Bacterial Anaerobic Phosphite Oxidation
Final Report Abstract
Development of a spectrophotometric assay for measurement of phosphite oxidizing enzyme activity in cell-free extracts of D. phosphitoxidans cells with 80% of the total phosphite oxidation activity measured in soluble fraction of proteins. Four phosphite - specific protein spots were found in the soluble fraction of proteins and three in the membrane fraction. Their N-terminal sequences were analyzed, and PMF search of the databases were performed, but we could not identify the proteins of interest - which indicated that these proteins are entirely new. Degenerate oligonucleotides/PCR primers were developed on the base of N-terminal sequences of phosphite-induced proteins, and in combination with Inverted PCR technique revealed the gene responsible for their synthesis - gene coding for a putative NAD(P)-binding epimerase/dehydratase was identified. Further, the partial operon structure coding for proteins involved in phosphite oxidation in our strain was discovered. This gene cluster is formed of 8 genes. The sequence has been submitted to GeneBank under accession number EU069391- Desulfotignum phosphitoxidans phosphite oxidation gene cluster, partial sequence. Due to the operon high plasticity with two pairs of inverted repeats our attempts to obtain random gene library expressed in E. coll as host, had failed. Therefore, genes 2 to 7 were amplified from genomic DNA of D. phosphitoxidans and cloned into pCR 2.1 vector, and then the fragment was subcloned into pBBR1MCS-5 vector.
