1887

Abstract

, the meningococcus, is naturally competent for transformation throughout its growth cycle. The uptake of exogenous DNA into the meningococcus cell during transformation is a multi-step process. Beyond the requirement for type IV pilus expression for efficient transformation, little is known about the neisserial proteins involved in DNA binding, uptake and genome integration. This study aimed to identify and characterize neisserial DNA binding proteins in order to further elucidate the multi-factorial transformation machinery. The meningococcus inner membrane and soluble cell fractions were searched for DNA binding components by employing 1D and 2D gel electrophoresis approaches in combination with a solid-phase overlay assay with DNA substrates. Proteins that bound DNA were identified by MS analysis. In the membrane fraction, multiple components bound DNA, including the neisserial competence lipoprotein ComL. In the soluble fraction, the meningococcus orthologue of the single-stranded DNA binding protein SSB was predominant. The DNA binding activity of the recombinant ComL and SSB proteins purified to homogeneity was verified by electromobility shift assay, and the ComL–DNA interaction was shown to be Mg-dependent. In 3D models of the meningococcus ComL and SSB predicted structures, potential DNA binding sites were suggested. ComL was found to co-purify with the outer membrane, directly interacting with the secretin PilQ. The combined use of 1D/2D solid-phase overlay assays with MS analysis was a useful strategy for identifying DNA binding components. The ComL DNA binding properties and outer membrane localization suggest that this lipoprotein plays a direct role in neisserial transformation, while neisserial SSB is a DNA binding protein that contributes to the terminal part of the transformation process.

Funding
This study was supported by the:
  • , Medical Research Curriculum
  • , Institute of Medical Basic Sciences at the University of Oslo
  • , Research Council of Norway
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2011-05-01
2021-03-09
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References

  1. Ambur O. H., Frye S. A., Tønjum T.( 2007). New functional identity for the DNA uptake sequence in transformation and its presence in transcriptional terminators. J Bacteriol 189:2077–2085 [CrossRef][PubMed]
    [Google Scholar]
  2. Ames G. F., Prody C., Kustu S.( 1984). Simple, rapid, and quantitative release of periplasmic proteins by chloroform. J Bacteriol 160:1181–1183[PubMed]
    [Google Scholar]
  3. Arnold K., Bordoli L., Kopp J., Schwede T.( 2006). The swiss-model workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201 [CrossRef][PubMed]
    [Google Scholar]
  4. Assalkhou R., Balasingham S., Collins R. F., Frye S. A., Davidsen T., Benam A. V., Bjørås M., Derrick J. P., Tønjum T.( 2007). The outer membrane secretin PilQ from Neisseria meningitidis binds DNA. Microbiology 153:1593–1603 [CrossRef][PubMed]
    [Google Scholar]
  5. Averhoff B.( 2004). DNA transport and natural transformation in mesophilic and thermophilic bacteria. J Bioenerg Biomembr 36:25–33 [CrossRef][PubMed]
    [Google Scholar]
  6. Babu M. M., Priya M. L., Selvan A. T., Madera M., Gough J., Aravind L., Sankaran K.( 2006). A database of bacterial lipoproteins (dolop) with functional assignments to predicted lipoproteins. J Bacteriol 188:2761–2773 [CrossRef][PubMed]
    [Google Scholar]
  7. Baker N. A., Sept D., Joseph S., Holst M. J., McCammon J. A.( 2001). Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci U S A 98:10037–10041 [CrossRef][PubMed]
    [Google Scholar]
  8. Balasingham S. V., Collins R. F., Assalkhou R., Homberset H., Frye S. A., Derrick J. P., Tønjum T.( 2007). Interactions between the lipoprotein PilP and the secretin PilQ in Neisseria meningitidis. J Bacteriol 189:5716–5727 [CrossRef][PubMed]
    [Google Scholar]
  9. Bateman A., Coin L., Durbin R., Finn R. D., Hollich V., Griffiths-Jones S., Khanna A., Marshall M., Moxon S. et al.( 2004). The Pfam protein families database. Nucleic Acids Res 32:Database issueD138–D141 [CrossRef][PubMed]
    [Google Scholar]
  10. Bennett-Lovsey R. M., Herbert A. D., Sternberg M. J., Kelley L. A.( 2008). Exploring the extremes of sequence/structure space with ensemble fold recognition in the program Phyre. Proteins 70:611–625 [CrossRef][PubMed]
    [Google Scholar]
  11. Boeckmann B., Bairoch A., Apweiler R., Blatter M. C., Estreicher A., Gasteiger E., Martin M. J., Michoud K., O’Donovan C. et al.( 2003). The swiss-prot protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Res 31:365–370 [CrossRef][PubMed]
    [Google Scholar]
  12. Bradley D. E.( 1980). A function of Pseudomonas aeruginosa PAO polar pili: twitching motility. Can J Microbiol 26:146–154 [CrossRef][PubMed]
    [Google Scholar]
  13. Buratowski S., Chodosh L. A.( 1996). Mobility shift DNA-binding assay using gel electrophoresis. In Current Protocols in Molecular Biology Chapter 12 12.2.1–12.2.11Edited by Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. New York: Wiley.;
    [Google Scholar]
  14. Carbonnelle E., Hélaine S., Prouvensier L., Nassif X., Pelicic V.( 2005). Type IV pilus biogenesis in Neisseria meningitidis: PilW is involved in a step occurring after pilus assembly, essential for fibre stability and function. Mol Microbiol 55:54–64 [CrossRef][PubMed]
    [Google Scholar]
  15. Caugant D. A.( 2008). Genetics and evolution of Neisseria meningitidis: importance for the epidemiology of meningococcal disease. Infect Genet Evol 8:558–565 [CrossRef][PubMed]
    [Google Scholar]
  16. Chen I., Gotschlich E. C.( 2001). ComE, a competence protein from Neisseria gonorrhoeae with DNA-binding activity. J Bacteriol 183:3160–3168 [CrossRef][PubMed]
    [Google Scholar]
  17. Cuff J. A., Clamp M. E., Siddiqui A. S., Finlay M., Barton G. J.( 1998). JPred: a consensus secondary structure prediction server. Bioinformatics 14:892–893 [CrossRef][PubMed]
    [Google Scholar]
  18. D'Andrea L. D., Regan L.( 2003). TPR proteins: the versatile helix. Trends Biochem Sci 28:655–662 [CrossRef][PubMed]
    [Google Scholar]
  19. Davidsen T., Tønjum T.( 2006). Meningococcal genome dynamics. Nat Rev Microbiol 4:11–22 [CrossRef][PubMed]
    [Google Scholar]
  20. Davidsen T., Rødland E. A., Lagesen K., Seeberg E., Rognes T., Tønjum T.( 2004). Biased distribution of DNA uptake sequences towards genome maintenance genes. Nucleic Acids Res 32:1050–1058 [CrossRef][PubMed]
    [Google Scholar]
  21. DeLano W. L. 2002; The PyMOL Molecular Graphics System. San Carlos, CA: DeLano Scientific LLC; http://www.pymol.org
  22. Facius D., Meyer T. F. ( 1993). A novel determinant (comA) essential for natural transformation competence in Neisseria gonorrhoeae and the effect of a comA defect on pilin variation. Mol Microbiol 10:699–712 [CrossRef][PubMed]
    [Google Scholar]
  23. Fanning E., Klimovich V., Nager A. R. ( 2006). A dynamic model for replication protein A (RPA) function in DNA processing pathways. Nucleic Acids Res 34:4126–4137 [CrossRef][PubMed]
    [Google Scholar]
  24. Fedorov R., Witte G., Urbanke C., Manstein D. J., Curth U. ( 2006). 3D structure of Thermus aquaticus single-stranded DNA-binding protein gives insight into the functioning of SSB proteins. Nucleic Acids Res 34:6708–6717 [CrossRef][PubMed]
    [Google Scholar]
  25. Fleckenstein B., Qiao S. W., Larsen M. R., Jung G., Roepstorff P., Sollid L. M. ( 2004). Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides. J Biol Chem 279:17607–17616 [CrossRef][PubMed]
    [Google Scholar]
  26. Frøholm L. O., Jyssum K., Bøvre K. ( 1973). Electron microscopical and cultural features of Neisseria meningitidis competence variants. Acta Pathol Microbiol Scand B Microbiol Immunol 81:525–537[PubMed]
    [Google Scholar]
  27. Frye S. A., Assalkhou R., Collins R. F., Ford R. C., Petersson C., Derrick J. P., Tønjum T. ( 2006). Topology of the outer-membrane secretin PilQ from Neisseria meningitidis . Microbiology 152:3751–3764 [CrossRef][PubMed]
    [Google Scholar]
  28. Fussenegger M., Facius D., Meier J., Meyer T. F. ( 1996). A novel peptidoglycan-linked lipoprotein (ComL) that functions in natural transformation competence of Neisseria gonorrhoeae . Mol Microbiol 19:1095–1105 [CrossRef][PubMed]
    [Google Scholar]
  29. Fussenegger M., Rudel T., Barten R., Ryll R., Meyer T. F. ( 1997). Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae – a review. Gene 192:125–134 [CrossRef][PubMed]
    [Google Scholar]
  30. Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M. R., Appel R. D., Bairoch A. ( 2005). Protein identification and analysis tools on the ExPASY server. In The Proteomics Protocols Handbook pp 571–607Edited by Walker J. M. Totowa, NJ: Humana Press; [CrossRef]
    [Google Scholar]
  31. Goodman S. D., Scocca J. J. ( 1988). Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae . Proc Natl Acad Sci U S A 85:6982–6986 [CrossRef][PubMed]
    [Google Scholar]
  32. Hulo N., Sigrist C. J., Le Saux V., Langendijk-Genevaux P. S., Bordoli L., Gattiker A., De Castro E., Bucher P., Bairoch A. ( 2004). Recent improvements to the prosite database. Nucleic Acids Res 32:Database issueD134–D137 [CrossRef][PubMed]
    [Google Scholar]
  33. Hwang S., Gou Z., Kuznetsov I. B. ( 2007). DP-Bind: a web server for sequence-based prediction of DNA-binding residues in DNA-binding proteins. Bioinformatics 23:634–636 [CrossRef][PubMed]
    [Google Scholar]
  34. Jones D. T., Taylor W. R., Thornton J. M. ( 1994). A model recognition approach to the prediction of all-helical membrane protein structure and topology. Biochemistry 33:3038–3049 [CrossRef][PubMed]
    [Google Scholar]
  35. Jose J., Otto G. W., Meyer T. F. ( 2003). The integration site of the iga gene in commensal Neisseria sp. Mol Genet Genomics 269:197–204[PubMed]
    [Google Scholar]
  36. Judd R. C., Porcella S. F. ( 1993). Isolation of the periplasm of Neisseria gonorrhoeae . Mol Microbiol 10:567–574 [CrossRef][PubMed]
    [Google Scholar]
  37. Jyssum K., Lie S. ( 1965). Genetic factors determining competence in transformation of Neisseria meningitidis. 1. A permanent loss of competence. Acta Pathol Microbiol Scand 63:306–316[PubMed]
    [Google Scholar]
  38. Kim K., Oh J., Han D., Kim E. E., Lee B., Kim Y. ( 2006). Crystal structure of PilF: functional implication in the type 4 pilus biogenesis in Pseudomonas aeruginosa . Biochem Biophys Res Commun 340:1028–1038 [CrossRef][PubMed]
    [Google Scholar]
  39. Knowles T. J., Scott-Tucker A., Overduin M., Henderson I. R. ( 2009). Membrane protein architects: the role of the BAM complex in outer membrane protein assembly. Nat Rev Microbiol 7:206–214 [CrossRef][PubMed]
    [Google Scholar]
  40. Koo J., Tammam S., Ku S. Y., Sampaleanu L. M., Burrows L. L., Howell P. L. ( 2008). PilF is an outer membrane lipoprotein required for multimerization and localization of the Pseudomonas aeruginosa Type IV pilus secretin. J Bacteriol 190:6961–6969 [CrossRef][PubMed]
    [Google Scholar]
  41. Koomey J. M., Falkow S. ( 1987). Cloning of the recA gene of Neisseria gonorrhoeae and construction of gonococcal recA mutants. J Bacteriol 169:790–795[PubMed]
    [Google Scholar]
  42. Lång E., Haugen K., Fleckenstein B., Homberset H., Frye S. A., Ambur O. H., Tønjum T. ( 2009). Identification of neisserial DNA binding components. Microbiology 155:852–862 [CrossRef][PubMed]
    [Google Scholar]
  43. Lindner C., Nijland R., van Hartskamp M., Bron S., Hamoen L. W., Kuipers O. P. ( 2004). Differential expression of two paralogous genes of Bacillus subtilis encoding single-stranded DNA binding protein. J Bacteriol 186:1097–1105 [CrossRef][PubMed]
    [Google Scholar]
  44. Makhov A. M., Griffith J. D. ( 2006). Visualization of the annealing of complementary single-stranded DNA catalyzed by the herpes simplex virus type 1 ICP8 SSB/recombinase. J Mol Biol 355:911–922 [CrossRef][PubMed]
    [Google Scholar]
  45. Makhov A. M., Sen A., Yu X., Simon M. N., Griffith J. D., Egelman E. H. ( 2009). The bipolar filaments formed by herpes simplex virus type 1 SSB/recombination protein (ICP8) suggest a mechanism for DNA annealing. J Mol Biol 386:273–279 [CrossRef][PubMed]
    [Google Scholar]
  46. Malinverni J. C., Werner J., Kim S., Sklar J. G., Kahne D., Misra R., Silhavy T. J. ( 2006). YfiO stabilizes the YaeT complex and is essential for outer membrane protein assembly in Escherichia coli . Mol Microbiol 61:151–164 [CrossRef][PubMed]
    [Google Scholar]
  47. Maniatis T., Fritsch E. F., Sambrook J. ( 1982). Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  48. Mapelli M., Panjikar S., Tucker P. A. ( 2005). The crystal structure of the herpes simplex virus 1 ssDNA-binding protein suggests the structural basis for flexible, cooperative single-stranded DNA binding. J Biol Chem 280:2990–2997 [CrossRef][PubMed]
    [Google Scholar]
  49. Masson L., Holbein B. E. ( 1983). Physiology of sialic acid capsular polysaccharide synthesis in serogroup B Neisseria meningitidis . J Bacteriol 154:728–736[PubMed]
    [Google Scholar]
  50. Monaco C., Talà A., Spinosa M. R., Progida C., De Nitto E., Gaballo A., Bruni C. B., Bucci C., Alifano P. ( 2006). Identification of a meningococcal l-glutamate ABC transporter operon essential for growth in low-sodium environments. Infect Immun 74:1725–1740 [CrossRef][PubMed]
    [Google Scholar]
  51. Obradovic Z., Peng K., Vucetic S., Radivojac P., Dunker A. K. ( 2005). Exploiting heterogeneous sequence properties improves prediction of protein disorder. Proteins 61 :Suppl. 7176–182 [CrossRef][PubMed]
    [Google Scholar]
  52. Ogura M., Yamaguchi H., Kobayashi K., Ogasawara N., Fujita Y., Tanaka T. ( 2002). Whole-genome analysis of genes regulated by the Bacillus subtilis competence transcription factor ComK. J Bacteriol 184:2344–2351 [CrossRef][PubMed]
    [Google Scholar]
  53. Raghunathan S., Kozlov A. G., Lohman T. M., Waksman G. ( 2000). Structure of the DNA binding domain of E. coli SSB bound to ssDNA. Nat Struct Biol 7:648–652 [CrossRef][PubMed]
    [Google Scholar]
  54. Redfield R. J., Cameron A. D., Qian Q., Hinds J., Ali T. R., Kroll J. S., Langford P. R. ( 2005). A novel CRP-dependent regulon controls expression of competence genes in Haemophilus influenzae . J Mol Biol 347:735–747 [CrossRef][PubMed]
    [Google Scholar]
  55. Rice P., Longden I., Bleasby A. ( 2000). EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 16:276–277 [CrossRef][PubMed]
    [Google Scholar]
  56. Sanner M. F. ( 1999). Python: a programming language for software integration and development. J Mol Graph Model 17:57–61[PubMed]
    [Google Scholar]
  57. Savvides S. N., Raghunathan S., Fütterer K., Kozlov A. G., Lohman T. M., Waksman G. ( 2004). The C-terminal domain of full-length E. coli SSB is disordered even when bound to DNA. Protein Sci 13:1942–1947 [CrossRef][PubMed]
    [Google Scholar]
  58. Shereda R. D., Kozlov A. G., Lohman T. M., Cox M. M., Keck J. L. ( 2008). SSB as an organizer/mobilizer of genome maintenance complexes. Crit Rev Biochem Mol Biol 43:289–318 [CrossRef][PubMed]
    [Google Scholar]
  59. Stephens D. S., Greenwood B., Brandtzaeg P. ( 2007). Epidemic meningitis, meningococcaemia, and Neisseria meningitidis . Lancet 369:2196–2210 [CrossRef][PubMed]
    [Google Scholar]
  60. Swanson J., Kraus S. J., Gotschlich E. C. ( 1971). Studies on gonococcus infection. I. Pili and zones of adhesion: their relation to gonococcal growth patterns. J Exp Med 134:886–906 [CrossRef][PubMed]
    [Google Scholar]
  61. Tibballs K. L., Ambur O. H., Alfsnes K., Homberset H., Frye S. A., Davidsen T., Tønjum T. ( 2009). Characterization of the meningococcal DNA glycosylase Fpg involved in base excision repair. BMC Microbiol 9:7 [CrossRef][PubMed]
    [Google Scholar]
  62. Tønjum T., Koomey M. ( 1997). The pilus colonization factor of pathogenic neisserial species: organelle biogenesis and structure/function relationships – a review. Gene 192:155–163 [CrossRef][PubMed]
    [Google Scholar]
  63. Tønjum T., Freitag N. E., Namork E., Koomey M. ( 1995). Identification and characterization of pilG, a highly conserved pilus-assembly gene in pathogenic Neisseria. Mol Microbiol 16:451–464 [CrossRef][PubMed]
    [Google Scholar]
  64. Tønjum T., Caugant D. A., Dunham S. A., Koomey M. ( 1998). Structure and function of repetitive sequence elements associated with a highly polymorphic domain of the Neisseria meningitidis PilQ protein. Mol Microbiol 29:111–124 [CrossRef][PubMed]
    [Google Scholar]
  65. Trindade M. B., Job V., Contreras-Martel C., Pelicic V., Dessen A. ( 2008). Structure of a widely conserved type IV pilus biogenesis factor that affects the stability of secretin multimers. J Mol Biol 378:1031–1039 [CrossRef][PubMed]
    [Google Scholar]
  66. Ward J. J., McGuffin L. J., Bryson K., Buxton B. F., Jones D. T. ( 2004). The DISOPRED server for the prediction of protein disorder. Bioinformatics 20:2138–2139 [CrossRef][PubMed]
    [Google Scholar]
  67. Wolfgang M., van Putten J. P., Hayes S. F., Koomey M. ( 1999). The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation. Mol Microbiol 31:1345–1357 [CrossRef][PubMed]
    [Google Scholar]
  68. Wu T., Malinverni J., Ruiz N., Kim S., Silhavy T. J., Kahne D. ( 2005). Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli . Cell 121:235–245 [CrossRef][PubMed]
    [Google Scholar]
  69. Yazdankhah S. P., Kriz P., Tzanakaki G., Kremastinou J., Kalmusova J., Musilek M., Alvestad T., Jolley K. A., Wilson D. J. et al. ( 2004). Distribution of serogroups and genotypes among disease-associated and carried isolates of Neisseria meningitidis from the Czech Republic, Greece, and Norway. J Clin Microbiol 42:5146–5153 [CrossRef][PubMed]
    [Google Scholar]
  70. Zou Y., Liu Y., Wu X., Shell S. M. ( 2006). Functions of human replication protein A (RPA): from DNA replication to DNA damage and stress responses. J Cell Physiol 208:267–273 [CrossRef][PubMed]
    [Google Scholar]
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