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. 1997 Apr;179(8):2668–2677. doi: 10.1128/jb.179.8.2668-2677.1997

Transposon mutagenesis reinforces the correlation between Mycoplasma pneumoniae cytoskeletal protein HMW2 and cytadherence.

D C Krause 1, T Proft 1, C T Hedreyda 1, H Hilbert 1, H Plagens 1, R Herrmann 1
PMCID: PMC179017  PMID: 9098066

Abstract

A new genetic locus associated with Mycoplasma pneumoniae cytadherence was previously identified by transposon mutagenesis with Tn4001. This locus maps approximately 160 kbp from the genes encoding cytadherence-associated proteins HMW1 and HMW3, and yet insertions therein result in loss of these proteins and a hemadsorption-negative (HA-) phenotype, prompting the designation cytadherence-regulatory locus (crl). In the current study, passage of transformants in the absence of antibiotic selection resulted in loss of the transposon, a wild-type protein profile, and a HA+ phenotype, underscoring the correlation between crl and M. pneumoniae cytadherence. Nucleotide sequence analysis of crl revealed open reading frames (ORFs) orfp65, orfp216, orfp41, and orfp24, arranged in tandem and flanked by a promoter-like and a terminator-like sequence, suggesting a single transcriptional unit, the P65 operon. The 5' end of orfp65 mRNA was mapped by primer extension, and a likely promoter was identified just upstream. The product of each ORF was identified by using antisera prepared against fusion proteins. The previously characterized surface protein P65 is encoded by orfp65, while the 190,000 Mr cytadherence-associated protein HMW2 is a product of orfp216. Proteins with sizes of 47,000 and 41,000 Mr and unknown function were identified for orfp41 and orfp24, respectively. Structural analyses of HMW2 predict a periodicity highly characteristic of a coiled-coil conformation and five leucine zipper motifs, indicating that HMW2 probably forms dimers in vivo, which is consistent with a structural role in cytadherence. Each transposon insertion mapped to orfp216 but affected the levels of all products of the P65 operon. HMW2 is thought to form a disulfide-linked dimer, formerly designated HMW5, and examination of an hmw2 deletion mutant confirms that HMW5 is a product of the hmw2 gene.

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Selected References

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  1. 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]
  2. 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]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bork P., Ouzounis C., Casari G., Schneider R., Sander C., Dolan M., Gilbert W., Gillevet P. M. Exploring the Mycoplasma capricolum genome: a minimal cell reveals its physiology. Mol Microbiol. 1995 Jun;16(5):955–967. doi: 10.1111/j.1365-2958.1995.tb02321.x. [DOI] [PubMed] [Google Scholar]
  5. Byrne M. E., Rouch D. A., Skurray R. A. Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001. Gene. 1989 Sep 30;81(2):361–367. doi: 10.1016/0378-1119(89)90197-2. [DOI] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [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. Cohen C., Parry D. A. Alpha-helical coiled coils and bundles: how to design an alpha-helical protein. Proteins. 1990;7(1):1–15. doi: 10.1002/prot.340070102. [DOI] [PubMed] [Google Scholar]
  9. Dirksen L. B., Proft T., Hilbert H., Plagens H., Herrmann R., Krause D. C. Sequence analysis and characterization of the hmw gene cluster of Mycoplasma pneumoniae. Gene. 1996 May 24;171(1):19–25. doi: 10.1016/0378-1119(96)00050-9. [DOI] [PubMed] [Google Scholar]
  10. Franzoso G., Hu P. C., Meloni G. A., Barile M. F. The immunodominant 90-kilodalton protein is localized on the terminal tip structure of Mycoplasma pneumoniae. Infect Immun. 1993 Apr;61(4):1523–1530. doi: 10.1128/iai.61.4.1523-1530.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fraser C. M., Gocayne J. D., White O., Adams M. D., Clayton R. A., Fleischmann R. D., Bult C. J., Kerlavage A. R., Sutton G., Kelley J. M. The minimal gene complement of Mycoplasma genitalium. Science. 1995 Oct 20;270(5235):397–403. doi: 10.1126/science.270.5235.397. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Guerin-Marchand C., Druilhe P., Galey B., Londono A., Patarapotikul J., Beaudoin R. L., Dubeaux C., Tartar A., Mercereau-Puijalon O., Langsley G. A liver-stage-specific antigen of Plasmodium falciparum characterized by gene cloning. Nature. 1987 Sep 10;329(6135):164–167. doi: 10.1038/329164a0. [DOI] [PubMed] [Google Scholar]
  14. Hahn T. W., Krebes K. A., Krause D. C. Expression in Mycoplasma pneumoniae of the recombinant gene encoding the cytadherence-associated protein HMW1 and identification of HMW4 as a product. Mol Microbiol. 1996 Mar;19(5):1085–1093. doi: 10.1046/j.1365-2958.1996.455985.x. [DOI] [PubMed] [Google Scholar]
  15. Hedreyda C. T., Krause D. C. Identification of a possible cytadherence regulatory locus in Mycoplasma pneumoniae. Infect Immun. 1995 Sep;63(9):3479–3483. doi: 10.1128/iai.63.9.3479-3483.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hilbert H., Himmelreich R., Plagens H., Herrmann R. Sequence analysis of 56 kb from the genome of the bacterium Mycoplasma pneumoniae comprising the dnaA region, the atp operon and a cluster of ribosomal protein genes. Nucleic Acids Res. 1996 Feb 15;24(4):628–639. doi: 10.1093/nar/24.4.628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Himmelreich R., Hilbert H., Plagens H., Pirkl E., Li B. C., Herrmann R. Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res. 1996 Nov 15;24(22):4420–4449. doi: 10.1093/nar/24.22.4420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Krause D. C., Lee K. K. Juxtaposition of the genes encoding Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW3. Gene. 1991 Oct 30;107(1):83–89. doi: 10.1016/0378-1119(91)90300-z. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Krause D. C. Mycoplasma pneumoniae cytadherence: unravelling the tie that binds. Mol Microbiol. 1996 Apr;20(2):247–253. doi: 10.1111/j.1365-2958.1996.tb02613.x. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. 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]
  27. 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]
  28. Ogle K. F., Lee K. K., Krause D. C. Cloning and analysis of the gene encoding the cytadherence phase-variable protein HMW3 from Mycoplasma pneumoniae. Gene. 1991 Jan 2;97(1):69–75. doi: 10.1016/0378-1119(91)90011-y. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Proft T., Hilbert H., Layh-Schmitt G., Herrmann R. 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. J Bacteriol. 1995 Jun;177(12):3370–3378. doi: 10.1128/jb.177.12.3370-3378.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Proft T., Hilbert H., Plagens H., Herrmann R. The P200 protein of Mycoplasma pneumoniae shows common features with the cytadherence-associated proteins HMW1 and HMW3. Gene. 1996 May 24;171(1):79–82. doi: 10.1016/0378-1119(96)00014-5. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Reddy S. P., Rasmussen W. G., Baseman J. B. Isolation and characterization of transposon Tn4001-generated, cytadherence-deficient transformants of Mycoplasma pneumoniae and Mycoplasma genitalium. FEMS Immunol Med Microbiol. 1996 Oct;15(4):199–211. doi: 10.1111/j.1574-695X.1996.tb00086.x. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  39. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Villarejo M. R., Zabin I. Beta-galactosidase from termination and deletion mutant strains. J Bacteriol. 1974 Oct;120(1):466–474. doi: 10.1128/jb.120.1.466-474.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wenzel R., Herrmann R. Physical mapping of the Mycoplasma pneumoniae genome. Nucleic Acids Res. 1988 Sep 12;16(17):8323–8336. doi: 10.1093/nar/16.17.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Yanagisawa M., Hamada Y., Katsuragawa Y., Imamura M., Mikawa T., Masaki T. Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence. Implications on topography and function of myosin. J Mol Biol. 1987 Nov 20;198(2):143–157. doi: 10.1016/0022-2836(87)90302-0. [DOI] [PubMed] [Google Scholar]
  44. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  45. Yen T. J., Li G., Schaar B. T., Szilak I., Cleveland D. W. CENP-E is a putative kinetochore motor that accumulates just before mitosis. Nature. 1992 Oct 8;359(6395):536–539. doi: 10.1038/359536a0. [DOI] [PubMed] [Google Scholar]

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