Aromatic compounds represent an important source of energy for soil-dwelling organisms. The ␤-ketoadipate pathway is a key metabolic pathway involved in the catabolism of the aromatic compounds protocatechuate and catechol, and here we show through enzymatic analysis and mutant analysis that genes required for growth and catabolism of protocatechuate in the soil-dwelling bacterium Sinorhizobium meliloti are organized on the pSymB megaplasmid in two transcriptional units designated pcaDCHGB and pcaIJF. The pcaD promoter was mapped by primer extension, and expression from this promoter is demonstrated to be regulated by the LysR-type protein PcaQ. ␤-Ketoadipate succinyl-coenzyme A (CoA) transferase activity in S. meliloti was shown to be encoded by SMb20587 and SMb20588, and these genes have been renamed pcaI and pcaJ, respectively. These genes are organized in an operon with a putative ␤-ketoadipyl-CoA thiolase gene (pcaF), and expression of the pcaIJF operon is shown to be regulated by an IclR-type transcriptional regulator, SMb20586, which we have named pcaR. We show that pcaR transcription is negatively autoregulated and that PcaR is a positive regulator of pcaIJF expression and is required for growth of S. meliloti on protocatechuate as the carbon source. The characterization of the protocatechuate catabolic pathway in S. meliloti offers an opportunity for comparison with related species, including Agrobacterium tumefaciens. Differences observed between S. meliloti and A. tumefaciens pcaIJ offer the first evidence of pca genes that may have been acquired after speciation in these closely related species.

A reduced exopolysaccharide phenotype is associated with inability to synthesize polyhydroxyalkanaote (PHA) stores in Sinorhizobium meliloti strain Rm1021. Loss of function mutations in phbB and phbC result in non-mucoid colony morphology on Yeast Mannitol Agar, compared to the mucoid phenotype exhibited by the parental strain. This phenotype is attributed to reduction in succinoglycan synthesis. We have used complementation of this phenotype and the previously described D-3-hydroxybutyrate/acetoacetate utilization phenotype to isolate a heterologous clone containing a Bradyrhizobium japonicum phbC gene. Sequence analysis confirmed that this clone contains one of the five predicted phbC genes in the B. japonicum genome. The described phenotypic complementation strategy should be useful for isolation of novel PHA synthesis genes of diverse origin.

The competitive abilities of Sinorhizobium meliloti mutant strains containing lesions in the PHB synthesis (phbC) and degradation (bdhA) pathways were compared. While the bdhA mutant showed no noticeable symbiotic defects on alfalfa host plants when inoculated alone, in mixed inoculation experiments it was found to be less competitive than the wild type for nodule occupancy. Long-term survival of the bdhA mutant on a carbon-limiting medium was not affected. However, when subjected to competition with the wild-type strain in periodic subculturing through alternating carbon-limiting and carbon-excess conditions, the bdhA mutant performed poorly. A more severe defect in competition for growth and nodule occupancy was observed with a mutant unable to synthesize PHB (phbC). These results indicate that the ability to efficiently deposit cellular PHB stores is a key factor influencing competitive survival under conditions of fluctuating nutrient carbon availability, whereas the ability to use these stores is less important.

We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer uponEscherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying alacZ transcriptional fusion to bdhAdemonstrated that gene expression is growth phase associated.