Phenotypic characterization and complementation analysis of Bacillus subtilis 6S RNA single and double deletion mutants

6S RNA, a global regulator of transcription in bacteria, binds to housekeeping RNA polymerase (RNAP) holoenzymes to competitively inhibit transcription from DNA promoters. Bacillus subtilis encodes two 6S RNA homologs whose differential functions are as yet unclear. We constructed derivative strains of B. subtilis PY79 lacking 6S-1 RNA (Delta bsrA), 6S-2 RNA (Delta bsrB) or both (Delta bsrAB) to study the physiological role of the two 6S RNAs. We observed two growth phenotypes of mutant strains: (i) accelerated decrease of optical density toward extended stationary phase and (ii) faster outgrowth from stationary phase under alkaline stress conditions (pH 9.8). The first phenotype was observed for bacteria lacking bsrA, and even more pronounced for Delta bsrAB bacteria, but not for those lacking bsrB. The magnitude of the second phenotype was relatively weak for Delta bsrB, moderate for Delta bsrA and again strongest for Delta bsrAB bacteria. Whereas Delta bsrAB bacteria complemented with bsrB or bsrA (strains Delta bsrAB + B and Delta bsrAB + A) mimicked the phenotypes of the Delta bsrA and tlbsrB strains, respectively, complementation with the gene ssrS encoding Escherichia coli 6S RNA failed to cure the "low stationary optical density" phenotype of the double mutant, despite ssrS expression, in line with previous findings. Finally, proteomics (twodimensional differential gel electrophoresis, 2D-DIGE) of B. subtilis 6S RNA deletion strains unveiled a set of proteins that were expressed at higher levels particularly during exponential growth and preferentially in mutant strains lacking 6S-2 RNA. Several of these proteins are involved in metabolism and stress responses. (C) 2015 Elsevier B.V. and Societe francaise de biochimie et biologie Moleculaire (SFBBM). All rights reserved.