The Blue-Light Receptor YtvA Acts in the Environmental Stress Signaling Pathway of Bacillus subtilis

ABSTRACT The general stress response of the bacterium Bacillus subtilis is regulated by a partner-switching mechanism in which serine and threonine phosphorylation controls protein interactions in the stress-signaling pathway. The environmental branch of this pathway contains a family of five paralogous proteins that function as negative regulators. Here we present genetic evidence that a sixth paralog, YtvA, acts as a positive regulator in the same environmental signaling branch. We also present biochemical evidence that YtvA and at least three of the negative regulators can be isolated from cell extracts in a large environmental signaling complex. YtvA differs from these associated negative regulators by its flavin mononucleotide (FMN)-containing light-oxygen-voltage domain. Others have shown that this domain has the photochemistry expected for a blue-light sensor, with the covalent linkage of the FMN chromophore to cysteine 62 composing a critical part of the photocycle. Consistent with the view that light intensity modifies the output of the environmental signaling pathway, we found that cysteine 62 is required for YtvA to exert its positive regulatory role in the absence of other stress. Transcriptional analysis of the ytvA structural gene indicated that it provides the entry point for at least one additional environmental input, mediated by the Spx global regulator of disulfide stress. These results support a model in which the large signaling complex serves to integrate multiple environmental signals in order to modulate the general stress response.

[1]  L. Tsakiris,et al.  Secondary Pancreatic Involvement by Diffuse Large B-Cell Lymphoma Presenting as Acute Pancreatitis: Treatment and Outcome , 2011, Pancreatology.

[2]  W. Eisenreich,et al.  An optomechanical transducer in the blue light receptor phototropin from Avena sativa , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Sébastien Rodrigue,et al.  Novel Mycobacterium tuberculosis anti‐σ factor antagonists control σF activity by distinct mechanisms , 2002 .

[4]  J. Christie,et al.  LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[5]  J. Christie,et al.  Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function. , 2002, The Plant journal : for cell and molecular biology.

[6]  R. Losick,et al.  Role of adenosine nucleotides in the regulation of a stress-response transcription factor in Bacillus subtilis. , 1996, Journal of molecular biology.

[7]  C. Kang,et al.  Serine kinase activity of a Bacillus subtilis switch protein is required to transduce environmental stress signals but not to activate its target PP2C phosphatase , 1998, Molecular microbiology.

[8]  C. Kang,et al.  Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress to activate a bacterial transcription factor. , 1996, Genes & Development.

[9]  Keith Moffat,et al.  The LOV domain family: photoresponsive signaling modules coupled to diverse output domains. , 2003, Biochemistry.

[10]  Keith Moffat,et al.  Photoexcited Structure of a Plant Photoreceptor Domain Reveals a Light-Driven Molecular Switch Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010475. , 2002, The Plant Cell Online.

[11]  M. Seibert,et al.  The effects of light intensity and spectral quality on growth and shoot initiation in tobacco callus. , 1975, Plant physiology.

[12]  R. Lewis,et al.  The RsbRST Stress Module in Bacteria: A Signalling System That May Interact with Different Output Modules , 2005, Journal of Molecular Microbiology and Biotechnology.

[13]  M. Perego Integrational Vectors for Genetic Manipulation in Bacillus subtilis , 1993 .

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

[15]  C. Kang,et al.  Homologous pairs of regulatory proteins control activity of Bacillus subtilis transcription factor sigma(b) in response to environmental stress , 1996, Journal of bacteriology.

[16]  W. Haldenwang,et al.  Interactions between a Bacillus subtilis anti-sigma factor (RsbW) and its antagonist (RsbV) , 1994, Journal of bacteriology.

[17]  Wolfgang Gärtner,et al.  First evidence for phototropin-related blue-light receptors in prokaryotes. , 2002, Biophysical journal.

[18]  U. Völker,et al.  Chill Induction of the SigB-Dependent General Stress Response in Bacillus subtilis and Its Contribution to Low-Temperature Adaptation , 2003, Journal of bacteriology.

[19]  K. Gardner,et al.  Disruption of the LOV-Jalpha helix interaction activates phototropin kinase activity. , 2004, Biochemistry.

[20]  K. Devine,et al.  New Family of Regulators in the Environmental Signaling Pathway Which Activates the General Stress Transcription Factor ςB of Bacillus subtilis , 2001, Journal of bacteriology.

[21]  Chien-Cheng Chen,et al.  A supramolecular complex in the environmental stress signalling pathway of Bacillus subtilis , 2003, Molecular microbiology.

[22]  Kevin H. Gardner,et al.  Structural Basis of a Phototropin Light Switch , 2003, Science.

[23]  P. Stragier,et al.  Plasmids for ectopic integration in Bacillus subtilis. , 1996, Gene.

[24]  J. Roe,et al.  Regulation of σB by an Anti- and an Anti-Anti-Sigma Factor in Streptomyces coelicolor in Response to Osmotic Stress , 2004, Journal of bacteriology.

[25]  Jeffrey H. Miller Experiments in molecular genetics , 1972 .

[26]  W. Haldenwang,et al.  Relative levels and fractionation properties of Bacillus subtilis sigma(B) and its regulators during balanced growth and stress , 1996, Journal of bacteriology.

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

[28]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[29]  G. Mittenhuber A phylogenomic study of the general stress response sigma factor sigmaB of Bacillus subtilis and its regulatory proteins. , 2002, Journal of molecular microbiology and biotechnology.

[30]  Jeff F. Miller,et al.  Interactions between Partner Switcher Orthologs BtrW and BtrV Regulate Type III Secretion in Bordetella , 2005, Journal of bacteriology.

[31]  A. Losi The bacterial counterparts of plant phototropins , 2004, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[32]  U Krauss,et al.  Initial characterization of a blue-light sensing, phototropin-related protein from Pseudomonas putida: a paradigm for an extended LOV construct. , 2005, Physical chemistry chemical physics : PCCP.

[33]  M. Hecker,et al.  Separate mechanisms activate sigma B of Bacillus subtilis in response to environmental and metabolic stresses , 1995, Journal of bacteriology.

[34]  Y. M. Lee,et al.  Threonine phosphorylation of modulator protein RsbR governs its ability to regulate a serine kinase in the environmental stress signaling pathway of Bacillus subtilis. , 1999, Journal of molecular biology.

[35]  M. Frohman,et al.  On beyond classic RACE (rapid amplification of cDNA ends). , 1994, PCR methods and applications.

[36]  A. Losi,et al.  Tryptophan Fluorescence in the Bacillus subtilis Phototropin-related Protein YtvA as a Marker of Interdomain Interaction¶ , 2004, Photochemistry and photobiology.

[37]  U. Völker,et al.  Functional and Structural Characterization of RsbU, a Stress Signaling Protein Phosphatase 2C* , 2004, Journal of Biological Chemistry.

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

[39]  I. Zhulin,et al.  PAS Domains: Internal Sensors of Oxygen, Redox Potential, and Light , 1999, Microbiology and Molecular Biology Reviews.

[40]  S. Nakano,et al.  A regulatory protein that interferes with activator-stimulated transcription in bacteria , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Klaas J. Hellingwerf,et al.  Blue Light Activates the σB-Dependent Stress Response of Bacillus subtilis via YtvA , 2006, Journal of bacteriology.

[42]  P. Zuber Spx-RNA Polymerase Interaction and Global Transcriptional Control during Oxidative Stress , 2004, Journal of bacteriology.

[43]  A. Benson,et al.  Bacillus subtilis sigma B is regulated by a binding protein (RsbW) that blocks its association with core RNA polymerase. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[44]  P. Stragier,et al.  Processing of a sporulation sigma factor in Bacillus subtilis: How morphological structure could control gene expression , 1988, Cell.

[45]  P. Hegemann,et al.  Functional variations among LOV domains as revealed by FT-IR difference spectroscopy , 2004, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[46]  J. D. Helmann,et al.  Compilation and analysis of Bacillus subtilis sigma A-dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA , 1995, Nucleic Acids Res..

[47]  C. Price,et al.  A multicomponent protein complex mediates environmental stress signaling in Bacillus subtilis. , 2004, Journal of molecular biology.

[48]  C. Price,et al.  Stress-induced activation of the sigma B transcription factor of Bacillus subtilis , 1993, Journal of bacteriology.

[49]  E. Koonin,et al.  The STAS domain — a link between anion transporters and antisigma-factor antagonists , 2000, Current Biology.

[50]  P. Oeller,et al.  Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. , 1997, Science.

[51]  C. Price,et al.  In Vivo Phosphorylation of Partner Switching Regulators Correlates with Stress Transmission in the Environmental Signaling Pathway of Bacillus subtilis , 2004, Journal of bacteriology.

[52]  Peter Zuber,et al.  Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in Bacillus subtilis , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[53]  C. Price,et al.  A PP2C phosphatase containing a PAS domain is required to convey signals of energy stress to the σB transcription factor of Bacillus subtilis , 2000, Molecular microbiology.

[54]  C. Price,et al.  General Stress Response , 2002 .

[55]  S. M. Thomas,et al.  Activation of Bacillus subtilis transcription factor sigma B by a regulatory pathway responsive to stationary-phase signals , 1992, Journal of bacteriology.

[56]  Lei Hua,et al.  Core of the partner switching signalling mechanism is conserved in the obligate intracellular pathogen Chlamydia trachomatis , 2006, Molecular microbiology.

[57]  R. Lewis,et al.  Structure of a nonheme globin in environmental stress signaling. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[58]  J. Christie,et al.  Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin. , 2000, Biochemistry.

[59]  A. Losi,et al.  Listening to the blue: the time-resolved thermodynamics of the bacterial blue-light receptor YtvA and its isolated LOV domain , 2003, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.