MgpS, a Complex Regulatory Locus Involved in the Transcriptional Control of the Puc and Puf Operons in Rhodobacter Sphaeroides 2.4.1

A new method has been developed in order to select mutants showing decreased puc operon transcription in Rhodobacter sphaeroides 2.4.1. A transcriptional fusion of a promoterless fragment derived from the sacB gene, encoding the levansucrase from Bacillus subtilis, to the upstream regulatory region of the puc operon has been constructed. With appropriate levels of exogenous sucrose, survivors of a sucrose killing challenge have been isolated. Subsequent analysis revealed the presence of both cis- and trans-acting "down" mutations in relation to puc operon expression. One of the trans-acting regulatory mutations was chosen for further study. The original mutation showed less than 2% of the level of puc operon transcription compared with the wild type under aerobic conditions and an 86% reduction under dark dimethyl sulfoxide conditions. This mutation can be complemented by a 3.9-kb BamHI DNA fragment derived from a cosmid contained within a genomic cosmid bank. DNA sequence analysis of this fragment revealed the presence of a 2.8-kb open reading frame, designated mgpS, which would encode a 930-amino-acid protein. The N-terminal portion of the putative protein product presents homologies to proteins of the RNA helicase family. Disruption of the chromosomal mgpS resulted in decreased transcription of both puc and puf, while the presence of mgpS in multicopy in the wild type, 2.4.1., increased puc expression by a factor of 2 under aerobic conditions. Structural analysis of the mgpS locus revealed that expression of mgpS was likely to be complex. A smaller protein containing the 472 C-terminal amino acids of MgpS is able to act by itself as an activator of puc transcription and is expressed independently of the large open reading frame in which it is contained.