1887

Abstract

Fresh isolates of the oral bacterial pathogen exhibit a fimbriated, rough colony phenotype. Evidence suggests that the fimbrial subunit gene is part of a cluster of 14 genes ( to ) thought to encode proteins involved in the synthesis, assembly and export of these fimbriae. To determine the transcriptional organization of the 5′ terminus of this gene cluster, total RNA from rough and smooth phenotype variants of strain 283 were analysed by RT-PCR. Primers designed to amplify regions spanning gene junctions or multiple genes yielded amplicons at each individual gene junction from to for both the rough and smooth variants. Semi-quantitative RT-PCR of the to amplicon revealed that significantly more mRNA was transcribed from the rough than the smooth variant. Longer amplicons encompassing to (3·9 kb) and to (2·1 kb) were also detected, but only from the rough variant. Rapid amplification of cDNA ends (RACE) was used to identify the 5′ end of the mRNA containing . Antisense primers located within , and enabled amplification of a RACE product that was subsequently isolated and subcloned into pGEM-T. DNA sequencing indicated that the 5′ end of the mRNA was located at a G or T nucleotide −102 to −101 nt upstream of . Corresponding σ consensus sequences were located at −10 (TATAAT) and −35 (TTGCAT) relative to the transcription start site. These data confirm that the gene cluster is an operon transcribed as a polycistronic message commencing from a G or T nucleotide located in the intergenic region upstream of . Promoter function of the upstream region was confirmed using a reporter gene construct transformed into . RT-PCR analysis further suggests that although transcription does occur in both the rough and smooth variants, full-length transcripts are rapidly degraded or are significantly downregulated in the smooth variant.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.25786-0
2003-01-01
2020-04-07
Loading full text...

Full text loading...

/deliver/fulltext/micro/149/1/mic149_21.html?itemId=/content/journal/micro/10.1099/mic.0.25786-0&mimeType=html&fmt=ahah

References

  1. Brogan J. M, Lally E. T, Poulsen K, Kilian M., Demuth D. R. 1994; Regulation of Actinobacillus actinomycetemcomitans leukotoxin expression: analysis of the promoter regions of leukotoxic and minimally leukotoxic strains. Infect Immun62:501–508
    [Google Scholar]
  2. Brown R. C., Taylor R. K. 1995; Organization of tcp , acf , and toxT genes within a ToxT-dependent operon. Mol Microbiol16:425–439
    [Google Scholar]
  3. Burgher L. W, Loomis G. W., Ware F. 1973; Systemic infection due to Actinobacillus actinomycetemcomitans. Am J Clin Pathol60:412–415
    [Google Scholar]
  4. Collison S. K, Clouthier S. C, Doran J. L, Banser P. A., Kay W. W. 1996; Salmonella enteritidis agfBAC operon encoding thin, aggregative fimbriae. J Bacteriol178:662–667
    [Google Scholar]
  5. Deretic V., Konyecsni W. M. 1989; Control of mucoidy in Pseudomonas aeruginosa : transcriptional regulation of algR and identification of the second regulatory gene, algQ. J Bacteriol171:3680–3688
    [Google Scholar]
  6. el Khizzi N, Kasab S. A., Osoba A. O. 1997; HACEK group endocarditis at the Riyadh Armed Forces Hospital. J Infect34:69–74
    [Google Scholar]
  7. Fine D. H, Furgang D, Schreiner H. C, Gonocharof P, Charlesworth J, Ghazwan G, Fitzgerald-Bocarsly P., Figurski D. H. 1999a; Phenotypic variation in Actinobacillus actinomycetemcomitans during laboratory growth: implications for virulence. Microbiology145:1335–1347
    [Google Scholar]
  8. Fine D. H, Furgang D, Kaplan J, Charlesworth J., Figurski D. H. 1999b; Tenacious adhesion of Actinobacillus actinomycetemcomitans strain CU1000 to salivary-coated hydroxyapatite. Arch Oral Biol44:1063–1076
    [Google Scholar]
  9. Fine D. H, Goncharoff P, Schreiner H, Chang K. M, Furgang D., Figurski D. 2001; Colonization and persistence of rough and smooth colony variants of Actinobacillus actinomycetemcomitans in the mouths of rats. Arch Oral Biol46:1065–1078
    [Google Scholar]
  10. Fives-Taylor P, Meyer D., Mintz P. 1995; Characteristics of Actinobacillus actinomycetemcomitans invasion of and adhesion to cultured epithelial cells. Adv Dent Res9:55–62
    [Google Scholar]
  11. Gally D. L, Bogan J. A, Eisenstein B. I., Blomfield I. C. 1993; Environmental regulation of the fim switch controlling type 1 fimbrial phase variation in Escherichia coli K-12; effects of temperature and media. J Bacteriol175:6186–6193
    [Google Scholar]
  12. Haase E. M, Zmuda J. L., Scannapieco F. A. 1999; Identification and molecular analysis of rough-colony-specific outer membrane proteins of Actinobacillus actinomycetemcomitans. Infect Immun67:2901–2908
    [Google Scholar]
  13. Haraszthy V. I, Sunday G. J, Bobek L. A, Motley T. S, Preus H., Zambon J. J. 1992; Identification and analysis of the gap region in the 23S ribosomal RNA from Actinobacillus actinomycetemcomitans. J Dent Res71:1561–1568
    [Google Scholar]
  14. Haraszthy V. I, Zambon J. J, Trevisan M, Zeid M., Genco R. J. 2000; Identification of periodontal pathogens in atheromatous plaques. J Periodontol71:1554–1560
    [Google Scholar]
  15. Hardie K. R, Seydel A, Guilvout I., Pugsley A. P. 1996; The secretin-specific, chaperone-like protein of the general secretory pathway: separation of proteolytic protection and piloting functions. Mol Microbiol22:967–976
    [Google Scholar]
  16. Hernandez V. J., Bremer H. 1990; Guanosine tetraphosphate (ppGpp) dependence of the growth rate control of rrnB P1 promoter activity in Escherichia coli. J Biol Chem265:11605–11614
    [Google Scholar]
  17. Inoue T, Tanimoto I, Ohta H, Kato K, Murayama Y., Kazuhiro F. 1998; Molecular characterization of low-molecular-weight component protein, Flp, in Actinobacillus actinomycetemcomitans fimbriae. Microbiol Immunol42:253–258
    [Google Scholar]
  18. Inouye T, Ohta H, Kokeguchi S, Fukui K., Kato K. 1990; Colonial variation and fimbriation of Actinobacillus actinomycetemcomitans. FEMS Microbiol Lett69:13–18
    [Google Scholar]
  19. Kachlany S. C, Planet P. J, Bhattacharjee M. K, Kollia E, DeSalle R, Fine D. H., Figurski D. H. 2000; Nonspecific adherence by Actinobacillus actinomycetemcomitans requires genes widespread in bacteria and archaea. J Bacteriol182:6169–6176
    [Google Scholar]
  20. Kachlany S. C, Planet P. J, Desalle R, Fine D. H, Figurski D. H., Kaplan J. B. 2001; flp-1 , the first representative of a new pilin gene subfamily, is required for non-specific adherence of Actinobacillus actinomycetemcomitans. Mol Microbiol40:542–554
    [Google Scholar]
  21. Klemm P. 1994; Fimbriae: Adhesion, Genetics, Biogenesis, and Vaccines Boca Raton, FL: CRC Press;
    [Google Scholar]
  22. Kolodrubetz D, Spitznagel J, Wang B, Phillips L. H, Jacobs C., Kraig E. 1996; Cis elements and trans factors are both important in strain-specific regulation of the leukotoxin gene in Actinobacillus actinomycetemcomitans. Infect Immun64:3451–3460
    [Google Scholar]
  23. Marrs E. F, Ruehl W. W, Schoolnik G. K., Falkow S. 1988; Pilin-gene phase variation of Moraxella bovis is caused by an inversion of the pilin genes. J Bacteriol170:3032–3039
    [Google Scholar]
  24. Martin B. F, Derby B. M, Budzilovich G. N., Ransohoff J. 1967; Brain abscesses due to Actinobacillus actinomycetemcomitans. Neurology17:833–837
    [Google Scholar]
  25. Meyer D. H, Sreenivasan P. K., Fives-Taylor P. M. 1991; Evidence for invasion of a human oral cell line by Actinobacillus actinomycetemcomitans. Infect Immun59:2719–2726
    [Google Scholar]
  26. Miller J. H. 1972; Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  27. Mims C. A. 1976; Host and microbial factors influencing susceptibility. In The Pathogenesis of Infectious Disease pp196–199 Edited by Mims C. A.. New York: Academic Press;
    [Google Scholar]
  28. Muhle I, Rau J., Ruskin J. 1979; Vertebral osteomyelitis due to Actinobacillus actinomycetemcomitans. J Am Med Assoc241:1824–1825
    [Google Scholar]
  29. Ogierman M. A, Zabihi S, Mourtzios L., Manning P. A. 1993; Genetic organization and sequence of the promoter-distal region of the tcp gene cluster of Vibrio cholerae. Gene 126:51–60
    [Google Scholar]
  30. Overholt B. F. 1966; Actinobacillus actinomycetemcomitans endocarditis. Arch Intern Med117:99–102
    [Google Scholar]
  31. Owen-Huphes T. A, Pavitt G. D, Santos D. S, Sidebotham J. M, Hulton C. S, Hinton J. C., Higgins C. F. 1992; The chromatin-associated protein H-NS interacts with curved DNA topology and gene expression. Cell71:255–265
    [Google Scholar]
  32. Page M. I., King E. O. 1966; Infection due to Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. N Engl J Med275:181–188
    [Google Scholar]
  33. Preus H. R, Sunday G. J, Haraszthy V. I., Zambon J. J. 1992; Rapid identification of Actinobacillus actinomycetemcomitans based on analysis of 23S ribosomal RNA. Oral Microbiol Immunol7:372–375
    [Google Scholar]
  34. Puente J. L, Bieber D, Ramer S. W, Murray W., Schoolnik G. K. 1996; The bundle-forming pili of enteropathogenic Escherichia coli : transcriptional regulation by environmental signals. Mol Microbiol20:87–100
    [Google Scholar]
  35. Pugsley A. P. 1993; The complete general secretory pathway in gram-negative bacteria. Microbiol Rev57:50–108
    [Google Scholar]
  36. Ramer S. W, Bieber D., Schoolnik G. K. 1996; BfpB, an outer membrane lipoprotein required for the biogenesis of bundle-forming pili in enteropathogenic Escherichia coli. J Bacteriol178:6555–6563
    [Google Scholar]
  37. Roosendaal B, van Bergen en Henegouwen P. M. P, Mooi F. R., de Graaf F. K. 1986; Regulatory aspects of K99 fimbriae synthesis. In Protein–Carbohydrate Interactions in Biological Systems: the Molecular Biology of Microbial Pathogenicity pp57–59 Edited by Normark S., Uhlin B. E., Wolf-Watz H.. New York: Academic Press;
    [Google Scholar]
  38. Rosan B, Slots J, Lamont R. J, Listgarten M. A., Nelson G. M. 1988; Actinobacillus actinomycetemcomitans fimbriae. Oral Microbiol Immunol3:58–63
    [Google Scholar]
  39. Ross W, Gosink K. K, Salomon J, Igarashi K, Zou C, Ishihama A, Severinov K., Gourse R. L. 1993; A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science262:1407–1413
    [Google Scholar]
  40. Rudney J. D, Chen R., Sedgewick G. J. 2001; Intracellular Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in buccal epithelial cells collected from human subjects. Infect Immun69:2700–2707
    [Google Scholar]
  41. Ruparelia S, McGill E. S., Spitznagel J. K. 2000; Pilin gene expression by Actinobacillus actinomycetemcomitans (abstract). J Dent Res79:393
    [Google Scholar]
  42. Scannapieco F. A, Millar S. J, Reynolds H. S, Zambon J. J., Levine M. J. 1987; Effect of anaerobiosis on the surface ultrastructure and surface proteins of Actinobacillus actinomycetemcomitans ( Haemophilus actinomycetemcomitans ). Infect Immun55:2320–2323
    [Google Scholar]
  43. Schwan W. R, Seifert H. S., Duncan J. L. 1992; Growth conditions mediate differential transcription of fim genes involved in phase variation of type I pili. J Bacteriol174:2367–2375
    [Google Scholar]
  44. Serra P., Tonato M. 1969; Subacute bacterial endocarditis due to Actinobacillus actinomycetemcomitans. Amer J Med47:809–812
    [Google Scholar]
  45. Simons R. W, Houman F., Kleckner N. 1987; Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene53:85–96
    [Google Scholar]
  46. Skerker J. M., Shapiro L. 2000; Identification and cell cycle control of a novel pilus system in Caulobacter crescentus. EMBO J19:3223–3234
    [Google Scholar]
  47. Slots J., Dahlen G. 1985; Subgingival microorganisms and bacterial virulence factors in periodontitis. Scand J Dent Res93:119–127
    [Google Scholar]
  48. Snellings N. J, Tall B. D., Venkatesan M. M. 1997; Characterization of Shigella type 1 fimbriae; expression, FimA sequence, and phase variation. Infect Immun65:2462–2467
    [Google Scholar]
  49. Steckelberg J. M, Melton L. J, Ilstrup D. M, Rouse M. S., Wilson W. R. 1990; Influence of referral bias on the apparent clinical spectrum of infective endocarditis. Am J Med88:582–588
    [Google Scholar]
  50. Stone K. D, Zhang H, Carlson L. K., Donnenberg M. S. 1996; A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus. Mol Microbiol20:325–337
    [Google Scholar]
  51. Strom M. S., Lory S. 1993; Structure-function and biogenesis of the type IV pili. Annu Rev Microbiol47:565–596
    [Google Scholar]
  52. Tanner A. C. R, Haffer C, Brathall G. T, Visconti R. A., Socransky S. S. 1979; A study of the bacteria associated with advancing periodontitis in man. J Clin Periodontol6:278–307
    [Google Scholar]
  53. Vandepitte J, DeGeest H., Jousten P. 1977; Subacute bacterial endocarditis due to Actinobacillus actinomycetemcomitans. J Clin Pathol30:842–846
    [Google Scholar]
  54. Villar M. T, Helber J. T, Hood B, Schaefer M. R., Hirschberg R. L. 1999; Eikenella corrodens phase variation involves a posttranslational event in pilus formation. J Bacteriol181:4154–4160
    [Google Scholar]
  55. Zambon J. J. 1985; Actinobacillus actinomycetemcomitans in human periodontal disease. J Clin Periodontol12:1–20
    [Google Scholar]
  56. Zambon J. J., Sunday G. J. 1989; Absence of the 23S ribosomal RNA subunit in Actinobacillus actinomycetemcomitans. J Dent Res68:218
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.25786-0
Loading
/content/journal/micro/10.1099/mic.0.25786-0
Loading

Data & Media loading...

Most cited this month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error