1887

Abstract

The ParB protein of is important for growth, cell division, nucleoid segregation and different types of motility. To further understand its function we have demonstrated a vital role of the hydrophobic residues in the C terminus of ParB. By modelling of the C-terminal domain (amino acids 242–290) the hydrophobic residues L282, V285 and I289 (but not L286) are engaged in leucine-zipper-like structure formation, whereas the charged residues R290 and Q266 are implicated in forming a salt bridge involved in protein stabilization. Five mutant alleles were constructed and their functionality was defined and . In agreement with model predictions, the substitution L286A had no effect on mutant protein activities. Two ParBs with single substitutions L282A or V285A and deletions of two or seven C-terminal amino acids were impaired in both dimerization and DNA binding and were not able to silence genes adjacent to , suggesting that dimerization through the C terminus is a prerequisite for spreading on DNA. The defect in dimerization also correlated with loss of ability to interact with partner protein ParA. Reverse genetics demonstrated that a mutant producing ParB lacking the two C-terminal amino acids as well as mutants producing ParB with single substitution L282A or V285A had defects similar to those of a null mutant. Thus so far all the properties of ParB seem to depend on dimerization.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.056234-0
2012-05-01
2019-12-11
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/5/1183.html?itemId=/content/journal/micro/10.1099/mic.0.056234-0&mimeType=html&fmt=ahah

References

  1. Autret S. , Errington J. . ( 2003; ). A role for division-site-selection protein MinD in regulation of internucleoid jumping of Soj (ParA) protein in Bacillus subtilis . . Mol Microbiol 47:, 159–169. [CrossRef] [PubMed]
    [Google Scholar]
  2. Bartosik A. A. , Lasocki K. , Mierzejewska J. , Thomas C. M. , Jagura-Burdzy G. . ( 2004; ). ParB of Pseudomonas aeruginosa: interactions with its partner ParA and its target parS and specific effects on bacterial growth. . J Bacteriol 186:, 6983–6998. [CrossRef] [PubMed]
    [Google Scholar]
  3. Bartosik A. A. , Mierzejewska J. , Thomas C. M. , Jagura-Burdzy G. . ( 2009; ). ParB deficiency in Pseudomonas aeruginosa destabilizes the partner protein ParA and affects a variety of physiological parameters. . Microbiology 155:, 1080–1092. [CrossRef] [PubMed]
    [Google Scholar]
  4. Bignell C. R. , Haines A. S. , Khare D. , Thomas C. M. . ( 1999; ). Effect of growth rate and incC mutation on symmetric plasmid distribution by the IncP-1 partitioning apparatus. . Mol Microbiol 34:, 205–216. [CrossRef] [PubMed]
    [Google Scholar]
  5. Bingle L. E. , Macartney D. P. , Fantozzi A. , Manzoor S. E. , Thomas C. M. . ( 2005; ). Flexibility in repression and cooperativity by KorB of broad host range IncP-1 plasmid RK2. . J Mol Biol 349:, 302–316. [CrossRef] [PubMed]
    [Google Scholar]
  6. Bowman G. R. , Comolli L. R. , Zhu J. , Eckart M. , Koenig M. , Downing K. H. , Moerner W. E. , Earnest T. , Shapiro L. . ( 2008; ). A polymeric protein anchors the chromosomal origin/ParB complex at a bacterial cell pole. . Cell 134:, 945–955. [CrossRef] [PubMed]
    [Google Scholar]
  7. 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. [CrossRef] [PubMed]
    [Google Scholar]
  8. Cervin M. A. , Spiegelman G. B. , Raether B. , Ohlsen K. , Perego M. , Hoch J. A. . ( 1998; ). A negative regulator linking chromosome segregation to developmental transcription in Bacillus subtilis . . Mol Microbiol 29:, 85–95. [CrossRef] [PubMed]
    [Google Scholar]
  9. Churchward G. , Belin D. , Nagamine Y. . ( 1984; ). A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors. . Gene 31:, 165–171. [CrossRef] [PubMed]
    [Google Scholar]
  10. Delbrück H. , Ziegelin G. , Lanka E. , Heinemann U. . ( 2002; ). An Src homology 3-like domain is responsible for dimerization of the repressor protein KorB encoded by the promiscuous IncP plasmid RP4. . J Biol Chem 277:, 4191–4198. [CrossRef] [PubMed]
    [Google Scholar]
  11. Donovan C. , Schwaiger A. , Krämer R. , Bramkamp M. . ( 2010; ). Subcellular localization and characterization of the ParAB system from Corynebacterium glutamicum . . J Bacteriol 192:, 3441–3451. [CrossRef] [PubMed]
    [Google Scholar]
  12. Ebersbach G. , Briegel A. , Jensen G. J. , Jacobs-Wagner C. . ( 2008; ). A self-associating protein critical for chromosome attachment, division, and polar organization in Caulobacter . . Cell 134:, 956–968. [CrossRef] [PubMed]
    [Google Scholar]
  13. El-Sayed A. K. , Hothersall J. , Thomas C. M. . ( 2001; ). Quorum-sensing-dependent regulation of biosynthesis of the polyketide antibiotic mupirocin in Pseudomonas fluorescens NCIMB 10586. . Microbiology 147:, 2127–2139.[PubMed]
    [Google Scholar]
  14. Figge R. M. , Easter J. Jr , Gober J. W. . ( 2003; ). Productive interaction between the chromosome partitioning proteins, ParA and ParB, is required for the progression of the cell cycle in Caulobacter crescentus . . Mol Microbiol 47:, 1225–1237. [CrossRef] [PubMed]
    [Google Scholar]
  15. Fogel M. A. , Waldor M. K. . ( 2006; ). A dynamic, mitotic-like mechanism for bacterial chromosome segregation. . Genes Dev 20:, 3269–3282. [CrossRef] [PubMed]
    [Google Scholar]
  16. Gerdes K. , Møller-Jensen J. , Jensen R. B. . ( 2000; ). Plasmid and chromosome partitioning: surprises from phylogeny. . Mol Microbiol 37:, 455–466. [CrossRef] [PubMed]
    [Google Scholar]
  17. Glaser P. , Sharpe M. E. , Raether B. , Perego M. , Ohlsen K. , Errington J. . ( 1997; ). Dynamic, mitotic-like behavior of a bacterial protein required for accurate chromosome partitioning. . Genes Dev 11:, 1160–1168. [CrossRef] [PubMed]
    [Google Scholar]
  18. Godfrin-Estevenon A.-M. , Pasta F. , Lane D. . ( 2002; ). The parAB gene products of Pseudomonas putida exhibit partition activity in both P. putida and Escherichia coli . . Mol Microbiol 43:, 39–49. [CrossRef] [PubMed]
    [Google Scholar]
  19. Gruber S. , Errington J. . ( 2009; ). Recruitment of condensin to replication origin regions by ParB/Spo0J promotes chromosome segregation in B. subtilis . . Cell 137:, 685–696. [CrossRef] [PubMed]
    [Google Scholar]
  20. Irani V. R. , Rowe J. J. . ( 1997; ). Enhancement of transformation in Pseudomonas aeruginosa PAO1 by Mg2+ and heat. . Biotechniques 22:, 54–56.[PubMed]
    [Google Scholar]
  21. Ireton K. , Gunther N. W. IV , Grossman A. D. . ( 1994; ). spo0J is required for normal chromosome segregation as well as the initiation of sporulation in Bacillus subtilis . . J Bacteriol 176:, 5320–5329.[PubMed]
    [Google Scholar]
  22. Jagura-Burdzy G. , Ibbotson J. P. , Thomas C. M. . ( 1991; ). The korF region of broad-host-range plasmid RK2 encodes two polypeptides with transcriptional repressor activity. . J Bacteriol 173:, 826–833. [CrossRef] [PubMed]
    [Google Scholar]
  23. Jagura-Burdzy G. , Thomas C. M. . ( 1995; ). Purification of KorA protein from broad host range plasmid RK2: definition of a hierarchy of KorA operators. . J Mol Biol 253:, 39–50. [CrossRef] [PubMed]
    [Google Scholar]
  24. Jakimowicz D. , Chater K. F. , Zakrzewska-Czerwínska J. . ( 2002; ). The ParB protein of Streptomyces coelicolor A3(2) recognizes a cluster of parS sequences within the origin-proximal region of the linear chromosome. . Mol Microbiol 45:, 1365–1377. [CrossRef] [PubMed]
    [Google Scholar]
  25. Jakimowicz D. , Mouz S. , Zakrzewska-Czerwinska J. , Chater K. F. . ( 2006; ). Developmental control of a parAB promoter leads to formation of sporulation-associated ParB complexes in Streptomyces coelicolor . . J Bacteriol 188:, 1710–1720. [CrossRef] [PubMed]
    [Google Scholar]
  26. Jakimowicz D. , Zydek P. , Kois A. , Zakrzewska-Czerwińska J. , Chater K. F. . ( 2007; ). Alignment of multiple chromosomes along helical ParA scaffolding in sporulating Streptomyces hyphae. . Mol Microbiol 65:, 625–641. [CrossRef] [PubMed]
    [Google Scholar]
  27. Kadoya R. , Baek J. H. , Sarker A. , Chattoraj D. K. . ( 2011; ). Participation of chromosome segregation protein ParAI of Vibrio cholerae in chromosome replication. . J Bacteriol 193:, 1504–1514. [CrossRef] [PubMed]
    [Google Scholar]
  28. Kahn M. R. , Kolter R. , Thomas C. M. , Figurski D. , Meyer R. , Remaut E. , Helinski D. R. . ( 1979; ). Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2. . Methods Enzymol 68:, 268–280. [CrossRef] [PubMed]
    [Google Scholar]
  29. Karimova G. , Pidoux J. , Ullmann A. , Ladant D. . ( 1998; ). A bacterial two-hybrid system based on a reconstituted signal transduction pathway. . Proc Natl Acad Sci U S A 95:, 5752–5756. [CrossRef] [PubMed]
    [Google Scholar]
  30. Katoh K. , Toh H. . ( 2008; ). Recent developments in the MAFFT multiple sequence alignment program. . Brief Bioinform 9:, 286–298. [CrossRef] [PubMed]
    [Google Scholar]
  31. Khare D. , Ziegelin G. , Lanka E. , Heinemann U. . ( 2004; ). Sequence-specific DNA binding determined by contacts outside the helix-turn-helix motif of the ParB homolog KorB. . Nat Struct Mol Biol 11:, 656–663. [CrossRef] [PubMed]
    [Google Scholar]
  32. Kim H.-J. , Calcutt M. J. , Schmidt F. J. , Chater K. F. . ( 2000; ). Partitioning of the linear chromosome during sporulation of Streptomyces coelicolor A3(2) involves an oriC-linked parAB locus. . J Bacteriol 182:, 1313–1320. [CrossRef] [PubMed]
    [Google Scholar]
  33. Kovach M. E. , Elzer P. H. , Hill D. S. , Robertson G. T. , Farris M. A. , Roop R. M. II , Peterson K. M. . ( 1995; ). Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. . Gene 166:, 175–176. [CrossRef] [PubMed]
    [Google Scholar]
  34. Kusiak M. , Gapczynska A. , Plochocka D. , Thomas C. M. , Jagura-Burdzy G. . ( 2011; ). Binding and spreading of ParB on DNA determine its biological function in Pseudomonas aeruginosa . . J Bacteriol 193:, 3342–3355. [CrossRef] [PubMed]
    [Google Scholar]
  35. Larkin M. A. , Blackshields G. , Brown N. P. , Chenna R. , McGettigan P. A. , McWilliam H. , Valentin F. , Wallace I. M. , Wilm A. . & other authors ( 2007; ). clustal w and clustal_x version 2.0. . Bioinformatics 23:, 2947–2948. [CrossRef] [PubMed]
    [Google Scholar]
  36. Lasocki K. , Bartosik A. A. , Mierzejewska J. , Thomas C. M. , Jagura-Burdzy G. . ( 2007; ). Deletion of the parA (soj) homologue in Pseudomonas aeruginosa causes ParB instability and affects growth rate, chromosome segregation, and motility. . J Bacteriol 189:, 5762–5772. [CrossRef] [PubMed]
    [Google Scholar]
  37. Lee P. S. , Grossman A. D. . ( 2006; ). The chromosome partitioning proteins Soj (ParA) and Spo0J (ParB) contribute to accurate chromosome partitioning, separation of replicated sister origins, and regulation of replication initiation in Bacillus subtilis . . Mol Microbiol 60:, 853–869. [CrossRef] [PubMed]
    [Google Scholar]
  38. Lee P. S. , Lin D. C.-H. , Moriya S. , Grossman A. D. . ( 2003; ). Effects of the chromosome partitioning protein Spo0J (ParB) on oriC positioning and replication initiation in Bacillus subtilis . . J Bacteriol 185:, 1326–1337. [CrossRef] [PubMed]
    [Google Scholar]
  39. Leonard T. A. , Butler P. J. , Löwe J. . ( 2004; ). Structural analysis of the chromosome segregation protein Spo0J from Thermus thermophilus . . Mol Microbiol 53:, 419–432. [CrossRef] [PubMed]
    [Google Scholar]
  40. Lewis R. A. , Bignell C. R. , Zeng W. , Jones A. C. , Thomas C. M. . ( 2002; ). Chromosome loss from par mutants of Pseudomonas putida depends on growth medium and phase of growth. . Microbiology 148:, 537–548.[PubMed]
    [Google Scholar]
  41. Lin D. C.-H. , Grossman A. D. . ( 1998; ). Identification and characterization of a bacterial chromosome partitioning site. . Cell 92:, 675–685. [CrossRef] [PubMed]
    [Google Scholar]
  42. Marston A. L. , Errington J. . ( 1999; ). Dynamic movement of the ParA-like Soj protein of B. subtilis and its dual role in nucleoid organization and developmental regulation. . Mol Cell 4:, 673–682. [CrossRef] [PubMed]
    [Google Scholar]
  43. Mohl D. A. , Gober J. W. . ( 1997; ). Cell cycle-dependent polar localization of chromosome partitioning proteins in Caulobacter crescentus . . Cell 88:, 675–684. [CrossRef] [PubMed]
    [Google Scholar]
  44. Mohl D. A. , Easter J. Jr , Gober J. W. . ( 2001; ). The chromosome partitioning protein, ParB, is required for cytokinesis in Caulobacter crescentus . . Mol Microbiol 42:, 741–755. [CrossRef] [PubMed]
    [Google Scholar]
  45. Mullis K. , Faloona F. , Scharf S. , Saiki R. , Horn G. , Erlich H. . ( 1986; ). Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. . Cold Spring Harb Symp Quant Biol 51:, 263–273. [CrossRef] [PubMed]
    [Google Scholar]
  46. Murray H. , Errington J. . ( 2008; ). Dynamic control of the DNA replication initiation protein DnaA by Soj/ParA. . Cell 135:, 74–84. [CrossRef] [PubMed]
    [Google Scholar]
  47. Notredame C. , Higgins D. G. , Heringa J. . ( 2000; ). T-Coffee: a novel method for fast and accurate multiple sequence alignment. . J Mol Biol 302:, 205–217. [CrossRef] [PubMed]
    [Google Scholar]
  48. Ogura Y. , Ogasawara N. , Harry E. J. , Moriya S. . ( 2003; ). Increasing the ratio of Soj to Spo0J promotes replication initiation in Bacillus subtilis . . J Bacteriol 185:, 6316–6324. [CrossRef] [PubMed]
    [Google Scholar]
  49. Ptacin J. L. , Lee S. F. , Garner E. C. , Toro E. , Eckart M. , Comolli L. R. , Moerner W. E. , Shapiro L. . ( 2010; ). A spindle-like apparatus guides bacterial chromosome segregation. . Nat Cell Biol 12:, 791–798. [CrossRef] [PubMed]
    [Google Scholar]
  50. Quisel J. D. , Grossman A. D. . ( 2000; ). Control of sporulation gene expression in Bacillus subtilis by the chromosome partitioning proteins Soj (ParA) and Spo0J (ParB). . J Bacteriol 182:, 3446–3451. [CrossRef] [PubMed]
    [Google Scholar]
  51. Quisel J. D. , Lin D. C. , Grossman A. D. . ( 1999; ). Control of development by altered localization of a transcription factor in B. subtilis . . Mol Cell 4:, 665–672. [CrossRef] [PubMed]
    [Google Scholar]
  52. Rashid M. H. , Kornberg A. . ( 2000; ). Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa . . Proc Natl Acad Sci U S A 97:, 4885–4890. [CrossRef] [PubMed]
    [Google Scholar]
  53. Real G. , Autret S. , Harry E. J. , Errington J. , Henriques A. O. . ( 2005; ). Cell division protein DivIB influences the Spo0J/Soj system of chromosome segregation in Bacillus subtilis . . Mol Microbiol 55:, 349–367. [CrossRef] [PubMed]
    [Google Scholar]
  54. Rodionov O. , Yarmolinsky M. . ( 2004; ). Plasmid partitioning and the spreading of P1 partition protein ParB. . Mol Microbiol 52:, 1215–1223. [CrossRef] [PubMed]
    [Google Scholar]
  55. Rodionov O. , Lobocka M. , Yarmolinsky M. . ( 1999; ). Silencing of genes flanking the P1 plasmid centromere. . Science 283:, 546–549. [CrossRef] [PubMed]
    [Google Scholar]
  56. Saint-Dic D. , Frushour B. P. , Kehrl J. H. , Kahng L. S. . ( 2006; ). A parA homolog selectively influences positioning of the large chromosome origin in Vibrio cholerae . . J Bacteriol 188:, 5626–5631. [CrossRef] [PubMed]
    [Google Scholar]
  57. Sambrook J. , Fritsch E. F. , Maniatis T. . ( 1989; ). Molecular Cloning: a Laboratory Manual, , 2nd edn.. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory;.
    [Google Scholar]
  58. Schofield W. B. , Lim H. Ch. , Jacobs-Wagner C. . ( 2010; ). Cell cycle coordination and regulation of bacterial chromosome segregation dynamics by polarly localized proteins. . EMBO J 29:, 3068–3081. [CrossRef] [PubMed]
    [Google Scholar]
  59. Scholefield G. , Whiting R. , Errington J. , Murray H. . ( 2011; ). Spo0J regulates the oligomeric state of Soj to trigger its switch from an activator to an inhibitor of DNA replication initiation. . Mol Microbiol 79:, 1089–1100. [CrossRef] [PubMed]
    [Google Scholar]
  60. Schumacher M. A. , Mansoor A. , Funnell B. E. . ( 2007; ). Structure of a four-way bridged ParB-DNA complex provides insight into P1 segrosome assembly. . J Biol Chem 282:, 10456–10464. [CrossRef] [PubMed]
    [Google Scholar]
  61. Schumacher M. A. , Piro K. M. , Xu W. . ( 2010; ). Insight into F plasmid DNA segregation revealed by structures of SopB and SopB-DNA complexes. . Nucleic Acids Res 38:, 4514–4526. [CrossRef] [PubMed]
    [Google Scholar]
  62. Sharpe M. E. , Errington J. . ( 1996; ). The Bacillus subtilis soj-spo0J locus is required for a centromere-like function involved in prespore chromosome partitioning. . Mol Microbiol 21:, 501–509. [CrossRef] [PubMed]
    [Google Scholar]
  63. Simon R. , O’Connell M. , Labes M. , Pühler A. . ( 1986; ). Plasmid vectors for the genetic analysis and manipulation of rhizobia and other Gram-negative bacteria. . Methods Enzymol 118:, 640–659. [CrossRef] [PubMed]
    [Google Scholar]
  64. Sullivan N. L. , Marquis K. A. , Rudner D. Z. . ( 2009; ). Recruitment of SMC by ParB–parS organizes the origin region and promotes efficient chromosome segregation. . Cell 137:, 697–707. [CrossRef] [PubMed]
    [Google Scholar]
  65. Thanbichler M. , Shapiro L. . ( 2006; ). MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter . . Cell 126:, 147–162. [CrossRef] [PubMed]
    [Google Scholar]
  66. Toro E. , Hong S.-H. , McAdams H. H. , Shapiro L. . ( 2008; ). Caulobacter requires a dedicated mechanism to initiate chromosome segregation. . Proc Natl Acad Sci U S A 105:, 15435–15440. [CrossRef] [PubMed]
    [Google Scholar]
  67. Viollier P. H. , Thanbichler M. , McGrath P. T. , West L. , Meewan M. , McAdams H. H. , Shapiro L. . ( 2004; ). Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication. . Proc Natl Acad Sci U S A 101:, 9257–9262. [CrossRef] [PubMed]
    [Google Scholar]
  68. Webb C. D. , Teleman A. , Gordon S. , Straight A. , Belmont A. , Lin D. C.-H. , Grossman A. D. , Wright A. , Losick R. . ( 1997; ). Bipolar localization of the replication origin regions of chromosomes in vegetative and sporulating cells of B. subtilis . . Cell 88:, 667–674. [CrossRef] [PubMed]
    [Google Scholar]
  69. Williams D. R. , Thomas C. M. . ( 1992; ). Active partitioning of bacterial plasmids. . J Gen Microbiol 138:, 1–16.[PubMed] [CrossRef]
    [Google Scholar]
  70. Yamaichi Y. , Niki H. . ( 2000; ). Active segregation by the Bacillus subtilis partitioning system in Escherichia coli . . Proc Natl Acad Sci U S A 97:, 14656–14661. [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.056234-0
Loading
/content/journal/micro/10.1099/mic.0.056234-0
Loading

Data & Media loading...

Supplements

Supplementary material 

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