Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Aug;63(8):2924–2930. doi: 10.1128/iai.63.8.2924-2930.1995

Construction and use of integration plasmids to generate site-specific mutations in the Actinomyces viscosus T14V chromosome.

M K Yeung 1
PMCID: PMC173398  PMID: 7622214

Abstract

Stable transformants of Actinomyces viscosus T14V carrying heterologous DNA were obtained with the aid of integration plasmids. These plasmids contained a kanamycin resistance (Kmr) gene flanked by A. viscosus T14V genomic DNA, including parts of the type 1 structural fimbrial subunit gene (fimP) on one or both sides of the antibiotic marker. Significantly more Kmr transformants were obtained with a plasmid carrying longer segments of homologous strain T14V DNA. Integration of this plasmid into the A. viscosus T14V genome affected the expression and function of type 1 fimbriae in the transformants. In the transformant strain designated A. viscosus MY50D, the inactivated fimP replaced the wild-type fimP via allelic replacement. A. viscosus MY51S and MY52S each contained a copy of the plasmid integrated into the genome by a Campbell-like insertion mechanism. A. viscosus MY50D and MY51S lacked type 1 fimbriae and did not bind to proline-rich proteins (the fimbrial receptors) immobilized on nitrocellulose. In contrast, strain MY52S synthesized the structural subunit protein, as detected by immunostaining with anti-A. viscosus T14V type 1 fimbria antibodies. However, the high-molecular-weight proteins observed in sodium dodecyl sulfate-polyacrylamide gels of fimbriae from the cell wall of the wild-type strain T14V were absent in cell wall preparations of this strain. Moreover, A. viscosus MY52S failed to bind, in vitro, to proline-rich proteins. Thus, these results demonstrate that insertion of heterologous DNA at specific sites of the Actinomyces genome can be facilitated with integratable plasmids and that the transformants and mutants generated will aid in the delineation of the roles and contributions of specific genes to the structure and function of any macromolecule produced by these organisms.

Full Text

The Full Text of this article is available as a PDF (368.2 KB).

Selected References

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

  1. Batteiger B., Newhall W. J., 5th, Jones R. B. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J Immunol Methods. 1982 Dec 30;55(3):297–307. doi: 10.1016/0022-1759(82)90089-8. [DOI] [PubMed] [Google Scholar]
  2. Cato A., Jr, Guild W. R. Transformation and DNA size. I. Activity of fragments of defined size and a fit to a random double cross-over model. J Mol Biol. 1968 Oct 14;37(1):157–178. doi: 10.1016/0022-2836(68)90080-6. [DOI] [PubMed] [Google Scholar]
  3. Cisar J. O., Barsumian E. L., Siraganian R. P., Clark W. B., Yeung M. K., Hsu S. D., Curl S. H., Vatter A. E., Sandberg A. L. Immunochemical and functional studies of Actinomyces viscosus T14V type 1 fimbriae with monoclonal and polyclonal antibodies directed against the fimbrial subunit. J Gen Microbiol. 1991 Aug;137(8):1971–1979. doi: 10.1099/00221287-137-8-1971. [DOI] [PubMed] [Google Scholar]
  4. Cisar J. O., Kolenbrander P. E., McIntire F. C. Specificity of coaggregation reactions between human oral streptococci and strains of Actinomyces viscosus or Actinomyces naeslundii. Infect Immun. 1979 Jun;24(3):742–752. doi: 10.1128/iai.24.3.742-752.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cisar J. O., Vatter A. E., Clark W. B., Curl S. H., Hurst-Calderone S., Sandberg A. L. Mutants of Actinomyces viscosus T14V lacking type 1, type 2, or both types of fimbriae. Infect Immun. 1988 Nov;56(11):2984–2989. doi: 10.1128/iai.56.11.2984-2989.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davison J., Heusterspreute M., Chevalier N., Ha-Thi V., Brunel F. Vectors with restriction site banks. V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites. Gene. 1987;51(2-3):275–280. doi: 10.1016/0378-1119(87)90316-7. [DOI] [PubMed] [Google Scholar]
  7. Doig P., Sastry P. A., Hodges R. S., Lee K. K., Paranchych W., Irvin R. T. Inhibition of pilus-mediated adhesion of Pseudomonas aeruginosa to human buccal epithelial cells by monoclonal antibodies directed against pili. Infect Immun. 1990 Jan;58(1):124–130. doi: 10.1128/iai.58.1.124-130.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fives-Taylor P. M., Thompson D. W. Surface properties of Streptococcus sanguis FW213 mutants nonadherent to saliva-coated hydroxyapatite. Infect Immun. 1985 Mar;47(3):752–759. doi: 10.1128/iai.47.3.752-759.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gibbons R. J., Hay D. I., Cisar J. O., Clark W. B. Adsorbed salivary proline-rich protein 1 and statherin: receptors for type 1 fimbriae of Actinomyces viscosus T14V-J1 on apatitic surfaces. Infect Immun. 1988 Nov;56(11):2990–2993. doi: 10.1128/iai.56.11.2990-2993.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gibbons R. J., Hay D. I. Human salivary acidic proline-rich proteins and statherin promote the attachment of Actinomyces viscosus LY7 to apatitic surfaces. Infect Immun. 1988 Feb;56(2):439–445. doi: 10.1128/iai.56.2.439-445.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Handley P. S., Jacob A. E. Some structural and physiological properties of fimbriae of Streptococcus faecalis. J Gen Microbiol. 1981 Dec;127(2):289–293. doi: 10.1099/00221287-127-2-289. [DOI] [PubMed] [Google Scholar]
  13. Hermans J., Martin C., Huijberts G. N., Goosen T., de Bont J. A. Transformation of Mycobacterium aurum and Mycobacterium smegmatis with the broad host-range gram-negative cosmid vector pJRD215. Mol Microbiol. 1991 Jun;5(6):1561–1566. doi: 10.1111/j.1365-2958.1991.tb00803.x. [DOI] [PubMed] [Google Scholar]
  14. Hultgren S. J., Lindberg F., Magnusson G., Kihlberg J., Tennent J. M., Normark S. The PapG adhesin of uropathogenic Escherichia coli contains separate regions for receptor binding and for the incorporation into the pilus. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4357–4361. doi: 10.1073/pnas.86.12.4357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hultgren S. J., Normark S., Abraham S. N. Chaperone-assisted assembly and molecular architecture of adhesive pili. Annu Rev Microbiol. 1991;45:383–415. doi: 10.1146/annurev.mi.45.100191.002123. [DOI] [PubMed] [Google Scholar]
  16. Jannière L., Niaudet B., Pierre E., Ehrlich S. D. Stable gene amplification in the chromosome of Bacillus subtilis. Gene. 1985;40(1):47–55. doi: 10.1016/0378-1119(85)90023-x. [DOI] [PubMed] [Google Scholar]
  17. Khan A. S., Schifferli D. M. A minor 987P protein different from the structural fimbrial subunit is the adhesin. Infect Immun. 1994 Oct;62(10):4233–4243. doi: 10.1128/iai.62.10.4233-4243.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krogfelt K. A., Bergmans H., Klemm P. Direct evidence that the FimH protein is the mannose-specific adhesin of Escherichia coli type 1 fimbriae. Infect Immun. 1990 Jun;58(6):1995–1998. doi: 10.1128/iai.58.6.1995-1998.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Lee K. K., Sheth H. B., Wong W. Y., Sherburne R., Paranchych W., Hodges R. S., Lingwood C. A., Krivan H., Irvin R. T. The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip-associated event involving the C-terminal region of the structural pilin subunit. Mol Microbiol. 1994 Feb;11(4):705–713. doi: 10.1111/j.1365-2958.1994.tb00348.x. [DOI] [PubMed] [Google Scholar]
  21. Leenhouts K. J., Kok J., Venema G. Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. lactis. Appl Environ Microbiol. 1989 Feb;55(2):394–400. doi: 10.1128/aem.55.2.394-400.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Oka A., Sugisaki H., Takanami M. Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol. 1981 Apr 5;147(2):217–226. doi: 10.1016/0022-2836(81)90438-1. [DOI] [PubMed] [Google Scholar]
  23. Piggot P. J., Curtis C. A., de Lencastre H. Use of integrational plasmid vectors to demonstrate the polycistronic nature of a transcriptional unit (spoIIA) required for sporulation of Bacillus subtilis. J Gen Microbiol. 1984 Aug;130(8):2123–2136. doi: 10.1099/00221287-130-8-2123. [DOI] [PubMed] [Google Scholar]
  24. Rothbard J. B., Fernandez R., Wang L., Teng N. N., Schoolnik G. K. Antibodies to peptides corresponding to a conserved sequence of gonococcal pilins block bacterial adhesion. Proc Natl Acad Sci U S A. 1985 Feb;82(3):915–919. doi: 10.1073/pnas.82.3.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Snapper S. B., Lugosi L., Jekkel A., Melton R. E., Kieser T., Bloom B. R., Jacobs W. R., Jr Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6987–6991. doi: 10.1073/pnas.85.18.6987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  27. Virji M., Saunders J. R., Sims G., Makepeace K., Maskell D., Ferguson D. J. Pilus-facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin. Mol Microbiol. 1993 Dec;10(5):1013–1028. doi: 10.1111/j.1365-2958.1993.tb00972.x. [DOI] [PubMed] [Google Scholar]
  28. Vosman B., Kooistra J., Olijve J., Venema G. Integration of vector-containing Bacillus subtilis chromosomal DNA by a Campbell-like mechanism. Mol Gen Genet. 1986 Sep;204(3):524–531. doi: 10.1007/BF00331035. [DOI] [PubMed] [Google Scholar]
  29. Wheeler T. T., Clark W. B., Birdsell D. C. Adherence of Actinomyces viscosus T14V and T14AV to hydroxyapatite surfaces in vitro and human teeth in vivo. Infect Immun. 1979 Sep;25(3):1066–1074. doi: 10.1128/iai.25.3.1066-1074.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yanagawa R., Honda E. Presence of pili in species of human and animal parasites and pathogens of the genuscorynebacterium. Infect Immun. 1976 Apr;13(4):1293–1295. doi: 10.1128/iai.13.4.1293-1295.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yeung M. K., Chassy B. M., Cisar J. O. Cloning and expression of a type 1 fimbrial subunit of Actinomyces viscosus T14V. J Bacteriol. 1987 Apr;169(4):1678–1683. doi: 10.1128/jb.169.4.1678-1683.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yeung M. K., Cisar J. O. Sequence homology between the subunits of two immunologically and functionally distinct types of fimbriae of Actinomyces spp. J Bacteriol. 1990 May;172(5):2462–2468. doi: 10.1128/jb.172.5.2462-2468.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yeung M. K. Conservation of an Actinomyces viscosus T14V type 1 fimbrial subunit homolog among divergent groups of Actinomyces spp. Infect Immun. 1992 Mar;60(3):1047–1054. doi: 10.1128/iai.60.3.1047-1054.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yeung M. K., Fernandez S. R. Isolation of a neuraminidase gene from Actinomyces viscosus T14V. Appl Environ Microbiol. 1991 Nov;57(11):3062–3069. doi: 10.1128/aem.57.11.3062-3069.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yeung M. K., Kozelsky C. S. Transformation of Actinomyces spp. by a gram-negative broad-host-range plasmid. J Bacteriol. 1994 Jul;176(13):4173–4176. doi: 10.1128/jb.176.13.4173-4176.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES