Molecular genetic analysis of acetoacetate metabolism in Sinorhizobium meliloti

Many bacteria accumulate carbon stores as poly-3-hydroxybutyrate (PHB) when growth is limited but carbon availability is not. This stored carbon can then be utilized during conditions of limited carbon availability. The net PHB accumulation in the cell is dependent on the balance between PHB synthesis and degradation. Sinorhizobium meliloti accumulates PHB in the free-living stage but not in the symbiotic stage. The physiological role of the PHB cycle in S. meliloti is unknown. As a first step to understand the genetics of PHB degradation, transposon-generated mutants that were not able to use PHB degradation intermediates, such as 3-hydroxybutyrate and acetoacetate, as a sole carbon source, were isolated. Genetic mapping revealed that there were at least three chromosomal loci involved in acetoacetate metabolism. Identification of these three loci determined that in S. meliloti: (1) acetoacetyl-CoA synthetase (AcsA), encoded by acsA2 gene, rather than the enzyme acetoacetate:succinyl-CoA transferase, is the enzyme that catalyzes activation of acetoacetate to acetoacetyl-CoA; (2) PHB synthase, encoded by phbC, is required for acetoacetate utilization; (3) a putative transporter protein encoding gene, aau-3, may also be involved in acetoacetate metabolism. acsA2 and aau-3 were 78% linked in co-transduction, while phbC was mapped to somewhere else on the chromosome. Biochemical analysis revealed that acsA2::Tn5 mutants lacked AcsA activity but not acetoacetate:succinyl-CoA transferase activity, while phbC::Tn5 maintained similar level of AcsA activity as wild type in vitro. PHB was absent in the phbC mutant.One transposon-generated mutant, age-1, showed enhanced growth rate on acetoacetate medium. Genetic mapping and transductional analysis indicated that the location of the mutation in age-1 is tightly linked to acsA2. Fine mapping with PCR and DNA sequence techniques showed that Tn5 in age-1 was located at 132 by upstream of the putative translation start site of acsA2. Gene expression analysis indicated that age-1 insertion results in elevated transcription of acsA2. Thus enhanced growth rate on acetoacetate was due to the increased gene expression. acsA2 transcription was induced by acetoacetate and 3-hydroxybutyrate, and repressed by glucose and acetate.All mutants formed root nodules that fixed nitrogen with varying decrease of impairment. Acetoacetate metabolism and the PHB degradation are not essential for symbiosis