ACETOACETYL COENZYME-A REDUCTASE AND POLYHYDROXYBUTYRATE SYNTHESIS INRHIZOBIUM (CICER) SP. STRAIN CC-1192

Biochemical controls that regulate the biosynthesis of poly-3-hydroxyb utyrate (PHB) were investigated in Rhizobium (Cicer) sp, strain CC 119 2, This species is of interest for studying PHB synthesis because the polymer accumulates to a large extent in free-living cells but not in bacteroids during nitrogen-fixing symbiosis with chickpea (Cicer ariet inum L.) plants. Evidence is presented that indicates that CC 1192 cel ls retain the enzymic capacity to synthesize PHB when they differentia te from the free-living state to the bacteroid state. This evidence in cludes the incorporation by CC 1192 bacteroids of radiolabel from [C-1 4]malate into 3-hydroxybutyrate which was derived by chemically degrad ing insoluble material from bacteroid pellets. Furthermore, the presen ce of an NADPH-dependent acetoacetyl coenzyme A (CoA) reductase, which was specific for R-(-)-3-hydroxybutyryl-CoA and NADP(+) in the oxidat ive direction, was demonstrated in extracts from free-living and bacte roid cells of CC 1192. Activity of this enzyme in the reductive direct ion appeared to be regulated at the biochemical level mainly by the av ailability of substrates. The CC 1192 cells also contained an NADH-spe cific acetoacetyl-CoA reductase which oxidized S-(+)-3-hydroxybutyryl- CoA. A membrane preparation from CC 1192 bacteroids readily oxidized N ADH but not NADPH, which is suggested to be a major source of reductan t for nitrogenase. Thus, a high ratio of NADPH to NADP+, which could e nhance delivery of reductant to nitrogenase, could also favor the redu ction of acetoacetyl-CoA for PHB synthesis. This would mean that fine controls that regulate the partitioning of acetyl-CoA between citrate synthase and 3-ketothiolase are important in determining whether PHB a ccumulates.