A novel gene whose expression is regulated by the response-regulator, SphR, in response to phosphate limitation in Synechococcus species PCC7942

Abstract

In Synechococcus species PCC7942, the production of a subset of proteins is induced when it is grown in a phosphate-limited medium. We previously suggested that a pair of cyanobacterial two-component regulatory proteins, SphS (sensory-kinase) and SphR (response-regulator), may be involved in this particular response to phosphate limitation. Here it was found that a protein with an apparent molecular mass of 33 kDa became particularly abundant when the total cellular proteins from cells grown in a phosphate-limited medium were analysed by SDS-PAGE. A deletion mutant lacking both the sphS and the sphR genes failed to produce this 33 kDa protein in response to phosphate limitation. Thus it was reasonable to assume that this protein is a member of the group of proteins involved in the Synechococcus phosphate regulatory circuit (hence, it was named SphX). The SphX protein was purified to near homogeneity, and the corresponding structural gene was cloned. The determined nucleotide sequence revealed that the sphX gene encodes a novel protein with a calculated molecular mass of 36,374 Da, which was demonstrated to be located in the cytoplasmic membrane. Structural features of the sphX promoter were then clarified by determining its transcription start site, from which transcription was triggered in response to phosphate limitation. Furthermore, the putative response-regulator, SphR, was demonstrated to bind to the upstream region of the sphX promoter by means of in vitro DNase I footprinting. From these results, we conclude that the sphX gene is a member of the Synechococcus phosphate regulatory circuit, in which the two signal-transduction components, SphS and SphR, are crucially involved as transcriptional regulators. The SphX protein may play a role in phosphate assimilation in Synechococcus.