1887

Abstract

Partition systems contribute to stable plasmid inheritance in bacteria through the active separation of DNA molecules to daughter cells, and the centromeric sequence located either upstream or downstream of canonical partition operons plays an important role in this process. A specific DNA-binding protein binds to this sequence and interacts with the motor NTPase protein to form a nucleoprotein complex. The inc18-family plasmid pSM19035 is partitioned by products of and genes, with encoding a Walker-type ATPase and encoding a DNA-binding protein. As the two genes are transcribed separately, this system differs from others in its organization; nonetheless, expression of these genes is regulated by Omega, which also regulates the copy number of the plasmid (by controlling gene expression). Protein Omega specifically recognizes WATCACW heptad repeats. In this study, we constructed a synthetic operon to enable an analysis of the centromeric functions of Omega-binding sites , and , discrete from their promoter functions. Our results show that these three regions do not support plasmid stabilization equally. We demonstrate that the site alone can simultaneously drive the expression of partition genes from the synthetic operon and act as a unique centromeric sequence to promote the most efficient plasmid partitioning. Moreover, can support the centromeric function in concert with the synthetic operon expressed from a heterologous promoter demonstrating that is the main centromeric sequence of the partition system. Additionally, the RNA polymerase-recognized sequence in is essential for its centromeric function.

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2016-07-01
2024-03-28
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References

  1. Bartosik A. A., Jagura-Burdzy G. 2005; Bacterial chromosome segregation. Acta Biochim Pol 52:1–34[PubMed]
    [Google Scholar]
  2. Breier A. M., Grossman A. D. 2007; Whole-genome analysis of the chromosome partitioning and sporulation protein Spo0J (ParB) reveals spreading and origin-distal sites on the Bacillus subtilis chromosome. Mol Microbiol 64:703–718 [View Article][PubMed]
    [Google Scholar]
  3. Cegłowski P., Boitsov A., Chai S., Alonso J. C. 1993; Analysis of the stabilization system of pSM19035-derived plasmid pBT233 in Bacillus subtilis . Gene 136:1–12 [View Article][PubMed]
    [Google Scholar]
  4. Chomczynski P. 1993; A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15:532–537[PubMed]
    [Google Scholar]
  5. de la Hoz A. B., Ayora S., Sitkiewicz I., Fernández S., Pankiewicz R., Alonso J. C., Ceglowski P. 2000; Plasmid copy-number control and better-than-random segregation genes of pSM19035 share a common regulator. Proc Natl Acad Sci U S A 97:728–733 [View Article][PubMed]
    [Google Scholar]
  6. de la Hoz A. B., Pratto F., Misselwitz R., Speck C., Weihofen W., Welfle K., Saenger W., Welfle H., Alonso J. C. 2004; Recognition of DNA by omega protein from the broad-host range Streptococcus pyogenes plasmid pSM19035: analysis of binding to operator DNA with one to four heptad repeats. Nucleic Acids Res 32:3136–3147 [View Article][PubMed]
    [Google Scholar]
  7. Dmowski M., Jagura-Burdzy G. 2013a; Active stable maintenance functions in low copy-number plasmids of Gram-positive bacteria I. Partition systems. Pol J Microbiol 62:3–16
    [Google Scholar]
  8. Dmowski M., Jagura-Burdzy G. 2013b; Active stable maintenance functions in low copy-number plasmids of Gram-positive bacteria II. Post-segregational killing systems. Pol J Microbiol 62:17–22
    [Google Scholar]
  9. Dmowski M., Sitkiewicz I., Ceglowski P. 2006; Characterization of a novel partition system encoded by the delta and omega genes from the streptococcal plasmid pSM19035. J Bacteriol 188:4362–4372 [View Article][PubMed]
    [Google Scholar]
  10. Dworkin J., Losick R. 2002; Does RNA polymerase help drive chromosome segregation in bacteria?. Proc Natl Acad Sci U S A 99:14089–14094 [View Article][PubMed]
    [Google Scholar]
  11. Friedman B. M., Yasbin R. E. 1983; The genetics and specificity of the constitutive excision repair system of Bacillus subtilis . Mol Gen Genet 190:481–486 [View Article][PubMed]
    [Google Scholar]
  12. Gerdes K., Møller-Jensen J., Bugge Jensen R. 2000; Plasmid and chromosome partitioning: surprises from phylogeny. Mol Microbiol 37:455–466 [View Article][PubMed]
    [Google Scholar]
  13. Gerdes K., Howard M., Szardenings F. 2010; Pushing and pulling in prokaryotic DNA segregation. Cell 141:927–942 [View Article][PubMed]
    [Google Scholar]
  14. Grigoriev P. S., Lobocka M. B. 2001; Determinants of segregational stability of the linear plasmid-prophage N15 of Escherichia coli . Mol Microbiol 42:355–368 [View Article][PubMed]
    [Google Scholar]
  15. Hanahan D., Harbor C. S. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580 [View Article][PubMed]
    [Google Scholar]
  16. Havey J. C., Vecchiarelli A. G., Funnell B. E. 2012; ATP-regulated interactions between P1 ParA, ParB and non-specific DNA that are stabilized by the plasmid partition site, parS . Nucleic Acids Res 40:801–812 [View Article][PubMed]
    [Google Scholar]
  17. Jecz P., Bartosik A. A., Glabski K., Jagura-Burdzy G. 2015; A single parS sequence from the cluster of four sites closest to oriC is necessary and sufficient for proper chromosome segregation in Pseudomonas aeruginosa . PLoS One 10:e0120867 [View Article][PubMed]
    [Google Scholar]
  18. Kjos M., Veening J. W. 2014; Tracking of chromosome dynamics in live Streptococcus pneumoniae reveals that transcription promotes chromosome segregation. Mol Microbiol 91:1088–1105 [View Article][PubMed]
    [Google Scholar]
  19. Koonin E. V. 1993; A superfamily of ATPases with diverse functions containing either classical or deviant ATP-binding motif. J Mol Biol 229:1165–1174 [View Article][PubMed]
    [Google Scholar]
  20. Kulinska A., Czeredys M., Hayes F., Jagura-Burdzy G. 2008; Genomic and functional characterization of the modular broad-host-range RA3 plasmid, the archetype of the IncU group. Appl Environ Microbiol 74:4119–4132 [View Article][PubMed]
    [Google Scholar]
  21. Kulinska A., Cao Y., Macioszek M., Hayes F., Jagura-Burdzy G. 2011; The centromere site of the segregation cassette of broad-host-range plasmid RA3 is located at the border of the maintenance and conjugative transfer modules. Appl Environ Microbiol 77:2414–2427 [View Article][PubMed]
    [Google Scholar]
  22. Le T. B., Imakaev M. V., Mirny L. A., Laub M. T. 2013; High-resolution mapping of the spatial organization of a bacterial chromosome. Science 342:731–734 [View Article][PubMed]
    [Google Scholar]
  23. Liu Y. F., Wang C. H., Janapatla R. P., Fu H. M., Wu H. M., Wu J. J. 2007; Presence of plasmid pA15 correlates with prevalence of constitutive MLS(B) resistance in group A streptococcal isolates at a university hospital in southern Taiwan. J Antimicrob Chemother 59:1167–1170 [View Article][PubMed]
    [Google Scholar]
  24. Livny J., Yamaichi Y., Waldor M. K. 2007; Distribution of centromere-like parS sites in bacteria: insights from comparative genomics. J Bacteriol 189:8693–8703 [View Article][PubMed]
    [Google Scholar]
  25. Matsumoto K., Hara H., Fishov I., Mileykovskaya E., Norris V. 2015; The membrane: transertion as an organizing principle in membrane heterogeneity. Microb Physiol Metab 6:572 [View Article][PubMed]
    [Google Scholar]
  26. Møller-Jensen J., Gerdes K. 2007; Plasmid segregation: spatial awareness at the molecular level. J Cell Biol 179:813–815 [View Article][PubMed]
    [Google Scholar]
  27. O'Connor E. B., O'Sullivan O., Stanton C., Danielsen M., Simpson P. J., Callanan M. J., Ross R. P., Hill C. 2007; pEOC01: a plasmid from Pediococcus acidilactici which encodes an identical streptomycin resistance (aadE) gene to that found in Campylobacter jejuni . Plasmid 58:115–126 [View Article][PubMed]
    [Google Scholar]
  28. Ravin N., Lane D. 1999; Partition of the linear plasmid N15: interactions of N15 partition functions with the sop locus of the F plasmid. J Bacteriol 181:6898–6906[PubMed]
    [Google Scholar]
  29. Rottländer E., Trautner T. A. 1970; Genetic and transfection studies with B.s ubtilis phage SP 50 . Mol Gen Genet 108:47–60 [CrossRef]
    [Google Scholar]
  30. Sambrook J., Russell D. W. 2001 Molecular Cloning: a Laboratory Manual , 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  31. Schumacher M. A. 2012; Bacterial plasmid partition machinery: a minimalist approach to survival. Curr Opin Struct Biol 22:72–79 [View Article][PubMed]
    [Google Scholar]
  32. Sengupta M., Austin S. 2011; Prevalence and significance of plasmid maintenance functions in the virulence plasmids of pathogenic bacteria. Infect Immun 79:2502–2509 [View Article][PubMed]
    [Google Scholar]
  33. Stracy M., Lesterlin C., Garza de Leon F., Uphoff S., Zawadzki P., Kapanidis A. N. 2015; Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid. Proc Natl Acad Sci U S A 112:E4390E4399 [View Article][PubMed]
    [Google Scholar]
  34. Taylor J. A., Pastrana C. L., Butterer A., Pernstich C., Gwynn E. J., Sobott F., Moreno-Herrero F., Dillingham M. S. 2015; Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation. Nucleic Acids Res 43:719–731 [View Article][PubMed]
    [Google Scholar]
  35. Vellanoweth R. L., Rabinowitz J. C. 1992; The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo . Mol Microbiol 6:1105–1114 [View Article][PubMed]
    [Google Scholar]
  36. Volante A., Carrasco B., Tabone M., Alonso J. C. 2015; The interaction of ω 2 with the RNA polymerase β’′ subunit functions as an activation to repression switch. Nucleic Acids Res 43:9249–9261 [View Article][PubMed]
    [Google Scholar]
  37. Williams D. R., Macartney D. P., Thomas C. M. 1998; The partitioning activity of the RK2 central control region requires only incC, korB and KorB-binding site OB3 but other KorB-binding sites form destabilizing complexes in the absence of OB3. Microbiology 144:3369–3378 [View Article][PubMed]
    [Google Scholar]
  38. Woldringh C. L. 2002; The role of co-transcriptional translation and protein translocation (transertion) in bacterial chromosome segregation. Mol Microbiol 45:17–29 [View Article][PubMed]
    [Google Scholar]
  39. Zhu W., Murray P. R., Huskins W. C., Jernigan J. A., McDonald L. C., Clark N. C., Anderson K. F., McDougal L. K., Hageman J. C. et al. 2010; Dissemination of an Enterococcus Inc18-like vanA plasmid associated with vancomycin-resistant Staphylococcus aureus . Antimicrob Agents Chemother 54:4314–4320 [View Article][PubMed]
    [Google Scholar]
  40. Zielenkiewicz U., Cegłowski P. 2005; The toxin-antitoxin system of the streptococcal plasmid pSM19035. J Bacteriol 187:6094–6105 [CrossRef]
    [Google Scholar]
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