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. 1996 Jul;178(13):3689–3694. doi: 10.1128/jb.178.13.3689-3694.1996

Cloning and transcriptional analysis of two threonine biosynthetic genes from Lactococcus lactis MG1614.

S M Madsen 1, B Albrechtsen 1, E B Hansen 1, H Israelsen 1
PMCID: PMC178148  PMID: 8682767

Abstract

Two genes, hom and thrB, involved in threonine biosynthesis in Lactococcus lactis MG1614, were cloned and sequenced. These genes, which encode homoserine dehydrogenase and homoserine kinase, were initially identified by the homology of their gene products with known homoserine dehydrogenases and homoserine kinases from other organisms. The identification was supported by construction of a mutant containing a deletion in hom and thrB that was unable to grow in a defined medium lacking threonine. Transcriptional analysis showed that the two genes were located in a bicistronic operon with the order 5' hom-thrB 3' and that transcription started 66 bp upstream of the translational start codon of the hom gene. A putative -10 promoter region (TATAAT) was located 6 bp upstream of the transcriptional start point, but no putative -35 region was identified. A DNA fragment covering 155 bp upstream of the hom translational start site was functional in pAK80, an L. lactis promoter probe vector. In addition, transcriptional studies showed no threonine-dependent regulation of hom-thrB transcription.

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Selected References

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  1. BERTANI G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol. 1951 Sep;62(3):293–300. doi: 10.1128/jb.62.3.293-300.1951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bidnenko E., Ehrlich D., Chopin M. C. Phage operon involved in sensitivity to the Lactococcus lactis abortive infection mechanism AbiD1. J Bacteriol. 1995 Jul;177(13):3824–3829. doi: 10.1128/jb.177.13.3824-3829.1995. [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. Chiaruttini C., Milet M. Gene organization, primary structure and RNA processing analysis of a ribosomal RNA operon in Lactococcus lactis. J Mol Biol. 1993 Mar 5;230(1):57–76. doi: 10.1006/jmbi.1993.1126. [DOI] [PubMed] [Google Scholar]
  5. Chopin A. Organization and regulation of genes for amino acid biosynthesis in lactic acid bacteria. FEMS Microbiol Rev. 1993 Sep;12(1-3):21–37. doi: 10.1111/j.1574-6976.1993.tb00011.x. [DOI] [PubMed] [Google Scholar]
  6. Clepet C., Borne F., Krishnapillai V., Baird C., Patte J. C., Cami B. Isolation, organization and expression of the Pseudomonas aeruginosa threonine genes. Mol Microbiol. 1992 Nov;6(21):3109–3119. doi: 10.1111/j.1365-2958.1992.tb01768.x. [DOI] [PubMed] [Google Scholar]
  7. Follettie M. T., Shin H. K., Sinskey A. J. Organization and regulation of the Corynebacterium glutamicum hom-thrB and thrC loci. Mol Microbiol. 1988 Jan;2(1):53–62. doi: 10.1111/j.1365-2958.1988.tb00006.x. [DOI] [PubMed] [Google Scholar]
  8. Gardner J. F. Regulation of the threonine operon: tandem threonine and isoleucine codons in the control region and translational control of transcription termination. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1706–1710. doi: 10.1073/pnas.76.4.1706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Godon J. J., Delorme C., Bardowski J., Chopin M. C., Ehrlich S. D., Renault P. Gene inactivation in Lactococcus lactis: branched-chain amino acid biosynthesis. J Bacteriol. 1993 Jul;175(14):4383–4390. doi: 10.1128/jb.175.14.4383-4390.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Han K. S., Archer J. A., Sinskey A. J. The molecular structure of the Corynebacterium glutamicum threonine synthase gene. Mol Microbiol. 1990 Oct;4(10):1693–1702. doi: 10.1111/j.1365-2958.1990.tb00546.x. [DOI] [PubMed] [Google Scholar]
  12. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  13. Holo H., Nes I. F. High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media. Appl Environ Microbiol. 1989 Dec;55(12):3119–3123. doi: 10.1128/aem.55.12.3119-3123.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Jensen P. R., Hammer K. Minimal Requirements for Exponential Growth of Lactococcus lactis. Appl Environ Microbiol. 1993 Dec;59(12):4363–4366. doi: 10.1128/aem.59.12.4363-4366.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Keilty S., Rosenberg M. Constitutive function of a positively regulated promoter reveals new sequences essential for activity. J Biol Chem. 1987 May 5;262(13):6389–6395. [PubMed] [Google Scholar]
  17. Komatsubara S., Kisumi M., Chibata I. Transductional construction of a threonine-producing strain of Serratia marcescens. Appl Environ Microbiol. 1979 Dec;38(6):1045–1051. doi: 10.1128/aem.38.6.1045-1051.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kumar A., Malloch R. A., Fujita N., Smillie D. A., Ishihama A., Hayward R. S. The minus 35-recognition region of Escherichia coli sigma 70 is inessential for initiation of transcription at an "extended minus 10" promoter. J Mol Biol. 1993 Jul 20;232(2):406–418. doi: 10.1006/jmbi.1993.1400. [DOI] [PubMed] [Google Scholar]
  19. Leenhouts K. J., Kok J., Venema G. Replacement recombination in Lactococcus lactis. J Bacteriol. 1991 Aug;173(15):4794–4798. doi: 10.1128/jb.173.15.4794-4798.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Macrina F. L., Evans R. P., Tobian J. A., Hartley D. L., Clewell D. B., Jones K. R. Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning. Gene. 1983 Nov;25(1):145–150. doi: 10.1016/0378-1119(83)90176-2. [DOI] [PubMed] [Google Scholar]
  21. Malumbres M., Mateos L. M., Lumbreras M. A., Guerrero C., Martín J. F. Analysis and expression of the thrC gene of Brevibacterium lactofermentum and characterization of the encoded threonine synthase. Appl Environ Microbiol. 1994 Jul;60(7):2209–2219. doi: 10.1128/aem.60.7.2209-2219.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mateos L. M., Pisabarro A., Pátek M., Malumbres M., Guerrero C., Eikmanns B. J., Sahm H., Martín J. F. Transcriptional analysis and regulatory signals of the hom-thrB cluster of Brevibacterium lactofermentum. J Bacteriol. 1994 Dec;176(23):7362–7371. doi: 10.1128/jb.176.23.7362-7371.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miyajima R., Otsuka S., Shiio I. Regulation of aspartate family amino acid biosynthesis in Brevibacterium flavum. I. Inhibition by amino acids of the enzymes in threonine biosynthesis. J Biochem. 1968 Feb;63(2):139–148. doi: 10.1093/oxfordjournals.jbchem.a128754. [DOI] [PubMed] [Google Scholar]
  24. O'sullivan D. J., Klaenhammer T. R. Rapid Mini-Prep Isolation of High-Quality Plasmid DNA from Lactococcus and Lactobacillus spp. Appl Environ Microbiol. 1993 Aug;59(8):2730–2733. doi: 10.1128/aem.59.8.2730-2733.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ochman H., Gerber A. S., Hartl D. L. Genetic applications of an inverse polymerase chain reaction. Genetics. 1988 Nov;120(3):621–623. doi: 10.1093/genetics/120.3.621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Omori K., Imai Y., Suzuki S., Komatsubara S. Nucleotide sequence of the Serratia marcescens threonine operon and analysis of the threonine operon mutations which alter feedback inhibition of both aspartokinase I and homoserine dehydrogenase I. J Bacteriol. 1993 Feb;175(3):785–794. doi: 10.1128/jb.175.3.785-794.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. PATTE J. C., LE BRAS G., LOVINY T., COHEN G. N. [Retro-inhibition and repression of the homoserine dehydrogenase of Escherichia coli]. Biochim Biophys Acta. 1963 Jan 8;67:16–30. doi: 10.1016/0006-3002(63)91793-1. [DOI] [PubMed] [Google Scholar]
  28. Peoples O. P., Liebl W., Bodis M., Maeng P. J., Follettie M. T., Archer J. A., Sinskey A. J. Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom-thrB operon. Mol Microbiol. 1988 Jan;2(1):63–72. doi: 10.1111/j.1365-2958.1988.tb00007.x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Thèze J., Saint-Girons I. Threonine locus of Escherichia coli K-12: genetic structure and evidence for an operon. J Bacteriol. 1974 Jun;118(3):990–998. doi: 10.1128/jb.118.3.990-998.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  32. van Asseldonk M., Rutten G., Oteman M., Siezen R. J., de Vos W. M., Simons G. Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363. Gene. 1990 Oct 30;95(1):155–160. doi: 10.1016/0378-1119(90)90428-t. [DOI] [PubMed] [Google Scholar]
  33. van Rooijen R. J., Gasson M. J., de Vos W. M. Characterization of the Lactococcus lactis lactose operon promoter: contribution of flanking sequences and LacR repressor to promoter activity. J Bacteriol. 1992 Apr;174(7):2273–2280. doi: 10.1128/jb.174.7.2273-2280.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. van de Guchte M., Kok J., Venema G. Gene expression in Lactococcus lactis. FEMS Microbiol Rev. 1992 Feb;8(2):73–92. doi: 10.1111/j.1574-6968.1992.tb04958.x. [DOI] [PubMed] [Google Scholar]

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