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. 1995 Sep;177(18):5254–5260. doi: 10.1128/jb.177.18.5254-5260.1995

Stress response in Lactococcus lactis: cloning, expression analysis, and mutation of the lactococcal superoxide dismutase gene.

J W Sanders 1, K J Leenhouts 1, A J Haandrikman 1, G Venema 1, J Kok 1
PMCID: PMC177316  PMID: 7665513

Abstract

In an analysis of the stress response of Lactococcus lactis, three proteins that were induced under low pH culture conditions were detected. One of these was identified as the lactococcal superoxide dismutase (SodA) by N-terminal amino acid sequence analysis. The gene encoding this protein, designated sodA, was cloned by the complementation of a sodA sodB Escherichia coli strain. The deduced amino acid sequence of L. lactis SodA showed the highest degree of similarity to the manganese-containing Sod (MnSod) of Bacillus stearothermophilus. A promoter upstream of the sodA gene was identified by primer extension analysis, and an inverted repeat surrounding the -35 hexanucleotide of this promoter is possibly involved in the regulation of the expression of sodA. The expression of sodA was analyzed by transcriptional fusions with a promoterless lacZ gene. The induction of beta-galactosidase activity occurred in aerated cultures. Deletion experiments revealed that a DNA fragment of more than 130 bp surrounding the promoter was needed for the induction of lacZ expression by aeration. The growth rate of an insertion mutant of sodA did not differ from that of the wild type in standing cultures but was decreased in aerated cultures.

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Selected References

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

  1. Anders R. F., Hogg D. M., Jago G. R. Formation of hydrogen peroxide by group N streptococci and its effect on their growth and metabolism. Appl Microbiol. 1970 Apr;19(4):608–612. doi: 10.1128/am.19.4.608-612.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Archibald F. S., Fridovich I. Manganese, superoxide dismutase, and oxygen tolerance in some lactic acid bacteria. J Bacteriol. 1981 Jun;146(3):928–936. doi: 10.1128/jb.146.3.928-936.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Artoni G., Gianazza E., Zanoni M., Gelfi C., Tanzi M. C., Barozzi C., Ferruti P., Righetti P. G. Fractionation techniques in a hydro-organic environment. II. Acryloyl-morpholine polymers as a matrix for electrophoresis in hydro-organic solvents. Anal Biochem. 1984 Mar;137(2):420–428. doi: 10.1016/0003-2697(84)90108-8. [DOI] [PubMed] [Google Scholar]
  4. Beauchamp C., Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem. 1971 Nov;44(1):276–287. doi: 10.1016/0003-2697(71)90370-8. [DOI] [PubMed] [Google Scholar]
  5. Bowler C., Van Kaer L., Van Camp W., Van Montagu M., Inzé D., Dhaese P. Characterization of the Bacillus stearothermophilus manganese superoxide dismutase gene and its ability to complement copper/zinc superoxide dismutase deficiency in Saccharomyces cerevisiae. J Bacteriol. 1990 Mar;172(3):1539–1546. doi: 10.1128/jb.172.3.1539-1546.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. Buchmeier N. A., Heffron F. Induction of Salmonella stress proteins upon infection of macrophages. Science. 1990 May 11;248(4956):730–732. doi: 10.1126/science.1970672. [DOI] [PubMed] [Google Scholar]
  8. Buist G., Kok J., Leenhouts K. J., Dabrowska M., Venema G., Haandrikman A. J. Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation. J Bacteriol. 1995 Mar;177(6):1554–1563. doi: 10.1128/jb.177.6.1554-1563.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carlioz A., Ludwig M. L., Stallings W. C., Fee J. A., Steinman H. M., Touati D. Iron superoxide dismutase. Nucleotide sequence of the gene from Escherichia coli K12 and correlations with crystal structures. J Biol Chem. 1988 Jan 25;263(3):1555–1562. [PubMed] [Google Scholar]
  10. Carlioz A., Touati D. Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? EMBO J. 1986 Mar;5(3):623–630. doi: 10.1002/j.1460-2075.1986.tb04256.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Church S. L. Manganese superoxide dismutase: nucleotide and deduced amino acid sequence of a cDNA encoding a new human transcript. Biochim Biophys Acta. 1990 Oct 23;1087(2):250–252. doi: 10.1016/0167-4781(90)90213-l. [DOI] [PubMed] [Google Scholar]
  13. Compan I., Touati D. Interaction of six global transcription regulators in expression of manganese superoxide dismutase in Escherichia coli K-12. J Bacteriol. 1993 Mar;175(6):1687–1696. doi: 10.1128/jb.175.6.1687-1696.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Eaton T., Shearman C., Gasson M. Cloning and sequence analysis of the dnaK gene region of Lactococcus lactis subsp. lactis. J Gen Microbiol. 1993 Dec;139(12):3253–3264. doi: 10.1099/00221287-139-12-3253. [DOI] [PubMed] [Google Scholar]
  15. Fahey R. C., Brown W. C., Adams W. B., Worsham M. B. Occurrence of glutathione in bacteria. J Bacteriol. 1978 Mar;133(3):1126–1129. doi: 10.1128/jb.133.3.1126-1129.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fee J. A. Regulation of sod genes in Escherichia coli: relevance to superoxide dismutase function. Mol Microbiol. 1991 Nov;5(11):2599–2610. doi: 10.1111/j.1365-2958.1991.tb01968.x. [DOI] [PubMed] [Google Scholar]
  17. Foster J. W., Hall H. K. Adaptive acidification tolerance response of Salmonella typhimurium. J Bacteriol. 1990 Feb;172(2):771–778. doi: 10.1128/jb.172.2.771-778.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fridovich I. Superoxide dismutases. Annu Rev Biochem. 1975;44:147–159. doi: 10.1146/annurev.bi.44.070175.001051. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Godon J. J., Jury K., Shearman C. A., Gasson M. J. The Lactococcus lactis sex-factor aggregation gene cluA. Mol Microbiol. 1994 May;12(4):655–663. doi: 10.1111/j.1365-2958.1994.tb01053.x. [DOI] [PubMed] [Google Scholar]
  21. Gough J. A., Murray N. E. Sequence diversity among related genes for recognition of specific targets in DNA molecules. J Mol Biol. 1983 May 5;166(1):1–19. doi: 10.1016/s0022-2836(83)80047-3. [DOI] [PubMed] [Google Scholar]
  22. Hassan H. M., Schrum L. W. Roles of manganese and iron in the regulation of the biosynthesis of manganese-superoxide dismutase in Escherichia coli. FEMS Microbiol Rev. 1994 Aug;14(4):315–323. doi: 10.1111/j.1574-6976.1994.tb00105.x. [DOI] [PubMed] [Google Scholar]
  23. Kim S. G., Batt C. A. Cloning and sequencing of the Lactococcus lactis subsp. lactis groESL operon. Gene. 1993 May 15;127(1):121–126. doi: 10.1016/0378-1119(93)90626-e. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Meister A. Glutathione metabolism and its selective modification. J Biol Chem. 1988 Nov 25;263(33):17205–17208. [PubMed] [Google Scholar]
  27. Nakayama K. Nucleotide sequence of Streptococcus mutans superoxide dismutase gene and isolation of insertion mutants. J Bacteriol. 1992 Aug;174(15):4928–4934. doi: 10.1128/jb.174.15.4928-4934.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nakayama K. Rapid viability loss on exposure to air in a superoxide dismutase-deficient mutant of Porphyromonas gingivalis. J Bacteriol. 1994 Apr;176(7):1939–1943. doi: 10.1128/jb.176.7.1939-1943.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Olson E. R. Influence of pH on bacterial gene expression. Mol Microbiol. 1993 Apr;8(1):5–14. doi: 10.1111/j.1365-2958.1993.tb01198.x. [DOI] [PubMed] [Google Scholar]
  30. Parker M. W., Blake C. C. Crystal structure of manganese superoxide dismutase from Bacillus stearothermophilus at 2.4 A resolution. J Mol Biol. 1988 Feb 20;199(4):649–661. doi: 10.1016/0022-2836(88)90308-7. [DOI] [PubMed] [Google Scholar]
  31. Parker M. W., Blake C. C. Iron- and manganese-containing superoxide dismutases can be distinguished by analysis of their primary structures. FEBS Lett. 1988 Mar 14;229(2):377–382. doi: 10.1016/0014-5793(88)81160-8. [DOI] [PubMed] [Google Scholar]
  32. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rottländer E., Trautner T. A. Genetic and transfection studies with B, subtilis phage SP 50. I. Phage mutants with restricted growth on B. subtilis strain 168. Mol Gen Genet. 1970;108(1):47–60. doi: 10.1007/BF00343184. [DOI] [PubMed] [Google Scholar]
  34. Sadosky A. B., Wilson J. W., Steinman H. M., Shuman H. A. The iron superoxide dismutase of Legionella pneumophila is essential for viability. J Bacteriol. 1994 Jun;176(12):3790–3799. doi: 10.1128/jb.176.12.3790-3799.1994. [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. Smart J. B., Thomas T. D. Effect of oxygen on lactose metabolism in lactic streptococci. Appl Environ Microbiol. 1987 Mar;53(3):533–541. doi: 10.1128/aem.53.3.533-541.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Steinman H. M. Construction of an Escherichia coli K-12 strain deleted for manganese and iron superoxide dismutase genes and its use in cloning the iron superoxide dismutase gene of Legionella pneumophila. Mol Gen Genet. 1992 Apr;232(3):427–430. doi: 10.1007/BF00266247. [DOI] [PubMed] [Google Scholar]
  38. Steinman H. M. The amino acid sequence of mangano superoxide dismutase from Escherichia coli B. J Biol Chem. 1978 Dec 25;253(24):8708–8720. [PubMed] [Google Scholar]
  39. Takeda Y., Avila H. Structure and gene expression of the E. coli Mn-superoxide dismutase gene. Nucleic Acids Res. 1986 Jun 11;14(11):4577–4589. doi: 10.1093/nar/14.11.4577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  42. Zabarovsky E. R., Winberg G. High efficiency electroporation of ligated DNA into bacteria. Nucleic Acids Res. 1990 Oct 11;18(19):5912–5912. doi: 10.1093/nar/18.19.5912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. van Asseldonk M., Simons A., Visser H., de Vos W. M., Simons G. Cloning, nucleotide sequence, and regulatory analysis of the Lactococcus lactis dnaJ gene. J Bacteriol. 1993 Mar;175(6):1637–1644. doi: 10.1128/jb.175.6.1637-1644.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. van de Guchte M., Kok J., Venema G. Distance-dependent translational coupling and interference in Lactococcus lactis. Mol Gen Genet. 1991 May;227(1):65–71. doi: 10.1007/BF00260708. [DOI] [PubMed] [Google Scholar]

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