Characterization of a Mannose Utilization System in Bacillus subtilis

Abstract

ABSTRACT The mannose operon of Bacillus subtilis consists of three genes, manP , manA , and yjdF , which are responsible for the transport and utilization of mannose. Upstream and in the same orientation as the mannose operon a regulatory gene, manR , codes for a transcription activator of the mannose operon, as shown in this study. Both mannose operon transcription and manR transcription are inducible by mannose. The presence of mannose resulted in a 4- to 7-fold increase in expression of lacZ from the manP promoter ( P manP ) and in a 3-fold increase in expression of lacZ from the manR promoter ( P manR ). The transcription start sites of manPA-yjdF and manR were determined to be a single A residue and a single G residue, respectively, preceded by −10 and −35 boxes resembling a vegetative σ A promoter structure. Through deletion analysis the target sequences of ManR upstream of P manP and P manR were identified between bp −80 and −35 with respect to the transcriptional start site of both promoters. Deletion of manP (mannose transporter) resulted in constitutive expression from both the P manP and P manR promoters, indicating that the phosphotransferase system (PTS) component EII Man has a negative effect on regulation of the mannose operon and manR . Moreover, both P manP and P manR are subject to carbon catabolite repression (CCR). By constructing protein sequence alignments a DNA binding motif at the N-terminal end, two PTS regulation domains (PRDs), and an EIIA- and EIIB-like domain were identified in the ManR sequence, indicating that ManR is a PRD-containing transcription activator. Like findings for other PRD regulators, the phosphoenolpyruvate (PEP)-dependent phosphorylation by the histidine protein HPr via His15 plays an essential role in transcriptional activation of P manP and P manR . Phosphorylation of Ser46 of HPr or of the homologous Crh protein by HPr kinase and formation of a repressor complex with CcpA are parts of the B. subtilis CCR system. Only in the double mutant with an HPr Ser46Ala mutation and a crh knockout mutation was CCR strongly reduced. In contrast, P manR and P manP were not inducible in a ccpA deletion mutant.