1887

Abstract

pBsph is a mosquitocidal plasmid first identified from , encoding binary toxins (Bin toxins) that are highly toxic to mosquito larvae. This plasmid plays an important role in the maintenance and evolution of the genes in . However, little is known about its replication and partitioning. Here, we identified a 2.4 kb minimal replicon of pBsph plasmid that contained an operon encoding TubR-Bs and TubZ-Bs, each of which was shown to be required for plasmid replication. TubR-Bs was shown to be a transcriptional repressor of -Bs genes and could bind cooperatively to the replication origin of eleven 12 bp degenerate repeats in three blocks, and this binding was essential for plasmid replication. TubZ-Bs exhibited GTPase activities and interacted with TubR-Bs : DNA complex to form a ternary nucleoprotein apparatus. Electron and fluorescence microscopy revealed that TubZ-Bs assembled filaments both and , and two point mutations in TubZ-Bs (T114A and Y260A) that severely impaired the GTPase and polymerization activities were found to be defective for plasmid maintenance. Further investigation demonstrated that overproduction of TubZ-Bs-GFP or its mutant forms significantly reduced the stability of pBsph. Taken together, these results suggested that TubR-Bs and TubZ-Bs are involved in the replication and probably in the partitioning of pBsph plasmid, increasing our understanding of the genetic particularity of TubZ systems.

Funding
This study was supported by the:
  • NFSC (Award 31272384 and 30800002)
  • 973 (Award 2009CB118902)
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.075465-0
2014-06-01
2021-08-05
Loading full text...

Full text loading...

/deliver/fulltext/micro/160/6/1112.html?itemId=/content/journal/micro/10.1099/mic.0.075465-0&mimeType=html&fmt=ahah

References

  1. Akhtar P., Khan S. A. ( 2012). Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis.. Plasmid 67:111–117 [View Article][PubMed]
    [Google Scholar]
  2. Akhtar P., Anand S. P., Watkins S. C., Khan S. A. ( 2009). The tubulin-like RepX protein encoded by the pXO1 plasmid forms polymers in vivo in Bacillus anthracis.. J Bacteriol 191:2493–2500 [View Article][PubMed]
    [Google Scholar]
  3. Anand S. P., Akhtar P., Tinsley E., Watkins S. C., Khan S. A. ( 2008). GTP-dependent polymerization of the tubulin-like RepX replication protein encoded by the pXO1 plasmid of Bacillus anthracis.. Mol Microbiol 67:881–890 [View Article][PubMed]
    [Google Scholar]
  4. Aylett C. H., Löwe J. ( 2012). Superstructure of the centromeric complex of TubZRC plasmid partitioning systems. Proc Natl Acad Sci U S A 109:16522–16527 [View Article][PubMed]
    [Google Scholar]
  5. Aylett C. H., Wang Q., Michie K. A., Amos L. A., Löwe J. ( 2010). Filament structure of bacterial tubulin homologue TubZ. Proc Natl Acad Sci U S A 107:19766–19771 [View Article][PubMed]
    [Google Scholar]
  6. Aylett C. H., Izoré T., Amos L. A., Löwe J. ( 2013). Structure of the tubulin/FtsZ-like protein TubZ from Pseudomonas bacteriophage ΦKZ. J Mol Biol 425:2164–2173 [View Article][PubMed]
    [Google Scholar]
  7. Barillà D. ( 2010). One-way ticket to the cell pole: plasmid transport by the prokaryotic tubulin homolog TubZ. Proc Natl Acad Sci U S A 107:12061–12062 [View Article][PubMed]
    [Google Scholar]
  8. Baumann L., Broadwell A. H., Baumann P. ( 1988). Sequence analysis of the mosquitocidal toxin genes encoding 51.4- and 41.9-kilodalton proteins from Bacillus sphaericus 2362 and 2297. J Bacteriol 170:2045–2050[PubMed]
    [Google Scholar]
  9. Berry C. ( 2012). The bacterium, Lysinibacillus sphaericus, as an insect pathogen. J Invertebr Pathol 109:1–10 [View Article][PubMed]
    [Google Scholar]
  10. Betteridge T., Yang J., Pittard A. J., Praszkier J. ( 2004). Role of RepA and DnaA proteins in the opening of the origin of DNA replication of an IncB plasmid. J Bacteriol 186:3785–3793 [View Article][PubMed]
    [Google Scholar]
  11. Chung K. M., Hsu H. H., Yeh H. Y., Chang B. Y. ( 2007). Mechanism of regulation of prokaryotic tubulin-like GTPase FtsZ by membrane protein EzrA. J Biol Chem 282:14891–14897 [View Article][PubMed]
    [Google Scholar]
  12. Gasset-Rosa F., Díaz-López T., Lurz R., Prieto A., Fernández-Tresguerres M. E., Giraldo R. ( 2008). Negative regulation of pPS10 plasmid replication: origin pairing by zipping-up DNA-bound RepA monomers. Mol Microbiol 68:560–572 [View Article][PubMed]
    [Google Scholar]
  13. Gayathri P., Fujii T., Møller-Jensen J., van den Ent F., Namba K., Löwe J. ( 2012). A bipolar spindle of antiparallel ParM filaments drives bacterial plasmid segregation. Science 338:1334–1337 [View Article][PubMed]
    [Google Scholar]
  14. Ge Y., Hu X. M., Zheng D. S., Wu Y. M., Yuan Z. M. ( 2011). Allelic diversity and population structure of Bacillus sphaericus as revealed by multilocus sequence typing. Appl Environ Microbiol 77:5553–5556 [View Article][PubMed]
    [Google Scholar]
  15. Gerdes K., Howard M., Szardenings F. ( 2010). Pushing and pulling in prokaryotic DNA segregation. Cell 141:927–942 [View Article][PubMed]
    [Google Scholar]
  16. Hindley J., Berry C. ( 1987). Identification, cloning and sequence analysis of the Bacillus sphaericus 1593 41.9 kD larvicidal toxin gene. Mol Microbiol 1:187–194 [View Article][PubMed]
    [Google Scholar]
  17. Ho T. D., Hastie J. L., Intile P. J., Ellermeier C. D. ( 2011). The Bacillus subtilis extracytoplasmic function σ factor σV is induced by lysozyme and provides resistance to lysozyme. J Bacteriol 193:6215–6222 [View Article][PubMed]
    [Google Scholar]
  18. Hoshino S., Hayashi I. ( 2012). Filament formation of the FtsZ/tubulin-like protein TubZ from the Bacillus cereus pXO1 plasmid. J Biol Chem 287:32103–32112 [View Article][PubMed]
    [Google Scholar]
  19. Hu X. M., Hansen B. M., Yuan Z. M., Johansen J. E., Eilenberg J., Hendriksen N. B., Smidt L., Jensen G. B. ( 2005). Transfer and expression of the mosquitocidal plasmid pBtoxis in Bacillus cereus group strains. FEMS Microbiol Lett 245:239–247 [View Article][PubMed]
    [Google Scholar]
  20. Hu X. M., Fan W., Han B., Liu H., Zheng D., Li Q., Dong W., Yan J., Gao M. & other authors ( 2008). Complete genome sequence of the mosquitocidal bacterium Bacillus sphaericus C3-41 and comparison with those of closely related Bacillus species. J Bacteriol 190:2892–2902 [View Article][PubMed]
    [Google Scholar]
  21. Huang J., Guo S., Mahillon J., Van der Auwera G. A., Wang L., Han D., Yu Z., Sun M. ( 2006). Molecular characterization of a DNA fragment harboring the replicon of pBMB165 from Bacillus thuringiensis subsp. tenebrionis.. BMC Genomics 7:270 [View Article][PubMed]
    [Google Scholar]
  22. Kraemer J. A., Erb M. L., Waddling C. A., Montabana E. A., Zehr E. A., Wang H., Nguyen K., Pham D. S. L., Agard D. A., Pogliano J. ( 2012). A phage tubulin assembles dynamic filaments by an atypical mechanism to center viral DNA within the host cell. Cell 149:1488–1499 [View Article][PubMed]
    [Google Scholar]
  23. Larsen R. A., Cusumano C., Fujioka A., Lim-Fong G., Patterson P., Pogliano J. ( 2007). Treadmilling of a prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis.. Genes Dev 21:1340–1352 [View Article][PubMed]
    [Google Scholar]
  24. Lee C., Kim J., Shin S. G., Hwang S. ( 2006). Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli.. J Biotechnol 123:273–280 [View Article][PubMed]
    [Google Scholar]
  25. Li J., Huang C. Y., Zheng D. S., Wang Y. L., Yuan Z. M. ( 2012). CcpA-mediated enhancement of sugar and amino acid metabolism in Lysinibacillus sphaericus by NMR-based metabolomics. J Proteome Res 11:4654–4661 [View Article][PubMed]
    [Google Scholar]
  26. Ni L. S., Xu W. J., Kumaraswami M., Schumacher M. A. ( 2010). Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition. Proc Natl Acad Sci U S A 107:11763–11768 [View Article][PubMed]
    [Google Scholar]
  27. Oliva M. A., Martin-Galiano A. J., Sakaguchi Y., Andreu J. M. ( 2012). Tubulin homolog TubZ in a phage-encoded partition system. Proc Natl Acad Sci U S A 109:7711–7716 [View Article][PubMed]
    [Google Scholar]
  28. Poncet S., Bernard C., Dervyn E., Cayley J., Klier A., Rapoport G. ( 1997). Improvement of Bacillus sphaericus toxicity against dipteran larvae by integration, via homologous recombination, of the Cry11A toxin gene from Bacillus thuringiensis subsp. israelensis.. Appl Environ Microbiol 63:4413–4420[PubMed]
    [Google Scholar]
  29. Réjasse A., Gilois N., Barbosa I., Huillet E., Bevilacqua C., Tran S., Ramarao N., Stenfors Arnesen L. P., Sanchis V. ( 2012). Temperature-dependent production of various PlcR-controlled virulence factors in Bacillus weihenstephanensis strain KBAB4. Appl Environ Microbiol 78:2553–2561 [View Article][PubMed]
    [Google Scholar]
  30. Tang M., Bideshi D. K., Park H. W., Federici B. A. ( 2006). Minireplicon from pBtoxis of Bacillus thuringiensis subsp. israelensis.. Appl Environ Microbiol 72:6948–6954 [View Article][PubMed]
    [Google Scholar]
  31. Tang M., Bideshi D. K., Park H. W., Federici B. A. ( 2007). Iteron-binding ORF157 and FtsZ-like ORF156 proteins encoded by pBtoxis play a role in its replication in Bacillus thuringiensis subsp. israelensis.. J Bacteriol 189:8053–8058 [View Article][PubMed]
    [Google Scholar]
  32. Tinsley E., Khan S. A. ( 2006). A novel FtsZ-like protein is involved in replication of the anthrax toxin-encoding pXO1 plasmid in Bacillus anthracis.. J Bacteriol 188:2829–2835 [View Article][PubMed]
    [Google Scholar]
  33. Vecchiarelli A. G., Hwang L. C., Mizuuchi K. ( 2013). Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism. Proc Natl Acad Sci U S A 110:E1390–E1397 [View Article][PubMed]
    [Google Scholar]
  34. Vimberg V., Tats A., Remm M., Tenson T. ( 2007). Translation initiation region sequence preferences in Escherichia coli.. BMC Mol Biol 8:100 [View Article][PubMed]
    [Google Scholar]
  35. Wu E. Y., Jun L., Yuan Y. M., Yan J. P., Berry C., Yuan Z. M. ( 2007). Characterization of a cryptic plasmid from Bacillus sphaericus strain LP1-G. Plasmid 57:296–305 [View Article][PubMed]
    [Google Scholar]
  36. Wu Y., Hu X., Ge Y., Zheng D., Yuan Z. ( 2012). Generation of mariner-based transposon insertion mutant library of Bacillus sphaericus 2297 and investigation of genes involved in sporulation and mosquito-larvicidal crystal protein synthesis. FEMS Microbiol Lett 330:105–112 [View Article][PubMed]
    [Google Scholar]
  37. Yamaichi Y., Gerding M. A., Davis B. M., Waldor M. K. ( 2011). Regulatory cross-talk links Vibrio cholerae chromosome II replication and segregation. PLoS Genet 7:e1002189 [View Article][PubMed]
    [Google Scholar]
  38. Zampini M., Derome A., Bailey S. E., Barillà D., Hayes F. ( 2009). Recruitment of the ParG segregation protein to different affinity DNA sites. J Bacteriol 191:3832–3841 [View Article][PubMed]
    [Google Scholar]
  39. Zhang W., Wang Y., Song Y., Wang T., Xu S., Peng Z., Lin X., Zhang L., Shen X. ( 2013). A type VI secretion system regulated by OmpR in Yersinia pseudotuberculosis functions to maintain intracellular pH homeostasis. Environ Microbiol 15:557–569 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.075465-0
Loading
/content/journal/micro/10.1099/mic.0.075465-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
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