Abstract
Two structural endoflagellar genes of Treponema pallidum that encode the 34.5- and 31.0-kilodalton (kDa) polypeptides as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were cloned, sequenced, and expressed. We designated these genes flaB1 and flaB3. A DNA sequence analysis of flaB1 and flaB3 showed that each gene possesses a single open reading frame that encodes a polypeptide; these polypeptides have molecular masses of 31.1 and 31.0 kDa, respectively. Shine-Dalgarno ribosome-binding sequences were identified upstream from the initiation codons of each gene. In addition, a single consensus promoter sequence was identified 121 base pairs upstream from the initiation codon of flaB1, suggesting polycistronic transcription of flaB1 and flaB3. Computer-induced alignment showed that the FlaB1 amino acid sequence was identical at 206 positions (72%) to the FlaB3 sequence. Both genes were subcloned into pATH vectors and were expressed under the control of the trpE promoter. The expression products of flaB1 and flaB3 revealed fusion proteins having molecular masses of 61.0 and 59.0 kDa, respectively, which were identified on immunoblots by using specific anti-T. pallidum endoflagellar serum.
Full text
PDF







Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Baseman J. B., Nichols J. C., Mogerley S. Capacity of virulent Treponema pallidum (Nichols) for deoxyribonucleic acid synthesis. Infect Immun. 1979 Feb;23(2):392–397. doi: 10.1128/iai.23.2.392-397.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Blanco D. R., Champion C. I., Miller J. N., Lovett M. A. Antigenic and structural characterization of Treponema pallidum (Nichols strain) endoflagella. Infect Immun. 1988 Jan;56(1):168–175. doi: 10.1128/iai.56.1.168-175.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blanco D. R., Walker E. M., Haake D. A., Champion C. I., Miller J. N., Lovett M. A. Complement activation limits the rate of in vitro treponemicidal activity and correlates with antibody-mediated aggregation of Treponema pallidum rare outer membrane protein. J Immunol. 1990 Mar 1;144(5):1914–1921. [PubMed] [Google Scholar]
- Brahamsha B., Greenberg E. P. Cloning and sequence analysis of flaA, a gene encoding a Spirochaeta aurantia flagellar filament surface antigen. J Bacteriol. 1989 Mar;171(3):1692–1697. doi: 10.1128/jb.171.3.1692-1697.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Canale-Parola E. Motility and chemotaxis of spirochetes. Annu Rev Microbiol. 1978;32:69–99. doi: 10.1146/annurev.mi.32.100178.000441. [DOI] [PubMed] [Google Scholar]
- Cockayne A., Bailey M. J., Penn C. W. Analysis of sheath and core structures of the axial filament of Treponema pallidum. J Gen Microbiol. 1987 Jun;133(6):1397–1407. doi: 10.1099/00221287-133-6-1397. [DOI] [PubMed] [Google Scholar]
- Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Freter R., Allweiss B., O'Brien P. C., Halstead S. A., Macsai M. S. Role of chemotaxis in the association of motile bacteria with intestinal mucosa: in vitro studies. Infect Immun. 1981 Oct;34(1):241–249. doi: 10.1128/iai.34.1.241-249.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Freter R., O'Brien P. C., Macsai M. S. Role of chemotaxis in the association of motile bacteria with intestinal mucosa: in vivo studies. Infect Immun. 1981 Oct;34(1):234–240. doi: 10.1128/iai.34.1.234-240.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanff P. A., Bishop N. H., Miller J. N., Lovett M. A. Humoral immune response in experimental syphilis to polypeptides of Treponema pallidum. J Immunol. 1983 Oct;131(4):1973–1977. [PubMed] [Google Scholar]
- Hanff P. A., Fehniger T. E., Miller J. N., Lovett M. A. Humoral immune response in human syphilis to polypeptides of Treponema pallidum. J Immunol. 1982 Sep;129(3):1287–1291. [PubMed] [Google Scholar]
- Hardy P. H., Jr, Fredericks W. R., Nell E. E. Isolation and antigenic characteristics of axial filaments from the Reiter Treponeme. Infect Immun. 1975 Feb;11(2):380–386. doi: 10.1128/iai.11.2.380-386.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Helmann J. D., Chamberlin M. J. DNA sequence analysis suggests that expression of flagellar and chemotaxis genes in Escherichia coli and Salmonella typhimurium is controlled by an alternative sigma factor. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6422–6424. doi: 10.1073/pnas.84.18.6422. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Liu S. L., Ezaki T., Miura H., Matsui K., Yabuuchi E. Intact motility as a Salmonella typhi invasion-related factor. Infect Immun. 1988 Aug;56(8):1967–1973. doi: 10.1128/iai.56.8.1967-1973.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lukehart S. A., Baker-Zander S. A., Lloyd R. M., Sell S. Effect of cortisone administration on host-parasite relationships in early experimental syphilis. J Immunol. 1981 Oct;127(4):1361–1368. [PubMed] [Google Scholar]
- MILLER J. N., WHANG S. J., FAZZAN F. P. STUDIES ON IMMUNITY IN EXPERIMENTAL SYPHILIS. I. IMMUNOLOGIC RESPONSE OF RABBITS IMMUNIZED WITH REITER PROTEIN ANTIGEN AND CHALLENGED WITH VIRULENT TREPONEMA PALLIDUM. Br J Vener Dis. 1963 Sep;39:195–198. doi: 10.1136/sti.39.3.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Magnarelli L. A., Anderson J. F., Johnson R. C. Cross-reactivity in serological tests for Lyme disease and other spirochetal infections. J Infect Dis. 1987 Jul;156(1):183–188. doi: 10.1093/infdis/156.1.183. [DOI] [PubMed] [Google Scholar]
- Marck C. 'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 1988 Mar 11;16(5):1829–1836. doi: 10.1093/nar/16.5.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Minnich S. A., Newton A. Promoter mapping and cell cycle regulation of flagellin gene transcription in Caulobacter crescentus. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1142–1146. doi: 10.1073/pnas.84.5.1142. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Norris S. J., Charon N. W., Cook R. G., Fuentes M. D., Limberger R. J. Antigenic relatedness and N-terminal sequence homology define two classes of periplasmic flagellar proteins of Treponema pallidum subsp. pallidum and Treponema phagedenis. J Bacteriol. 1988 Sep;170(9):4072–4082. doi: 10.1128/jb.170.9.4072-4082.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pallesen L., Hindersson P. Cloning and sequencing of a Treponema pallidum gene encoding a 31.3-kilodalton endoflagellar subunit (FlaB2). Infect Immun. 1989 Jul;57(7):2166–2172. doi: 10.1128/iai.57.7.2166-2172.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pedersen N. S., Petersen C. S., Axelsen N. H. Enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody against the Reiter treponeme flagellum in syphilis. J Clin Microbiol. 1982 Oct;16(4):608–614. doi: 10.1128/jcm.16.4.608-614.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pedersen N. S., Petersen C. S., Vejtorp M., Axelsen N. H. Serodiagnosis of syphilis by an enzyme-linked immunosorbent assay for IgG antibodies against the Reiter treponeme flagellum. Scand J Immunol. 1982 Apr;15(4):341–348. doi: 10.1111/j.1365-3083.1982.tb00657.x. [DOI] [PubMed] [Google Scholar]
- Penn C. W., Bailey M. J., Cockayne A. The axial filament antigen of Treponema pallidum. Immunology. 1985 Apr;54(4):635–641. [PMC free article] [PubMed] [Google Scholar]
- Radolf J. D., Blanco D. R., Miller J. N., Lovett M. A. Antigenic interrelationship between endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum (Nichols): molecular characterization of endoflagellar proteins. Infect Immun. 1986 Dec;54(3):626–634. doi: 10.1128/iai.54.3.626-634.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Thomas D. D., Navab M., Haake D. A., Fogelman A. M., Miller J. N., Lovett M. A. Treponema pallidum invades intercellular junctions of endothelial cell monolayers. Proc Natl Acad Sci U S A. 1988 May;85(10):3608–3612. doi: 10.1073/pnas.85.10.3608. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Uhlman D. L., Jones G. W. Chemotaxis as a factor in interactions between HeLa cells and Salmonella typhimurium. J Gen Microbiol. 1982 Feb;128(2):415–418. doi: 10.1099/00221287-128-2-415. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]