Detection of small RNAs in Pseudomonas aeruginosa by RNomics and structure-based bioinformatic tools.
Inactivation of the Pseudomonas aeruginosa (PAO1) hfq gene, encoding the Sm-like Hfq protein, resulted in pleiotropic effects that included an attenuated virulence. As regulation by Hfq often involves the action of small regulatory RNAs (sRNAs), we have used a shotgun cloning approach (RNomics) and bioinformatic tools to identify sRNAs in strain PAO1. For cDNA library construction, total RNA was extracted from PAO1 cultures either grown to stationary phase or exposed to human serum. The cDNA libraries were generated from small-sized RNAs of PAO1 after co-immunoprecipitation with Hfq. Of 400 sequenced cDNA clones, 11 mapped to intergenic regions. Band-shift assays and Northern blot analyses performed with two selected sRNAs confirmed that Hfq binds to and affects the steady-state levels of these RNAs. A proteome study performed upon overproduction of one sRNA, PhrS, implicated it in riboregulation. PhrS contains an ORF, and evidence for its translation is presented. In addition, based on surveys with structure-based bioinformatic tools, we provide an electronic compilation of putative sRNA and non-coding RNA genes of PAO1 based on their evolutionarily conserved structure.
Sonnleitner
Elisabeth
E
Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.
Sorger-Domenigg
Theresa
T
Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.
Madej
Monika J
MJ
Innsbruck Biocenter, Division of Genomics and RNomics, Medical University Innsbruck, Fritz-Pregl Str. 3, A-6020 Innsbruck, Austria.
Findeiss
Sven
S
Bioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, University of Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Germany.
Hackermüller
Jörg
J
Bioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, University of Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Germany.
Hüttenhofer
Alexander
A
Innsbruck Biocenter, Division of Genomics and RNomics, Medical University Innsbruck, Fritz-Pregl Str. 3, A-6020 Innsbruck, Austria.
Stadler
Peter F
PF
Bioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, University of Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Germany.
Bläsi
Udo
U
Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.
Moll
Isabella
I
Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D019295:Computational Biology / Q000379:methods; D024202:Electrophoretic Mobility Shift Assay; D015723:Gene Library; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods; D035001:Host Factor 1 Protein / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D020543:Proteome / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics*; D022661:RNA, Untranslated / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment
D001426:Bacterial Proteins; D035001:Host Factor 1 Protein; D020543:Proteome; D012329:RNA, Bacterial; D022661:RNA, Untranslated