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. 1995 Jun;177(12):3370–3378. doi: 10.1128/jb.177.12.3370-3378.1995

The proline-rich P65 protein of Mycoplasma pneumoniae is a component of the Triton X-100-insoluble fraction and exhibits size polymorphism in the strains M129 and FH.

T Proft 1, H Hilbert 1, G Layh-Schmitt 1, R Herrmann 1
PMCID: PMC177038  PMID: 7768845

Abstract

Previously, we described the identification of a novel Mycoplasma pneumoniae M129 protein, named P65 because of its apparent molecular mass of 65 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (T. Proft and R. Herrmann, Mol. Microbiol. 13:337-348, 1994). DNA sequence analysis of the P65 open reading frame (orfp65), however, revealed an ORF encoding a protein with a molecular weight of 47,034. This discrepancy can be explained by the unusual amino acid composition of this protein. According to the deduced amino acid sequence, the N-terminal half of P65 contains several penta- and hexapeptides (DPNAY and DPNQAY) forming a proline-rich acidic domain. Secondary-structure predictions indicated beta-sheets and turns within that region, suggesting an extended and rigid conformation. Near the C terminus of P65 the tripeptide Arg-Gly-Asp (RGD) was found. This motif is known to play an important role in binding of extracellular matrix proteins to integrins. P65 could be located exclusively to the Triton X-100-insoluble cell fraction. The results of immunofluorescence microscopy and of immunoadsorption experiments indicated that P65 carries surface-exposed regions. Mild treatment of whole cells with proteases resulted in cleavage of a limited amount of P65 molecules, suggesting either that only a small percentage of P65 molecules are exposed on the surface or that protease cleavage is hampered by a compact protein conformation or by binding of an unknown component to P65. P65 exhibits size polymorphism in M. pneumoniae M129 and FH. This is caused by an intragenetic duplication of a 54-bp sequence within the FH orfp65. As a consequence, the number of DPNAY pentapeptides increased from 9 to 12 repeats in the FH strain.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bairoch A. PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res. 1992 May 11;20 (Suppl):2013–2018. doi: 10.1093/nar/20.suppl.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barile M. F., Chandler D. K., Yoshida H., Grabowski M. W., Harasawa R., Razin S. Parameters of Mycoplasma pneumoniae infection in Syrian hamsters. Infect Immun. 1988 Sep;56(9):2443–2449. doi: 10.1128/iai.56.9.2443-2449.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baseman J. B., Cole R. M., Krause D. C., Leith D. K. Molecular basis for cytadsorption of Mycoplasma pneumoniae. J Bacteriol. 1982 Sep;151(3):1514–1522. doi: 10.1128/jb.151.3.1514-1522.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baseman J. B., Morrison-Plummer J., Drouillard D., Puleo-Scheppke B., Tryon V. V., Holt S. C. Identification of a 32-kilodalton protein of Mycoplasma pneumoniae associated with hemadsorption. Isr J Med Sci. 1987 May;23(5):474–479. [PubMed] [Google Scholar]
  5. Bennett V. Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm. Physiol Rev. 1990 Oct;70(4):1029–1065. doi: 10.1152/physrev.1990.70.4.1029. [DOI] [PubMed] [Google Scholar]
  6. Brewer S., Tolley M., Trayer I. P., Barr G. C., Dorman C. J., Hannavy K., Higgins C. F., Evans J. S., Levine B. A., Wormald M. R. Structure and function of X-Pro dipeptide repeats in the TonB proteins of Salmonella typhimurium and Escherichia coli. J Mol Biol. 1990 Dec 20;216(4):883–895. doi: 10.1016/S0022-2836(99)80008-4. [DOI] [PubMed] [Google Scholar]
  7. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  8. Cowan S. W., Rosenbusch J. P. Folding pattern diversity of integral membrane proteins. Science. 1994 May 13;264(5161):914–916. doi: 10.1126/science.8178151. [DOI] [PubMed] [Google Scholar]
  9. D'Souza S. E., Ginsberg M. H., Plow E. F. Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif. Trends Biochem Sci. 1991 Jul;16(7):246–250. doi: 10.1016/0968-0004(91)90096-e. [DOI] [PubMed] [Google Scholar]
  10. Dallo S. F., Chavoya A., Baseman J. B. Characterization of the gene for a 30-kilodalton adhesion-related protein of Mycoplasma pneumoniae. Infect Immun. 1990 Dec;58(12):4163–4165. doi: 10.1128/iai.58.12.4163-4165.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dallo S. F., Horton J. R., Su C. J., Baseman J. B. Restriction fragment length polymorphism in the cytadhesin P1 gene of human clinical isolates of Mycoplasma pneumoniae. Infect Immun. 1990 Jun;58(6):2017–2020. doi: 10.1128/iai.58.6.2017-2020.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dame J. B., Williams J. L., McCutchan T. F., Weber J. L., Wirtz R. A., Hockmeyer W. T., Maloy W. L., Haynes J. D., Schneider I., Roberts D. Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum. Science. 1984 Aug 10;225(4662):593–599. doi: 10.1126/science.6204383. [DOI] [PubMed] [Google Scholar]
  13. Emini E. A., Hughes J. V., Perlow D. S., Boger J. Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide. J Virol. 1985 Sep;55(3):836–839. doi: 10.1128/jvi.55.3.836-839.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Engleberg N. C., Pearlman E., Eisenstein B. I. Legionella pneumophila surface antigens cloned and expressed in Escherichia coli are translocated to the host cell surface and interact with specific anti-Legionella antibodies. J Bacteriol. 1984 Oct;160(1):199–203. doi: 10.1128/jb.160.1.199-203.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Feldner J., Göbel U., Bredt W. Mycoplasma pneumoniae adhesin localized to tip structure by monoclonal antibody. Nature. 1982 Aug 19;298(5876):765–767. doi: 10.1038/298765a0. [DOI] [PubMed] [Google Scholar]
  16. Foy H. M., Kenny G. E., Cooney M. K., Allan I. D. Long-term epidemiology of infections with Mycoplasma pneumoniae. J Infect Dis. 1979 Jun;139(6):681–687. doi: 10.1093/infdis/139.6.681. [DOI] [PubMed] [Google Scholar]
  17. Franzoso G., Hu P. C., Meloni G. A., Barile M. F. Immunoblot analyses of chimpanzee sera after infection and after immunization and challenge with Mycoplasma pneumoniae. Infect Immun. 1994 Mar;62(3):1008–1014. doi: 10.1128/iai.62.3.1008-1014.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Furthmayr H., Timpl R. Characterization of collagen peptides by sodium dodecylsulfate-polyacrylamide electrophoresis. Anal Biochem. 1971 Jun;41(2):510–516. doi: 10.1016/0003-2697(71)90173-4. [DOI] [PubMed] [Google Scholar]
  19. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  20. Göbel U., Speth V., Bredt W. Filamentous structures in adherent Mycoplasma pneumoniae cells treated with nonionic detergents. J Cell Biol. 1981 Nov;91(2 Pt 1):537–543. doi: 10.1083/jcb.91.2.537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hayflick L. Tissue cultures and mycoplasmas. Tex Rep Biol Med. 1965 Jun;23(Suppl):285+–285+. [PubMed] [Google Scholar]
  22. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  23. Hixon W. G., Searcy D. G. Cytoskeleton in the archaebacterium Thermoplasma acidophilum? Viscosity increase in soluble extracts. Biosystems. 1993;29(2-3):151–160. doi: 10.1016/0303-2647(93)90091-p. [DOI] [PubMed] [Google Scholar]
  24. Hu P. C., Cole R. M., Huang Y. S., Graham J. A., Gardner D. E., Collier A. M., Clyde W. A., Jr Mycoplasma pneumoniae infection: role of a surface protein in the attachment organelle. Science. 1982 Apr 16;216(4543):313–315. doi: 10.1126/science.6801766. [DOI] [PubMed] [Google Scholar]
  25. Hu P. C., Collier A. M., Baseman J. B. Surface parasitism by Mycoplasma pneumoniae of respiratory epithelium. J Exp Med. 1977 May 1;145(5):1328–1343. doi: 10.1084/jem.145.5.1328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Inamine J. M., Ho K. C., Loechel S., Hu P. C. Evidence that UGA is read as a tryptophan codon rather than as a stop codon by Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma gallisepticum. J Bacteriol. 1990 Jan;172(1):504–506. doi: 10.1128/jb.172.1.504-506.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Inamine J. M., Loechel S., Hu P. C. Analysis of the nucleotide sequence of the P1 operon of Mycoplasma pneumoniae. Gene. 1988 Dec 15;73(1):175–183. doi: 10.1016/0378-1119(88)90323-x. [DOI] [PubMed] [Google Scholar]
  28. Kahane I., Tucker S., Leith D. K., Morrison-Plummer J., Baseman J. B. Detection of the major adhesin P1 in triton shells of virulent Mycoplasma pneumoniae. Infect Immun. 1985 Dec;50(3):944–946. doi: 10.1128/iai.50.3.944-946.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Krause D. C., Leith D. K., Baseman J. B. Reacquisition of specific proteins confers virulence in Mycoplasma pneumoniae. Infect Immun. 1983 Feb;39(2):830–836. doi: 10.1128/iai.39.2.830-836.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Krause D. C., Leith D. K., Wilson R. M., Baseman J. B. Identification of Mycoplasma pneumoniae proteins associated with hemadsorption and virulence. Infect Immun. 1982 Mar;35(3):809–817. doi: 10.1128/iai.35.3.809-817.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  32. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  33. Layh-Schmitt G., Herrmann R. Localization and biochemical characterization of the ORF6 gene product of the Mycoplasma pneumoniae P1 operon. Infect Immun. 1992 Jul;60(7):2906–2913. doi: 10.1128/iai.60.7.2906-2913.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Layh-Schmitt G., Herrmann R. Spatial arrangement of gene products of the P1 operon in the membrane of Mycoplasma pneumoniae. Infect Immun. 1994 Mar;62(3):974–979. doi: 10.1128/iai.62.3.974-979.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  36. Lupas A., Van Dyke M., Stock J. Predicting coiled coils from protein sequences. Science. 1991 May 24;252(5009):1162–1164. doi: 10.1126/science.252.5009.1162. [DOI] [PubMed] [Google Scholar]
  37. Maniloff J. Cytoskeletal elements in mycoplasmas and other prokaryotes. Biosystems. 1981;14(3-4):305–312. doi: 10.1016/0303-2647(81)90037-x. [DOI] [PubMed] [Google Scholar]
  38. Marrs J. A., Bouck G. B. The two major membrane skeletal proteins (articulins) of Euglena gracilis define a novel class of cytoskeletal proteins. J Cell Biol. 1992 Sep;118(6):1465–1475. doi: 10.1083/jcb.118.6.1465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  40. Meng K. E., Pfister R. M. Intracellular structures of Mycoplasma pneumoniae revealed after membrane removal. J Bacteriol. 1980 Oct;144(1):390–399. doi: 10.1128/jb.144.1.390-399.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Needleman S. B., Wunsch C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970 Mar;48(3):443–453. doi: 10.1016/0022-2836(70)90057-4. [DOI] [PubMed] [Google Scholar]
  42. Nikaido H. Porins and specific channels of bacterial outer membranes. Mol Microbiol. 1992 Feb;6(4):435–442. doi: 10.1111/j.1365-2958.1992.tb01487.x. [DOI] [PubMed] [Google Scholar]
  43. Ogle K. F., Lee K. K., Krause D. C. Nucleotide sequence analysis reveals novel features of the phase-variable cytadherence accessory protein HMW3 of Mycoplasma pneumoniae. Infect Immun. 1992 Apr;60(4):1633–1641. doi: 10.1128/iai.60.4.1633-1641.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Proft T., Herrmann R. Identification and characterization of hitherto unknown Mycoplasma pneumoniae proteins. Mol Microbiol. 1994 Jul;13(2):337–348. doi: 10.1111/j.1365-2958.1994.tb00427.x. [DOI] [PubMed] [Google Scholar]
  45. Razin S., Jacobs E. Mycoplasma adhesion. J Gen Microbiol. 1992 Mar;138(3):407–422. doi: 10.1099/00221287-138-3-407. [DOI] [PubMed] [Google Scholar]
  46. Ruland K., Wenzel R., Herrmann R. Analysis of three different repeated DNA elements present in the P1 operon of Mycoplasma pneumoniae: size, number and distribution on the genome. Nucleic Acids Res. 1990 Nov 11;18(21):6311–6317. doi: 10.1093/nar/18.21.6311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sperker B., Hu P., Herrmann R. Identification of gene products of the P1 operon of Mycoplasma pneumoniae. Mol Microbiol. 1991 Feb;5(2):299–306. doi: 10.1111/j.1365-2958.1991.tb02110.x. [DOI] [PubMed] [Google Scholar]
  49. Stevens M. K., Krause D. C. Disulfide-linked protein associated with Mycoplasma pneumoniae cytadherence phase variation. Infect Immun. 1990 Oct;58(10):3430–3433. doi: 10.1128/iai.58.10.3430-3433.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stevens M. K., Krause D. C. Localization of the Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW4 in the cytoskeletonlike Triton shell. J Bacteriol. 1991 Feb;173(3):1041–1050. doi: 10.1128/jb.173.3.1041-1050.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Stevens M. K., Krause D. C. Mycoplasma pneumoniae cytadherence phase-variable protein HMW3 is a component of the attachment organelle. J Bacteriol. 1992 Jul;174(13):4265–4274. doi: 10.1128/jb.174.13.4265-4274.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Su C. J., Dallo S. F., Alderman H., Baseman J. B. Distinctions in DNA and protein profiles among clinical isolates of Mycoplasma pneumoniae. J Gen Microbiol. 1991 Dec;137(12):2727–2732. doi: 10.1099/00221287-137-12-2727. [DOI] [PubMed] [Google Scholar]
  53. Vu A. C., Foy H. M., Cartwright F. D., Kenny G. E. The principal protein antigens of isolates of Mycoplasma pneumoniae as measured by levels of immunoglobulin G in human serum are stable in strains collected over a 10-year period. Infect Immun. 1987 Aug;55(8):1830–1836. doi: 10.1128/iai.55.8.1830-1836.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wenzel R., Herrmann R. Cloning of the complete Mycoplasma pneumoniae genome. Nucleic Acids Res. 1989 Sep 12;17(17):7029–7043. doi: 10.1093/nar/17.17.7029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wenzel R., Pirkl E., Herrmann R. Construction of an EcoRI restriction map of Mycoplasma pneumoniae and localization of selected genes. J Bacteriol. 1992 Nov;174(22):7289–7296. doi: 10.1128/jb.174.22.7289-7296.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yu Y. G., King D. S., Shin Y. K. Insertion of a coiled-coil peptide from influenza virus hemagglutinin into membranes. Science. 1994 Oct 14;266(5183):274–276. doi: 10.1126/science.7939662. [DOI] [PubMed] [Google Scholar]

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