Abstract
New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains of Streptococcus mutans(pTV1-OK) and subsequent recovery of interrupted genes in Escherichia coli (pT21delta2TetM). In this report, we demonstrate the utility of Tn917 mutagenesis of a poorly transformable strain of S. mutans (JH1005) by showing (i) the conditional replication of pTV1-OK, a repA(Ts) derivative of the broad-host-range plasmid pWVO1 harboring Tn9l7, in JH1005 at the permissive temperature (30 degrees C) versus that at the nonpermissive temperature (45 degrees C); (ii) transposition frequencies similar to those reported for Bacillus subtilis (10(-5) to 10(-4)) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant survivors following a temperature shift to 42 to 45 degrees C; and (iii) the apparent randomness of Tn917 insertion as determined by Southern hybridization analysis and the ability to isolate nutritional mutants, mutants in acid tolerance, and mutants in bacteriocin production, at frequencies ranging from 0.1 to 0.7%. Recovery of transposon-interrupted genes was achieved by two methods: (i) marker rescue in E. coli with the recovery vector pTV21delta2TetM, a tetracycline-resistant and ampicillin-sensitive Tn9l7-pBR322 hybrid, and (ii) "shotgun" cloning of genomic libraries of Tn917 mutants into pUC19. Sequence analyses revealed insertions at five different genetic loci in sequences displaying homologies to Clostridium spp.fhs (66% identity), E. coli dfp (43% identity), and B. subtilis ylxM-ffh (58% identity), icd (citC [69% identity]), and argD (61% identity). Insertions in icd and argD caused nutritional requirements; the one in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caused both acid sensitivity and nutritional requirements. This paper describes the construction of pTV1-OK and demonstrates that it can be efficiently employed to deliver Tn917 into S. mutans for genetic analyses with some degree of randomness and that insertions in the chromosome can be easily recovered for subsequent characterization. This represents the first published report of successful Tn9l7 mutagenesis in the genus Streptococcus.
Full Text
The Full Text of this article is available as a PDF (452.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Babb B. L., Collett H. J., Reid S. J., Woods D. R. Transposon mutagenesis of Clostridium acetobutylicum P262: isolation and characterization of solvent deficient and metronidazole resistant mutants. FEMS Microbiol Lett. 1993 Dec 15;114(3):343–348. doi: 10.1111/j.1574-6968.1993.tb06596.x. [DOI] [PubMed] [Google Scholar]
- Burne R. A., Quivey R. G., Jr Use of transposons to dissect pathogenic strategies of gram-positive bacteria. Methods Enzymol. 1994;235:405–426. doi: 10.1016/0076-6879(94)35158-9. [DOI] [PubMed] [Google Scholar]
- Byström A. S., Hjalmarsson K. J., Wikström P. M., Björk G. R. The nucleotide sequence of an Escherichia coli operon containing genes for the tRNA(m1G)methyltransferase, the ribosomal proteins S16 and L19 and a 21-K polypeptide. EMBO J. 1983;2(6):899–905. doi: 10.1002/j.1460-2075.1983.tb01519.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Camilli A., Portnoy A., Youngman P. Insertional mutagenesis of Listeria monocytogenes with a novel Tn917 derivative that allows direct cloning of DNA flanking transposon insertions. J Bacteriol. 1990 Jul;172(7):3738–3744. doi: 10.1128/jb.172.7.3738-3744.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carlsson J. Nutritional requirements of Streptococcus mutans. Caries Res. 1970;4(4):305–320. doi: 10.1159/000259653. [DOI] [PubMed] [Google Scholar]
- Cheung A. L., Koomey J. M., Butler C. A., Projan S. J., Fischetti V. A. Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6462–6466. doi: 10.1073/pnas.89.14.6462. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ciampi M. S., Schmid M. B., Roth J. R. Transposon Tn10 provides a promoter for transcription of adjacent sequences. Proc Natl Acad Sci U S A. 1982 Aug;79(16):5016–5020. doi: 10.1073/pnas.79.16.5016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clewell D. B., Tomich P. K., Gawron-Burke M. C., Franke A. E., Yagi Y., An F. Y. Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917. J Bacteriol. 1982 Dec;152(3):1220–1230. doi: 10.1128/jb.152.3.1220-1230.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cossart P., Vicente M. F., Mengaud J., Baquero F., Perez-Diaz J. C., Berche P. Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation. Infect Immun. 1989 Nov;57(11):3629–3636. doi: 10.1128/iai.57.11.3629-3636.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
- Hamilton I. R., Buckley N. D. Adaptation by Streptococcus mutans to acid tolerance. Oral Microbiol Immunol. 1991 Apr;6(2):65–71. doi: 10.1111/j.1399-302x.1991.tb00453.x. [DOI] [PubMed] [Google Scholar]
- Heimberg H., Boyen A., Crabeel M., Glansdorff N. Escherichia coli and Saccharomyces cerevisiae acetylornithine aminotransferase: evolutionary relationship with ornithine aminotransferase. Gene. 1990 May 31;90(1):69–78. doi: 10.1016/0378-1119(90)90440-3. [DOI] [PubMed] [Google Scholar]
- Hillman J. D., Chen A., Duncan M., Lee S. W. Evidence that L-(+)-lactate dehydrogenase deficiency is lethal in Streptococcus mutans. Infect Immun. 1994 Jan;62(1):60–64. doi: 10.1128/iai.62.1.60-64.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hillman J. D., Dzuback A. L., Andrews S. W. Colonization of the human oral cavity by a Streptococcus mutans mutant producing increased bacteriocin. J Dent Res. 1987 Jun;66(6):1092–1094. doi: 10.1177/00220345870660060101. [DOI] [PubMed] [Google Scholar]
- Hillman J. D., Johnson K. P., Yaphe B. I. Isolation of a Streptococcus mutans strain producing a novel bacteriocin. Infect Immun. 1984 Apr;44(1):141–144. doi: 10.1128/iai.44.1.141-144.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Honda K., Nakamura K., Nishiguchi M., Yamane K. Cloning and characterization of a Bacillus subtilis gene encoding a homolog of the 54-kilodalton subunit of mammalian signal recognition particle and Escherichia coli Ffh. J Bacteriol. 1993 Aug;175(15):4885–4894. doi: 10.1128/jb.175.15.4885-4894.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Horinouchi S., Weisblum B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol. 1982 May;150(2):815–825. doi: 10.1128/jb.150.2.815-825.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Iordănescu S. Three distinct plasmids originating in the same Staphylococcus aureus strain. Arch Roum Pathol Exp Microbiol. 1976 Jan-Jun;35(1-2):111–118. [PubMed] [Google Scholar]
- Israelsen H., Madsen S. M., Vrang A., Hansen E. B., Johansen E. Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80. Appl Environ Microbiol. 1995 Jul;61(7):2540–2547. doi: 10.1128/aem.61.7.2540-2547.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jack R. W., Tagg J. R., Ray B. Bacteriocins of gram-positive bacteria. Microbiol Rev. 1995 Jun;59(2):171–200. doi: 10.1128/mr.59.2.171-200.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jin S., Sonenshein A. L. Identification of two distinct Bacillus subtilis citrate synthase genes. J Bacteriol. 1994 Aug;176(15):4669–4679. doi: 10.1128/jb.176.15.4669-4679.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kok J., van der Vossen J. M., Venema G. Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl Environ Microbiol. 1984 Oct;48(4):726–731. doi: 10.1128/aem.48.4.726-731.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lovell C. R., Przybyla A., Ljungdahl L. G. Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase. Arch Microbiol. 1988;149(4):280–285. doi: 10.1007/BF00411642. [DOI] [PubMed] [Google Scholar]
- Luirink J., Dobberstein B. Mammalian and Escherichia coli signal recognition particles. Mol Microbiol. 1994 Jan;11(1):9–13. doi: 10.1111/j.1365-2958.1994.tb00284.x. [DOI] [PubMed] [Google Scholar]
- Lundberg L. G., Thoresson H. O., Karlström O. H., Nyman P. O. Nucleotide sequence of the structural gene for dUTPase of Escherichia coli K-12. EMBO J. 1983;2(6):967–971. doi: 10.1002/j.1460-2075.1983.tb01529.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lunsford R. D. A Tn4001 delivery system for Streptococcus gordonii (Challis). Plasmid. 1995 Mar;33(2):153–157. doi: 10.1006/plas.1995.1016. [DOI] [PubMed] [Google Scholar]
- Maguin E., Duwat P., Hege T., Ehrlich D., Gruss A. New thermosensitive plasmid for gram-positive bacteria. J Bacteriol. 1992 Sep;174(17):5633–5638. doi: 10.1128/jb.174.17.5633-5638.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- O'Reilly M., Devine K. M. Sequence and analysis of the citrulline biosynthetic operon argC-F from Bacillus subtilis. Microbiology. 1994 May;140(Pt 5):1023–1025. doi: 10.1099/13500872-140-5-1023. [DOI] [PubMed] [Google Scholar]
- Parrot M., Caufield P. W., Lavoie M. C. Preliminary characterization of four bacteriocins from Streptococcus mutans. Can J Microbiol. 1990 Feb;36(2):123–130. doi: 10.1139/m90-022. [DOI] [PubMed] [Google Scholar]
- Perez-Casal J., Price J. A., Maguin E., Scott J. R. An M protein with a single C repeat prevents phagocytosis of Streptococcus pyogenes: use of a temperature-sensitive shuttle vector to deliver homologous sequences to the chromosome of S. pyogenes. Mol Microbiol. 1993 May;8(5):809–819. doi: 10.1111/j.1365-2958.1993.tb01628.x. [DOI] [PubMed] [Google Scholar]
- Perry D., Kuramitsu H. K. Genetic transformation of Streptococcus mutans. Infect Immun. 1981 Jun;32(3):1295–1297. doi: 10.1128/iai.32.3.1295-1297.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Phillips G. J., Silhavy T. J. The E. coli ffh gene is necessary for viability and efficient protein export. Nature. 1992 Oct 22;359(6397):744–746. doi: 10.1038/359744a0. [DOI] [PubMed] [Google Scholar]
- Rankin C. A., Haslam G. C., Himes R. H. Sequence and expression of the gene for N10-formyltetrahydrofolate synthetase from Clostridium cylindrosporum. Protein Sci. 1993 Feb;2(2):197–205. doi: 10.1002/pro.5560020208. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ribes V., Römisch K., Giner A., Dobberstein B., Tollervey D. E. coli 4.5S RNA is part of a ribonucleoprotein particle that has properties related to signal recognition particle. Cell. 1990 Nov 2;63(3):591–600. doi: 10.1016/0092-8674(90)90454-m. [DOI] [PubMed] [Google Scholar]
- Saunders C. W., Guild W. R. Monomer plasmid DNA transforms Streptococcus pneumoniae. Mol Gen Genet. 1981;181(1):57–62. doi: 10.1007/BF00339005. [DOI] [PubMed] [Google Scholar]
- Smith H. E., Wisselink H. J., Vecht U., Gielkens A. L., Smits M. A. High-efficiency transformation and gene inactivation in Streptococcus suis type 2. Microbiology. 1995 Jan;141(Pt 1):181–188. doi: 10.1099/00221287-141-1-181. [DOI] [PubMed] [Google Scholar]
- Spatafora G., Rohrer K., Barnard D., Michalek S. A Streptococcus mutans mutant that synthesizes elevated levels of intracellular polysaccharide is hypercariogenic in vivo. Infect Immun. 1995 Jul;63(7):2556–2563. doi: 10.1128/iai.63.7.2556-2563.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spitzer E. D., Jimenez-Billini H. E., Weiss B. beta-Alanine auxotrophy associated with dfp, a locus affecting DNA synthesis in Escherichia coli. J Bacteriol. 1988 Feb;170(2):872–876. doi: 10.1128/jb.170.2.872-876.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spitzer E. D., Weiss B. dfp Gene of Escherichia coli K-12, a locus affecting DNA synthesis, codes for a flavoprotein. J Bacteriol. 1985 Dec;164(3):994–1003. doi: 10.1128/jb.164.3.994-1003.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Terleckyj B., Shockman G. D. Amino acid requirements of Streptococcus mutans and other oral streptococci. Infect Immun. 1975 Apr;11(4):656–664. doi: 10.1128/iai.11.4.656-664.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas W. D., Jr, Archer G. L. Identification and cloning of the conjugative transfer region of Staphylococcus aureus plasmid pGO1. J Bacteriol. 1989 Feb;171(2):684–691. doi: 10.1128/jb.171.2.684-691.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thorsness P. E., Koshland D. E., Jr Inactivation of isocitrate dehydrogenase by phosphorylation is mediated by the negative charge of the phosphate. J Biol Chem. 1987 Aug 5;262(22):10422–10425. [PubMed] [Google Scholar]
- Tobian J. A., Cline M. L., Macrina F. L. Characterization and expression of a cloned tetracycline resistance determinant from the chromosome of Streptococcus mutans. J Bacteriol. 1984 Nov;160(2):556–563. doi: 10.1128/jb.160.2.556-563.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tomich P. K., An F. Y., Clewell D. B. A transposon (Tn917) in Streptococcus faecalis that exhibits enhanced transposition during induction of drug resistance. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):1217–1221. doi: 10.1101/sqb.1979.043.01.138. [DOI] [PubMed] [Google Scholar]
- Trieu-Cuot P., Courvalin P. Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III. Gene. 1983 Sep;23(3):331–341. doi: 10.1016/0378-1119(83)90022-7. [DOI] [PubMed] [Google Scholar]
- Villafane R., Bechhofer D. H., Narayanan C. S., Dubnau D. Replication control genes of plasmid pE194. J Bacteriol. 1987 Oct;169(10):4822–4829. doi: 10.1128/jb.169.10.4822-4829.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Whitehead T. R., Rabinowitz J. C. Cloning and expression in Escherichia coli of the gene for 10-formyltetrahydrofolate synthetase from Clostridium acidiurici ("Clostridium acidi-urici"). J Bacteriol. 1986 Jul;167(1):205–209. doi: 10.1128/jb.167.1.205-209.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamashita Y., Takehara T., Kuramitsu H. K. Molecular characterization of a STreptococcus mutans mutant altered in environmental stress responses. J Bacteriol. 1993 Oct;175(19):6220–6228. doi: 10.1128/jb.175.19.6220-6228.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Youngman P. J., Perkins J. B., Losick R. Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcus faecalis transposon Tn917. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2305–2309. doi: 10.1073/pnas.80.8.2305. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Youngman P., Perkins J. B., Losick R. A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn917 insertions. Mol Gen Genet. 1984;195(3):424–433. doi: 10.1007/BF00341443. [DOI] [PubMed] [Google Scholar]