Characterization of bdhA, encoding the enzyme D-3-hydroxybutyrate dehydrogenase, from Sinorhizobium sp. strain NGR234.
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 EcoRI 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 OmegaSmSp cassette at a unique EcoRV 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.
Aneja
Punita
P
Department of Natural Resource Sciences, McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Que., Canada H9X 3V9.
Charles
Trevor C
TC
D020155:3-Hydroxybutyric Acid / Q000378:metabolism; D000595:Amino Acid Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D007887:Fabaceae / Q000382:microbiology; D023061:Gene Order; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D016366:Open Reading Frames; D009876:Operon; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D020579:Sinorhizobium / Q000201:enzymology* / Q000235:genetics; D013559:Symbiosis / Q000235:genetics; D026801:Synteny
D004269:DNA, Bacterial; D006884:Hydroxybutyrate Dehydrogenase; D020155:3-Hydroxybutyric Acid