Leptospira spp. are spirochete bacteria comprising both pathogenic and free-living species. The saprophyte L. biflexa is a model bacterium for studying leptospiral biology due to relative ease of culturing and genetic manipulation. In this study, we constructed a library of 4,996 random transposon mutants in L. biflexa. We screened the library for increased susceptibility to the DNA intercalating agent, ethidium bromide (EtBr), in order to identify genetic determinants that reduce L. biflexa susceptibility to antimicrobial agents. By phenotypic screening, using subinhibitory EtBr concentrations, we identified 29 genes that, when disrupted via transposon insertion, led to increased sensitivity of the bacteria to EtBr. At the functional level, these genes could be categorized by function as follows: regulation and signaling (n=11), transport (n=6), membrane structure (n=5), stress response (n=2), DNA damage repair (n=1), and other processes (n=3), while 1 gene had no predicted function. Genes involved in transport (including efflux pumps) and regulation (two-component systems, anti-sigma factor antagonists, etc.) were overrepresented, demonstrating that these genes are major contributors to EtBr tolerance. This finding suggests that transport genes which would prevent EtBr to enter the cell cytoplasm are critical for EtBr resistance. We identified genes required for the growth of L. biflexa in the presence of sublethal EtBr concentration and characterized their potential as antibiotic resistance determinants. This study will help to delineate mechanisms of adaptation to toxic compounds, as well as potential mechanisms of antibiotic resistance development in pathogenic L. interrogans.

Institut Pasteur Biology of Spirochetes Unit Paris France

Masaryk University Faculty of Medicine Department of Biology Brno Czech Republic Institut Pasteur Biology of Spirochetes Unit Paris France

Zobrazit více v PubMed

Levett PN. 2001. Leptospirosis. Clin. Microbiol. Rev. 14:296–326. 10.1128/CMR.14.2.296-326.2001 PubMed DOI PMC

Bulach DM, Zuerner RL, Wilson P, Seemann T, McGrath A, Cullen PA, Davis J, Johnson M, Kuczek E, Alt DP, Peterson-Burch B, Coppel RL, Rood JI, Davies JK, Adler B. 2006. Genome reduction in Leptospira borgpetersenii reflects limited transmission potential. Proc. Natl. Acad. Sci. U. S. A. 103:14560–14565. 10.1073/pnas.0603979103 PubMed DOI PMC

Picardeau M. 2008. Conjugative transfer between Escherichia coli and Leptospira spp. as a new genetic tool. Appl. Environ. Microbiol. 74:319–322. 10.1128/AEM.02172-07 PubMed DOI PMC

Louvel H, Picardeau M. 2007. Genetic manipulation of Leptospira biflexa. Curr. Protoc. Microbiol. Chapter 12:Unit 12E.4. 10.1002/9780471729259.mc12e04s05 PubMed DOI

Picardeau M, Bulach DM, Bouchier C, Zuerner RL, Zidane N, Wilson PJ, Creno S, Kuczek ES, Bommezzadri S, Davis JC, McGrath A, Johnson MJ, Boursaux-Eude C, Seemann T, Rouy Z, Coppel RL, Rood JI, Lajus A, Davies JK, Médigue C, Adler B. 2008. Genome sequence of the saprophyte Leptospira biflexa provides insights into the evolution of Leptospira and the pathogenesis of leptospirosis. PLoS One 3:e1607. 10.1371/journal.pone.0001607 PubMed DOI PMC

Waring MJ. 1965. Complex formation between ethidium bromide and nucleic acids. J. Mol. Biol. 13:269–282. 10.1016/S0022-2836(65)80096-1 PubMed DOI

Mesak LR, Miao V, Davies J. 2008. Effects of subinhibitory concentrations of antibiotics on SOS and DNA repair gene expression in Staphylococcus aureus. Antimicrob. Agents Chemother. 52:3394–3397. 10.1128/AAC.01599-07 PubMed DOI PMC

Nakamura SI, Oda Y, Shimada T, Oki I, Sugimoto K. 1987. SOS-inducing activity of chemical carcinogens and mutagens in Salmonella typhimurium TA1535/pSK1002: examination with 151 chemicals. Mutat. Res. 192:239–246. 10.1016/0165-7992(87)90063-7 PubMed DOI

Kuzminov A. 1999. Recombinational repair of DNA damage in Escherichia coli and bacteriophage λ. Microbiol. Mol. Biol. Rev. 63:751–813 PubMed PMC

Xue F, Yan J, Picardeau M. 2009. Evolution and pathogenesis of Leptospira spp.: lessons learned from the genomes. Microbes Infect. 11:328–333. 10.1016/j.micinf.2008.12.007 PubMed DOI

Nikaido H. 2001. Preventing drug access to targets: cell surface permeability barriers and active efflux in bacteria. Semin. Cell Dev. Biol. 12:215–223. 10.1006/scdb.2000.0247 PubMed DOI

Tegos G, Mylonakis E. 2012. Antimicrobial drug discovery: emerging strategies. CABI, Wallingford, United Kingdom

Putman M, van Veen HW, Konings WN. 2000. Molecular properties of bacterial multidrug transporters. Microbiol. Mol. Biol. Rev. 64:672–693. 10.1128/MMBR.64.4.672-693.2000 PubMed DOI PMC

Nikaido H, Pagès J-M. 2012. Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. FEMS Microbiol. Rev. 36:340–363. 10.1111/j.1574-6976.2011.00290.x PubMed DOI PMC

Sánchez P, Linares JF, Ruiz-Díez B, Campanario E, Navas A, Baquero F, Martínez JL. 2002. Fitness of in vitro selected Pseudomonas aeruginosa nalB and nfxB multidrug resistant mutants. J. Antimicrob. Chemother. 50:657–664. 10.1093/jac/dkf185 PubMed DOI

Sherman DJ, Lazarus MB, Murphy L, Liu C, Walker S, Ruiz N, Kahne D. 2014. Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport. Proc. Natl. Acad. Sci. U. S. A. 111:4982–4987. 10.1073/pnas.1323516111 PubMed DOI PMC

Deng X, Sun F, Ji Q, Liang H, Missiakas D, Lan L, He C. 2012. Expression of multidrug resistance efflux pump gene norA is iron responsive in Staphylococcus aureus. J. Bacteriol. 194:1753–1762. 10.1128/JB.06582-11 PubMed DOI PMC

Piddock LJV. 2006. Multidrug-resistance efflux pumps: not just for resistance. Nat. Rev. Microbiol. 4:629–636. 10.1038/nrmicro1464 PubMed DOI

Vallenet D, Labarre L, Rouy Z, Barbe V, Bocs S, Cruveiller S, Lajus A, Pascal G, Scarpelli C, Médigue C. 2006. MaGe: a microbial genome annotation system supported by synteny results. Nucleic Acids Res. 34:53–65. 10.1093/nar/gkj406 PubMed DOI PMC

Tatusov RL, Koonin EV, Lipman DJ. 1997. A genomic perspective on protein families. Science 278:631–637. 10.1126/science.278.5338.631 PubMed DOI

Viveiros M, Dupont M, Rodrigues L, Couto I, Davin-Regli A, Martins M, Pagès J-M, Amaral L. 2007. Antibiotic stress, genetic response, and altered permeability of Escherichia coli. PLoS One 2:e365. 10.1371/journal.pone.0000365 PubMed DOI PMC

Fernández L, Hancock REW. 2012. Adaptive and mutational resistance: role of porins and efflux pumps in drug resistance. Clin. Microbiol. Rev. 25:661–681. 10.1128/CMR.00043-12 PubMed DOI PMC

Terekhova D, Sartakova ML, Wormser GP, Schwartz I, Cabello FC. 2002. Erythromycin resistance in Borrelia burgdorferi. Antimicrob. Agents Chemother. 46:3637–3640. 10.1128/AAC.46.11.3637-3640.2002 PubMed DOI PMC

Stamm LV, Stapleton JT, Bassford PJ., Jr 1988. In vitro assay to demonstrate high-level erythromycin resistance of a clinical isolate of Treponema pallidum. Antimicrob. Agents Chemother. 32:164–169. 10.1128/AAC.32.2.164 PubMed DOI PMC

Criswell D, Tobiason VL, Lodmell JS, Samuels DS. 2006. Mutations conferring aminoglycoside and spectinomycin resistance in Borrelia burgdorferi. Antimicrob. Agents Chemother. 50:445–452. 10.1128/AAC.50.2.445-452.2006 PubMed DOI PMC

Malmström J, Beck M, Schmidt A, Lange V, Deutsch EW, Aebersold R. 2009. Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans. Nature 460:762–765. 10.1038/nature08184 PubMed DOI PMC

Neyfakh AA, Bidnenko VE, Chen LB. 1991. Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system. Proc. Natl. Acad. Sci. U. S. A. 88:4781–4785. 10.1073/pnas.88.11.4781 PubMed DOI PMC

Slamti L, Picardeau M. 2012. Construction of a library of random mutants in the spirochete Leptospira biflexa using a mariner transposon, p 169–176 In Bigot Y. (ed), Mobile genetic elements. Humana Press, Totowa, NJ PubMed

Rozen S, Skaletsky H. 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132:365–386 PubMed

Murray CK, Hospenthal DR. 2004. Broth microdilution susceptibility testing for Leptospira spp. Antimicrob. Agents Chemother. 48:1548–1552. 10.1128/AAC.48.5.1548-1552.2004 PubMed DOI PMC

Marchler-Bauer A, Bryant SH. 2004. CD-Search: protein domain annotations on the fly. Nucleic Acids Res. 32:W327–W331. 10.1093/nar/gkh454 PubMed DOI PMC

Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, Heger A, Hetherington K, Holm L, Mistry J, Sonnhammer ELL, Tate J, Punta M. 2014. Pfam: the protein families database. Nucleic Acids Res. 42:D222–D230. 10.1093/nar/gkt1223 PubMed DOI PMC

Sigrist CJA, Cerutti L, de Castro E, Langendijk-Genevaux PS, Bulliard V, Bairoch A, Hulo N. 2010. PROSITE, a protein domain database for functional characterization and annotation. Nucleic Acids Res. 38:D161–D166. 10.1093/nar/gkp885 PubMed DOI PMC

Dassa E, Bouige P. 2001. The ABC of ABCs: a phylogenetic and functional classification of ABC systems in living organisms. Res. Microbiol. 152:211–229. 10.1016/S0923-2508(01)01194-9 PubMed DOI

Rawlings ND, Barrett AJ, Bateman A. 2012. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 40:D343–D350. 10.1093/nar/gkr987 PubMed DOI PMC

Yu NY, Wagner JR, Laird MR, Melli G, Rey S, Lo R, Dao P, Sahinalp SC, Ester M, Foster LJ, Brinkman FSL. 2010. PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26:1608–1615. 10.1093/bioinformatics/btq249 PubMed DOI PMC

Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8:785–786. 10.1038/nmeth.1701 PubMed DOI

de Jong A, Pietersma H, Cordes M, Kuipers OP, Kok J. 2012. PePPER: a webserver for prediction of prokaryote promoter elements and regulons. BMC Genomics 13:299. 10.1186/1471-2164-13-299 PubMed DOI PMC

Zuerner RL, Knudtson W, Bolin CA, Trueba G. 1991. Characterization of outer membrane and secreted proteins of Leptospira interrogans serovar Pomona. Microb. Pathog. 10:311–322. 10.1016/0882-4010(91)90014-2 PubMed DOI

Eshghi A, Lourdault K, Murray GL, Bartpho T, Sermswan RW, Picardeau M, Adler B, Snarr B, Zuerner RL, Cameron CE. 2012. Leptospira interrogans catalase is required for resistance to H2O2 and for virulence. Infect. Immun. 80:3892–3899. 10.1128/IAI.00466-12 PubMed DOI PMC

Stewart PE, Carroll JA, Dorward DW, Stone HH, Sarkar A, Picardeau M, Rosa PA. 2012. Characterization of the Bat proteins in the oxidative stress response of Leptospira biflexa. BMC Microbiol. 12:290. 10.1186/1471-2180-12-290 PubMed DOI PMC

Azad MA, Wright GD. 2012. Determining the mode of action of bioactive compounds. Bioorg. Med. Chem. 20:1929–1939. 10.1016/j.bmc.2011.10.088 PubMed DOI

Lampe DJ, Grant TE, Robertson HM. 1998. Factors affecting transposition of the Himar1 mariner transposon in vitro. Genetics 149:179–187 PubMed PMC

Llama-Palacios A, López-Solanilla E, Rodríguez-Palenzuela P. 2002. The ybiT gene of Erwinia chrysanthemi codes for a putative ABC transporter and is involved in competitiveness against endophytic bacteria during infection. Appl. Environ. Microbiol. 68:1624–1630. 10.1128/AEM.68.4.1624-1630.2002 PubMed DOI PMC

Green DH, Cutting SM. 2000. Membrane topology of the Bacillus subtilis pro-sigma(K) processing complex. J. Bacteriol. 182:278–285. 10.1128/JB.182.2.278-285.2000 PubMed DOI PMC

Akiyama Y, Kanehara K, Ito K. 2004. RseP (YaeL), an Escherichia coli RIP protease, cleaves transmembrane sequences. EMBO J. 23:4434–4442. 10.1038/sj.emboj.7600449 PubMed DOI PMC

Murray GL, Morel V, Cerqueira GM, Croda J, Srikram A, Henry R, Ko AI, Dellagostin OA, Bulach DM, Sermswan RW, Adler B, Picardeau M. 2009. Genome-wide transposon mutagenesis in pathogenic Leptospira species. Infect. Immun. 77:810–816. 10.1128/IAI.01293-08 PubMed DOI PMC

Caimano MJ, Sivasankaran SK, Allard A, Hurley D, Hokamp K, Grassmann AA, Hinton JCD, Nally JE. 2014. A model system for studying the transcriptomic and physiological changes associated with mammalian host-adaptation by Leptospira interrogans serovar Copenhageni. PLoS Pathog. 10:e1004004. 10.1371/journal.ppat.1004004 PubMed DOI PMC

Gardan R, Cossart P, Labadie J. 2003. Identification of Listeria monocytogenes genes involved in salt and alkaline-pH tolerance. Appl. Environ. Microbiol. 69:3137–3143. 10.1128/AEM.69.6.3137-3143.2003 PubMed DOI PMC

Reddy M, Gowrishankar J. 2000. Characterization of the uup locus and its role in transposon excisions and tandem repeat deletions in Escherichia coli. J. Bacteriol. 182:1978–1986. 10.1128/JB.182.7.1978-1986.2000 PubMed DOI PMC

Murat D, Goncalves L, Dassa E. 2008. Deletion of the Escherichia coli uup gene encoding a protein of the ATP binding cassette superfamily affects bacterial competitiveness. Res. Microbiol. 159:671–677. 10.1016/j.resmic.2008.09.004 PubMed DOI

Geistlich M, Losick R, Turner JR, Rao RN. 1992. Characterization of a novel regulatory gene governing the expression of a polyketide synthase gene in Streptomyces ambofaciens. Mol. Microbiol. 6:2019–2029. 10.1111/j.1365-2958.1992.tb01374.x PubMed DOI

Peschke U, Schmidt H, Zhang HZ, Piepersberg W. 1995. Molecular characterization of the lincomycin-production gene cluster of Streptomyces lincolnensis 78-11. Mol. Microbiol. 16:1137–1156. 10.1111/j.1365-2958.1995.tb02338.x PubMed DOI

Allignet J, Loncle V, el Sohl N. 1992. Sequence of a staphylococcal plasmid gene, vga, encoding a putative ATP-binding protein involved in resistance to virginiamycin A-like antibiotics. Gene 117:45–51. 10.1016/0378-1119(92)90488-B PubMed DOI

Davidson AL, Dassa E, Orelle C, Chen J. 2008. Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol. Mol. Biol. Rev. 72:317–364. 10.1128/MMBR.00031-07 PubMed DOI PMC

Missiakas D, Raina S. 1998. The extracytoplasmic function sigma factors: role and regulation. Mol. Microbiol. 28:1059–1066. 10.1046/j.1365-2958.1998.00865.x PubMed DOI

Kim T-J, Gaidenko TA, Price CW. 2004. In vivo phosphorylation of partner switching regulators correlates with stress transmission in the environmental signaling pathway of Bacillus subtilis. J. Bacteriol. 186:6124–6132. 10.1128/JB.186.18.6124-6132.2004 PubMed DOI PMC

Sharma AK, Rigby AC, Alper SL. 2011. STAS domain structure and function. Cell. Physiol. Biochem. 28:407–422. 10.1159/000335104 PubMed DOI PMC

Clarke L, Carbon J. 1976. A colony bank containing synthetic CoI EI hybrid plasmids representative of the entire Escherichia coli genome. Cell 9:91–99. 10.1016/0092-8674(76)90055-6 PubMed DOI

Palma M, Cheung AL. 2001. Sigma(B) activity in Staphylococcus aureus is controlled by RsbU and an additional factor(s) during bacterial growth. Infect. Immun. 69:7858–7865. 10.1128/IAI.69.12.7858-7865.2001 PubMed DOI PMC

Delumeau O, Dutta S, Brigulla M, Kuhnke G, Hardwick SW, Völker U, Yudkin MD, Lewis RJ. 2004. Functional and structural characterization of RsbU, a stress signaling protein phosphatase 2C. J. Biol. Chem. 279:40927–40937. 10.1074/jbc.M405464200 PubMed DOI

Hirakawa H, Nishino K, Hirata T, Yamaguchi A. 2003. Comprehensive studies of drug resistance mediated by overexpression of response regulators of two-component signal transduction systems in Escherichia coli. J. Bacteriol. 185:1851–1856. 10.1128/JB.185.6.1851-1856.2003 PubMed DOI PMC

Eguchi Y, Oshima T, Mori H, Aono R, Yamamoto K, Ishihama A, Utsumi R. 2003. Transcriptional regulation of drug efflux genes by EvgAS, a two-component system in Escherichia coli. Microbiology 149:2819–2828. 10.1099/mic.0.26460-0 PubMed DOI

Römling U, Amikam D. 2006. Cyclic di-GMP as a second messenger. Curr. Opin. Microbiol. 9:218–228. 10.1016/j.mib.2006.02.010 PubMed DOI

Fineran PC, Williamson NR, Lilley KS, Salmond GPC. 2007. Virulence and prodigiosin antibiotic biosynthesis in Serratia are regulated pleiotropically by the GGDEF/EAL domain protein, PigX. J. Bacteriol. 189:7653–7662. 10.1128/JB.00671-07 PubMed DOI PMC

Sulavik MC, Houseweart C, Cramer C, Jiwani N, Murgolo N, Greene J, DiDomenico B, Shaw KJ, Miller GH, Hare R, Shimer G. 2001. Antibiotic susceptibility profiles of Escherichia coli Strains lacking multidrug efflux pump genes. Antimicrob. Agents Chemother. 45:1126–1136. 10.1128/AAC.45.4.1126-1136.2001 PubMed DOI PMC

Nishino K, Yamaguchi A. 2001. Analysis of a complete library of putative drug transporter genes in Escherichia coli. J. Bacteriol. 183:5803–5812. 10.1128/JB.183.20.5803-5812.2001 PubMed DOI PMC

Bassford PJ, Jr, Bradbeer C, Kadner RJ, Schnaitman CA. 1976. Transport of vitamin B12 in tonB mutants of Escherichia coli. J. Bacteriol. 128:242–247 PubMed PMC

Louvel H, Bommezzadri S, Zidane N, Boursaux-Eude C, Creno S, Magnier A, Rouy Z, Médigue C, Saint Girons I, Bouchier C, Picardeau M. 2006. Comparative and functional genomic analyses of iron transport and regulation in Leptospira spp. J. Bacteriol. 188:7893–7904. 10.1128/JB.00711-06 PubMed DOI PMC

Zhao Q, Li XZ, Mistry A, Srikumar R, Zhang L, Lomovskaya O, Poole K. 1998. Influence of the TonB energy-coupling protein on efflux-mediated multidrug resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 42:2225–2231 PubMed PMC

Alberts AW, Majerus PW, Talamo B, Vagelos PR. 1964. Acyl-carrier protein. II. Intermediary reactions of fatty acid synthesis. Biochemistry 3:1563–1571 PubMed

Zhang Y, Cronan JE., Jr 1998. Transcriptional analysis of essential genes of the Escherichia coli fatty acid biosynthesis gene cluster by functional replacement with the analogous Salmonella typhimurium gene cluster. J. Bacteriol. 180:3295–3303 PubMed PMC

Lai C-Y, Cronan JE. 2004. Isolation and characterization of β-ketoacyl-acyl carrier protein reductase (fabG) mutants of Escherichia coli and Salmonella enterica serovar Typhimurium. J. Bacteriol. 186:1869–1878. 10.1128/JB.186.6.1869-1878.2004 PubMed DOI PMC

Wang H, Cronan JE. 2004. Only one of the two annotated Lactococcus lactis fabG genes encodes a functional beta-ketoacyl-acyl carrier protein reductase. Biochemistry 43:11782–11789. 10.1021/bi0487600 PubMed DOI

Kabanov DS, Prokhorenko IR. 2010. Structural analysis of lipopolysaccharides from Gram-negative bacteria. Biochemistry Mosc. 75:383–404. 10.1134/S0006297910040012 PubMed DOI

Matsunaga J, Lo M, Bulach DM, Zuerner RL, Adler B, Haake DA. 2007. Response of Leptospira interrogans to physiologic osmolarity: relevance in signaling the environment-to-host transition. Infect. Immun. 75:2864–2874. 10.1128/IAI.01619-06 PubMed DOI PMC

Johnson DR, Coronado E, Moreno-Forero SK, Heipieper HJ, van der Meer JR. 2011. Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii. BMC Microbiol. 11:250. 10.1186/1471-2180-11-250 PubMed DOI PMC

Tsilibaris V, Maenhaut-Michel G, Van Melderen L. 2006. Biological roles of the Lon ATP-dependent protease. Res. Microbiol. 157:701–713. 10.1016/j.resmic.2006.05.004 PubMed DOI

Riethdorf S, Völker U, Gerth U, Winkler A, Engelmann S, Hecker M. 1994. Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene. J. Bacteriol. 176:6518–6527 PubMed PMC

Pfennig PL, Flower AM. 2001. BipA is required for growth of Escherichia coli K12 at low temperature. Mol. Genet. Genomics 266:313–317. 10.1007/s004380100559 PubMed DOI

Neidig A, Yeung ATY, Rosay T, Tettmann B, Strempel N, Rueger M, Lesouhaitier O, Overhage J. 2013. TypA is involved in virulence, antimicrobial resistance and biofilm formation in Pseudomonas aeruginosa. BMC Microbiol. 13:77. 10.1186/1471-2180-13-77 PubMed DOI PMC

Petit M-A, Ehrlich D. 2002. Essential bacterial helicases that counteract the toxicity of recombination proteins. EMBO J. 21:3137–3147. 10.1093/emboj/cdf317 PubMed DOI PMC

Tchamedeu Kameni A-P, Couture-Tosi E, Saint-Girons I, Picardeau M. 2002. Inactivation of the spirochete recA gene results in a mutant with low viability and irregular nucleoid morphology. J. Bacteriol. 184:452–458. 10.1128/JB.184.2.452-458.2002 PubMed DOI PMC

Nascimento ALTO, Verjovski-Almeida S, Van Sluys MA, Monteiro-Vitorello CB, Camargo LEA, Digiampietri LA, Harstkeerl RA, Ho PL, Marques MV, Oliveira MC, Setubal JC, Haake DA, Martins EL. 2004. Genome features of Leptospira interrogans serovar Copenhageni. Braz. J. Med. Biol. Res. 37:459–477. 10.1590/S0100-879X2004000400003 PubMed DOI PMC

Demarre G, Guérout A-M, Matsumoto-Mashimo C, Rowe-Magnus DA, Marlière P, Mazel D. 2005. A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPα) conjugative machineries and their cognate Escherichia coli host strains. Res. Microbiol. 156:245–255. 10.1016/j.resmic.2004.09.007 PubMed DOI

Simon R, Priefer U, Pühler A. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Nat. Biotechnol. 1:784–791. 10.1038/nbt1183-784 DOI

King AM, Pretre G, Bartpho T, Sermswan RW, Toma C, Suzuki T, Eshghi A, Picardeau M, Adler B, Murray GL. 2014. High-temperature protein G is an essential virulence factor of Leptospira interrogans. Infect. Immun. 82:1123–1131. 10.1128/IAI.01546-13 PubMed DOI PMC