in polymer accumulation in addition to control of global protein expression (6). We found that aniA of S. meliloti is involved in carbon/energy regulation under normal growth conditions, but especially under low oxygen conditions it seems to modulate polymer production in rhizobia. Since an efficient control of carbon/energy flux is one of the requisites to increase PHB production, we studied the role of aniA in EPS synthesis by transferring an aniA::Km mutation to different S. meliloti strains carrying exp-lacZ and exo-lacZ fusions. -galactosidase activities were assayed in the double mutants and compared to the wild-type background. The results indicated that aniA regulates some exp and exo genes. The type of EPS produced by aniA-negative mutant and the parental strain was also analyzed. Finally, the role of aniA in the regulation of PHB accumulation in S. meliloti was studied and the related results will be presented.