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. 1996 Jun;178(11):3177–3187. doi: 10.1128/jb.178.11.3177-3187.1996

Characterization of the cytoplasmic filament protein gene (cfpA) of Treponema pallidum subsp. pallidum.

Y You 1, S Elmore 1, L L Colton 1, C Mackenzie 1, J K Stoops 1, G M Weinstock 1, S J Norris 1
PMCID: PMC178068  PMID: 8655496

Abstract

Treponema pallidum and other members of the genera Treponema, Spirochaeta, and Leptonema contain multiple cytoplasmic filaments that run the length of the organism just underneath the cytoplasmic membrane. These cytoplasmic filaments have a ribbon-like profile and consist of a major cytoplasmic filament protein subunit (CfpA, formerly called TpN83) with a relative molecular weight of approximately 80,000. Degenerate DNA primers based on N-terminal and CNBr cleavage fragment amino acid sequences of T. pallidum subsp. pallidum (Nichols) CfpA were utilized to amplify a fragment of the encoding gene (cfpA). A 6.8-kb EcoRI fragment containing all but the 5' end of cfpA was identified by hybridization with the resulting PCR product and cloned into Lambda ZAP II. The 5' region was obtained by inverse PCR, and the complete gene sequence was determined. The cfpA sequence contained a 2,034-nucleotide coding region, a putative promoter with consensus sequences (5'-TTTACA-3' for -35 and 5'-TACAAT-3' for -10) similar to the sigma70 recognition sequence of Escherichia coli and other organisms, and a putative ribosome-binding site (5'-AGGAG-3'). The deduced amino acid sequence of CfpA indicated a protein of 678 residues with a calculated molecular mass of 78.5 kDa and an estimated pI of 6.15. No significant homology to known proteins or structural motifs was found among known prokaryotic or eukaryotic sequences. Expression of a LacZ-CfpA fusion protein in E. coli was detrimental to survival and growth of the host strain and resulted in the formation of short, irregular filaments suggestive of partial self-assembly of CfpA. The cytoplasmic filaments of T. pallidum and other spirochetes appear to represent a unique form of prokaryotic intracytoplasmic inclusions.

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Selected References

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  1. Bermudes D., Hinkle G., Margulis L. Do prokaryotes contain microtubules? Microbiol Rev. 1994 Sep;58(3):387–400. doi: 10.1128/mr.58.3.387-400.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Casaregola S., Norris V., Goldberg M., Holland I. B. Identification of a 180 kD protein in Escherichia coli related to a yeast heavy-chain myosin. Mol Microbiol. 1990 Mar;4(3):505–511. doi: 10.1111/j.1365-2958.1990.tb00617.x. [DOI] [PubMed] [Google Scholar]
  3. Casarégola S., Jacq A., Laoudj D., McGurk G., Margarson S., Tempête M., Norris V., Holland I. B. Cloning and analysis of the entire Escherichia coli ams gene. ams is identical to hmp1 and encodes a 114 kDa protein that migrates as a 180 kDa protein. J Mol Biol. 1992 Nov 5;228(1):30–40. doi: 10.1016/0022-2836(92)90489-7. [DOI] [PubMed] [Google Scholar]
  4. Eipert S. R., Black S. H. Characterization of the cytoplasmic fibrils of Treponema refringens (Nichols). Arch Microbiol. 1979 Mar 12;120(3):205–214. doi: 10.1007/BF00423067. [DOI] [PubMed] [Google Scholar]
  5. Hanff P. A., Norris S. J., Lovett M. A., Miller J. N. Purification of Treponema pallidum, Nichols strain, by Percoll density gradient centrifugation. Sex Transm Dis. 1984 Oct-Dec;11(4):275–286. doi: 10.1097/00007435-198410000-00003. [DOI] [PubMed] [Google Scholar]
  6. Hardham J. M., Frye J. G., Stamm L. V. Identification and sequences of the Treponema pallidum fliM', fliY, fliP, fliQ, fliR and flhB' genes. Gene. 1995 Dec 1;166(1):57–64. doi: 10.1016/0378-1119(95)00583-x. [DOI] [PubMed] [Google Scholar]
  7. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Helmann J. D., Chamberlin M. J. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–872. doi: 10.1146/annurev.bi.57.070188.004203. [DOI] [PubMed] [Google Scholar]
  9. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  10. Hougen K. H., Birch-Andersen A. Electron microscopy of endoflagella and microtubules in Treponema reiter. Acta Pathol Microbiol Scand B Microbiol Immunol. 1971;79(1):37–50. doi: 10.1111/j.1699-0463.1971.tb00031.x. [DOI] [PubMed] [Google Scholar]
  11. Hougen K. H. Further observations on the ultrastructure of Treponema pallidum nichols. Acta Pathol Microbiol Scand B Microbiol Immunol. 1972;80(2):297–304. doi: 10.1111/j.1699-0463.1972.tb00163.x. [DOI] [PubMed] [Google Scholar]
  12. Hovind-Hougen K., Birch-Andersen A., Jensen H. J. Ultrastructure of cells of Treponema pertenue obtained from experimentally infected hamsters. Acta Pathol Microbiol Scand B. 1976 Apr;84(2):101–108. doi: 10.1111/j.1699-0463.1976.tb01909.x. [DOI] [PubMed] [Google Scholar]
  13. Hovind-Hougen K. Determination by means of electron microscopy of morphological criteria of value for classification of some spirochetes, in particular treponemes. Acta Pathol Microbiol Scand Suppl. 1976;(255):1–41. [PubMed] [Google Scholar]
  14. Isaacs R. D., Hanke J. H., Guzman-Verduzco L. M., Newport G., Agabian N., Norgard M. V., Lukehart S. A., Radolf J. D. Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum. Infect Immun. 1989 Nov;57(11):3403–3411. doi: 10.1128/iai.57.11.3403-3411.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Isaacs R. D., Radolf J. D. Expression in Escherichia coli of the 37-kilodalton endoflagellar sheath protein of Treponema pallidum by use of the polymerase chain reaction and a T7 expression system. Infect Immun. 1990 Jul;58(7):2025–2034. doi: 10.1128/iai.58.7.2025-2034.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jackson S., Black S. H. Ultrastructure of Treponema pallidum Nichols following lysis by physical and chemical methods. I. Envelope, wall, membrane and fibrils. Arch Mikrobiol. 1971;76(4):308–324. doi: 10.1007/BF00408528. [DOI] [PubMed] [Google Scholar]
  17. Limberger R. J., Charon N. W. Treponema phagedenis has at least two proteins residing together on its periplasmic flagella. J Bacteriol. 1986 Apr;166(1):105–112. doi: 10.1128/jb.166.1.105-112.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lupas A. Prediction and analysis of coiled-coil structures. Methods Enzymol. 1996;266:513–525. doi: 10.1016/s0076-6879(96)66032-7. [DOI] [PubMed] [Google Scholar]
  19. Masuda K., Kawata T. Isolation and characterization of cytoplasmic fibrils from treponemes. Microbiol Immunol. 1989;33(8):619–630. doi: 10.1111/j.1348-0421.1989.tb02012.x. [DOI] [PubMed] [Google Scholar]
  20. Matsudaira P. Limited N-terminal sequence analysis. Methods Enzymol. 1990;182:602–613. doi: 10.1016/0076-6879(90)82047-6. [DOI] [PubMed] [Google Scholar]
  21. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  22. McClure W. R. Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem. 1985;54:171–204. doi: 10.1146/annurev.bi.54.070185.001131. [DOI] [PubMed] [Google Scholar]
  23. Norris S. J. Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Treponema Pallidum Polypeptide Research Group. Microbiol Rev. 1993 Sep;57(3):750–779. doi: 10.1128/mr.57.3.750-779.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Okada Y., Wachi M., Hirata A., Suzuki K., Nagai K., Matsuhashi M. Cytoplasmic axial filaments in Escherichia coli cells: possible function in the mechanism of chromosome segregation and cell division. J Bacteriol. 1994 Feb;176(3):917–922. doi: 10.1128/jb.176.3.917-922.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ovcinnikov N. M., Delektorskij V. V. Current concepts of the morphology and biology of Treponema pallidum based on electron microscopy. Br J Vener Dis. 1971 Oct;47(5):315–328. doi: 10.1136/sti.47.5.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Penn C. W. The eighth C. L. Oakley lecture. Pathogenicity and immunobiology of Treponema pallidum. J Med Microbiol. 1987 Aug;24(1):1–9. doi: 10.1099/00222615-24-1-1. [DOI] [PubMed] [Google Scholar]
  27. Radolf J. D., Norgard M. V., Schulz W. W. Outer membrane ultrastructure explains the limited antigenicity of virulent Treponema pallidum. Proc Natl Acad Sci U S A. 1989 Mar;86(6):2051–2055. doi: 10.1073/pnas.86.6.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sweeney F. P., Watts F. Z., Pocklington M. J., Orr E. The MYO1 gene from Saccharomyces cerevisiae: its complete nucleotide sequence. Nucleic Acids Res. 1990 Dec 11;18(23):7147–7147. doi: 10.1093/nar/18.23.7147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Triglia T., Peterson M. G., Kemp D. J. A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 1988 Aug 25;16(16):8186–8186. doi: 10.1093/nar/16.16.8186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wachi M., Doi M., Ueda T., Ueki M., Tsuritani K., Nagai K., Matsuhashi M. Sequence of the downstream flanking region of the shape-determining genes mreBCD of Escherichia coli. Gene. 1991 Sep 30;106(1):135–136. doi: 10.1016/0378-1119(91)90578-y. [DOI] [PubMed] [Google Scholar]
  32. Walker E. M., Howell J. K., You Y., Hoffmaster A. R., Heath J. D., Weinstock G. M., Norris S. J. Physical map of the genome of Treponema pallidum subsp. pallidum (Nichols). J Bacteriol. 1995 Apr;177(7):1797–1804. doi: 10.1128/jb.177.7.1797-1804.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Walker E. M., Zampighi G. A., Blanco D. R., Miller J. N., Lovett M. A. Demonstration of rare protein in the outer membrane of Treponema pallidum subsp. pallidum by freeze-fracture analysis. J Bacteriol. 1989 Sep;171(9):5005–5011. doi: 10.1128/jb.171.9.5005-5011.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wiegand S. E., Strobel P. L., Glassman L. H. Electron microscopic anatomy of pathogenic Treponema pallidum. J Invest Dermatol. 1972 Apr;58(4):186–204. doi: 10.1111/1523-1747.ep12539907. [DOI] [PubMed] [Google Scholar]
  35. de Boer P., Crossley R., Rothfield L. The essential bacterial cell-division protein FtsZ is a GTPase. Nature. 1992 Sep 17;359(6392):254–256. doi: 10.1038/359254a0. [DOI] [PubMed] [Google Scholar]
  36. von Hippel P. H., Bear D. G., Morgan W. D., McSwiggen J. A. Protein-nucleic acid interactions in transcription: a molecular analysis. Annu Rev Biochem. 1984;53:389–446. doi: 10.1146/annurev.bi.53.070184.002133. [DOI] [PubMed] [Google Scholar]

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