Regulation of Neurotoxin Production and Sporulation by a Putative agrBD Signaling System in Proteolytic Clostridium botulinum

ABSTRACT A significant number of genome sequences of Clostridium botulinum and related species have now been determined. In silico analysis of these data revealed the presence of two distinct agr loci (agr-1 and agr-2) in all group I strains, each encoding putative proteins with similarity to AgrB and AgrD of the well-studied Staphylococcus aureus agr quorum sensing system. In S. aureus, a small diffusible autoinducing peptide is generated from AgrD in a membrane-located processing event that requires AgrB. Here the characterization of both agr loci in the group I strain C. botulinum ATCC 3502 and of their homologues in a close relative, Clostridium sporogenes NCIMB 10696, is reported. In C. sporogenes NCIMB 10696, agr-1 and agr-2 appear to form transcriptional units that consist of agrB, agrD, and flanking genes of unknown function. Several of these flanking genes are conserved in Clostridium perfringens. In agreement with their proposed role in quorum sensing, both loci were maximally expressed during late-exponential-phase growth. Modulation of agrB expression in C. sporogenes was achieved using antisense RNA, whereas in C. botulinum, insertional agrD mutants were generated using ClosTron technology. In comparison to the wild-type strains, these strains exhibited drastically reduced sporulation and, for C. botulinum, also reduced production of neurotoxin, suggesting that both phenotypes are controlled by quorum sensing. Interestingly, while agr-1 appeared to control sporulation, agr-2 appeared to regulate neurotoxin formation.

[1]  J. Heap,et al.  The ClosTron: a universal gene knock-out system for the genus Clostridium. , 2007, Journal of microbiological methods.

[2]  M. Peck.  Biology and genomic analysis of Clostridium botulinum. , 2009, Advances in microbial physiology.

[3]  C. Tomas,et al.  Overexpression of groESL in Clostridium acetobutylicum Results in Increased Solvent Production and Tolerance, Prolonged Metabolism, and Changes in the Cell's Transcriptional Program , 2003, Applied and Environmental Microbiology.

[4]  N. Minton,et al.  The development of Clostridium difficile genetic systems. , 2004, Anaerobe.

[5]  E. Papoutsakis,et al.  Metabolic flux analysis elucidates the importance of the acid-formation pathways in regulating solvent production by Clostridium acetobutylicum. , 1999, Metabolic engineering.

[6]  A. T. Carter,et al.  Effects of Carbon Dioxide on Growth of Proteolytic Clostridium botulinum, Its Ability To Produce Neurotoxin, and Its Transcriptome , 2009, Applied and Environmental Microbiology.

[7]  M. Kleerebezem,et al.  An agr-Like Two-Component Regulatory System in Lactobacillus plantarum Is Involved in Production of a Novel Cyclic Peptide and Regulation of Adherence , 2005, Journal of bacteriology.

[8]  F. Vandenesch,et al.  The agr P2 operon: an autocatalytic sensory transduction system in Staphylococcus aureus. , 1995, Molecular & general genetics : MGG.

[9]  K. Scott,et al.  Section 14 - Biotechnology and medical applications , 1995 .

[10]  B. Lazazzera,et al.  The extracellular Phr peptide-Rap phosphatase signaling circuit of Bacillus subtilis. , 2003, Frontiers in bioscience : a journal and virtual library.

[11]  Eric A. Johnson,et al.  Recombinant Holotoxoid Vaccine against Botulism , 2007, Infection and Immunity.

[12]  B. McClane,et al.  Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13 , 2009, PloS one.

[13]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[14]  Wei-Jen Lin,et al.  Variations in expression and release of botulinum neurotoxin in Clostridium botulinum type A strains. , 2007, Foodborne pathogens and disease.

[15]  E. Holst,et al.  Relative Neurotoxin Gene Expression in Clostridium botulinum Type B, Determined Using Quantitative Reverse Transcription-PCR , 2004, Applied and Environmental Microbiology.

[16]  S. Arvidson,et al.  The role of the delta‐lysin gene (hld) in the regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus. , 1990, The EMBO journal.

[17]  K. Winzer,et al.  Look who's talking: communication and quorum sensing in the bacterial world , 2007, Philosophical Transactions of the Royal Society B: Biological Sciences.

[18]  Eric A. Johnson,et al.  Regulation of neurotoxin complex expression in Clostridium botulinum strains 62A, Hall A-hyper, and NCTC 2916. , 2004, Anaerobe.

[19]  Julian Parkhill,et al.  The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome , 2006, Nature Genetics.

[20]  Eleftherios T. Papoutsakis,et al.  Antisense RNA Strategies for Metabolic Engineering of Clostridium acetobutylicum , 1999, Applied and Environmental Microbiology.

[21]  M. Popoff,et al.  Expression of botulinum neurotoxins A and E, and associated non-toxin genes, during the transition phase and stability at high temperature: analysis by quantitative reverse transcription-PCR. , 2006, Microbiology.

[22]  K. Inoue,et al.  botR/A is a positive regulator of botulinum neurotoxin and associated non‐toxin protein genes in Clostridium botulinum A , 1998, Molecular microbiology.

[23]  S. Elliott,et al.  Botulism: the many faces of botulinum toxin and its potential for bioterrorism. , 2006, Infectious disease clinics of North America.

[24]  E. Papoutsakis,et al.  Design of Antisense RNA Constructs for Downregulation of the Acetone Formation Pathway of Clostridium acetobutylicum , 2003, Journal of bacteriology.

[25]  A. T. Carter,et al.  Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes. , 2007, Genome research.

[26]  C. Livesey,et al.  Diagnosis and implications of botulism , 2008, In Practice.

[27]  P. Dürre,et al.  Initiation of endospore formation in Clostridium acetobutylicum. , 2004, Anaerobe.

[28]  Tohru Shimizu,et al.  Virulence Gene Regulation by the agr System in Clostridium perfringens , 2009, Journal of bacteriology.

[29]  M. Sarker,et al.  Antisense-RNA-Mediated Decreased Synthesis of Small, Acid-Soluble Spore Proteins Leads to Decreased Resistance of Clostridium perfringens Spores to Moist Heat and UV Radiation , 2007, Applied and Environmental Microbiology.

[30]  A. Charbit,et al.  Identification of the agr Locus of Listeria monocytogenes: Role in Bacterial Virulence , 2003, Infection and Immunity.

[31]  N. Minton,et al.  Development of a transformation and gene reporter system for group II, non-proteolytic Clostridium botulinum type B strains. , 2000, Journal of molecular microbiology and biotechnology.

[32]  C. Shone,et al.  Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay , 1985, Applied and environmental microbiology.

[33]  S. Junne,et al.  Antisense RNA Downregulation of Coenzyme A Transferase Combined with Alcohol-Aldehyde Dehydrogenase Overexpression Leads to Predominantly Alcohologenic Clostridium acetobutylicum Fermentations , 2003, Journal of bacteriology.

[34]  R. Novick.  Autoinduction and signal transduction in the regulation of staphylococcal virulence , 2003, Molecular microbiology.

[35]  J. M. Dow,et al.  A two‐component system involving an HD‐GYP domain protein links cell–cell signalling to pathogenicity gene expression in Xanthomonas campestris , 2000, Molecular microbiology.

[36]  A. McLeod,et al.  Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier , 2002, Molecular microbiology.

[37]  A. T. Carter,et al.  Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum , 2009, BMC Genomics.

[38]  E J Schantz,et al.  Properties and use of botulinum toxin and other microbial neurotoxins in medicine. , 1992, Microbiological reviews.