Skip to main content
SpringerLink
Log in

Characterisation of oxidative stress-resistant mutants of Streptococcus thermophilus CNRZ368

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

During industrial processes, the dairy organism Streptococcus thermophilus is exposed to stress conditions. Its ability to survive and grow in an aerobic environment indicates that it must possess defensive mechanisms against reactive oxygen species. To identify the genes involved in oxidative stress defence, a collection of mutants was generated by random insertional mutagenesis and screened for menadione sensitivity and resistance. Results obtained for resistant clones allowed the identification of eight loci. The insertions affected genes whose homologues in other bacteria were previously identified as being involved in stress response (deoB, gst) or transcription regulation (rggC) and five ORFs of unknown function. The tolerance of the eight mutants to air-exposure, methyl viologen and H2O2 was studied. Real-time quantitative PCR was used to analyse the transcript level of mutated genes and revealed that most were down-regulated during oxidative stress.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Berks BC, Sargent F, Palmer T (2000) The Tat protein export pathway. Mol Microbiol 35:260–274

    Article  CAS  PubMed  Google Scholar 

  • Bracquart P (1981) An agar medium for the differential enumeration of Streptococcus thermophilus and Lactobacillus bulgaricus in yogurt. J Appl Bacteriol 51:303–305

    Google Scholar 

  • Burrus V, Pavlovic G, Decaris B, Guedon G (2002) The ICESt1 element of Streptococcus thermophilus belongs to a large family of integrative and conjugative elements that exchange modules and change their specificity of integration. Plasmid 48:77–97

    Article  CAS  PubMed  Google Scholar 

  • Chaussee MS, Ajdic D, Ferretti JJ (1999) The rgg gene of Streptococcus pyogenes NZ131 positively influences extracellular SPE B production. Infect Immun 67:1715–1722

    CAS  PubMed  Google Scholar 

  • Chaussee MS, Watson RO, Smoot JC, Musser JM (2001) Identification of Rgg-regulated exoproteins of Streptococcus pyogenes. Infect Immun 69:822–831

    Article  CAS  PubMed  Google Scholar 

  • Chia JS, Lee YY, Huang PT, Chen JY (2001) Identification of stress-responsive genes in Streptococcus mutans by differential display reverse transcription–PCR. Infect Immun 69:2493–2501

    Article  CAS  PubMed  Google Scholar 

  • Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    Article  CAS  PubMed  Google Scholar 

  • Duwat P, Cochu A, Ehrlich SD, Gruss A (1997) Characterization of Lactococcus lactis UV-sensitive mutants obtained by ISS1 transposition. J Bacteriol 179:4473–4479

    CAS  PubMed  Google Scholar 

  • Duwat P, Ehrlich SD, Gruss A (1999) Effect of metabolic flux on stress response pathways in Lactococcus lactis. Mol Microbiol 31:845–858

    Article  CAS  PubMed  Google Scholar 

  • Fridovich I (1997) Superoxide anion radical (O2•−), superoxide dismutases, and related matters. J Biol Chem 272:18515–18517

    Article  CAS  PubMed  Google Scholar 

  • Gibson CM, Mallett TC, Claiborne A, Caparon MG (2000) Contribution of NADH oxidase to aerobic metabolism of Streptococcus pyogenes. J Bacteriol 182:448–455

    Article  CAS  PubMed  Google Scholar 

  • Higuchi M, Yamamoto Y, Poole LB, Shimada M, Sato Y, Takahashi N, Kamio Y (1999) Functions of two types of NADH oxidases in energy metabolism and oxidative stress of Streptococcus mutans. J Bacteriol 181:5940–5947

    CAS  PubMed  Google Scholar 

  • Hoskins J, Alborn WE Jr, Arnold J, Blaszczak LC, Burgett S, DeHoff BS, Estrem ST, Fritz L, Fu DJ, Fuller W, Geringer C, Gilmour R, Glass JS, Khoja H, Kraft AR, Lagace RE, LeBlanc DJ, Lee LN, Lefkowitz EJ, Lu J, Matsushima P, McAhren SM, McHenney M, McLeaster K, Mundy CW, Nicas TI, Norris FH, O’Gara M, Peery RB, Robertson GT, Rockey P, Sun PM, Winkler ME, Yang Y, Young-Bellido M, Zhao G, Zook CA, Baltz RH, Jaskunas SR, Rosteck PR Jr, Skatrud PL, Glass JI (2001) Genome of the bacterium Streptococcus pneumoniae strain R6. J Bacteriol 183:5709–5717

    Article  CAS  PubMed  Google Scholar 

  • Jakubovics N, Smith A, Jenkinson H (2002) Oxidative stress tolerance is manganese (Mn2+) regulated in Streptococcus gordonii. Microbiology 148:3255–3263

    CAS  PubMed  Google Scholar 

  • Kang WK, Icho T, Isono S, Kitakawa M, Isono K (1989) Characterization of the gene rimK responsible for the addition of glutamic acid residues to the C-terminus of ribosomal protein S6 in Escherichia coli K12. Mol Gen Genet 217:281–288

    CAS  PubMed  Google Scholar 

  • King KY, Horenstein JA, Caparon MG (2000) Aerotolerance and peroxide resistance in peroxidase and PerR mutants of Streptococcus pyogenes. J Bacteriol 182:5290–5299

    Article  CAS  PubMed  Google Scholar 

  • Koonin EV, Aravind L, Galperin YA (2000) A comparative-genomic view of the microbial stress response. In: Storz G, Henge-Aronis R (eds) Bacterial stress response. American Society of Microbiology, Washington, D.C.

    Google Scholar 

  • Larsen B, Wills NM, Nelson C, Atkins JF, Gesteland RF (2000) Nonlinearity in genetic decoding: homologous DNA replicase genes use alternatives of transcriptional slippage or translational frameshifting. Proc Natl Acad Sci USA 97:1683–1688

    Article  CAS  PubMed  Google Scholar 

  • Leenhouts K (1995) Integration strategies and vectors. Dev Biol Stand 85:523–530

    CAS  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔC(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Maguin E, Prevost H, Ehrlich SD, Gruss A (1996) Efficient insertional mutagenesis in lactococci and other gram-positive bacteria. J Bacteriol 178:931–935

    CAS  PubMed  Google Scholar 

  • Niven DF, Ekins A (2001) Iron content of Streptococcus suis and evidence for a dpr homologue. Can J Microbiol 47:412–416

    Article  CAS  PubMed  Google Scholar 

  • Nygaard P (1993) Purine and pyrimidine salvage pathways. In: Sonenshein AL, Hoch JA, Losick R (eds) Bacillus subtillis and other gram-positive bacteria. American Society for Microbiology, Washington, D.C.

    Google Scholar 

  • Pericone CD, Park S, Imlay JA, Weiser JN (2003) Factors contributing to hydrogen peroxide resistance in Streptococcus pneumoniae include pyruvate oxidase (SpxB) and avoidance of the toxic effects of the fenton reaction. J Bacteriol 185:6815–6825

    Article  CAS  PubMed  Google Scholar 

  • Poyart C, Pellegrini E, Gaillot O, Boumaila C, Baptista M, Trieu-Cuot P (2001) Contribution of Mn-cofactored superoxide dismutase (SodA) to the virulence of Streptococcus agalactiae. Infect Immun 69:5098–5106

    Article  CAS  PubMed  Google Scholar 

  • Rallu F, Gruss A, Ehrlich SD, Maguin E (2000) Acid- and multistress-resistant mutants of Lactococcus lactis: identification of intracellular stress signals. Mol Microbiol 35:517–528

    Article  CAS  PubMed  Google Scholar 

  • Rawlinson EL, Nes IF, Skaugen M (2002) LasX, a transcriptional regulator of the lactocin S biosynthetic genes in Lactobacillus sakei L45, acts both as an activator and a repressor. Biochimie 84:559–567

    Article  CAS  PubMed  Google Scholar 

  • Ricci S, Janulczyk R, Bjorck L (2002) The regulator PerR is involved in oxidative stress response and iron homeostasis and is necessary for full virulence of Streptococcus pyogenes. Infect Immun 70:4968–4976

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Schell MA (1993) Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol 47:597–626

    Article  CAS  PubMed  Google Scholar 

  • Smoot LM, Smoot JC, Graham MR, Somerville GA, Sturdevant DE, Migliaccio CA, Sylva GL, Musser JM (2001) Global differential gene expression in response to growth temperature alteration in group A Streptococcus. Proc Natl Acad Sci USA 98:10416–10421

    Article  CAS  PubMed  Google Scholar 

  • Sonnhammer ELL, Von Heijne G, Krogh A (1998) A hidden Markov model for predicting transmembrane helices in protein sequences. In: Glasgow J, Littlejohn T, Major F, Sankoff D, R L, Sensen C (eds) Proceedings of the sixth international conference on intelligent systems for molecular microbiology. AAAI, Menlo Park, pp 175–182

    Google Scholar 

  • Spatafora G, Van Hoeven N, Wagner K, Fives-Taylor P (2002) Evidence that ORF3 at the Streptococcus parasanguis fimA locus encodes a thiol-specific antioxidant. Microbiology 148:755–762

    CAS  PubMed  Google Scholar 

  • Storck T, Schulte S, Hofmann K, Stoffel W (1992) Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain. Proc Natl Acad Sci USA 89:10955–10959

    CAS  PubMed  Google Scholar 

  • Sugano Y, Nakano R, Sasaki K, Shoda M (2000) Efficient heterologous expression in Aspergillus oryzae of a unique dye-decolorizing peroxidase, DyP, of Geotrichum candidum Dec 1. Appl Environ Microbiol 66:1754–1758

    Article  CAS  PubMed  Google Scholar 

  • Sulavik MC, Clewell DB (1996) Rgg is a positive transcriptional regulator of the Streptococcus gordonii gtfG gene. J Bacteriol 178:5826–5830

    CAS  PubMed  Google Scholar 

  • Terzaghi B, Sandine W (1975) Improved medium for lactic streptococci and their bacteriophages. Appl Environ Microbiol 29:807–813

    CAS  Google Scholar 

  • Tettelin H, Nelson KE, Paulsen IT, Eisen JA, Read TD, Peterson S, Heidelberg J, DeBoy RT, Haft DH, Dodson RJ, Durkin AS, Gwinn M, Kolonay JF, Nelson WC, Peterson JD, Umayam LA, White O, Salzberg SL, Lewis MR, Radune D, Holtzapple E, Khouri H, Wolf AM, Utterback TR, Hansen CL, McDonald LA, Feldblyum TV, Angiuoli S, Dickinson T, Hickey EK, Holt IE, Loftus BJ, Yang F, Smith HO, Venter JC, Dougherty BA, Morrison DA, Hollingshead SK, Fraser CM (2001) Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293:498–506

    Article  CAS  PubMed  Google Scholar 

  • Thibessard A, Fernandez A, Gintz B, Decaris B, Leblond-Bourget N (2002) Transposition of pGh9:ISS1 is random and efficient in Streptococcus thermophilus CNRZ368. Can J Microbiol 48:473–478

    Article  CAS  PubMed  Google Scholar 

  • Thibessard A, Borges F, Fernandez A, Gintz B, Decaris B, Leblond-Bourget N (2004) Identification of Streptococcus thermophilus CNRZ368 genes involved in defense against superoxide stress. Appl Environ Microbiol 70:2220–2229

    Article  CAS  PubMed  Google Scholar 

  • Tusnady GE, Simon I (1998) Principles governing amino acid composition of integral membrane proteins: application to topology prediction. J Mol Biol 283:489–506

    Article  CAS  PubMed  Google Scholar 

  • Varcamonti M, Graziano MR, Pezzopane R, Naclerio G, Arsenijevic S, De Felice M (2003) Impaired temperature stress response of a Streptococcus thermophilus deoD mutant. Appl Environ Microbiol 69:1287–1289

    Article  CAS  PubMed  Google Scholar 

  • Voyich JM, Sturdevant DE, Braughton KR, Kobayashi SD, Lei B, Virtaneva K, Dorward DW, Musser JM, DeLeo FR (2003) Genome-wide protective response used by group A Streptococcusto evade destruction by human polymorphonuclear leukocytes. Proc Natl Acad Sci USA 100:1996–2001

    Article  CAS  PubMed  Google Scholar 

  • Yamamoto Y, Higuchi M, Poole LB, Kamio Y (2000) Role of the dpr product in oxygen tolerance in Streptococcus mutans. J Bacteriol 182:3740–3747

    Article  CAS  PubMed  Google Scholar 

  • Yesilkaya H, Kadioglu A, Gingles N, Alexander JE, Mitchell TJ, Andrew PW (2000) Role of manganese-containing superoxide dismutase in oxidative stress and virulence of Streptococcus pneumoniae. Infect Immun 68:2819–2826

    Article  CAS  PubMed  Google Scholar 

  • Zheng M, Wang X, Templeton LJ, Smulski DR, LaRossa RA, Storz G (2001) DNA microarray-mediated transcriptional profiling of the Escherichia coli response to hydrogen peroxide. J Bacteriol 183:4562–4570

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

A.F. and A.T. were supported by grants from the Ministère de la recherche. F.B. was supported by a grant from the Institut National de la Recherche Agronomique. We are grateful to Paul Hoskisson for his advice regarding the English formulation of the manuscript. Sequence data for S. thermophilus LMG18311 were obtained from the UCL Life Sciences Institute (ISV) website at http://www.biol.ucl.ac.be/gene/genome/. Sequencing of S. thermophilus was supported by the Walloon Region (BIOVAL grant 9813866).

Author information

Authors and Affiliations

  1. Laboratoire de Génétique et Microbiologie, UMR INRA 1128, IFR 110, Faculté des Sciences de l’Université Henri Poincaré Nancy 1, BP 238, 54506, Vandoeuvre-lès-Nancy, France

    Annabelle Fernandez, Annabelle Thibessard, Frédéric Borges, Brigitte Gintz, Bernard Decaris & Nathalie Leblond–Bourget

Authors
  1. Annabelle Fernandez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Annabelle Thibessard
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Frédéric Borges
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Brigitte Gintz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bernard Decaris
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nathalie Leblond–Bourget
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nathalie Leblond–Bourget.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fernandez, A., Thibessard, A., Borges, F. et al. Characterisation of oxidative stress-resistant mutants of Streptococcus thermophilus CNRZ368. Arch Microbiol 182, 364–372 (2004). https://doi.org/10.1007/s00203-004-0712-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-004-0712-2

Keywords

Search

Navigation