Gene cloning and regulation of gene expression of the puc operon from Rhodovulum sulfidophilum

摘要

Rhodovulum (Rhv.) sulfidophilum, unlike other nonsulfur purple bacteria, is able to synthesize the peripheral antenna complex even under fully aerobic conditions in the dark. We have obtained strong evidence that Rhv. sulfidophilum encodes only one copy of the puc operon, comprising pucB, pucA and pucC. pucB and pucA encode the β- and α-polypeptides. The third ORF (pucC), downstream of pucA, has a strong homology to pucC of Rhodobacter (Rb.) capsulatus. Deletion mutation analysis indicated that the requirement for the pucC gene product for LH II expression was less strict than in Rb. capsulatus. Comparison of the deduced α and β polypeptide sequences with the directly determined primary structure revealed a C-terminal processing of the α-subunit. Primer extension analysis showed that the pucBAC is transcribed from a σ70-type promoter 130 bases upstream of the translational start of pucB. Transcriptional expression of the pucBAC operon in Rhv. sulfidophilum is higher, the lower the light intensity is, and is not reduced to a ground-level by the presence of oxygen. Based on lacZ fusions the relative promoter activities were, for dark aerobic:dark semiaerobic:low light anaerobic:medium light anaerobic:high light anaerobic, 5.5:7.0:2.0:1.0:0.78. Still unidentified cis-regulatory elements or binding sites of trans-regulatory elements are apparently localized in two distinct upstream regions. Furthermore, comparison of the promoter region of the Rhv. sulfidophilum pucBAC with the promoter regions of puc operons in related species showed distinct differences in the regulatory elements. The significance of these results with respect to the regulation of transcription and the oxygen-independent synthesis of LH II from Rhv. sulfidophilum is discussed.