Tools for Functional Postgenomic Analysis of Listeria monocytogenes

ABSTRACT We describe the development of genetic tools for regulated gene expression, the introduction of chromosomal mutations, and improved plasmid transfer by electroporation in the food-borne pathogen Listeria monocytogenes. pIMK, a kanamycin-resistant, site-specific, integrative listeriophage vector was constructed and then modified for overexpression (pIMK2) or for isopropyl-β-d-thiogalactopyranoside (IPTG)-regulated expression (pIMK3 and pIMK4). The dynamic range of promoters was assessed by determining luciferase activity, P60 secretion, and internalin A-mediated invasion. These analyses demonstrated that pIMK4 and pIMK3 have a stringently controlled dynamic range of 540-fold. Stable gene overexpression was achieved with pIMK2, giving a range of expression for the three vectors of 1,350-fold. The lactococcal pORI280 system was optimized for the generation of chromosomal mutations and used to create five new prfA star mutants. The combination of pIMK4 and pORI280 allowed streamlined creation of “IPTG-dependent” mutants. This was exemplified by creation of a clean deletion mutant with deletion of the universally essential secA gene, and this mutant exhibited a rapid loss of viability upon withdrawal of IPTG. We also improved plasmid transfer by electroporation into three commonly used laboratory strains of L. monocytogenes. A 125-fold increase in transformation efficiency for EGDe compared with the widely used protocol of Park and Stewart (S. F. Park and G. S. Stewart, Gene 94:129-132, 1990) was observed. Maximal transformation efficiencies of 5.7 × 106 and 6.7 × 106 CFU per μg were achieved for EGDe and 10403S, respectively, with a replicating plasmid. An efficiency of 2 × 107 CFU per μg is the highest efficiency reported thus far for L. monocytogenes F2365.

[1]  S. Ho,et al.  Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. , 2013, BioTechniques.

[2]  E. Domann,et al.  Novel Bacterial Artificial Chromosome Vector pUvBBAC for Use in Studies of the Functional Genomics of Listeria spp , 2008, Applied and Environmental Microbiology.

[3]  W. Eisenreich,et al.  Pathogenomics of Listeria spp. , 2007, International journal of medical microbiology : IJMM.

[4]  T. Luong,et al.  Improved single-copy integration vectors for Staphylococcus aureus. , 2007, Journal of microbiological methods.

[5]  M. Braunstein,et al.  Silencing Essential Protein Secretion in Mycobacterium smegmatis by Using Tetracycline Repressors , 2007 .

[6]  Yin Li,et al.  Bacteriocin production as a mechanism for the antiinfective activity of Lactobacillus salivarius UCC118 , 2007, Proceedings of the National Academy of Sciences.

[7]  C. Hill,et al.  Improved Luciferase Tagging System for Listeria monocytogenes Allows Real-Time Monitoring In Vivo and In Vitro , 2007, Applied and Environmental Microbiology.

[8]  M. Cao,et al.  A mariner-Based Transposition System for Listeria monocytogenes , 2007, Applied and Environmental Microbiology.

[9]  D. Higgins,et al.  Differential function of Listeria monocytogenes listeriolysin O and phospholipases C in vacuolar dissolution following cell‐to‐cell spread , 2007, Cellular microbiology.

[10]  A. Shen,et al.  A bifunctional O-GlcNAc transferase governs flagellar motility through anti-repression. , 2006, Genes & development.

[11]  P. Cossart,et al.  Control of Listeria Superoxide Dismutase by Phosphorylation* , 2006, Journal of Biological Chemistry.

[12]  H. Marquis,et al.  Listeria monocytogenes Flagella Are Used for Motility, Not as Adhesins, To Increase Host Cell Invasion , 2006, Infection and Immunity.

[13]  V. Lazarevic,et al.  Identification of an Essential Gene of Listeria monocytogenes Involved in Teichoic Acid Biogenesis , 2006, Journal of bacteriology.

[14]  C. Hill,et al.  Novel Luciferase Reporter System for In Vitro and Organ-Specific Monitoring of Differential Gene Expression in Listeria monocytogenes , 2006, Applied and Environmental Microbiology.

[15]  J. Vaissaire,et al.  Bmc Microbiology , 2006 .

[16]  O. Kuipers,et al.  To have neighbour's fare: extending the molecular toolbox for Streptococcus pneumoniae. , 2006, Microbiology.

[17]  J. Swanson,et al.  Cytolysin‐dependent delay of vacuole maturation in macrophages infected with Listeria monocytogenes , 2006, Cellular microbiology.

[18]  M. Rohde,et al.  Simultaneous Deficiency of both MurA and p60 Proteins Generates a Rough Phenotype in Listeria monocytogenes , 2005, Journal of bacteriology.

[19]  H. Goldfine,et al.  Listeria monocytogenes phosphatidylinositol-specific phospholipase C has evolved for virulence by greatly reduced activity on GPI anchors. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Michiel Kleerebezem,et al.  10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis , 2005, Applied Microbiology and Biotechnology.

[21]  P. Cossart,et al.  Gp96 is a receptor for a novel Listeria monocytogenes virulence factor, Vip, a surface protein , 2005, The EMBO journal.

[22]  Zhongxia Li,et al.  Conditional Lethality Yields a New Vaccine Strain of Listeria monocytogenes for the Induction of Cell-Mediated Immunity , 2005, Infection and Immunity.

[23]  R. Sleator,et al.  Contribution of Three Bile-Associated Loci, bsh, pva, and btlB, to Gastrointestinal Persistence and Bile Tolerance of Listeria monocytogenes , 2005, Infection and Immunity.

[24]  P. Bremer,et al.  Morphotypic Conversion in Listeria monocytogenes Biofilm Formation: Biological Significance of Rough Colony Isolates , 2004, Applied and Environmental Microbiology.

[25]  M. Arnaud,et al.  New Vector for Efficient Allelic Replacement in Naturally Nontransformable, Low-GC-Content, Gram-Positive Bacteria , 2004, Applied and Environmental Microbiology.

[26]  M. Giedlin,et al.  Listeria-based cancer vaccines that segregate immunogenicity from toxicity. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Kendy K. Y. Wong,et al.  A Novel Mutation within the Central Listeria monocytogenes Regulator PrfA That Results in Constitutive Expression of Virulence Gene Products , 2004, Journal of bacteriology.

[28]  F. Lecointe,et al.  Vectors for regulated gene expression in the radioresistant bacterium Deinococcus radiodurans. , 2004, Gene.

[29]  W. Goebel,et al.  New Listeria monocytogenes prfA* mutants, transcriptional properties of PrfA* proteins and structure–function of the virulence regulator PrfA , 2004, Molecular microbiology.

[30]  David A Rasko,et al.  Whole genome comparisons of serotype 4b and 1/2a strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species. , 2004, Nucleic acids research.

[31]  P. Cossart,et al.  Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence , 2004, Molecular microbiology.

[32]  C. Buchrieser,et al.  New Aspects Regarding Evolution and Virulence of Listeria monocytogenes Revealed by Comparative Genomics and DNA Arrays , 2004, Infection and Immunity.

[33]  C. Hill,et al.  Disruption of Putative Regulatory Loci in Listeria monocytogenes Demonstrates a Significant Role for Fur and PerR in Virulence , 2004, Infection and Immunity.

[34]  Michele P Calos,et al.  Phage integrases: biology and applications. , 2004, Journal of molecular biology.

[35]  L. Lenz,et al.  SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Shruti Jain,et al.  Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene , 2003, Molecular microbiology.

[37]  W. Goebel,et al.  Deletion of the Gene Encoding p60 in Listeria monocytogenes Leads to Abnormal Cell Division and Loss of Actin-Based Motility , 2003, Infection and Immunity.

[38]  S. Kathariou,et al.  An Improved Cloning Vector for Construction of Gene Replacements in Listeria monocytogenes , 2003, Applied and Environmental Microbiology.

[39]  M. Wiedmann,et al.  Pathogen, host and environmental factors contributing to the pathogenesis of listeriosis , 2003, Cellular and Molecular Life Sciences CMLS.

[40]  D. Portnoy,et al.  Construction, Characterization, and Use of Two Listeria monocytogenes Site-Specific Phage Integration Vectors , 2003 .

[41]  D. Portnoy,et al.  Inducible Control of Virulence Gene Expression in Listeria monocytogenes: Temporal Requirement of Listeriolysin O during Intracellular Infection , 2002, Journal of bacteriology.

[42]  C. Hill,et al.  The LisRK Signal Transduction System Determines the Sensitivity of Listeria monocytogenes to Nisin and Cephalosporins , 2002, Antimicrobial Agents and Chemotherapy.

[43]  D. Portnoy,et al.  Construction, Characterization, and Use of Two Listeria monocytogenes Site-Specific Phage Integration Vectors , 2002, Journal of bacteriology.

[44]  W. Goebel,et al.  InlA‐ but not InlB‐mediated internalization of Listeria monocytogenes by non‐phagocytic mammalian cells needs the support of other internalins , 2002, Molecular microbiology.

[45]  A. Grossman,et al.  In Vivo Effects of Sporulation Kinases on Mutant Spo0A Proteins in Bacillus subtilis , 2001, Journal of bacteriology.

[46]  L. Gautier,et al.  Comparative Genomics of Listeria Species , 2001, Science.

[47]  H. Čelešnik,et al.  Regulated ectopic expression and allelic-replacement mutagenesis as a method for gene essentiality testing in Staphylococcus aureus. , 2001, Plasmid.

[48]  W. Goebel,et al.  Listeria Pathogenesis and Molecular Virulence Determinants , 2001, Clinical Microbiology Reviews.

[49]  Kevin P. Francis,et al.  Monitoring Bioluminescent Staphylococcus aureusInfections in Living Mice Using a Novel luxABCDEConstruct , 2000, Infection and Immunity.

[50]  K. B. Kiser,et al.  SecA: the ubiquitous component of preprotein translocase in prokaryotes. , 1999, Microbes and infection.

[51]  P. Dabert,et al.  Gene replacement with linear DNA in electroporated wild-type Escherichia coli. , 1999, Nucleic acids research.

[52]  Rino Rappuoli,et al.  Counterselectable Markers: Untapped Tools for Bacterial Genetics and Pathogenesis , 1998, Infection and Immunity.

[53]  G. Venemâ,et al.  A lactococcal pWV01-based integration toolbox for bacteria , 1998 .

[54]  J. Vázquez-Boland,et al.  A Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes , 1997, Journal of bacteriology.

[55]  G. Venema,et al.  A general system for generating unlabelled gene replacements in bacterial chromosomes , 1996, Molecular and General Genetics MGG.

[56]  P. Youngman,et al.  Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements , 1996, Journal of bacteriology.

[57]  J. Wehland,et al.  Hyperexpression of listeriolysin in the nonpathogenic species Listeria innocua and high yield purification. , 1995, Journal of biotechnology.

[58]  G. Venema,et al.  A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes , 1995, Journal of bacteriology.

[59]  B. Birren,et al.  Transformation of Escherichia coli with large DNA molecules by electroporation. , 1995, Nucleic acids research.

[60]  P. Cossart,et al.  Entry of Listeria monocytogenes into hepatocytes requires expression of InIB, a surface protein of the internalin multigene family , 1995, Molecular microbiology.

[61]  B. Boizet-Bonhoure,et al.  Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum , 1995, Journal of bacteriology.

[62]  B. Müller-Hill,et al.  Quality and position of the three lac operators of E. coli define efficiency of repression. , 1994, The EMBO journal.

[63]  E. Maguin,et al.  New thermosensitive plasmid for gram-positive bacteria , 1992, Journal of bacteriology.

[64]  P. Youngman,et al.  Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene. , 1992, Biochimie.

[65]  W. Goebel,et al.  Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene , 1992, Journal of bacteriology.

[66]  G. Stewart,et al.  High-efficiency transformation of Listeria monocytogenes by electroporation of penicillin-treated cells. , 1990, Gene.

[67]  B. E. Davidson,et al.  A Simple and Rapid Method for Genetic Transformation of Lactic Streptococci by Electroporation , 1988, Applied and environmental microbiology.

[68]  D. Hinrichs,et al.  Adoptive transfer of immunity to Listeria monocytogenes. The influence of in vitro stimulation on lymphocyte subset requirements. , 1987, Journal of immunology.

[69]  R. Schoenfeld,et al.  Comparative Genomics of Listeria Species , 1976 .

[70]  M. Braunstein,et al.  Silencing Mycobacterium smegmatis by using tetracycline repressors. , 2007, Journal of bacteriology.

[71]  Shruti Jain,et al.  Mycobacteriophage Bxb 1 integrates into the Mycobacterium smegmatis groEL 1 gene , 2003 .

[72]  C. Hill,et al.  Identification and disruption of btlA, a locus involved in bile tolerance and general stress resistance in Listeria monocytogenes. , 2003, FEMS microbiology letters.

[73]  M. Gilmore,et al.  Electroporation and efficient transformation of Enterococcus faecalis grown in high concentrations of glycine. , 1995, Methods in molecular biology.

[74]  D. Henner,et al.  Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. , 1984, Proceedings of the National Academy of Sciences of the United States of America.