Isolation and sequence analysis of the propanediol dehydratase genes of propionibacterium freudenreichii

Abstract

In many Gram-negative enteric bacteria, vitamin B12-dependent reactions aid in the metabolism of small molecules and provide ATP for the bacteria. Propanediol dehydratase is such an enzyme that catalyzes the conversion of 1,2-propanediol to propionaldehyde, the initial step of the 1,2- propanediol degradation pathway. The presence of this enzyme has been reported in several species of enteric bacteria, although not in Escherichia coli, and the molecular structure, function and genetic regulation of the enzyme has been characterized extensively in Klebsiella oxytoca and Salmonella enterica. The existence of propanediol dehydratase activity has also been reported in some Gram-positive bacteria, including Propionibacterium freudenreichii, a member of the propionic acid bacteria. In this study, several strategies were taken to clone the genes encoding propanediol dehydratase from P. freudenreichii using the information already known about the large subunit of the enzyme from S. enterica. A genomic library of P. freudenreichii was constructed, but phenotypic screening for a clone harboring the genes was unsuccessful. Polymerase chain reaction with degenerate primers and the genomic DNA of P. freudenreichii as template did not produce any amplified product. Southern hybridization using the gene encoding the large subunit of propanediol dehydratase from S. enterica as the probe gave a positive signal with the genomic DNA of P. freudenreichii. The isolated genomic DNA fragment was cloned and named A63. Analysis of both the nucleotide sequences and deduced amino acid sequences of the open reading frames in A63 did not reveal any identity with known B12-dependent dehydratases. Instead, the best homologies were found with non-B12-dependent dehydratases and pyruvate formate-lyases from other bacteria. In addition to the putative propanediol dehydratase genes, the carbon utilization and antibiotic susceptibility patterns of P. freudenreichii were also characterized in this study.