title: Phenotypic characterization and complementation analysis of Bacillus subtilis 6S RNA single and double deletion mutants.
abstract: 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 (ΔbsrA), 6S-2 RNA (ΔbsrB) or both (ΔbsrAB) 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 ΔbsrAB bacteria, but not for those lacking bsrB. The magnitude of the second phenotype was relatively weak for ΔbsrB, moderate for ΔbsrA and again strongest for ΔbsrAB bacteria. Whereas ΔbsrAB bacteria complemented with bsrB or bsrA (strains ΔbsrAB + B and ΔbsrAB + A) mimicked the phenotypes of the ΔbsrA and ΔbsrB 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 (two-dimensional 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.
authors.affiliation: Philipps-Universität Marburg, Fachbereich Pharmazie, Institut für Pharmazeutische Chemie, Marbacher Weg 6, D-35037 Marburg, Germany. Electronic address: hoch@staff.uni-marburg.de.
authors.forename: Philipp G
authors.initials: PG
authors.lastname: Hoch
authors.affiliation: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia. Electronic address: alunita@rambler.ru.
authors.forename: Olga Y
authors.initials: OY
authors.lastname: Burenina
authors.affiliation: Philipps-Universität Marburg, Fachbereich Pharmazie, Institut für Pharmazeutische Chemie, Marbacher Weg 6, D-35037 Marburg, Germany. Electronic address: weberm@staff.uni-marburg.de.
authors.forename: Michael H W
authors.initials: MH
authors.lastname: Weber
authors.affiliation: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia. Electronic address: d.jolk@yahoo.com.
authors.forename: Daria A
authors.initials: DA
authors.lastname: Elkina
authors.affiliation: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia. Electronic address: nesterchuk.mihail@gmail.com.
authors.forename: Mikhail V
authors.initials: MV
authors.lastname: Nesterchuk
authors.affiliation: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia. Electronic address: petya@mail.genebee.msu.ru.
authors.forename: Petr V
authors.initials: PV
authors.lastname: Sergiev
authors.affiliation: Philipps-Universität Marburg, Fachbereich Pharmazie, Institut für Pharmazeutische Chemie, Marbacher Weg 6, D-35037 Marburg, Germany. Electronic address: roland.hartmann@staff.uni-marburg.de.
authors.forename: Roland K
authors.initials: RK
authors.lastname: Hartmann
authors.affiliation: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia. Electronic address: kubareva@belozersky.msu.ru.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Kubareva
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010641:Phenotype; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D022661:RNA, Untranslated / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
chemical_list: C411065:6S RNA; D001426:Bacterial Proteins; D020543:Proteome; D012329:RNA, Bacterial; D022661:RNA, Untranslated
keywords: 6S-1 RNA; 6S-2 RNA; Bacillus subtilis PY79; Deletion strains; Non-coding RNA