A Comprehensive Proteomics and Transcriptomics Analysis of Bacillus subtilis Salt Stress Adaptation

ABSTRACT In its natural habitats, Bacillus subtilis is exposed to changing osmolarity, necessitating adaptive stress responses. Transcriptomic and proteomic approaches can provide a picture of the dynamic changes occurring in salt-stressed B. subtilis cultures because these studies provide an unbiased view of cells coping with high salinity. We applied whole-genome microarray technology and metabolic labeling, combined with state-of-the-art proteomic techniques, to provide a global and time-resolved picture of the physiological response of B. subtilis cells exposed to a severe and sudden osmotic upshift. This combined experimental approach provided quantitative data for 3,961 mRNA transcription profiles, 590 expression profiles of proteins detected in the cytosol, and 383 expression profiles of proteins detected in the membrane fraction. Our study uncovered a well-coordinated induction of gene expression subsequent to an osmotic upshift that involves large parts of the SigB, SigW, SigM, and SigX regulons. Additionally osmotic upregulation of a large number of genes that do not belong to these regulons was observed. In total, osmotic upregulation of about 500 B. subtilis genes was detected. Our data provide an unprecedented rich basis for further in-depth investigation of the physiological and genetic responses of B. subtilis to hyperosmotic stress.

[1]  M. Hagemann,et al.  Proteomic screening of salt‐stress‐induced changes in plasma membranes of Synechocystis sp. strain PCC 6803 , 2006, Proteomics.

[2]  T. Hirokawa,et al.  Physicochemical factors for discriminating between soluble and membrane proteins: hydrophobicity of helical segments and protein length. , 1999, Protein engineering.

[3]  Michael Hecker,et al.  From complementarity to comprehensiveness – targeting the membrane proteome of growing Bacillus subtilis by divergent approaches , 2008, Proteomics.

[4]  C. Anagnostopoulos,et al.  REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS , 1961, Journal of bacteriology.

[5]  Makoto Fujisawa,et al.  Three two-component transporters with channel-like properties have monovalent cation/proton antiport activity , 2007, Proceedings of the National Academy of Sciences.

[6]  M. Hecker,et al.  Global expression profiling of Bacillus subtilis cells during industrial-close fed-batch fermentations with different nitrogen sources. , 2005, Biotechnology and bioengineering.

[7]  A. Krogh,et al.  Prediction of lipoprotein signal peptides in Gram‐negative bacteria , 2003, Protein science : a publication of the Protein Society.

[8]  Jörg Bernhardt,et al.  Salt stress adaptation of Bacillus subtilis: A physiological proteomics approach , 2006, Proteomics.

[9]  S. Ruzal,et al.  High salt stress in Bacillus subtilis: involvement of PBP4* as a peptidoglycan hydrolase. , 2009, Research in microbiology.

[10]  Cathy H. Wu,et al.  The Universal Protein Resource (UniProt) , 2004, Nucleic Acids Res..

[11]  M. Hecker,et al.  Complementary Analysis of the Vegetative Membrane Proteome of the Human Pathogen Staphylococcus aureus*S , 2008, Molecular & Cellular Proteomics.

[12]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[13]  M. Hecker,et al.  Temporal activation of beta-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool. , 1993, Journal of general microbiology.

[14]  Joshua E. Elias,et al.  Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. , 2003, Journal of proteome research.

[15]  David Bryant,et al.  DAVID Bioinformatics Resources: expanded annotation database and novel algorithms to better extract biology from large gene lists , 2007, Nucleic Acids Res..

[16]  J A Chudek,et al.  The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis. , 1990, Journal of general microbiology.

[17]  Shigeki Mitaku,et al.  Amphiphilicity index of polar amino acids as an aid in the characterization of amino acid preference at membrane-water interfaces , 2002, Bioinform..

[18]  P Youngman,et al.  Genome‐wide analysis of the general stress response in Bacillus subtilis , 2001, Molecular microbiology.

[19]  J. Errington Regulation of endospore formation in Bacillus subtilis , 2003, Nature Reviews Microbiology.

[20]  István Simon,et al.  The HMMTOP transmembrane topology prediction server , 2001, Bioinform..

[21]  Makoto Fujisawa,et al.  NhaK, a novel monovalent cation/H+ antiporter of Bacillus subtilis , 2005, Archives of Microbiology.

[22]  Sanying Wang,et al.  Proteomic analysis of salt-sensitive outer membrane proteins of Vibrio parahaemolyticus. , 2004, Research in microbiology.

[23]  W. Schumann FtsH--a single-chain charonin? , 1999, FEMS microbiology reviews.

[24]  Brad T. Sherman,et al.  DAVID: Database for Annotation, Visualization, and Integrated Discovery , 2003, Genome Biology.

[25]  Erik L. L. Sonnhammer,et al.  A Hidden Markov Model for Predicting Transmembrane Helices in Protein Sequences , 1998, ISMB.

[26]  S. Brunak,et al.  SHORT COMMUNICATION Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites , 1997 .

[27]  U. Völker,et al.  Genome-Wide Transcriptional Profiling Analysis of Adaptation of Bacillus subtilis to High Salinity , 2003, Journal of bacteriology.

[28]  E. Bremer,et al.  KtrAB and KtrCD: Two K+ Uptake Systems in Bacillus subtilis and Their Role in Adaptation to Hypertonicity , 2003, Journal of bacteriology.

[29]  R. Losick,et al.  Bacillus Subtilis and Its Closest Relatives: From Genes to Cells , 2001 .

[30]  A. Moir,et al.  σM, an ECF RNA polymerase sigma factor of Bacillus subtilis 168, is essential for growth and survival in high concentrations of salt , 1999, Molecular microbiology.

[31]  Peter J Lewis,et al.  Molecular Architecture of the "Stressosome," a Signal Integration and Transduction Hub , 2008, Science.

[32]  S. Ruzal,et al.  Variations of the Envelope Composition of Bacillus subtilis During Growth in Hyperosmotic Medium , 1998, Current Microbiology.

[33]  Charles O. Rock,et al.  β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is a Determining Factor in Branched-Chain Fatty Acid Biosynthesis , 2000, Journal of bacteriology.

[34]  J. Helmann,et al.  The Bacillus subtilis Extracytoplasmic-Function σX Factor Regulates Modification of the Cell Envelope and Resistance to Cationic Antimicrobial Peptides , 2004, Journal of bacteriology.

[35]  M. Hecker,et al.  General stress response of Bacillus subtilis and other bacteria. , 2001, Advances in microbial physiology.

[36]  Shigeki Mitaku,et al.  SOSUI: classification and secondary structure prediction system for membrane proteins , 1998, Bioinform..

[37]  Sung Kyu Park,et al.  A quantitative analysis software tool for mass spectrometry–based proteomics , 2008, Nature Methods.

[38]  E. Bremer Adaptation to Changing Osmolanty , 2002 .

[39]  Michael Hecker,et al.  Comprehensive Characterization of the Contribution of Individual SigB-Dependent General Stress Genes to Stress Resistance of Bacillus subtilis , 2005, Journal of bacteriology.

[40]  T. Mascher,et al.  Regulatory Overlap and Functional Redundancy among Bacillus subtilis Extracytoplasmic Function σ Factors , 2007, Journal of bacteriology.

[41]  A. Alice,et al.  Role of anionic phospholipids in the adaptation of Bacillus subtilis to high salinity. , 2006, Microbiology.

[42]  J. Yates,et al.  An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database , 1994, Journal of the American Society for Mass Spectrometry.

[43]  J. Cronan,et al.  A Bacillus subtilis Gene Induced by Cold Shock Encodes a Membrane Phospholipid Desaturase , 1998, Journal of bacteriology.

[44]  J. Helmann,et al.  The Bacillus subtilis σM regulon and its contribution to cell envelope stress responses , 2008, Molecular microbiology.

[45]  A I Saeed,et al.  TM4: a free, open-source system for microarray data management and analysis. , 2003, BioTechniques.

[46]  J. Hoheisel,et al.  Global Analysis of the General Stress Response ofBacillus subtilis , 2001, Journal of bacteriology.

[47]  G. Tusnády,et al.  Principles governing amino acid composition of integral membrane proteins: application to topology prediction. , 1998, Journal of molecular biology.

[48]  P. Setlow,et al.  Growth, osmotic downshock resistance and differentiation of Bacillus subtilis strains lacking mechanosensitive channels , 2007, Archives of Microbiology.

[49]  M. Hecker,et al.  Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis , 2002, Journal of bacteriology.

[50]  J. Sekiguchi,et al.  Complex Formation by the mrpABCDEFG Gene Products, Which Constitute a Principal Na+/H+ Antiporter in Bacillus subtilis , 2007, Journal of bacteriology.

[51]  U. Völker,et al.  High-Salinity-Induced Iron Limitation in Bacillus subtilis , 2002, Journal of bacteriology.

[52]  Lina Wu,et al.  Proteomic analysis on the expression of outer membrane proteins of Vibrio alginolyticus at different sodium concentrations , 2005, Proteomics.

[53]  Jan Maarten van Dijl,et al.  A proteomic view on genome-based signal peptide predictions. , 2001, Genome research.

[54]  Uwe Völker,et al.  A comprehensive proteome map of growing Bacillus subtilis cells , 2004, Proteomics.

[55]  E. Bremer,et al.  Responses of Bacillus subtilis to Hypotonic Challenges: Physiological Contributions of Mechanosensitive Channels to Cellular Survival , 2008, Applied and Environmental Microbiology.

[56]  V. Paakkarinen,et al.  Towards Functional Proteomics of Membrane Protein Complexes in Synechocystis sp. PCC 68031 , 2004, Plant Physiology.

[57]  G. Hambraeus,et al.  Genome-wide survey of mRNA half-lives in Bacillus subtilis identifies extremely stable mRNAs , 2003, Molecular Genetics and Genomics.

[58]  L. Cybulski,et al.  Mechanism of membrane fluidity optimization: isothermal control of the Bacillus subtilis acyl‐lipid desaturase , 2002, Molecular microbiology.

[59]  Jörg Bernhardt,et al.  Proteome signatures for stress and starvation in Bacillus subtilis as revealed by a 2‐D gel image color coding approach , 2006, Proteomics.

[60]  A. Moir,et al.  SigM-Responsive Genes of Bacillus subtilis and Their Promoters , 2007, Journal of bacteriology.

[61]  J. Yates,et al.  DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. , 2002, Journal of proteome research.

[62]  A. Krogh,et al.  A combined transmembrane topology and signal peptide prediction method. , 2004, Journal of molecular biology.

[63]  A. Sonenshein,et al.  Multiple Genes for the Last Step of Proline Biosynthesis in Bacillus subtilis , 2001, Journal of bacteriology.

[64]  T. A. Krulwich,et al.  mrp, a Multigene, Multifunctional Locus inBacillus subtilis with Roles in Resistance to Cholate and to Na+ and in pH Homeostasis , 1999, Journal of bacteriology.

[65]  Kenta Nakai,et al.  DBTBS: a database of transcriptional regulation in Bacillus subtilis containing upstream intergenic conservation information , 2007, Nucleic Acids Res..

[66]  J. Maupin-Furlow,et al.  Proteomic analysis of Haloferax volcanii reveals salinity-mediated regulation of the stress response protein PspA. , 2008, Microbiology.

[67]  S. Ruzal,et al.  Biochemical and biophysical studies of Bacillus subtilis envelopes under hyperosmotic stress. , 2000, International journal of food microbiology.

[68]  Erik L. L. Sonnhammer,et al.  Advantages of combined transmembrane topology and signal peptide prediction—the Phobius web server , 2007, Nucleic Acids Res..

[69]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.