Requirement for the Enzymes Acetoacetyl Coenzyme A Synthetase and Poly-3-Hydroxybutyrate (PHB) Synthase for Growth of Sinorhizobium meliloti on PHB Cycle Intermediates

Abstract

We have identified two Sinorhizobium meliloti chromosomal loci affecting the poly-3-hydroxybutyrate degradation pathway. One locus was identified as the gene acsA, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase. Analysis of the acsA nucleotide sequence revealed that this gene encodes a putative protein with a molecular weight of 72,000 that shows similarity to acetyl-CoA synthetase in other organisms. Acetyl-CoA synthetase activity was not affected in cell extracts of glucose-grown acsA::Tn5 mutants; instead, acetoacetyl-CoA synthetase activity was drastically reduced. These findings suggest that acetoacetyl-CoA synthetase, rather than CoA transferase, activates acetoacetate to acetoacetyl-CoA in the S. meliloti poly-3-hydroxybutyrate cycle. The second locus was identified as phbC, encoding poly-3-hydroxybutyrate synthase, and was found to be required for synthesis of poly-3-hydroxybutyrate deposits.The catabolism of intracellular carbon stores is a strategy employed by many bacterial species to survive nutritionally suboptimal conditions. Polyhydroxyalkanoates (PHA), such as poly-3-hydroxybutyrate (PHB), accumulate in cells when growth is limited but carbon availability is not (2). This stored carbon can then be utilized during conditions of otherwise limiting carbon availability (45). PHB synthesis and degradation are collectively referred to as the PHB cycle. The extent of PHB accumulation is dependent on the relative rates of synthesis and degradation, which in turn are controlled by growth conditions (36,41).PHA have attracted substantial industrial interest for their use as high-quality, biodegradable plastics (2). This interest has driven research efforts directed at PHA biosynthesis. The genes encoding the enzymes responsible for PHA synthesis have been isolated and characterized from a number of bacterial species (33, 43), including Sinorhizobium meliloti strains Rm41 (49) and Rm1021 (51). The degradative portion of the cycle has not been subjected to similar attention, and it is less well defined both genetically and biochemically.We have recently isolated a number of S. meliloti mutants that are affected in the ability to utilize PHB cycle intermediates as sole carbon sources to support growth (11). The mutations mapped to loci on both the chromosome and the pRmeSU47b (pEXO) megaplasmid. Two loci on the megaplasmid have been identified via enzymatic and nucleotide sequence analyses. One encodes the enzyme 3-hydroxybutyrate dehydrogenase (3), and the other encodes methylmalonyl coenzyme A (methylmalonyl-CoA) mutase (10). In this paper, we further characterize and identify two of the chromosomal loci. MATERIALS AND METHODSStrains, plasmids, and culture conditions. Bacterial strains and plasmids are listed in Table 1. The construction of new strains is described in the text. Bacterial culture in Luria-Bertani (LB) and TY complex media and modified M9 minimal salts medium and antibiotic selection were carried out as previously described (12). Modified M9 minimal salts medium was supplemented...

Human selection and the relaxation of legume defences against ineffective rhizobia

“…For rhizobia, trade-offs exist between fixing nitrogen for the benefit of the plant versus hoarding resources like polyhydroxybutyrate (Hahn & Studer 1986;Cevallos et al 1996;Denison & Kiers 2004a,b;Akçay & Roughgarden 2007), which can enhance future survival or reproduction (Cai et al 2000).…”

Identification of an Acetoacetyl Coenzyme A Synthetase-Dependent Pathway for Utilization of l -(+)-3-Hydroxybutyrate in Sinorhizobium meliloti

“…Preparation of cell extracts, protein determination, and assays for BDH activity (NADH formation) and for AACS and acetyl-CoA synthetase activities (acetyl-CoA formation coupled to reduction of NAD ϩ via malate dehydrogenase and citrate synthase) were carried out as described previously (5,8). NADH formation was measured using an Ultrospec 2000 spectrophotometer (Amersham Pharmacia Biotech, Baie d'Urfé, Canada).…”

“…Although there is a considerable body of research on the biochemistry and genetics of bacterial PHB synthesis, apart from our recent studies on the PHB degradation pathway of S. meliloti (1,5,8), not much is known about the genetics of the bacterial PHB degradation pathway(s) or assimilation of 3-hydroxybutyrate (3-HB). Our studies of the PHB cycle have included the isolation and characterization of transposon Tn5 insertion mutants that show altered growth on minimal media containing DL-3-hydroxybutyrate (DLHB) and/or AA as a sole carbon source (8).…”

“…A central enzyme in the PHB cycle is D-(Ϫ)-3-hydroxybutyrate dehydrogenase (BDH; EC 1.1.1.30; encoded by bdhA), which catalyzes the oxidation of the immediate PHB depolymerization product D-(Ϫ)-3-hydroxybutyrate (DHB) to acetoacetate (AA). The AA product of this reaction is activated to acetoacetyl-CoA by the enzyme acetoacetyl-CoA synthetase (AACS; EC 6.2.1.16), encoded by acsA2 in Sinorhizobium meliloti (5,7,15). The final step in the degradation portion of the PHB cycle yields two molecules of acetyl-CoA from one molecule of acetoacetylCoA via ketothiolase (EC 2.3.1.9).…”

Composition of mixed culture PHA biopolymers and implications for downstream processing

“…In Zoogloea ramigera, however, no CoA transferase activity was detected. It was reasoned that in this organism, acetoacetyl-CoA synthase might be responsible for acetoacetate activation (Cai et al 2000). (Lee et al 1995).…”