Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: When exposed to antibiotics and other cell wall stress agents Bacillus subtilis displays complex adaptive responses coordinated by a variety of regulatory proteins including the extracytoplasmic function (ECF) sigma factors (SigX, SigW, and SigM) and several two-component regulatory systems (TCS). During this last project period, we characterized genes regulated by the ECF sigma factor, Sigma-M and show that this factor regulates a large number of genes involved in cell envelope synthesis and cell division. We have also characterized a novel antibiotic-inducible, two-component sigma factor comprised of the YvrI and YvrHa proteins. We have also continued to investigate stress responses elicited by antibiotic exposure including those induced by daptomycin, a lipopeptide antibiotic used to treat Gram positive bacterial infections, moenomycin, and ramoplanin. In related work, we are investigating the effects of mutations that alter membrane lipid synthesis pathways on the generation of, and response to, cell envelope stresses. This work has been widely disseminated by peer-reviewed publications, conference presentations, and invited seminars at various Universities. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Resistance to antibiotics is a problem of grave medical concern. While it is widely appreciated that resistance genes are frequently found on transmissable genetic elements, and can spread rapidly within and among patients in clinical settings, the origins of such resistance genes are still debated. We suggest that many resistance genes evolved in soil bacteria that are themselves antibiotic producers or which cohabit with known antibiotic producers such as the actinomycetes. B. subtilis provides a model system for the analysis of the genetic responses of soil bacteria to antibacterial agents. We have discovered a number of resistance determinants that are important for antibiotic resistance and defined the corresponding regulatory systems. Promoters that are strongly induced by specific classes of antibiotics are also of use in antibiotic screening programs. Finally, we are developing B. subtilis as a model system for the analysis of cell-cell interactions among soil bacteria
Publications
- Gebhard, S, Gaballa, A., Helmann, J.D. and G.M. Cook. Direct stimulus perception and transcription activation by a membrane-bound DNA binding protein. Mol Microbiol. 2009 Aug;73(3):482-91.
- Luo Y, Helmann JD. Extracytoplasmic function factors with overlapping promoter specificity regulate sublancin production in Bacillus subtilis. J Bacteriol. 2009 191:4951-8.
- Maclellan, S.R., W. Eiamphungporn, and J.D. Helmann, ROMA: an in vitro approach to defining target genes for transcription regulators. Methods. 2009. 47:73-7.
- MacLellan, S.R., J.D. Helmann, and H. Antelmann, The YvrI alternative sigma factor is essential for acid stress induction of oxalate decarboxylase in Bacillus subtilis. J Bacteriol, 2009. 191:931-9.
- Helmann, J.D., RNA polymerase: a nexus of gene regulation. Methods, 2009. 47:1-5.
- Hachmann, A.B., E.R. Angert, and J.D. Helmann, Genetic Analysis of Factors Affecting Susceptibility of Bacillus subtilis to Daptomycin. Antimicrob Agents Chemother, 2009.
- Eiamphungporn, W. and J.D. Helmann, Extracytoplasmic function sigma factors regulate expression of the Bacillus subtilis yabE gene via a cis-acting antisense RNA. J Bacteriol, 2009. 191:1101-5.
- MacLellan, S.R., T. Wecke, and J.D. Helmann, A previously unidentified sigma factor and two accessory proteins regulate oxalate decarboxylase expression in Bacillus subtilis. Mol Microbiol, 2008. 69(4): p. 954-67.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: When exposed to antibiotics and other cell wall stress agents Bacillus subtilis displays complex adaptive responses coordinated by a variety of regulatory proteins including the extracytoplasmic function (ECF) sigma factors (SigX, SigW, and SigM) and several two-component regulatory systems (TCS). During this last project period, we characterized genes regulated by the ECF sigma factor, Sigma-M and show that this factor regulates a large number of genes involved in cell envelope synthesis and cell division. We have also characterized a novel antibiotic-inducible, two-component sigma factor comprised of the YvrI and YvrHa proteins. We have also continued to investigate stress responses elicited by antibiotic exposure including those induced by daptomycin, a lipopeptide antibiotic used to treat Gram positive bacterial infections, moenomycin, and ramoplanin. In related work, we are investigating the effects of mutations that alter membrane lipid synthesis pathways on the generation of, and response to, cell envelope stresses. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Resistance to antibiotics is a problem of grave medical concern. While it is widely appreciated that resistance genes are frequently found on transmissable genetic elements, and can spread rapidly within and among patients in clinical settings, the origins of such resistance genes are still debated. We suggest that many resistance genes evolved in soil bacteria that are themselves antibiotic producers or which cohabit with known antibiotic producers such as the actinomycetes. B. subtilis provides a model system for the analysis of the genetic responses of soil bacteria to antibacterial agents. We have discovered a number of resistance determinants that are important for antibiotic resistance and defined the corresponding regulatory systems. Promoters that are strongly induced by specific classes of antibiotics are also of use in antibiotic screening programs. Finally, we are developing B. subtilis as a model system for the analysis of cell-cell interactions among soil bacteria.
Publications
- Bordi, C., Butcher, B.G., Shi, Q., Hachmann, A.B., Peters, J.E., and Helmann, J.D. (2008) In vitro mutagenesis of Bacillus subtilis by using a modified Tn7 transposon with an outward-facing inducible promoter. Appl Environ Microbiol 74: 3419-3425.
- Eiamphungporn, W., and Helmann, J.D. (2008) The Bacillus subtilis sigma(M) regulon and its contribution to cell envelope stress responses. Mol Microbiol 67: 830-848.
- MacLellan, S.R., Wecke, T., and Helmann, J.D. (2008) A previously unidentified sigma factor and two accessory proteins regulate oxalate decarboxylase expression in Bacillus subtilis. Mol Microbiol 69: 954-967.
- Salzberg, L.I., and Helmann, J.D. (2008) Phenotypic and Transcriptomic Characterization of Bacillus subtilis Mutants with Grossly Altered Membrane Composition. J Bacteriol. 190: 7797-807.
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Progress 10/01/06 to 09/30/07
Outputs
When exposed to antibiotics and other cell wall stress agents Bacillus subtilis displays complex adaptive responses coordinated by a variety of regulatory proteins including the extracytoplasmic function (ECF) sigma factors (SigX, SigW, and SigM) and several two-component regulatory systems (TCS). During this last project period, we characterized genes regulated by the ECF sigma factor, Sigma-M and show that this factor regulates a large number of genes involved in cell envelope synthesis and cell division. We have also characterized a novel antibiotic-inducible, two-component sigma factor comprised of the YvrI and YvrHa proteins. We have also continued to investigate stress responses elicited by antibiotic exposure including those induced by daptomycin, a lipopeptide antibiotic used to treat Gram positive bacterial infections, moenomycin, and ramoplanin. In related work, we are investigating the effects of mutations that alter membrane lipid synthesis pathways on the
generation of, and response to, cell envelope stresses.
Impacts
Resistance to antibiotics is a problem of grave medical concern. While it is widely appreciated that resistance genes are frequently found on transmissable genetic elements, and can spread rapidly within and among patients in clinical settings, the origins of such resistance genes are still debated. We suggest that many resistance genes evolved in soil bacteria that are themselves antibiotic producers or which cohabit with known antibiotic producers such as the actinomycetes. B. subtilis provides a model system for the analysis of the genetic responses of soil bacteria to antibacterial agents. We have discovered a number of resistance determinants that are important for antibiotic resistance and defined the corresponding regulatory systems. Promoters that are strongly induced by specific classes of antibiotics are also of use in antibiotic screening programs. Finally, we are developing B. subtilis as a model system for the analysis of cell-cell interactions among soil
bacteria.
Publications
- Butcher B.G., Y.-P. Lin and J.D. Helmann. 2007. The yydFGHIJ operon of Bacillus subtilis encodes a peptide that induces the LiaRS two-component system. J Bacteriol. Oct 5; [Epub ahead of print].
- Mascher, T., A. Hachmann and J.D. Helmann. 2007. Regulatory Overlap and Functional Redundancy Among Bacillus subtilis Extracytoplasmic Function (ECF) -Factors. J Bacteriol. 189:6919-27.
- Jordan, S., E. Rietkotter, M.A. Strauch, F. Kalamorz, B.G. Butcher, J.D. Helmann and T. Mascher. 2007. LiaRS-dependent gene expression is embedded in transition state regulation in Bacillus subtilis. Microbiology. 153:2530-40.
- Butcher, B.G., T. Mascher and J.D. Helmann. 2007. Environmental sensing and the role of extracytoplasmic function (ECF) factors in Bacterial Physiology- A Molecular Approach, Walid M. El-Sharoud (Ed.). Springer-Verlag GmbH Berlin Heidelberg.
- Salzberg, L. and J.D. Helmann. 2007. An antibiotic-inducible cell-wall associated protein that protects Bacillus subtilis from autolysis. J. Bacteriol. 189:4671-80.
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Progress 01/01/06 to 12/31/06
Outputs
When exposed to antibiotics and other cell wall stress agents Bacillus subtilis displays complex adaptive responses coordinated by a variety of regulatory proteins including the extracytoplasmic function (ECF) sigma factors (SigX, SigW, and SigM) and several two-component regulatory systems (TCS). During this last project period, we characterized genes induced by daptomycin, a lipopeptide antibiotic used to treat Gram positive bacterial infections. We have also used genetic methods to identify genes contributing to the emergence of daptomycin resistance including mprF (encoding a lysyl-phosphatidylglycerol synthase) and des (encoding a lipid desaturase). In related work, we are investigating the effects of mutations that alter membrane lipid synthesis pathways on the generation of, and response to, cell envelope stresses. A second major focus has been to define the roles of bacteriocins produced by Bacillus subtilis. In published work, we have dissected the role of
the SigW regulator in intraspecies competition. The ability of one bacterium (the predator) to lyse another bacterial strain (the prey), and grow using the released nutrients as a food source, depends on the production of lytic bacteriocins, the absence of defense mechanisms (some controlled by SigW) in the prey bacteria, and the swarming ability of the predator strain. In an extension of this work, we have identified a toxic peptide produced by B. subtilis strains that can induce the LiaRS TCS
Impacts
Resistance to antibiotics is a problem of grave medical concern. While it is widely appreciated that resistance genes are frequently found on transmissable genetic elements, and can spread rapidly within and among patients in clinical settings, the origins of such resistance genes are still debated. We suggest that many resistance genes evolved in soil bacteria that are themselves antibiotic producers or which cohabit with known antibiotic producers such as the actinomycetes. B. subtilis provides a model system for the analysis of the genetic responses of soil bacteria to antibacterial agents. We have discovered a number of resistance determinants that are important for antibiotic resistance and defined the corresponding regulatory systems. Promoters that are strongly induced by specific classes of antibiotics are also of use in antibiotic screening programs. Finally, we are developing B. subtilis as a model system for the analysis of cell-cell interactions among soil
bacteria.
Publications
- B. Butcher and J.D. Helmann. 2006. Identification of Bacillus subtilis W-dependent genes that provide intrinsic resistance to antimicrobial compounds produced by Bacilli. Mol. Microbiol. 60:765-782.
- S. Jordan, A. Junker, J.D. Helmann, and T. Mascher. 2006. Regulation of LiaRS-Dependent Gene Expression in Bacillus subtilis: Identification of Inhibitor Proteins, Regulator Binding Sites, and Target Gene of a Conserved Cell Envelope Stress-Sensing Two Component System. J. Bacteriol. 188:5153-5166.
- T. Mascher, J.D. Helmann, G. Unden. 2006. Stimulus perception in bacterial signal-transducing histidine kinases. Microbiol Mol Biol Rev. 70:910-938.
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Progress 01/01/05 to 12/31/05
Outputs
We are investigating the genetic responses of Bacillus subtilis to cell wall antibiotics and other cell wall stress agents. Genes activated by cell wall damaging agents are controlled by a variety of regulatory proteins including the extracytoplasmic function (ECF) sigma factors (SigX, SigW, and SigM) and several two-component regulatory systems (TCS). Analysis of genes induced by bacitracin, a peptide antibiotic commonly used as a topical antibiotic and a feed additive, has led to the identification of a new family of TCS sensor kinases that lack an extracellular signal-sensing domain. In related work, we are investigating the effects of mutations that alter membrane lipid synthesis pathways on the generation of, and response to, cell envelope stresses. A major focus has been to define the role of SigW regulon in resistance to bacteriocins produced by other Bacilli or other soil bacteria. Using overlay experiments, we have dissected the genetic factors that determine
the outcome of competition between related bacterial species. The ability of one bacterium (the predator) to lyse another bacterial strain (the prey), and grow using the released nutrients as a food source, depends on the production of lytic bacteriocins, the absence of defense mechanisms (some controlled by SigW) in the prey bacteria, and the swarming ability of the predator strain.
Impacts
Resistance to antibiotics is a problem of grave medical concern. While it is widely appreciated that resistance genes are frequently found on transmissable genetic elements, and can spread rapidly within and among patients in clinical settings, the origins of such resistance genes are still debated. We suggest that many resistance genes evolved in soil bacteria that are themselves antibiotic producers or which cohabit with known antibiotic producers such as the actinomycetes. B. subtilis provides a model system for the analysis of the genetic responses of soil bacteria to antibacterial agents. We have discovered a number of resistance determinants that are important for antibiotic resistance and defined the corresponding regulatory systems. Promoters that are strongly induced by specific classes of antibiotics are also of use in antibiotic screening programs. Finally, we are developing B. subtilis as a model system for the analysis of cell-cell interactions among soil
bacteria.
Publications
- Zhou, X., X. He, J. Liang, A. Li, T. Xu, T. Keiser, J.D. Helmann, and Z. Deng. 2005. A novel DNA modification by sulphur. Mol. Microbiol. 57:1428-1438.
- Cao, M., C.M. Moore, and J.D. Helmann. 2005. Bacillus subtilis paraquat resistance is directed by SigM, an extracytoplasmic function sigma factor, and is conferred by YqjL and BcrC. J. Bacteriol. 187:2948-2956.
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Progress 01/01/04 to 12/31/04
Outputs
We are investigating the genetic responses of Bacillus subtilis to cell wall antibiotics and other cell wall stress agents. Genes activated by cell wall damaging agents are controlled by a variety of regulatory proteins including the extracytoplasmic function (ECF) sigma factors (SigX, SigW, and SigM) and several two-component regulatory systems (TCS). Analysis of genes induced by bacitracin, a peptide antibiotic commonly used as a topical antibiotic and a feed additive, has led to the identification of a new family of TCS sensor kinases that lack an extracellular signal-sensing domain. In related work, we are investigating the effects of mutations that alter membrane lipid synthesis pathways on the generation of, and response to, cell envelope stresses. In other work, we are currently testing the hypothesis that the SigW regulon contributes to resistance to bacteriocins produced by other Bacilli or other soil bacteria. Using overlay experiments, we have begun to
dissect the genetic factors that determine the outcome of competition between related bacterial species. The ability of one bacterium (the predator) to lyse another bacterial strain (the prey), and grow using the released nutrients as a food source, depends on the production of lytic bacteriocins, the absence of defense mechanisms (some controlled by SigW) in the prey bacteria, and the swarming ability of the predator strain.
Impacts
Resistance to antibiotics is a problem of grave medical concern. While it is widely appreciated that resistance genes are frequently found on transmissable genetic elements, and can spread rapidly within and among patients in clinical settings, the origins of such resistance genes are still debated. We suggest that many resistance genes evolved in soil bacteria that are themselves antibiotic producers or which cohabit with known antibiotic producers such as the actinomycetes. B. subtilis provides a model system for the analysis of the genetic responses of soil bacteria to antibacterial agents. We have discovered a number of resistance determinants that are important for antibiotic resistance and defined the corresponding regulatory systems. Promoters that are strongly induced by specific classes of antibiotics are also of use in antibiotic screening programs. Finally, we are developing B. subtilis as a model system for the analysis of cell-cell interactions among soil
bacteria.
Publications
- Mascher, T., Zimmer, S.L., Smith, T.-A., Helmann, J.D. 2004. Characterization and evaluation of an antibiotic-inducible promoter that is regulated by the cell envelope stress sensing two-component system LiaRS of Bacillus subtilis. Antimicrobial Agents and Chemotherapy. 48:2888-2896.
- Cao, M. and Helmann, J. D. 2004. The Bacillus subtilis extracytoplasmic-function sigmaX factor regulates modification of the cell envelope and resistance to cationic antimicrobial peptides. Journal of Bacteriology 186:1136-1146.
- Helmann, J.D. 2004. Signma factors. In: Encyclopedia of Biological Chemistry. Mordich, P. (ed.) Elsevier Academic Press. San Diego, CA.
- Helmann, J. D. 2004. Antibiotics and Intercellular Signaling in Bacillus subtilis. ASM Conference on Cell-Cell Communication in Bacteria. Banff, Alberta, Canada. July 23-27, 2004.
- Helmann, J.D. 2004. Predicting Function from Conserved Sequence Motifs: Networks of Gene Regulation in Bacillus subtilis. Proceedings of Bioinformatics 2004: sponsored by Society for Bioinformatics in Nordic Countries (SOCBIN). Linkoping, Sweden. June 3-6, 2004.
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Progress 01/01/03 to 12/31/03
Outputs
We have continued our investigation of the genetic responses of Bacillus subtilis to cell wall antibiotics. In addition to the previously described extracytoplasmic function (ECF) sigma factors (SigX, SigW, and SigM), we have now identified several two-component regulatory systems that also control regulons activated by cell wall antibiotics. Our recent work has focused on those genes induced by bacitracin, a peptide antibiotic commonly used as a topical antibiotic and a feed additive. Comparisons of the bacitracin stimulon with the stimulons induced by vancomycin and ramoplanin reveals complex and overlapping signal transduction networks that control cell wall stress responses. In addition, we continue to investigate modifications of the cell envelope that contribute to antibiotic resistance including D-alanylation of teichoic acids and membrane lipid synthesis pathways.
Impacts
Resistance to antibiotics is a problem of grave medical concern. While it is widely appreciated that resistance genes are frequently found on transmissable genetic elements, and can spread rapidly within and among patients in clinical settings, the origins of such resistance genes are still debated. We suggest that many resistance genes evolved in soil bacteria that are themselves antibiotic producers or which cohabit with known antibiotic producers such as the actinomycetes. B. subtilis provides a model system for the analysis of the genetic responses of soil bacteria to antibacterial agents. We have discovered a number of resistance determinants that are important for antibiotic resistance and defined the corresponding regulatory systems. Promoters that are strongly induced by specific classes of antibiotics are also of use in antibiotic screening programs.
Publications
- Mascher, T., N. G. Margulis, T. Wang, R. W. Ye, J.D. Helmann. 2003. Cell wall stress responses in Bacillus subtilis: the regulatory network of the bacitracin stimulon. Mol. Microbiol. 50: 1591-1604.
- Cao, M., L. Salzberg, C.S. Tsai, T. Mascher, C. Bonilla, T. Wang, R.W. Ye , L. Marquez-Magana, J.D. Helmann. 2003. Regulation of the Bacillus subtilis extracytoplasmic function protein sigma(Y) and its target promoters. J. Bacteriol. 185:4883-4890.
- Paget, M.S.B. and J.D. Helmann. 2003. The Sig70 family of sigma factors. Genome Biology 4:203.
- PATENT: U.S. Patent 6,635,475 (Oct. 21, 2003) 'Bacillus subtilis Extracytoplasmic Function s Factor.'
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Progress 01/01/02 to 12/31/02
Outputs
Our goal is to define the physiological roles of seven extracytoplasmic function (ECF) sigma factors in the model organism, Bacillus subtilis. During this project period we have continued our analysis of the SigW, SigX, and SigM regulons. Significant results include the finding that these regulons play a key role in coordinating the response of B. subtilis to antibiotics. Our major emphasis has been on the effects of cell wall antibiotics, such as vancomycin and bacitracin, on gene expression. The emerging view is that B. subtilis has evolved complex adaptive responses to protect against antibacterial agents produced by other soil microbes and some of these responses are coordinated by alternative sigma factors. During this project period we have continued our analysis of regulons induced by antibiotics using DNA microarray technnology. In addition, we have begun to explore the roles of ECF sigma factors in a wild isolate of B. subtilis that retains many phenotypes
that have been lost in the "domesticated" laboratory strains. Preliminary results indicate that ECF sigma factors contribute to formation of pellicles (biofilms) in a complex way that is not yet well understood.
Impacts
Resistance to antibiotics is a problem of great medical significance, yet for many bacterial species the mechanisms governing resistance are not well understood. Our results with B. subtilis will likely have relevance to closely related organisms that are important human and animal pathogens such as the Stapylococci and Streptococci. In addition, the techniques we are developing will increase our ability to assign function to genes of unknown function by linking them to known regulators and regulons.
Publications
- M. Cao and J.D. Helmann. 2002. Regulation of the Bacillus subtilis bcrC bacitracin resistance gene by two ECF s factors. J. Bacteriol. 184:6123-6129.
- Q. Qian, C. Y. Lee, J. D. Helmann and M. A. Strauch. 2002. AbrB is a Regulator of the sW Regulon in Bacillus subtilis. FEMS Micro. Lett. 211:219-223.
- M. Cao, T. Wang, R. Ye, and J. D. Helmann. 2002. Antibiotics That Inhibit Cell Wall Biosynthesis Induce Expression of the Bacillus subtilis sW and sM Regulons. Mol. Microbiol. 45:1267-1276.
- M. Cao, M.M. Morshedi, P.A. Kobel, M.F.W. Wu, C. Paddon, and J.D. Helmann. 2002. Defining the Bacillus subtilis sigma(W) regulon: A comparative analysis of promoter consensus search, run-off transcription/macroarray analysis (ROMA), and transcriptional profiling approaches. J. Mol. Biol. 316:443-457.
- J.D. Helmann. 2002. ECF sigma factors in gram positive bacteria. In Advances in Microbial Physiology, Vol. 46:47-110.
- J.D. Helmann and C.P. Moran, Jr. 2002. RNA Polymerase and Sigma Factors. In Bacillus subtilis and Its Relatives: From Genes to Cells. ASM Press. Washington D.C. pp. 289-312.
- J.D. Helmann. 2002. Gene Expression and Its Regulation. In Modern Microbial Genetics, 2nd edition, (U.N. Streips and R.E. Yasbin, eds.) Wiley-Liss. New York, New York. pp. 47-84.
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Progress 01/01/01 to 12/31/01
Outputs
Our goal is to define the physiological roles of seven extracytoplasmic function (ECF) sigma factors in the model organism, Bacillus subtilis. During this project period we have completed a thorough analysis of the SigW and SigX regulons and expanded our studies to include the other members of this regulatory family. Significant results include the finding that these regulons play a key role in coordinating the response of B. subtilis to antibiotics. The emerging view is that B. subtilis has evolved complex adaptive responses to protect against antibacterial agents produced by other soil microbes and some of these responses are coordinated by alternative sigma factors. For example, we have shown that one member of the SigW regulon is the major determinant of fosfomycin resistance in B. subtilis. The SigX regulon includes operons that modify the cell envelope composition to render the bacteria more resistance to cationic antimicrobial peptides. These operons include
the dlt operon governing D-alanylation of teichoic acids and the operon controlling synthesis of phosphatidyl ethanolamine in the membrane. Both of these envelope components carry positive charges and thereby reduce the net negative charge of the cell surface. We have also found that both the SigW and SigX regulon are induced by antibiotics and we are currently exploring these responses using DNA microarray technnology.
Impacts
Resistance to antibiotics is a problem of great medical significance, yet for many bacterial species the mechanisms governing resistance are not well understood. Our results with B. subtilis will likely have relevance to closely related organisms that are important human and animal pathogens such as the Stapylococci and Streptococci. In addition, the techniques we are developing will increase our ability to assign function to genes of unknown function by linking them to known regulators and regulons.
Publications
- Cao, M., B.A. Bernat, Z. Wang, R.N. Armstrong, and J.D. Helmann. 2001. FosB, a cysteine-dependent fosfomycin resistance protein under the control of sW, an extracytoplasmic function s factor in Bacillus subtilis. J. Bacteriol. 183:2380-2383.
- Qiu, J. and J.D. Helmann. 2001. The -10 region is a key promoter specificity determinant for the Bacillus subtilis extracytoplasmic function s factors, sX and sW J. Bacteriol. 183:1921-1927.
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