Axe–Txe, a broad‐spectrum proteic toxin–antitoxin system specified by a multidrug‐resistant, clinical isolate of Enterococcus faecium

Enterococcal species of bacteria are now acknowledged as leading causes of bacteraemia and other serious nosocomial infections. However, surprisingly little is known about the molecular mechanisms that promote the segregational stability of antibiotic resistance and other plasmids in these bacteria. Plasmid pRUM (24 873 bp) is a multidrug resistance plasmid identified in a clinical isolate of Enterococcus faecium. A novel proteic‐based toxin–antitoxin cassette identified on pRUM was demonstrated to be a functional segregational stability module in both its native host and evolutionarily diverse bacterial species. Induced expression of the toxin protein (Txe) of this system resulted in growth inhibition in Escherichia coli. The toxic effect of Txe was alleviated by co‐expression of the antitoxin protein, Axe. Homologues of the axe and txe genes are present in the genomes of a diversity of Eubacteria. These homologues (yefM–yoeB) present in the E. coli chromosome function as a toxin–antitoxin mechanism, although the Axe and YefM antitoxin components demonstrate specificity for their cognate toxin proteins in vivo. Axe–Txe is one of the first functional proteic toxin–antitoxin systems to be accurately described for Gram‐positive bacteria.

[1]  I. Golovliov,et al.  Genetic organization of the Francisella plasmid pFNL10. , 2001, Plasmid.

[2]  M. Gilmore,et al.  Multiple-drug resistant enterococci: the nature of the problem and an agenda for the future. , 1998, Emerging infectious diseases.

[3]  G. Mittenhuber,et al.  Occurrence of mazEF-like antitoxin/toxin systems in bacteria. , 1999, Journal of molecular microbiology and biotechnology.

[4]  P. Kuhnert,et al.  Molecular epidemiology and genetic linkage of macrolide and aminoglycoside resistance in Staphylococcus intermedius of canine origin. , 2001, Veterinary microbiology.

[5]  M. P. Cummings PHYLIP (Phylogeny Inference Package) , 2004 .

[6]  F. Hayes A Family of Stability Determinants in Pathogenic Bacteria , 1998, Journal of bacteriology.

[7]  K. Gerdes,et al.  Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins , 2002, Molecular microbiology.

[8]  A. Wright,et al.  DNA segregation in bacteria. , 2000, Annual review of microbiology.

[9]  P. Renault,et al.  Plasmid vectors for gram-positive bacteria switching from high to low copy number. , 1996, Gene.

[10]  M. Yarmolinsky,et al.  Plasmid addiction genes of bacteriophage P1: doc, which causes cell death on curing of prophage, and phd, which prevents host death when prophage is retained. , 1993, Journal of molecular biology.

[11]  U. Zielenkiewicz,et al.  Mechanisms of plasmid stable maintenance with special focus on plasmid addiction systems. , 2001, Acta biochimica Polonica.

[12]  R. Wenzel,et al.  Vancomycin-resistant enterococcal bacteremia: natural history and attributable mortality. , 1996, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[13]  K. Weaver,et al.  Identification, characterization, and nucleotide sequence of a region of Enterococcus faecalis pheromone-responsive plasmid pAD1 capable of autonomous replication , 1993, Journal of bacteriology.

[14]  Neville Firth,et al.  Complete Nucleotide Sequence of pSK41: Evolution of Staphylococcal Conjugative Multiresistance Plasmids , 1998, Journal of bacteriology.

[15]  P. Thompson,et al.  Molecular Mechanism of Aminoglycoside Antibiotic Kinase APH(3′)-IIIa , 2001, The Journal of Biological Chemistry.

[16]  A. Pekhov [Evolution of bacterial plasmids]. , 1980, Uspekhi sovremennoi biologii.

[17]  H. Pearson 'Superbug' hurdles key drug barrier , 2002, Nature.

[18]  G. Weinstock,et al.  Conjugal transfer of plasmid DNA from Escherichia coli to enterococci: a method to make insertion mutations. , 1998, Plasmid.

[19]  I. Paulsen,et al.  Characterisation of sin, a potential recombinase-encoding gene from Staphylococcus aureus. , 1994, Gene.

[20]  J. Inamine,et al.  Broad geographical distribution of homologous erythromycin, kanamycin, and streptomycin resistance determinants among group D streptococci of human and animal origin , 1986, Antimicrobial Agents and Chemotherapy.

[21]  L. Rice,et al.  Transferable, Plasmid-Mediated VanB-Type Glycopeptide Resistance in Enterococcus faecium , 1998, Antimicrobial Agents and Chemotherapy.

[22]  Christopher M Thomas,et al.  The bacterial ParA-ParB partitioning proteins. , 2001, Journal of biotechnology.

[23]  K. Gerdes,et al.  Antisense RNA regulation of the par post‐segregational killing system: structural analysis and mechanism of binding of the antisense RNA, RNAII and its target, RNAI , 2001, Molecular microbiology.

[24]  D. Rawlings Proteic toxin-antitoxin, bacterial plasmid addiction systems and their evolution with special reference to the pas system of pTF-FC2. , 1999, FEMS microbiology letters.

[25]  Roderic D. M. Page,et al.  TreeView: an application to display phylogenetic trees on personal computers , 1996, Comput. Appl. Biosci..

[26]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

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

[28]  J. Piškur,et al.  Bacterial Toxin-Antitoxin Gene System as Containment Control in Yeast Cells , 2000, Applied and Environmental Microbiology.

[29]  Elliot J. Lefkowitz,et al.  Genome of the Bacterium Streptococcus pneumoniae Strain R6 , 2001, Journal of bacteriology.

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

[31]  H. Engelberg-Kulka,et al.  Postsegregational Killing Mediated by the P1 Phage “Addiction Module” phd-doc Requires the Escherichia coli Programmed Cell Death SystemmazEF , 2001, Journal of bacteriology.

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

[33]  S. Austin,et al.  Plasmid-partition functions of the P7 prophage. , 1989, Journal of molecular biology.

[34]  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.

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

[36]  W. Saenger,et al.  Crystallization and preliminary X-ray diffraction studies of the ∊ζ addiction system encoded by Streptococcus pyogenes plasmid pSM19035 , 2001 .

[37]  H. Engelberg-Kulka,et al.  Addiction modules and programmed cell death and antideath in bacterial cultures. , 1999, Annual review of microbiology.

[38]  S. Ehrlich,et al.  DNA restriction-modification systems mediate plasmid maintenance , 1995, Journal of bacteriology.

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

[40]  M. Gilmore,et al.  High efficiency introduction of plasmid DNA into glycine treated Enterococcus faecalis by electroporation , 1990, Molecular and General Genetics MGG.

[41]  M. Teuber,et al.  Sequence of the 50-kb conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25. , 2001, Plasmid.

[42]  R. Novick,et al.  Comparative analysis of five related Staphylococcal plasmids. , 1988, Plasmid.

[43]  K. Gerdes,et al.  Bacterial toxin RelE induces apoptosis in human cells , 2002, FEBS letters.

[44]  P. Courvalin,et al.  Characterization of Tn1547, a composite transposon flanked by the IS16 and IS256-like elements, that confers vancomycin resistance in Enterococcus faecalis BM4281. , 1996, Gene.

[45]  I. Kobayashi Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution. , 2001, Nucleic acids research.

[46]  W. Witte,et al.  Aminoglycoside-Streptothricin Resistance Gene Cluster aadE–sat4–aphA-3 Disseminated among Multiresistant Isolates of Enterococcus faecium , 2001, Antimicrobial Agents and Chemotherapy.

[47]  R. Gaynes,et al.  An overview of nosocomial infections, including the role of the microbiology laboratory , 1993, Clinical Microbiology Reviews.

[48]  S. S. Smith,et al.  Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. , 1989, Nucleic acids research.