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. 1990 Jul;56(7):2156–2163. doi: 10.1128/aem.56.7.2156-2163.1990

Cloning and characterization of the thymidylate synthase gene from Lactococcus lactis subsp. lactis.

P Ross 1, F O'Gara 1, S Condon 1
PMCID: PMC184576  PMID: 2117882

Abstract

The thymidylate synthase (thyA) gene has been isolated from Lactococcus lactis subsp. lactis. The cloned gene was strongly expressed in Escherichia coli both in vivo and in vitro (maxicells and cell-free transcription and translation systems) and complemented E. coli thyA mutants. DNA-DNA hybridizations demonstrated that the thyA gene is encoded by the chromosome of L. lactis subsp. lactis. By sequential deletion of DNA outside the complementing region, the thyA gene was localized to a 1.1-kilobase DNA fragment. The nucleotide sequence of the lactococcal thyA gene was determined by the dideoxy-chain termination technique. The derived amino acid sequence indicated a protein size of 32,580 daltons, which is in good agreement with results obtained from maxicell and in vitro transcription and translation experiments. The primary sequence is homologous to 12 other thyA proteins from a variety of other organisms. Upstream from the structural gene, -10 and -35 promoter sequences which were almost canonical sigma-70 promoter sequences were identified, which may explain the strong expression of the thyA gene observed in E. coli. An A-T-rich sequence characteristic of gram-positive promoters was also noted adjacent to the -35 region. The thyA gene has potential as a marker for plasmid maintenance and selection in food systems.

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Selected References

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  1. Belfort M., Maley G., Pedersen-Lane J., Maley F. Primary structure of the Escherichia coli thyA gene and its thymidylate synthase product. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4914–4918. doi: 10.1073/pnas.80.16.4914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beverley S. M., Ellenberger T. E., Cordingley J. S. Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2584–2588. doi: 10.1073/pnas.83.8.2584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  5. Bzik D. J., Li W. B., Horii T., Inselburg J. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8360–8364. doi: 10.1073/pnas.84.23.8360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chu F. K., Maley G. F., Maley F., Belfort M. Intervening sequence in the thymidylate synthase gene of bacteriophage T4. Proc Natl Acad Sci U S A. 1984 May;81(10):3049–3053. doi: 10.1073/pnas.81.10.3049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crusberg T. C., Leary R., Kisliuk R. L. Properties of thymidylate synthetase from dichloromethotrexate-resistant Lactobacillus casei. J Biol Chem. 1970 Oct 25;245(20):5292–5296. [PubMed] [Google Scholar]
  9. Danenberg P. V. Thymidylate synthetase - a target enzyme in cancer chemotherapy. Biochim Biophys Acta. 1977 Dec 23;473(2):73–92. doi: 10.1016/0304-419x(77)90001-4. [DOI] [PubMed] [Google Scholar]
  10. Dunlap R. B., Harding N. G., Huennekens F. M. Thymidylate synthetase from amethopterin-resistant Lactobacillus casei. Biochemistry. 1971 Jan 5;10(1):88–97. doi: 10.1021/bi00777a014. [DOI] [PubMed] [Google Scholar]
  11. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  12. Gasson M. J., Davies F. L. High-frequency conjugation associated with Streptococcus lactis donor cell aggregation. J Bacteriol. 1980 Sep;143(3):1260–1264. doi: 10.1128/jb.143.3.1260-1264.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gasson M. J. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol. 1983 Apr;154(1):1–9. doi: 10.1128/jb.154.1.1-9.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goelling D., Stahl U. Cloning and expression of an alpha-acetolactate decarboxylase gene from Streptococcus lactis subsp. diacetylactis in Escherichia coli. Appl Environ Microbiol. 1988 Jul;54(7):1889–1891. doi: 10.1128/aem.54.7.1889-1891.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hardy L. W., Finer-Moore J. S., Montfort W. R., Jones M. O., Santi D. V., Stroud R. M. Atomic structure of thymidylate synthase: target for rational drug design. Science. 1987 Jan 23;235(4787):448–455. doi: 10.1126/science.3099389. [DOI] [PubMed] [Google Scholar]
  16. Herman R. E., McKay L. L. Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli. Appl Environ Microbiol. 1986 Jul;52(1):45–50. doi: 10.1128/aem.52.1.45-50.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Higgins D. G., Sharp P. M. CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene. 1988 Dec 15;73(1):237–244. doi: 10.1016/0378-1119(88)90330-7. [DOI] [PubMed] [Google Scholar]
  18. Honess R. W., Bodemer W., Cameron K. R., Niller H. H., Fleckenstein B., Randall R. E. The A+T-rich genome of Herpesvirus saimiri contains a highly conserved gene for thymidylate synthase. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3604–3608. doi: 10.1073/pnas.83.11.3604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Iwakura M., Kawata M., Tsuda K., Tanaka T. Nucleotide sequence of the thymidylate synthase B and dihydrofolate reductase genes contained in one Bacillus subtilis operon. Gene. 1988 Apr 15;64(1):9–20. doi: 10.1016/0378-1119(88)90476-3. [DOI] [PubMed] [Google Scholar]
  20. Kenny E., Atkinson T., Hartley B. S. Nucleotide sequence of the thymidylate synthetase gene (thyP3) from the Bacillus subtilis phage phi 3T. Gene. 1985;34(2-3):335–342. doi: 10.1016/0378-1119(85)90142-8. [DOI] [PubMed] [Google Scholar]
  21. Kieny M. P., Lathe R., Lecocq J. P. New versatile cloning and sequencing vectors based on bacteriophage M13. Gene. 1983 Dec;26(1):91–99. doi: 10.1016/0378-1119(83)90039-2. [DOI] [PubMed] [Google Scholar]
  22. Kok J., Leenhouts K. J., Haandrikman A. J., Ledeboer A. M., Venema G. Nucleotide sequence of the cell wall proteinase gene of Streptococcus cremoris Wg2. Appl Environ Microbiol. 1988 Jan;54(1):231–238. doi: 10.1128/aem.54.1.231-238.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  24. Ludwig W., Seewaldt E., Kilpper-Bälz R., Schleifer K. H., Magrum L., Woese C. R., Fox G. E., Stackebrandt E. The phylogenetic position of Streptococcus and Enterococcus. J Gen Microbiol. 1985 Mar;131(3):543–551. doi: 10.1099/00221287-131-3-543. [DOI] [PubMed] [Google Scholar]
  25. Maley G. F., Maley F., Baugh C. M. Studies on identifying the folylpolyglutamate binding sites of Lactobacillus casei thymidylate synthetase. Arch Biochem Biophys. 1982 Jul;216(2):551–558. doi: 10.1016/0003-9861(82)90244-2. [DOI] [PubMed] [Google Scholar]
  26. Perryman S. M., Rossana C., Deng T. L., Vanin E. F., Johnson L. F. Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme. Mol Biol Evol. 1986 Jul;3(4):313–321. doi: 10.1093/oxfordjournals.molbev.a040400. [DOI] [PubMed] [Google Scholar]
  27. Pinter K., Davisson V. J., Santi D. V. Cloning, sequencing, and expression of the Lactobacillus casei thymidylate synthase gene. DNA. 1988 May;7(4):235–241. doi: 10.1089/dna.1988.7.235. [DOI] [PubMed] [Google Scholar]
  28. Poolman B., Royer T. J., Mainzer S. E., Schmidt B. F. Lactose transport system of Streptococcus thermophilus: a hybrid protein with homology to the melibiose carrier and enzyme III of phosphoenolpyruvate-dependent phosphotransferase systems. J Bacteriol. 1989 Jan;171(1):244–253. doi: 10.1128/jb.171.1.244-253.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Porter E. V., Chassy B. M. Nucleotide sequence of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei: comparison to analogous pbg genes of other gram-positive organisms. Gene. 1988;62(2):263–276. doi: 10.1016/0378-1119(88)90564-1. [DOI] [PubMed] [Google Scholar]
  30. Renault P., Gaillardin C., Heslot H. Product of the Lactococcus lactis gene required for malolactic fermentation is homologous to a family of positive regulators. J Bacteriol. 1989 Jun;171(6):3108–3114. doi: 10.1128/jb.171.6.3108-3114.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ross P., O'Gara F., Condon S. Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes. Appl Environ Microbiol. 1990 Jul;56(7):2164–2169. doi: 10.1128/aem.56.7.2164-2169.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ross R., O'Gara F., Condon S. Cloning of chromosomal genes of Lactococcus by heterologous complementation: partial characterisation of a putative lactose transport gene. FEMS Microbiol Lett. 1989 Oct 1;52(1-2):183–187. doi: 10.1016/0378-1097(89)90193-6. [DOI] [PubMed] [Google Scholar]
  33. STACEY K. A., SIMSON E. IMPROVED METHOD FOR THE ISOLATION OF THYMINE-REQUIRING MUTANTS OF ESCHERICHIA COLI. J Bacteriol. 1965 Aug;90:554–555. doi: 10.1128/jb.90.2.554-555.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  37. Takeishi K., Kaneda S., Ayusawa D., Shimizu K., Gotoh O., Seno T. Nucleotide sequence of a functional cDNA for human thymidylate synthase. Nucleic Acids Res. 1985 Mar 25;13(6):2035–2043. doi: 10.1093/nar/13.6.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Taylor G. R., Lagosky P. A., Storms R. K., Haynes R. H. Molecular characterization of the cell cycle-regulated thymidylate synthase gene of Saccharomyces cerevisiae. J Biol Chem. 1987 Apr 15;262(11):5298–5307. [PubMed] [Google Scholar]
  39. Thompson R., Honess R. W., Taylor L., Morran J., Davison A. J. Varicella-zoster virus specifies a thymidylate synthetase. J Gen Virol. 1987 May;68(Pt 5):1449–1455. doi: 10.1099/0022-1317-68-5-1449. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Wirth R., An F. Y., Clewell D. B. Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector. J Bacteriol. 1986 Mar;165(3):831–836. doi: 10.1128/jb.165.3.831-836.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. de Bruijn F. J., Lupski J. R. The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids--a review. Gene. 1984 Feb;27(2):131–149. doi: 10.1016/0378-1119(84)90135-5. [DOI] [PubMed] [Google Scholar]
  43. von Wright A., Tynkkynen S., Suominen M. Cloning of a Streptococcus lactis subsp. lactis Chromosomal Fragment Associated with the Ability To Grow in Milk. Appl Environ Microbiol. 1987 Jul;53(7):1584–1588. doi: 10.1128/aem.53.7.1584-1588.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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