1887

Abstract

serovar is the most widely used natural biopesticide against mosquito larvae worldwide. Its lineage has been actively studied and a plasmid-free strain, . serovar BGSC 4Q7 (4Q7), has been produced. Previous sequencing of the genome of this strain has revealed the persistent presence of a 235 kb extrachromosomal element, pBtic235, which has been shown to be an inducible prophage, although three putative chromosomal prophages have been lost. Moreover, a 492 kb region, potentially including the standard replication terminus, has also been deleted in the 4Q7 strain, indicating an absence of essential genes in this area. We reanalysed the genome coverage distribution of reads for the previously sequenced variant strain, and sequenced two independently maintained samples of the 4Q7 strain. A 553 kb area, close to the 492 kb deletion, was found to be duplicated. This duplication presumably restored the equal sizes of the replichores, and a balanced functioning of replication termination. An analysis of genome assembly graphs revealed a transient association of the host chromosome with the pBtic235 element. This association may play a functional role in the replication of the bacterial chromosome, and the termination of this process in particular. The genome-restructuring events detected may modify the genetic status of cytotoxic or haemolytic toxins, potentially influencing strain virulence. Twelve of the single-nucleotide variants identified in 4Q7 were probably due to the procedure used for strain construction or were present in the precursor of this strain. No sequence variants were found in pBtic235, but the distribution of the corresponding 4Q7 reads indicates a significant difference from counterparts in natural serovar strains, suggesting a duplication or over-replication in 4Q7. Thus, the 4Q7 strain is not a pure plasmid-less offshoot, but a highly genetically modified derivative of its natural ancestor. In addition to potentially influencing virulence, genome-restructuring events can modify the replication termination machinery. These findings have potential implications for the conclusions of virulence studies on 4Q7 as a model, but they also raise interesting fundamental questions about the functioning of the genome.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.000468
2020-11-12
2021-10-21
Loading full text...

Full text loading...

/deliver/fulltext/mgen/6/12/mgen000468.html?itemId=/content/journal/mgen/10.1099/mgen.0.000468&mimeType=html&fmt=ahah

References

  1. Jeong H, Park S-H, Choi S-K. Genome sequence of the acrystalliferous Bacillus thuringiensis serovar israelensis strain 4Q7, widely used as a recombination host. Genome Announc 2014; 2:e00231-14 [View Article][PubMed]
    [Google Scholar]
  2. Poornima K, Saranya V, Abirami P, Binuramesh C, Suguna P et al. Phenotypic and genotypic characterization of B.t.LDC-391 strain that produce cytocidal proteins against human cancer cells. Bioinformation 2012; 8:461–465 [View Article][PubMed]
    [Google Scholar]
  3. Poornima K, Selvanayagam P, Shenbagarathai R. Identification of native Bacillus thuringiensis strain from South India having specific cytocidal activity against cancer cells. J Appl Microbiol 2010; 109:348–354 [View Article][PubMed]
    [Google Scholar]
  4. Raymond B, Federici BA. In defense of Bacillus thuringiensis, the safest and most successful microbial insecticide available to humanity - a response to EFSA. FEMS Microbiol Ecol 2017; 93:fix084 [View Article][PubMed]
    [Google Scholar]
  5. Ben-Dov E. Bacillus thuringiensis subsp. israelensis and its dipteran-specific toxins. Toxins 2014; 6:1222–1243 [View Article][PubMed]
    [Google Scholar]
  6. Margalit J, Dean D. The story of Bacillus thuringiensis var. israelensis (B.t.i.). J Am Mosq Control Assoc 1985; 1:1–7[PubMed]
    [Google Scholar]
  7. Otieno-Ayayo ZN, Zaritsky A, Wirth MC, Manasherob R, Khasdan V et al. Variations in the mosquito larvicidal activities of toxins from Bacillus thuringiensis ssp. israelensis . Environ Microbiol 2008; 10:2191–2199 [View Article][PubMed]
    [Google Scholar]
  8. Monnerat R, Pereira E, Teles B, Martins E, Praça L et al. Synergistic activity of Bacillus thuringiensis toxins against Simulium spp. larvae. J Invertebr Pathol 2014; 121:70–73 [View Article][PubMed]
    [Google Scholar]
  9. Hu X, Hansen BM, Yuan Z, Johansen JE, Eilenberg J et al. Transfer and expression of the mosquitocidal plasmid pBtoxis in Bacillus cereus group strains. FEMS Microbiol Lett 2005; 245:239–247 [View Article][PubMed]
    [Google Scholar]
  10. Jensen GB, Wilcks A, Petersen SS, Damgaard J, Baum JA et al. The genetic basis of the aggregation system in Bacillus thuringiensis subsp. israelensis is located on the large conjugative plasmid pXO16. J Bacteriol 1995; 177:2914–2917 [View Article][PubMed]
    [Google Scholar]
  11. Makart L, Gillis A, Hinnekens P, Mahillon J. A novel T4SS-mediated DNA transfer used by pXO16, a conjugative plasmid from Bacillus thuringiensis serovar israelensis . Environ Microbiol 2018; 20:1550–1561 [View Article][PubMed]
    [Google Scholar]
  12. Bolotin A, Gillis A, Sanchis V, Nielsen-LeRoux C, Mahillon J et al. Comparative genomics of extrachromosomal elements in Bacillus thuringiensis subsp. israelensis . Res Microbiol 2017; 168:331–344 [View Article][PubMed]
    [Google Scholar]
  13. Gillis A, Guo S, Bolotin A, Makart L, Sorokin A et al. Detection of the cryptic prophage-like molecule pBtic235 in Bacillus thuringiensis subsp. israelensis . Res Microbiol 2017; 168:319–330 [View Article][PubMed]
    [Google Scholar]
  14. Gillis A, Fayad N, Makart L, Bolotin A, Sorokin A et al. Role of plasmid plasticity and mobile genetic elements in the entomopathogen Bacillus thuringiensis serovar israelensis . FEMS Microbiol Rev 2018; 42:829–856 [View Article][PubMed]
    [Google Scholar]
  15. Doggett NA, Stubben CJ, Chertkov O, Bruce DC, Detter JC et al. Complete genome sequence of Bacillus thuringiensis serovar israelensis strain HD-789. Genome Announc 2013; 1:e01023-13 [View Article][PubMed]
    [Google Scholar]
  16. Johnson SL, Daligault HE, Davenport KW, Jaissle J, Frey KG et al. Complete genome sequences for 35 biothreat assay-relevant Bacillus species. Genome Announc 2015; 3:e00151-15 [View Article][PubMed]
    [Google Scholar]
  17. Dohm JC, Lottaz C, Borodina T, Himmelbauer H. Substantial biases in ultra-short read data sets from high-throughput DNA sequencing. Nucleic Acids Res 2008; 36:e105 [View Article][PubMed]
    [Google Scholar]
  18. Minoche AE, Dohm JC, Himmelbauer H. Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and genome analyzer systems. Genome Biol 2011; 12:R112 [View Article][PubMed]
    [Google Scholar]
  19. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012; 19:455–477 [View Article][PubMed]
    [Google Scholar]
  20. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009; 25:2078–2079 [View Article][PubMed]
    [Google Scholar]
  21. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 2017; 13:e1005595 [View Article][PubMed]
    [Google Scholar]
  22. Wattam AR, Davis JJ, Assaf R, Boisvert S, Brettin T et al. Improvements to PATRIC, the all-bacterial bioinformatics database and analysis resource center. Nucleic Acids Res 2017; 45:D535–D542 [View Article][PubMed]
    [Google Scholar]
  23. Afgan E, Baker D, Batut B, van den Beek M, Bouvier D et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res 2018; 46:W537–W544 [View Article][PubMed]
    [Google Scholar]
  24. Milne I, Stephen G, Bayer M, Cock PJA, Pritchard L et al. Using Tablet for visual exploration of second-generation sequencing data. Brief Bioinform 2013; 14:193–202 [View Article][PubMed]
    [Google Scholar]
  25. Grant JR, Arantes AS, Stothard P. Comparing thousands of circular genomes using the CGView comparison tool. BMC Genomics 2012; 13:202 [View Article][PubMed]
    [Google Scholar]
  26. Wick RR, Schultz MB, Zobel J, Holt KE. Bandage: interactive visualization of de novo genome assemblies. Bioinformatics 2015; 31:3350–3352 [View Article][PubMed]
    [Google Scholar]
  27. R Core Team R: a Language and Environment for Statistical Computing 2014 http://www.R-project.org
  28. Markowitz VM, Chen I-MA, Palaniappan K, Chu K, Szeto E et al. The integrated microbial genomes system: an expanding comparative analysis resource. Nucleic Acids Res 2010; 38:D382–D390 [View Article][PubMed]
    [Google Scholar]
  29. Rocha EPC. The replication-related organization of bacterial genomes. Microbiology 2004; 150:1609–1627 [View Article][PubMed]
    [Google Scholar]
  30. Merrikh H, Zhang Y, Grossman AD, Wang JD. Replication-transcription conflicts in bacteria. Nat Rev Microbiol 2012; 10:449–458 [View Article][PubMed]
    [Google Scholar]
  31. Merrikh H, Machón C, Grainger WH, Grossman AD, Soultanas P. Co-directional replication-transcription conflicts lead to replication restart. Nature 2011; 470:554–557 [View Article][PubMed]
    [Google Scholar]
  32. Hendrickson H, Lawrence JG. Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites. Mol Microbiol 2007; 64:42–56 [View Article][PubMed]
    [Google Scholar]
  33. Cui T, Moro-oka N, Ohsumi K, Kodama K, Ohshima T et al. Escherichia coli with a linear genome. EMBO Rep 2007; 8:181–187 [View Article][PubMed]
    [Google Scholar]
  34. Clark BD, Boyle TM, Chu CY, Dean DH. Restriction endonuclease mapping of three plasmids from Bacillus thuringiensis var. israelensis . Gene 1985; 36:169–171 [View Article][PubMed]
    [Google Scholar]
  35. Goldberg LJ, Margalit J. A bacterial spore demonstrating rapid larvicidal activity against Anopheles sargentii, Uranotaenia ungiculata, Culex univittatus, Aedes aegypti, and Culex pipiens . Mosq News 1977; 37:355–358
    [Google Scholar]
  36. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 2017; 27:722–736 [View Article][PubMed]
    [Google Scholar]
  37. Fayad N, Patiño-Navarrete R, Kambris Z, Antoun M, Osta M et al. Characterization and whole genome sequencing of AR23, a highly toxic Bacillus thuringiensis strain isolated from Lebanese soil. Curr Microbiol 2019; 76:15031511 [View Article][PubMed]
    [Google Scholar]
  38. Dewar JM, Walter JC. Mechanisms of DNA replication termination. Nat Rev Mol Cell Biol 2017; 18:507–516 [View Article][PubMed]
    [Google Scholar]
  39. Wake RG. Replication fork arrest and termination of chromosome replication in Bacillus subtilis . FEMS Microbiol Lett 1997; 153:247–254 [View Article][PubMed]
    [Google Scholar]
  40. El Kafsi H, Loux V, Mariadassou M, Blin C, Chiapello H et al. Unprecedented large inverted repeats at the replication terminus of circular bacterial chromosomes suggest a novel mode of chromosome rescue. Sci Rep 2017; 7:44331 [View Article][PubMed]
    [Google Scholar]
  41. Moumen B, Nguen-The C, Sorokin A. Sequence analysis of inducible prophage phIS3501 integrated into the haemolysin II gene of Bacillus thuringiensis var israelensis ATCC35646. Genet Res Int 2012; 2012:543286 [View Article][PubMed]
    [Google Scholar]
  42. Granum PE, O'Sullivan K, Lund T. The sequence of the non-haemolytic enterotoxin operon from Bacillus cereus . FEMS Microbiol Lett 1999; 177:225–229 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/mgen/10.1099/mgen.0.000468
Loading
/content/journal/mgen/10.1099/mgen.0.000468
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error