Characterization of bdhA , encoding the enzyme d ‐3‐hydroxybutyrate dehydrogenase, from Sinorhizobium sp. strain NGR234

Abstract A genomic library of Sinorhizobium sp. strain NGR234 was introduced into Escherichia coli LS5218, a strain with a constitutively active pathway for acetoacetate degradation, and clones that confer the ability to utilize d ‐3‐hydroxybutyrate as a sole carbon source were isolated. Subcloning experiments identified a 2.3 kb Eco RI fragment that retained complementing ability, and an ORF that appeared orthologous with known bdhA genes was located within this fragment. The deduced NGR234 BdhA amino acid sequence revealed 91% identity to the Sinorhizobium meliloti BdhA. Site‐directed insertion mutagenesis was performed by introduction of a ΩSmSp cassette at a unique Eco RV site within the bdhA coding region. A NGR234 bdhA mutant , NGRPA2, was generated by homogenotization, utilizing the sacB gene‐based lethal selection procedure. This mutant was devoid of d ‐3‐hydroxybutyrate dehydrogenase activity, and was unable to grow on d ‐3‐hydroxybutyrate as sole carbon source. NGRPA2 exhibited symbiotic defects on Leucaena but not on Vigna , Macroptilium or Tephrosia host plants. Furthermore, the d ‐3‐hydroxybutyrate utilization phenotype of NGRPA2 was suppressed by presence of plasmid‐encoded multiple copies of the S. meliloti acsA2 gene. The glpK – bdhA – xdhA gene organization and the bdhA – xdhA operon arrangement observed in S. meliloti are also conserved in NGR234.