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Characterization and Whole Genome Sequencing of AR23, a Highly Toxic Bacillus thuringiensis Strain Isolated from Lebanese Soil

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Abstract

The demand for sustainable and eco-friendly control methods of pests and insects is increasing worldwide. From this came the interest in Bacillus thuringiensis, an entomopathogenic bacterium capable of replacing chemical pesticides. However, the possibility of pests developing resistance to a particular strain may impair its use, and there is a need to identify novel strains of this species as potential commercial biopesticides. B. thuringiensis sv. israelensis is one of the most successful serovars, widely commercialized for its activity against black fly and mosquito larvae. In this study, we isolated, characterized, and sequenced a new Lebanese B. thuringiensis sv. israelensis isolate, strain AR23. Compared to the commercialized reference strain AM65-52 (Vectobac®, Sumitomo), AR23 showed an increased activity against several mosquito species. The genomic analysis revealed that this strain, compared to AM65-52, possesses a simplified plasmid content and an additional functional cry4Ba coding gene that most likely accounts for the increased effectiveness of this strain in mosquito larvae killing.

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References

  1. Regis L, Silva-Filha MH, Nielsen-LeRoux C, Charles J-F (2001) Bacteriological larvicides of dipteran disease vectors. Trends Parasitol 17:377–380. https://doi.org/10.1016/S1471-4922(01)01953-5

    Article  CAS  PubMed  Google Scholar 

  2. Schnepf E, Crickmore N, Rie JVAN et al (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Van Frankenhuyzen K (2009) Insecticidal activity of Bacillus thuringiensis crystal proteins. J Invertebr Pathol 101:1–16. https://doi.org/10.1016/j.jip.2009.02.009

    Article  CAS  PubMed  Google Scholar 

  4. Van Frankenhuyzen K (2013) Cross-order and cross-phylum activity of Bacillus thuringiensis pesticidal proteins. J Invertebr Pathol 114:76–85. https://doi.org/10.1016/j.jip.2013.05.010

    Article  CAS  PubMed  Google Scholar 

  5. Palma L, Muñoz D, Berry C et al (2014) Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins 6:3296–3325. https://doi.org/10.3390/toxins6123296

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Glare TR, O’Callaghan M (2000) Bacillus thuringiensis: biology, ecology and safety. Wiley, New York, pp 2–80

    Google Scholar 

  7. Roh JY, Choi JY, Li MS et al (2007) Bacillus thuringiensis as a specific, safe, and effective tool for insect pest control. J Microbiol Biotechnol 17:547–559

    CAS  PubMed  Google Scholar 

  8. De Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet 17:193–199. https://doi.org/10.1016/S0168-9525(01)02237-5

    Article  PubMed  Google Scholar 

  9. Gammon K, Jones GW, Hope SJ et al (2006) Conjugal transfer of a toxin-coding megaplasmid from Bacillus thuringiensis subsp. israelensis to mosquitocidal strains of Bacillus thuringiensis. Appl Environ Microbiol 72:1766–1770. https://doi.org/10.1128/AEM.72.3.1766

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Leonard C, Chene Y, Mahillon J (1997) Diversity and differential distribution of IS231, IS232 and IS240 among Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides. Microbiology 143:2537–2547

    Article  CAS  Google Scholar 

  11. Rang J, He H, Wang T et al (2015) Comparative analysis of genomics and proteomics in Bacillus thuringiensis 4.0718. PLoS ONE 10:e0119065. https://doi.org/10.1371/journal.pone.0119065

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Berry C, O’Neil S, Ben-dov E et al (2002) Complete sequence and organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 68:5082–5095. https://doi.org/10.1128/AEM.68.10.5082

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bravo A, Gill SS, Soberon M (2007) Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49:423–435

    Article  CAS  Google Scholar 

  14. Lacey LA (2007) Bacillus thuringiensis serovariety israelensis and Bacillus sphaericus for mosquito control. J Am Mosq Control Assoc 23:133–163. https://doi.org/10.2987/8756-971X(2007)23[133:BTSIAB]2.0.CO;2

    Article  CAS  PubMed  Google Scholar 

  15. Pérez C, Fernandez LE, Sun J et al (2005) Bacillus thuringiensis subsp. israelensis Cyt1Aa synergizes Cry11Aa toxin by functioning as a membrane-bound receptor. Proc Natl Acad Sci USA 102:18303–18308. https://doi.org/10.1073/pnas.0505494102

    Article  CAS  PubMed  Google Scholar 

  16. Poncet S, Delécluse A, Klier A, Rapoport G (1995) Evaluation of synergistic interactions between the CryIVA, CryIVB and CryIVD toxic components of Bacillus thuringiensis subsp. israelensis crystals. J Invertebr Pathol 66:131–135

    Article  CAS  Google Scholar 

  17. Wirth MC, Georghiou GP, Federici BA (1997) CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus. Proc Natl Acad Sci USA 94(10536):10540. https://doi.org/10.1073/pnas.94.20.10536

    Article  Google Scholar 

  18. Wirth MC, Delécluse A, Federici BA, Walton WE (1998) Variable cross-resistance to Cry11B from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) resistant to single or multiple toxins of Bacillus thuringienisis subsp. israelensis. Appl Environ Microbiol 64:4174–4179. https://doi.org/10.1128/AEM.67.4.1956-1958.2001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Brar SK, Verma M, Tyagi RD, Valéro JR (2006) Recent advances in downstream processing and formulations of Bacillus thuringiensis based biopesticides. Process Biochem 41:323–342

    Article  CAS  Google Scholar 

  20. He J, Wang J, Yin W et al (2011) Complete genome sequence of Bacillus thuringiensis subsp chinensis strain CT-43. J Bacteriol 193:3407–3408. https://doi.org/10.1128/JB.05085-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Doggett NA, Stubben CJ, Chertkov O et al (2013) Complete genome sequence of Bacillus thuringiensis serovar israelensis strain HD-789. Genome Announc 1:1–2. https://doi.org/10.1128/genomeA.01023-13.Copyright

    Article  Google Scholar 

  22. Liu G, Song L, Shu C et al (2013) Complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73. Genome Announc 1:2–3. https://doi.org/10.1128/genomeA.00080-13.Copyright

    Article  Google Scholar 

  23. Murawska E, Fiedoruk K, Bideshi DK, Swiecicka I (2013) Complete genome sequence of Bacillus thuringiensis subsp. thuringiensis strain IS5056, an isolate highly toxic to Trichoplusia ni. Genome Announc 1:e0010813. https://doi.org/10.1128/genomeA.00108-13

    Article  PubMed  Google Scholar 

  24. Johnson SL, Daligault HE, Davenport KW et al (2015) Complete genome sequences for 35 biothreat assay-relevant Bacillus species. Genome Announc 3(2):e00151. https://doi.org/10.1128/genomeA.00151-15

    Article  PubMed  PubMed Central  Google Scholar 

  25. Bolotin A, Gillis A, Sanchis V et al (2017) Comparative genomics of extrachromosomal elements in Bacillus thuringiensis subsp. israelensis. Res Microbiol 168:331–344. https://doi.org/10.1016/j.resmic.2016.10.008

    Article  CAS  PubMed  Google Scholar 

  26. Travers RS, Martin PAW, Reichelderfer CF (1987) Selective process for efficient isolation of soil Bacillus spp. Appl Environ Microbiol 53:1263–1266

    CAS  PubMed  PubMed Central  Google Scholar 

  27. El Khoury M, Azzouz H, Chavanieu A et al (2014) Isolation and characterization of a new Bacillus thuringiensis strain Lip harboring a new cry1Aa gene highly toxic to Ephestia kuehniella (Lepidoptera: Pyralidae) larvae. Arch Microbiol 196:435–445

    Article  Google Scholar 

  28. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  29. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  30. Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379

    Article  Google Scholar 

  31. Thomas WE, Ellar DJ (1983) Mechanism of action of Bacillus thuringiensis var israelensis insecticidal δ-endotoxin. FEBS Lett 154:362–368. https://doi.org/10.1016/0014-5793(83)80183-5

    Article  CAS  PubMed  Google Scholar 

  32. Bertani G (1951) A method for detection of mutations, using streptomycin dependence in Escherichia coli. Genetics 36:598

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Schmieder R, Edwards R (2011) Quality control and preprocessing of metagenomic datasets. Bioinformatics 27:863–864. https://doi.org/10.1093/bioinformatics/btr026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zerbino DR, Birney E (2008) Velvet: Algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829. https://doi.org/10.1101/gr.074492.107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Boetzer M, Henkel CV, Jansen HJ et al (2011) Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27:578–579. https://doi.org/10.1093/bioinformatics/btq683

    Article  CAS  PubMed  Google Scholar 

  36. Darling AE, Mau B, Perna NT (2010) Progressivemauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS ONE 5(6):e11147. https://doi.org/10.1371/journal.pone.0011147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Altschul SF, Madden TL, Schäffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. https://doi.org/10.1093/nar/25.17.3389

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760. https://doi.org/10.1093/bioinformatics/btp324

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Mckenna A, Hanna M, Banks E et al (2009) The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303. https://doi.org/10.1101/gr.107524.110.20

    Article  Google Scholar 

  40. Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu153

    Article  CAS  Google Scholar 

  41. Nawrocki EP, Kolbe DL, Eddy SR (2009) Infernal 1.0: inference of RNA alignments. Bioinformatics 25:1335–1337. https://doi.org/10.1093/bioinformatics/btp157

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Nawrocki EP, Burge SW, Bateman A et al (2015) Rfam 12.0: updates to the RNA families database. Nucleic Acids Res 43:D130–D137. https://doi.org/10.1093/nar/gku1063

    Article  CAS  PubMed  Google Scholar 

  43. Varani AM, Siguier P, Gourbeyre E et al (2011) ISsaga is an ensemble of web-based methods for high throughput identification and semi-automatic annotation of insertion sequences in prokaryotic genomes. Genome Biol 12:R30. https://doi.org/10.1186/gb-2011-12-3-r30

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Crickmore N, Zeigler DR, Schnepf E, van Rie J, Lereclus D, Baum J, Bravo A, Dean DH (1998) Bacillus thuringiensis toxin nomenclature. Microbiol Mol Biol Rev 62:807–813

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Böhm M-E, Huptas C, Krey VM, Scherer S (2015) Massive horizontal gene transfer, strictly vertical inheritance and ancient duplications differentially shape the evolution of Bacillus cereus enterotoxin operons hbl, cytK and nhe. BMC Evol Biol 15:246. https://doi.org/10.1186/s12862-015-0529-4

    Article  PubMed  PubMed Central  Google Scholar 

  46. Price MN, Dehal PS, Arkin AP (2010) FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS ONE 5:e9490. https://doi.org/10.1371/journal.pone.0009490

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Treangen TJ, Ondov BD, Koren S, Phillippy AM (2014) The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes. Genome Biol 15:524. https://doi.org/10.1186/s13059-014-0524-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Croucher NJ, Page AJ, Connor TR et al (2015) Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. Nucleic Acids Res 43:e15. https://doi.org/10.1093/nar/gku1196

    Article  CAS  PubMed  Google Scholar 

  49. Stamatakis A, Ludwig T, Meier H (2005) RAxML-III: a fast program for maximum likelihood-based inference of large phylogenetic trees. Bioinformatics 21:456–463. https://doi.org/10.1093/bioinformatics/bti191

    Article  CAS  PubMed  Google Scholar 

  50. Mahmood F (1998) Laboratory bioassay to compare susceptibilities of Aedes aegypti and Anopheles albimanus to Bacillus thuringiensis var. israelensis as affected by their feeding rates. J Am Mosq Control Assoc 14:69–71

    CAS  PubMed  Google Scholar 

  51. Crickmore N, Bone EJ, Williams JA, Ellar DJ (1995) Contribution of the individual components of the δ-endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis. FEMS Microbiol Lett 131:249–254. https://doi.org/10.1016/0378-1097(95)00264-6

    Article  CAS  Google Scholar 

  52. Zghal RZ, Tounsi S, Jaoua S (2006) Characterization of a cry4Ba-type gene of Bacillus thuringiensis israelensis and evidence of the synergistic larvicidal activity of its encoded protein with Cry2A delta-endotoxin of B. thuringiensis kurstaki on Culex pipiens (common house mosquito). Biotechnol Appl Biochem 44:19–25. https://doi.org/10.1042/BA20050134

    Article  CAS  PubMed  Google Scholar 

  53. Gillis A, Fayad N, Makart L et al (2018) Role of plasmid plasticity and mobile genetic elements in the entomopathogen Bacillus thuringiensis serovar israelensis. FEMS Microbiol Rev 42:829–856. https://doi.org/10.1093/femsre/fuy034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Makart L, Gillis A, Mahillon J (2015) PXO16 from Bacillus thuringiensis serovar israelensis: almost 350 kb of terra incognita. Plasmid 80:8–15. https://doi.org/10.1016/j.plasmid.2015.03.002

    Article  CAS  PubMed  Google Scholar 

  55. Gillis A, Guo S, Bolotin A et al (2017) Detection of the cryptic prophage-like molecule pBtic235 in Bacillus thuringiensis subsp. israelensis. Res Microbiol 168:319–330. https://doi.org/10.1016/j.resmic.2016.10.004

    Article  CAS  PubMed  Google Scholar 

  56. Makart L, Gillis A, Hinnekens P, Mahillon J (2018) A novel T4SS-mediated DNA transfer used by pXO16, a conjugative plasmid from Bacillus thuringiensis serovar israelensis. Environ Microbiol 20:1550–1561. https://doi.org/10.1111/1462-2920.14084

    Article  CAS  PubMed  Google Scholar 

  57. San Millan A, Toll-Riera M, Qi Q et al (2018) Integrative analysis of fitness and metabolic effects of plasmids in Pseudomonas aeruginosa PAO1. ISME J. https://doi.org/10.1038/s41396-018-0224-8

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors would like to thank Mr. Benoit Queffelec for his assistance in the statistical analysis of the bioassays.

Funding

This study was funded by the Lebanese national council for scientific research (CNRS-L) and Université Montpellier 2, through Grants to Nancy Fayad and Mandy Antoun and the research council of Saint-Joseph University through Grant FS59 funded the experimental work. Zakaria Kambris acknowledges AUB-URB support.

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    Authors and Affiliations

    1. Laboratory of Biodiversity and Functional Genomics, Faculty of Science, Université Saint-Joseph de Beyrouth, Beirut, Lebanon

      Nancy Fayad, Mandy Antoun & Mireille Kallassy Awad

    2. Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350, Jouy-en-Josas, France

      Rafael Patiño-Navarrete & Vincent Sanchis

    3. Biology Department, Faculty of Arts and Sciences, American University of Beirut, Beirut, Lebanon

      Zakaria Kambris & Mike Osta

    4. Institut Charles Gerhardt de Montpellier (ICGM), CNRS UMR 5253/UM/ENSCM Université de Montpellier Campus Triolet, Place Eugène Bataillon, 34095, Montpellier Cedex 5, France

      Mandy Antoun & Joel Chopineau

    5. Laboratory of Food and Environmental Microbiology, Earth and Life Institute, Université Catholique de Louvain, Louvain-la-Neuve, Belgium

      Nancy Fayad & Jacques Mahillon

    6. Génétique de La Drosophile Et Virulence Microbienne (GDVM), Faculty of Science, Université Saint-Joseph de Beyrouth, Beirut, Lebanon

      Laure El Chamy

    7. Université de Nîmes, Rue Georges Salan, 30000, Nîmes, France

      Mandy Antoun & Joel Chopineau

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    1. Nancy Fayad
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    Fayad, N., Patiño-Navarrete, R., Kambris, Z. et al. Characterization and Whole Genome Sequencing of AR23, a Highly Toxic Bacillus thuringiensis Strain Isolated from Lebanese Soil. Curr Microbiol 76, 1503–1511 (2019). https://doi.org/10.1007/s00284-019-01775-9

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