Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis

During bacterial cannibalism, a differentiated subpopulation harvests nutrients from their genetically identical siblings to allow continued growth in nutrient-limited conditions. Hypothesis-driven imaging mass spectrometry (IMS) was used to identify metabolites active in a Bacillus subtilis cannibalism system in which sporulating cells lyse nonsporulating siblings. Two candidate molecules with sequences matching the products of skfA and sdpC, genes for the proposed cannibalistic factors sporulation killing factor (SKF) and sporulation delaying protein (SDP), respectively, were identified and the structures of the final products elucidated. SKF is a cyclic 26-amino acid (aa) peptide that is posttranslationally modified with one disulfide and one cysteine thioether bridged to the α-position of a methionine, a posttranslational modification not previously described in biology. SDP is a 42-residue peptide with one disulfide bridge. In spot test assays on solid medium, overproduced SKF and SDP enact a cannibalistic killing effect with SDP having higher potency. However, only purified SDP affected B. subtilis cells in liquid media in fluorescence microscopy and growth assays. Specifically, SDP treatment delayed growth in a concentration-dependent manner, caused increases in cell permeability, and ultimately caused cell lysis accompanied by the production of membrane tubules and spheres. Similarly, SDP but not SKF was able to inhibit the growth of the pathogens Staphylococcus aureus and Staphylococcus epidermidis with comparable IC50 to vancomycin. This investigation, with the identification of SKF and SDP structures, highlights the strength of IMS in investigations of metabolic exchange of microbial colonies and also demonstrates IMS as a promising approach to discover novel biologically active molecules.

[1]  H. Engelberg-Kulka,et al.  Bacterial Programmed Cell Death and Multicellular Behavior in Bacteria , 2006, PLoS genetics.

[2]  H. Vlamakis,et al.  Thinking about Bacillus subtilis as a multicellular organism. , 2007, Current opinion in microbiology.

[3]  Nuno Bandeira,et al.  Dereplication and De Novo Sequencing of Nonribosomal Peptides , 2009, Nature Methods.

[4]  Michelle L. Reyzer,et al.  MALDI imaging mass spectrometry: molecular snapshots of biochemical systems , 2007, Nature Methods.

[5]  Bastien Chevreux,et al.  The Origins of 168, W23, and Other Bacillus subtilis Legacy Strains , 2008, Journal of bacteriology.

[6]  J. Hoch,et al.  Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay , 1991, Cell.

[7]  M. Gross,et al.  Multistep tandem mass spectrometry for Sequencing Cyclic Peptides in an Ion-Trap Mass Spectrometer , 1999, Journal of the American Society for Mass Spectrometry.

[8]  Jorge F. Reyes-Spindola,et al.  Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. , 2001, Nucleic acids research.

[9]  T. Stein,et al.  Rapid typing of Bacillus subtilis strains by their secondary metabolites using matrix‐assisted laser desorption/ionization mass spectrometry of intact cells , 1999 .

[10]  P Youngman,et al.  Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene. , 1984, Plasmid.

[11]  K. Pogliano,et al.  Cell‐specific SpoIIIE assembly and DNA translocation polarity are dictated by chromosome orientation , 2007, Molecular microbiology.

[12]  M. Erhard,et al.  Rapid typing and elucidation of new secondary metabolites of intact cyanobacteria using MALDI-TOF mass spectrometry , 1997, Nature Biotechnology.

[13]  M. Donia,et al.  Ribosomal peptide natural products: bridging the ribosomal and nonribosomal worlds. , 2009, Natural product reports.

[14]  J. Cronan,et al.  Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein. , 2000, Biochemistry.

[15]  K. Venkatesh,et al.  Sporulating bacteria prefers predation to cannibalism in mixed cultures , 2007, FEBS letters.

[16]  R. Losick,et al.  A Three-Protein Signaling Pathway Governing Immunity to a Bacterial Cannibalism Toxin , 2006, Cell.

[17]  R. Losick,et al.  The transcriptional profile of early to middle sporulation in Bacillus subtilis. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[18]  S. Bron,et al.  Thiol-Disulfide Oxidoreductases Are Essential for the Production of the Lantibiotic Sublancin 168* , 2002, The Journal of Biological Chemistry.

[19]  Roberto Kolter,et al.  Cannibalism enhances biofilm development in Bacillus subtilis , 2009, Molecular microbiology.

[20]  L. Aravind.  Type II CAAX prenyl endopeptidases belong to a novel superfamily of putative membrane-bound metalloproteases , 2001 .

[21]  Richard M. Caprioli,et al.  Solvent-free matrix dry-coating for MALDI imaging of phospholipids , 2008, Journal of the American Society for Mass Spectrometry.

[22]  J. Vederas,et al.  Drug Discovery and Natural Products: End of an Era or an Endless Frontier? , 2009, Science.

[23]  Richard M Caprioli,et al.  Molecular imaging of proteins in tissues by mass spectrometry , 2008, Proceedings of the National Academy of Sciences.

[24]  Pieter C. Dorrestein,et al.  Translating metabolic exchange with imaging mass spectrometry , 2009, Nature chemical biology.

[25]  Shane T. Jensen,et al.  The Spo0A regulon of Bacillus subtilis , 2003, Molecular microbiology.

[26]  K. Pogliano,et al.  Partitioning of Chromosomal DNA during Establishment of Cellular Asymmetry in Bacillus subtilis , 2002, Journal of bacteriology.

[27]  R. Losick,et al.  Cannibalism by Sporulating Bacteria , 2003, Science.

[28]  A. Grossman,et al.  Integration of multiple developmental signals in Bacillus subtilis through the Spo0A transcription factor. , 1993, Genes & development.

[29]  Ashlee M Earl,et al.  Ecology and genomics of Bacillus subtilis. , 2008, Trends in microbiology.

[30]  J. Vederas,et al.  Structure of subtilosin A, a cyclic antimicrobial peptide from Bacillus subtilis with unusual sulfur to alpha-carbon cross-links: formation and reduction of alpha-thio-alpha-amino acid derivatives. , 2004, Biochemistry.

[31]  Roberto Kolter,et al.  New developments in microbial interspecies signaling. , 2009, Current opinion in microbiology.

[32]  Jean-Pierre Claverys,et al.  Cannibalism and fratricide: mechanisms and raisons d'être , 2007, Nature Reviews Microbiology.

[33]  J. Jarrett,et al.  Crystal Structure of Biotin Synthase, an S-Adenosylmethionine-Dependent Radical Enzyme , 2004, Science.

[34]  A. Henriques,et al.  The Intestinal Life Cycle of Bacillus subtilis and Close Relatives , 2006, Journal of bacteriology.

[35]  H. Gu,et al.  A 3·1‐kb genomic fragment of Bacillus subtilis encodes the protein inhibiting growth of Xanthomonas oryzae pv. oryzae , 2001, Journal of applied microbiology.

[36]  R. Losick,et al.  Fruiting body formation by Bacillus subtilis , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[37]  A. Goffeau,et al.  The complete genome sequence of the Gram-positive bacterium Bacillus subtilis , 1997, Nature.

[38]  Erik L. L. Sonnhammer,et al.  Kalign, Kalignvu and Mumsa: web servers for multiple sequence alignment , 2006, Nucleic Acids Res..

[39]  W. A. van der Donk,et al.  Follow the leader: the use of leader peptides to guide natural product biosynthesis. , 2010, Nature chemical biology.