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Identification of Genes Required by Bacillus thuringiensis for Survival in Soil by Transposon-Directed Insertion Site Sequencing

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Abstract

Transposon-directed insertion site sequencing was used to identify genes required by Bacillus thuringiensis to survive in non-axenic plant/soil microcosms. A total of 516 genetic loci fulfilled the criteria as conferring survival characteristics. Of these, 127 (24.6 %) were associated with uptake and transport systems; 227 loci (44.0 %) coded for enzymatic properties; 49 (9.5 %) were gene regulation or sensory loci; 40 (7.8 %) were structural proteins found in the cell envelope or had enzymatic activities related to it and 24 (4.7 %) were involved in the production of antibiotics or resistance to them. Eighty-three (16.1 %) encoded hypothetical proteins or those of unknown function. The ability to form spores was a key survival characteristic in the microcosms: bacteria, inoculated in either spore or vegetative form, were able to multiply and colonise the soil, whereas a sporulation-deficient mutant was not. The presence of grass seedlings was critical to colonisation. Bacteria labelled with green fluorescent protein were observed to adhere to plant roots. The sporulation-specific promoter of spo0A, the key regulator of sporulation, was strongly activated in the rhizosphere. In contrast, the vegetative-specific promoters of spo0A and PlcR, a pleiotropic regulator of genes with diverse activities, were only very weakly activated.

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References

  1. Bais HP, Weir TF, Perry LG, Gilroy S, Vivanco JM (2006) The role of root exudates in rhizosphere interactions with plants and other organisms. Ann Rev Plant Biol 57:233–266

    Article  CAS  Google Scholar 

  2. Bishop AH (2002) The insecticidal proteins of Bacillus thuringiensis. In: Hendrickx M (ed) Applications and systematics of the genus bacillus and related organisms. Federation of European Microbiological Societies. Blackwell, Oxford, pp 160–175

    Chapter  Google Scholar 

  3. Bizzarri MF, Bishop AH (2007) Recovery of Bacillus thuringiensis in vegetative form from the phylloplane of clover (Trifolium hybridum) during a growing season. J Inv Pathol 94:38–47

    Article  Google Scholar 

  4. Bizzarri MF, Bishop AH (2008) The ecology of Bacillus thuringiensis on the phylloplane: colonisation from soil, conjugation and interaction with larvae of Pieris brassicae. Microbiol Ecol 56:133–139

    Article  CAS  Google Scholar 

  5. Bizzarri MF, Prabhakar A, Bishop AH (2008) Multiple-locus sequence typing analysis of Bacillus thuringiensis recovered from the phylloplane of clover (Trifolium hybridum) in vegetative form. Microbiol Ecol 55:619–625

    Article  CAS  Google Scholar 

  6. Bone EJ, Ellar DJ (1989) Transformation of Bacillus thuringiensis by electroporation. FEMS Microbiol Lett 58:171–178

    Article  CAS  Google Scholar 

  7. Degnan BA, Fontaine MC, Doebereiner AH, Lee JJ, Mastroeni P, Dougan G, Goodacre G, Kehoe MA (2000) Characterization of an isogenic mutant of Streptococcus pyogenes Manfredo lacking the ability to make Streptococcal acid glycoprotein. Infect Immun 68:241–248

    Article  Google Scholar 

  8. Deutschbauer A, Price MN, Wetmore KM, Shao W, Baumohl JK, Xu Z, Nguyen M, Tamse R, Davis RW, Arkin AP (2011) Evidence-based annotation of gene function in Shewanella oneidensis MR-1 using genome-wide fitness profiling across 121 conditions. PLoS Genet 11:e1002385

    Article  Google Scholar 

  9. Dziva F, van Diemen PM, Stevens MP, Smith AJ, Wallis TM (2004) Identification of Escherichia coli O157: H7 genes influencing colonization of the bovine gastrointestinal tract using signature-tagged mutagenesis. Microbiology 150:3631–3645

    Article  CAS  PubMed  Google Scholar 

  10. Eckert SE, Dziva F, Chaudhuri RR, Langridge GC, Turner DJ, Pickard DJ, Maskell DJ, Thompson NR, Stevens MP (2011) Retrospective application of transposon-directed insertion site sequencing to a library of signature-tagged mini-Tn5Km2 mutants of Escherichia coli O157:H7 screened in cattle. J Bacteriol 193:1771–1776

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Ellwood DC, Tempest DA (1969) Control of teichoic acid and teichuronic acid biosynthesis in chemostat cultures of Bacillus subtilis var. niger. Biochem J 111:1–5

    CAS  PubMed  Google Scholar 

  12. Gawronski JD, Wonga SMS, Giannoukos G, Ward DV, Akerley BJ (2009) Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung. Proc Natl Acad Sci USA 106:16422–16427

    Article  CAS  PubMed  Google Scholar 

  13. Gohar M, Faegri K, Perchat S, Ravnum S, Økstad OE, Gominet M, Gilois N, Kolstø AB, Lereclus D (2008) The PlcR virulence regulon of Bacillus cereus. PLoS ONE 3:e2793

    Article  PubMed Central  PubMed  Google Scholar 

  14. Groh JL, Luo Q, Ballard JD, Krumholz LR (2007) Genes that enhance the ecological fitness of Shewanella oneidensis MR-1 in sediments reveal the value of antibiotic resistance. Appl Environ Microbiol 73:492–498

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Helgason E, Økstad OA, Caugant DA, Johansen HA, Fouet A, Mock M, Hegna I, Kolstø A-B (2000) Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—one species on the basis of genetic evidence. Appl Environ Microbiol 66:2627–2630

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Hensel M, Shea JE, Gleeson C, Jones MD, Dalton E, Holden DW (1995) Simultaneous identification of bacterial virulence genes by negative selection. Science 269:400–403

    Article  CAS  PubMed  Google Scholar 

  17. He J, Shao X, Zheng H, Li M, Wang J, Zang Q, Li L, Liu Z, Sun M, Wang S, Yu Z (2010) Complete genome sequence of Bacillus thuringiensis mutant strain BMB171. J Bacteriol 192:4074–4075

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Hugh-Jones M, Blackburn J (2009) The ecology of Bacillus anthracis. Mol Aspects Med 30:356–367

    Article  PubMed  Google Scholar 

  19. Ivanova N, Sorokin A, Anderson I, Galleron N, Candelon B, Kapatral V (2003) Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423:87–91

    Article  CAS  PubMed  Google Scholar 

  20. Jensen GB, Hansen BM, Eilenberg J, Mahillon J (2003) The hidden lifestyles of Bacillus cereus and relatives. Environ Microbiol 5:631–640

    Article  CAS  PubMed  Google Scholar 

  21. Ju C, Gu H, Lu C (2012) Characterization and functional analysis of Atl, a novel gene encoding an autolysin in Streptococcus suis. J Bacteriol 194:1463–1473

    Article  Google Scholar 

  22. Keim P, Gruendike JM, Klevytska AM, Schupp JM, Challacombe J, Okinaka R (2009) The genome and variation of Bacillus cereus. Mol Aspects Med 30:397–405

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Kotiranta A, Haapasalo M, Kari K, Kerosuo E, Olsen I, Sorsa T, Meurman JH, Lounatmaa K (1998) Surface structure, hydrophobicity, phagocytosis, and adherence to matrix proteins of Bacillus cereus cells with and without the crystalline surface protein layer. Infect Immun 66:4895–4902

    CAS  PubMed Central  PubMed  Google Scholar 

  24. Langridge GC, Phan M-D, Turner DJ (2010) Simultaneous assay of every Salmonella typhi gene using one million transposon mutants. Genom Res 19:2308–2316

    Article  Google Scholar 

  25. Lee K, Costerton JW, Ravel J, Auerbach RK, Wagner DM, Keim P, Leid JG (2007) Phenotypic and functional characterization of Bacillus anthracis biofilms. Microbiology 153:1693–1701

    Article  CAS  PubMed  Google Scholar 

  26. Lereclus D, Agaisse H, Gominet M, Chaufaux J (1995) Overproduction of encapsulated insecticidal crystal proteins in a Bacillus thuringiensis mutant. Nat Biotechnol 17:67–71

    Article  Google Scholar 

  27. Lereclus D, Agaisse H, Gominet M, Salamitou S, Sanchis V (1996) Identification of a Bacillus thuringiensis gene that positively regulates transcription of the phosphatidylinositol-specific phospholipase C gene at the onset of the stationary phase. J Bacteriol 178:2749–2756

    CAS  PubMed Central  PubMed  Google Scholar 

  28. Luo Q, Groh JL, Ballard JD, Krumholz LR (2007) Identification of genes that confer sediment fitness to Deslfovibrio desulfuricans G20. Appl Environ Microbiol 73:6305–6312

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Mecsas J (2002) Use of signature tagged mutagenesis in pathogenesis studies. Curr Opin Microbiol 5:33–37

    Article  CAS  PubMed  Google Scholar 

  30. Mei JM, Nourbakhsh F, Ford CW, Holden DW (1997) Identification of Staphylococcus aureus virulence in a murine model of bacteraemia using signature-tagged mutagenesis. Mol Microbiol 26:399–407

    Article  CAS  PubMed  Google Scholar 

  31. Menedez A, Fernandez L, Reimundo X, Guijarro JA (2007) Genes required for Lactococcus garvieae survival in a fish host. Microbiology 153:3286–3294

    Article  Google Scholar 

  32. Nickerson KW, Bulla LA (1974) Physiology of spore forming bacteria associated with insects: minimal nutritional requirements for growth, sporulation, and parasporal crystal formation of Bacillus thuringiensis. Appl Microbiol 28:124–128

    CAS  PubMed Central  PubMed  Google Scholar 

  33. O’ Sullivan LA, Weightman AJ, Hefin Jones T, Marchbank AM, Tiedje JM, Mahenthiralingam E (2007) Identifying the genetic basis of ecologically and biotechnologically useful functions of the bacterium Burkholderia vietnamiensis. Environ Microbiol 9:1017–1034

    Article  Google Scholar 

  34. Perehinec TM, Qazi SNA, Gaddipati SR, Salisbury V, Rees CED, Hill PJ (2007) Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria. BMC Mol Biol 8:80–91

    Article  PubMed Central  PubMed  Google Scholar 

  35. Prabhakar A, Bishop AH (2012) Invertebrate pathogenicity and toxin-producing potential of strains of Bacillus thuringiensis endemic to Antarctica. J Inv Pathol 107:132–138

    Article  Google Scholar 

  36. Quisel JD, Grossman AD (2000) Control of sporulation gene expression in Bacillus subtilis by the chromosome partitioning proteins Soj (ParA) and Spo0J (ParB). J Bacteriol 182:3446–3451

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Ryan G, Sinclair RG, Rose JB, Hashsham SA, Gerba CP, Haas CN (2012) Criteria for selection of surrogates used to study the fate and control of pathogens in the environment. Appl Environ Microbiol 78:1969–1977

    Article  Google Scholar 

  39. Saile E, Koehler TM (2006) Bacillus anthracis multiplication, persistence, and genetic exchange in the rhizosphere of grass plants. Appl Environ Microbiol 72:3168–3174

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Sambrook J, Fritsch EF, Maniatis T (1987) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  41. Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J et al (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–790

    CAS  PubMed Central  PubMed  Google Scholar 

  42. Schuch R, Pelzek AJ, Kan S, Fischetti VA (2010) Prevalence of Bacillus anthracis-like organisms and bacteriophages in the intestinal tract of the earthworm Eisenia fetida. Appl Environ Microbiol 76:2286–2294

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  43. Seleem MN, Vemulappalli R, Boyle SM, Schurig GG, Sriranganathan N (2004) Improved expression vector for Brucella species. Biotechniques 37:740–744

    CAS  PubMed  Google Scholar 

  44. Song Y, Xie C, Ong YM, Gan YH, Chua KL (2005) The BpsIR quorum-sensing system of Burkholderia pseudomallei. J Bacteriol 187:785–790

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. van Opijnen T, Bodi KL, Camilli A (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6:767–772

    Article  PubMed Central  PubMed  Google Scholar 

  46. Villafane R, Bechhofer DH, Narayanan CS, Dubnau D (1987) Replication control genes of plasmid pE194. J Bacteriol 169:4822–4829

    CAS  PubMed Central  PubMed  Google Scholar 

  47. Vilain S, Luo Y, Hildreth MB, Brozel SV (2006) Analysis of the life cycle of the soil saprophyte Bacillus cereus in liquid soil extract and in the soil. Appl Environ Microbiol 72:4970–4977

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  48. Wilson A, Perego M, Hoch JA (2007) New transposon delivery plasmids for insertional mutagenesis in Bacillus anthracis. J Microbiol Methods 71:332–335

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

The authors are very grateful to The Defense Threat Reduction Agency (DTRA) for funding. AHB was also supported by funding from the Ministry of Defence, UK. We also acknowledge the help from Gill Hartley (Dstl) for confocal microscopy and Bry Lingard (Dstl) for assistance with sequence analysis. We thank Dr Sari Paikoff (DTRA) and Prof. Petra Oyston for support and advice.

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  1. Detection Department, Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire, SP4 0JQ, UK

    Alistair H. Bishop, Phillip A. Rachwal & Alka Vaid

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  1. Alistair H. Bishop
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Correspondence to Alistair H. Bishop.

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Bishop, A.H., Rachwal, P.A. & Vaid, A. Identification of Genes Required by Bacillus thuringiensis for Survival in Soil by Transposon-Directed Insertion Site Sequencing. Curr Microbiol 68, 477–485 (2014). https://doi.org/10.1007/s00284-013-0502-7

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