Regulation of PSTSCAB-PHOUB Genes in Sinorhizobium Meliloti

Abstract

Previous studies in this laboratory have identified two phosphate transport systems in Sinorhizobium meliloti encoded by the phoCDET and orfA-pit genes respectively. The PhoB regulatory protein is required for transcriptional activation of the phoCDET genes but repress the transcription of orfA-pit. Determination of the DNA sequence upstream of phoU-phoB revealed the presence of genes homologous to the pstA-pstB genes, which encode components of an ABC-type high affinity Pi specific transport system in E. coli. Further analysis of sequence from the S. meliloti genome project (unpublished) revealed the phoR-pstS-pstC genes upstream of pstA-pstB. Using an R-prime approach, we cloned a 7.5 kb Hindlll gene fragment which included the above phoR-pstS-pstC-pstA-pstB and partial phoU genes. Using Tn5-B20 and lacz-aacc1 cassette gene disruption/fusions, we mutated pstA, pstB and phoR gene respectively. We found that: a) pstA-pstB-phoU-phoB are in one operon, b) pstB expression is not regulated by the media phosphate concentration and is independent of phoB, c) in free-living cells, pstB mutants, like phoU or phoB mutants, exhibit alkaline phosphatase negative phenotypes, d) in plant tests, a pstB mutant had normal nitrogen fixation ability and like phoB mutations, the pstB mutation suppressed the Fix- phenotype of phoCDET mutants, e) phoB expression is neither regulated by phosphate concentration nor does its expression appear to be auto-regulated, and f) a phoR mutant exhibited an alkaline phosphatase negative phenotype. Sequence analysis showed that there is no pho box in the upstream of pstA-pstB-phoU-phoB operon and the phoR, but pstS gene has one putative pho box in its promoter region. Also discussion and some ideas for future study were presented.