Escherichia coli toxin/antitoxin pair MqsR/MqsA regulate toxin CspD.

Previously we identified that the Escherichia coli protein MqsR (YgiU) functions as a toxin and that it is involved in the regulation of motility by quorum sensing signal autoinducer-2 (AI-2). Furthermore, MqsR is directly associated with biofilm development and is linked to the development of persister cells. Here we show that MqsR and MqsA (YgiT) are a toxin/antitoxin (TA) pair, which, in significant difference to other TA pairs, regulates additional loci besides its own. We have recently identified that MqsR functions as an RNase. However, using three sets of whole-transcriptome studies and two nickel-enrichment DNA binding microarrays coupled with cell survival studies in which MqsR was overproduced in isogenic mutants, we identified eight genes (cspD, clpX, clpP, lon, yfjZ, relB, relE and hokA) that are involved in a mode of MqsR toxicity in addition to its RNase activity. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) showed that (i) the MqsR/MqsA complex (and MqsA alone) represses the toxin gene cspD, (ii) MqsR overproduction induces cspD, (iii) stress induces cspD, and (iv) stress fails to induce cspD when MqsR/MqsA are overproduced or when mqsRA is deleted. Electrophoretic mobility shift assays show that the MqsA/MqsR complex binds the promoter of cspD. In addition, proteases Lon and ClpXP are necessary for MqsR toxicity. Together, these results indicate the MqsR/MqsA complex represses cspD which may be derepressed by titrating MqsA with MqsR or by degrading MqsA via stress conditions through proteases Lon and ClpXP. Hence, we demonstrate that the MqsR/MqsA TA system controls cell physiology via its own toxicity as well as through its regulation of another toxin, CspD.

[1]  R. Kolter,et al.  A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli. , 1992, Genes & development.

[2]  A. Matin,et al.  Molecular and functional characterization of a carbon starvation gene of Escherichia coli. , 1991, Journal of molecular biology.

[3]  L. Wyns,et al.  Rejuvenation of CcdB-poisoned gyrase by an intrinsically disordered protein domain. , 2009, Molecular cell.

[4]  T. Wood,et al.  Control and benefits of CP4-57 prophage excision in Escherichia coli biofilms , 2009, The ISME Journal.

[5]  D. Weichart,et al.  Dynamic control of Dps protein levels by ClpXP and ClpAP proteases in Escherichia coli , 2003, Molecular microbiology.

[6]  S. Gottesman,et al.  Overproduction of the Lon protease triggers inhibition of translation in Escherichia coli: involvement of the yefM‐yoeB toxin‐antitoxin system , 2004, Molecular microbiology.

[7]  S. Kjelleberg,et al.  Ecological Advantages of Autolysis during the Development and Dispersal of Pseudoalteromonas tunicata Biofilms , 2006, Applied and Environmental Microbiology.

[8]  Makino Kozo,et al.  Cloning and characterization of the umu operon responsible for inducible mutagenesis in Escherichia coli , 1983 .

[9]  S. Kjelleberg,et al.  Bacteriophage and Phenotypic Variation in Pseudomonas aeruginosa Biofilm Development , 2004, Journal of bacteriology.

[10]  A. Gultyaev,et al.  Antisense RNA-regulated programmed cell death. , 1997, Annual review of genetics.

[11]  S. Gottesman,et al.  The RssB response regulator directly targets sigma(S) for degradation by ClpXP. , 2001, Genes & development.

[12]  E. Gazit,et al.  The yefM-yoeB Toxin-Antitoxin Systems of Escherichia coli and Streptococcus pneumoniae: Functional and Structural Correlation , 2006, Journal of bacteriology.

[13]  K. Makino,et al.  Cloning and characterization of the umu operon responsible for inducible mutagenesis in Escherichia coli. , 1983, Gene.

[14]  T. Wood,et al.  Gene expression in Escherichia coli biofilms , 2004, Applied Microbiology and Biotechnology.

[15]  Thomas K. Wood,et al.  Autoinducer 2 Controls Biofilm Formation in Escherichia coli through a Novel Motility Quorum-Sensing Regulator (MqsR, B3022) , 2006, Journal of bacteriology.

[16]  N. W. Davis,et al.  The complete genome sequence of Escherichia coli K-12. , 1997, Science.

[17]  Martin Overgaard,et al.  Messenger RNA interferase RelE controls relBE transcription by conditional cooperativity , 2008, Molecular microbiology.

[18]  Ö. Melefors,et al.  The RNA binding protein CsrA controls cyclic di‐GMP metabolism by directly regulating the expression of GGDEF proteins , 2008, Molecular microbiology.

[19]  R. Magnuson.  Hypothetical Functions of Toxin-Antitoxin Systems , 2007, Journal of bacteriology.

[20]  S. Kjelleberg,et al.  Cell Death in Pseudomonas aeruginosa Biofilm Development , 2003, Journal of bacteriology.

[21]  M. Inouye,et al.  MqsR, a Crucial Regulator for Quorum Sensing and Biofilm Formation, Is a GCU-specific mRNA Interferase in Escherichia coli* , 2009, The Journal of Biological Chemistry.

[22]  Qun Ma,et al.  Toxin-Antitoxin Systems in Escherichia coli Influence Biofilm Formation through YjgK (TabA) and Fimbriae , 2008, Journal of bacteriology.

[23]  A. Lupas,et al.  prlF and yhaV encode a new toxin-antitoxin system in Escherichia coli. , 2007, Journal of molecular biology.

[24]  T. Wood,et al.  Structure and function of the Escherichia coli protein YmgB: a protein critical for biofilm formation and acid-resistance. , 2007, Journal of molecular biology.

[25]  K. Gerdes,et al.  Prokaryotic toxin–antitoxin stress response loci , 2005, Nature Reviews Microbiology.

[26]  K. Bayles.  The biological role of death and lysis in biofilm development , 2007, Nature Reviews Microbiology.

[27]  K. Gerdes.  Toxin-Antitoxin Modules May Regulate Synthesis of Macromolecules during Nutritional Stress , 2000, Journal of bacteriology.

[28]  Jeremy D. Glasner,et al.  Systematic Mutagenesis of the Escherichia coli Genome , 2004, Journal of bacteriology.

[29]  K. Gerdes,et al.  Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon. , 1986, The EMBO journal.

[30]  G. Klein.  Studies on the Epstein-Barr Virus genome and the EBV-determined nuclear antigen in human malignant disease. , 1975, Cold Spring Harbor symposia on quantitative biology.

[31]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[32]  R. Hengge-aronis,et al.  Identification of a central regulator of stationary‐phase gene expression in Escherichia coli , 1991, Molecular microbiology.

[33]  Thomas K. Wood,et al.  Directed Evolution of Toluene ortho-Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation , 2002, Journal of bacteriology.

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

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

[36]  Robert A. LaRossa,et al.  DNA Microarray-Mediated Transcriptional Profiling of the Escherichia coli Response to Hydrogen Peroxide , 2001, Journal of bacteriology.

[37]  Ehud Gazit,et al.  The YefM Antitoxin Defines a Family of Natively Unfolded Proteins , 2004, Journal of Biological Chemistry.

[38]  H. Engelberg-Kulka,et al.  An Escherichia coli chromosomal "addiction module" regulated by guanosine [corrected] 3',5'-bispyrophosphate: a model for programmed bacterial cell death. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[39]  M. Vulić,et al.  GlpD and PlsB Participate in Persister Cell Formation in Escherichia coli , 2006, Journal of bacteriology.

[40]  H. Mori,et al.  Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. , 2006, DNA research : an international journal for rapid publication of reports on genes and genomes.

[41]  T. Wood,et al.  Exclusion of T4 phage by the hok/sok killer locus from plasmid R1 , 1996, Journal of bacteriology.

[42]  Arkady Khodursky,et al.  Persisters: a distinct physiological state of E. coli , 2006, BMC Microbiology.

[43]  T. Wood,et al.  Toxins Hha and CspD and small RNA regulator Hfq are involved in persister cell formation through MqsR in Escherichia coli. , 2010, Biochemical and biophysical research communications.

[44]  L. Van Melderen,et al.  What Is the Benefit to Escherichia coli of Having Multiple Toxin-Antitoxin Systems in Its Genome? , 2007, Journal of bacteriology.

[45]  R. Motiejūnaitė,et al.  Escherichia coli dinJ-yafQ genes act as a toxin-antitoxin module. , 2007, FEMS microbiology letters.

[46]  Thomas K. Wood,et al.  YliH (BssR) and YceP (BssS) Regulate Escherichia coli K-12 Biofilm Formation by Influencing Cell Signaling , 2006, Applied and Environmental Microbiology.

[47]  E. Ohtsubo,et al.  chpA and chpB, Escherichia coli chromosomal homologs of the pem locus responsible for stable maintenance of plasmid R100 , 1993, Journal of bacteriology.

[48]  R. Kolter,et al.  Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili , 1998, Molecular microbiology.

[49]  J. T. Grace.  STUDIES OF EPSTEIN‐BARR VIRUS , 1970, Annals of the New York Academy of Sciences.

[50]  R. B. Jensen,et al.  Programmed cell death in bacteria: proteic plasmid stabilization systems , 1995, Molecular microbiology.

[51]  R. Seidler,et al.  Evaluation of Methods for Sampling, Recovery, and Enumeration of Bacteria Applied to the Phylloplane , 1991, Applied and environmental microbiology.

[52]  Thomas K. Wood,et al.  Protein Translation and Cell Death: The Role of Rare tRNAs in Biofilm Formation and in Activating Dormant Phage Killer Genes , 2008, PloS one.

[53]  Dacheng Ren,et al.  Differential gene expression shows natural brominated furanones interfere with the autoinducer-2 bacterial signaling system of Escherichia coli. , 2004, Biotechnology and bioengineering.

[54]  M. Inouye,et al.  CspD, a novel DNA replication inhibitor induced during the stationary phase in Escherichia coli , 2001, Molecular microbiology.

[55]  Jason M. Brown,et al.  A Novel Family of Escherichia coli Toxin-Antitoxin Gene Pairs , 2003, Journal of bacteriology.

[56]  T. Wood,et al.  Temporal gene-expression in Escherichia coli K-12 biofilms. , 2007, Environmental microbiology.

[57]  P. Rather,et al.  Evidence for extracellular control of RpoS proteolysis in Escherichia coli. , 2008, FEMS microbiology letters.

[58]  M. Waldor,et al.  Characterization of a higBA Toxin-Antitoxin Locus in Vibrio cholerae , 2006, Journal of bacteriology.

[59]  D. McDougald,et al.  The biofilm life cycle and virulence of Pseudomonas aeruginosa are dependent on a filamentous prophage , 2009, The ISME Journal.

[60]  K. Gerdes,et al.  RelE, a global inhibitor of translation, is activated during nutritional stress , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[61]  T. Wood,et al.  Escherichia coli transcription factor YncC (McbR) regulates colanic acid and biofilm formation by repressing expression of periplasmic protein YbiM (McbA) , 2008, The ISME Journal.

[62]  K. Gerdes,et al.  The Escherichia coli relBE genes belong to a new toxin–antitoxin gene family , 1998, Molecular microbiology.

[63]  K. Gerdes,et al.  Multiple hok genes on the chromosome of Escherichia coli , 1999, Molecular microbiology.

[64]  J. Glasner,et al.  Genome-wide expression profiling in Escherichia coli K-12. , 1999, Nucleic acids research.

[65]  K. Gerdes,et al.  Toxin–antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes , 2005, Nucleic acids research.

[66]  T. Wood,et al.  Three Dimensional Structure of the MqsR:MqsA Complex: A Novel TA Pair Comprised of a Toxin Homologous to RelE and an Antitoxin with Unique Properties , 2009, PLoS pathogens.