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Volume 157, Issue 9

Disruption of the operon of impairs growth on pyruvate at low pH Free

Abstract

Diacetyl and acetoin are pyruvate-derived metabolites excreted by many micro-organisms, and are important in their physiology. Although generation of these four-carbon (C4) compounds in is a well-documented phenotype, little is known about the gene regulation of their biosynthetic pathway and the physiological role of the pathway in this bacterium. In this work, we identified the genes involved in C4 compound biosynthesis in and report their transcriptional analysis. These genes are part of the bicistronic operon, which encodes α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD). Our studies showed that operon transcription levels are maximal during the exponential phase of growth, decreasing thereafter. Furthermore, we found that this transcription is enhanced upon addition of pyruvate to the growth medium. In order to study the functional role of the operon, an isogenic mutant strain was constructed. This strain lost its capacity to generate C4 compounds, confirming the role of genes in this metabolic pathway. In contrast to the wild-type strain, the -deficient strain was unable to grow in LB medium supplemented with pyruvate at an initial pH of 4.5. This dramatic reduction in growth parameters for the mutant strain was simultaneously accompanied by the inability to alkalinize the internal and external medium under these conditions. In sum, these results suggest that the decarboxylation reactions related to the C4 biosynthetic pathway give enterococcal cells a competitive advantage during pyruvate metabolism at low pH.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Funding
This study was supported by the:
  • Agencia Nacional de Promoción Científica y Tecnológica
  • ANPCyT (Award PICT 2010-1828 and 15-38025)
  • European Union
  • BIAMFood (Award KBBE-211441)
  • Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina)
© 2011 SGM
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2011-09-01
2024-04-20
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References

  1. Ahn J. S., Chandramohan L., Liou L. E., Bayles K. W. ( 2006). Characterization of CidR-mediated regulation in Bacillus anthracis reveals a previously undetected role of S-layer proteins as murein hydrolases. Mol Microbiol 62:1158–1169 [View Article][PubMed]
     [Google Scholar]
  2. Breeuwer P., Drocourt J., Rombouts F. M., Abee T. ( 1996). A novel method for continuous determination of the intracellular pH in bacteria with the internally conjugated fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester. Appl Environ Microbiol 62:178–183[PubMed]
     [Google Scholar]
  3. Cotter P. D., Hill C. ( 2003). Surviving the acid test: responses of Gram-positive bacteria to low pH. Microbiol Mol Biol Rev 67:429–453 [View Article][PubMed]
     [Google Scholar]
  4. Deibel R. H., Niven C. F. Jr ( 1964). Pyruvate fermentation by Streptococcus faecalis . J Bacteriol 88:4–10[PubMed]
     [Google Scholar]
  5. Fertally S. S., Facklam R. ( 1987). Comparison of physiologic tests used to identify non-beta-hemolytic aerococci, enterococci, and streptococci. J Clin Microbiol 25:1845–1850[PubMed]
     [Google Scholar]
  6. Foulquié Moreno M. R., Sarantinopoulos P., Tsakalidou E., De Vuyst L. ( 2006). The role and application of enterococci in food and health. Int J Food Microbiol 106:1–24 [View Article][PubMed]
     [Google Scholar]
  7. Franz C. M. A. P., Stiles M. E., Schleifer K. H., Holzapfel W. H. ( 2003). Enterococci in foods – a conundrum for food safety. Int J Food Microbiol 88:105–122 [View Article][PubMed]
     [Google Scholar]
  8. Friesenegger A., Fiedler S., Devriese L. A., Wirth R. ( 1991). Genetic transformation of various species of Enterococcus by electroporation. FEMS Microbiol Lett 79:323–328 [View Article][PubMed]
     [Google Scholar]
  9. García-Quintáns N., Repizo G., Martín M., Magni C., López P. ( 2008). Activation of the diacetyl/acetoin pathway in Lactococcus lactis subsp. lactis bv. diacetylactis CRL264 by acidic growth. Appl Environ Microbiol 74:1988–1996 [View Article][PubMed]
     [Google Scholar]
  10. Giraffa G. ( 2003). Functionality of enterococci in dairy products. Int J Food Microbiol 88:215–222 [View Article][PubMed]
     [Google Scholar]
  11. Hanahan D. ( 1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580 [View Article][PubMed]
     [Google Scholar]
  12. Hugenholtz J., Starrenburg M. J. C. ( 1992). Diacetyl production by different strains of Lactococcus lactis subsp. lactis var. diacetylactis and Leuconostoc spp. Appl Microbiol Biotechnol 38:17–22 [CrossRef]
     [Google Scholar]
  13. Israelsen H., Madsen S. M., Vrang A., Hansen E. B., Johansen E. ( 1995). Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80. Appl Environ Microbiol 61:2540–2547[PubMed]
     [Google Scholar]
  14. Jacob A. E., Hobbs S. J. ( 1974). Conjugal transfer of plasmid-borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes . J Bacteriol 117:360–372[PubMed]
     [Google Scholar]
  15. Jarmer H., Larsen T. S., Krogh A., Saxild H. H., Brunak S., Knudsen S. ( 2001). Sigma A recognition sites in the Bacillus subtilis genome. Microbiology 147:2417–2424[PubMed]
     [Google Scholar]
  16. Jönsson M., Saleihan Z., Nes I. F., Holo H. ( 2009). Construction and characterization of three lactate dehydrogenase-negative Enterococcus faecalis V583 mutants. Appl Environ Microbiol 75:4901–4903 [View Article][PubMed]
     [Google Scholar]
  17. Kinsinger R. F., Kearns D. B., Hale M., Fall R. ( 2005). Genetic requirements for potassium ion-dependent colony spreading in Bacillus subtilis . J Bacteriol 187:8462–8469 [View Article][PubMed]
     [Google Scholar]
  18. Kovacikova G., Lin W., Skorupski K. ( 2005). Dual regulation of genes involved in acetoin biosynthesis and motility/biofilm formation by the virulence activator AphA and the acetate-responsive LysR-type regulator AlsR in Vibrio cholerae . Mol Microbiol 57:420–433 [View Article][PubMed]
     [Google Scholar]
  19. Law J., Buist G., Haandrikman A., Kok J., Venema G., Leenhouts K. ( 1995). A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes. J Bacteriol 177:7011–7018[PubMed]
     [Google Scholar]
  20. Leblanc D. J. ( 2006). Enterococcus. Prokaryotes vol. 4175–204 Dworkin M., Falkow S., Rosenberg E., Schleifer K., Stackenbrandt E. New York: Springer Science+Business Media; [View Article]
     [Google Scholar]
  21. Lolkema J. S., Poolman B., Konings W. N. ( 1995). Role of scalar protons in metabolic energy generation in lactic acid bacteria. J Bioenerg Biomembr 27:467–473 [View Article][PubMed]
     [Google Scholar]
  22. Maguin E., Prévost H., Ehrlich S. D., Gruss A. ( 1996). Efficient insertional mutagenesis in lactococci and other Gram-positive bacteria. J Bacteriol 178:931–935[PubMed]
     [Google Scholar]
  23. Marelli B. E., Magni C. ( 2010). A simple expression system for Lactococcus lactis and Enterococcus faecalis . J Microbiol Biotechnol 26:999–1007 [View Article]
     [Google Scholar]
  24. Martín M. G., Sender P. D., Peirú S., de Mendoza D., Magni C. ( 2004). Acid-inducible transcription of the operon encoding the citrate lyase complex of Lactococcus lactis biovar diacetylactis CRL264. J Bacteriol 186:5649–5660 [View Article][PubMed]
     [Google Scholar]
  25. Mathews D. H., Sabina J., Zuker M., Turner D. H. ( 1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 288:911–940 [View Article][PubMed]
     [Google Scholar]
  26. Mayer D., Schlensog V., Böck A. ( 1995). Identification of the transcriptional activator controlling the butanediol fermentation pathway in Klebsiella terrigena . J Bacteriol 177:5261–5269[PubMed]
     [Google Scholar]
  27. Molenaar D., Abee T., Konings W. N. ( 1991). Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator. Biochim Biophys Acta 1115:75–83[PubMed] [CrossRef]
     [Google Scholar]
  28. Nallapareddy S. R., Duh R. W., Singh K. V., Murray B. E. ( 2002). Molecular typing of selected Enterococcus faecalis isolates: pilot study using multilocus sequence typing and pulsed-field gel electrophoresis. J Clin Microbiol 40:868–876 [View Article][PubMed]
     [Google Scholar]
  29. Ogier J. C., Serror P. ( 2008). Safety assessment of dairy microorganisms: the Enterococcus genus. Int J Food Microbiol 126:291–301 [View Article][PubMed]
     [Google Scholar]
  30. Poyart C., Trieu-Cuot P. ( 1997). A broad-host-range mobilizable shuttle vector for the construction of transcriptional fusions to β-galactosidase in Gram-positive bacteria. FEMS Microbiol Lett 156:193–198 [View Article][PubMed]
     [Google Scholar]
  31. Renna M. C., Najimudin N., Winik L. R., Zahler S. A. ( 1993). Regulation of the Bacillus subtilis alsS, alsD, and alsR genes involved in post-exponential-phase production of acetoin. J Bacteriol 175:3863–3875[PubMed]
     [Google Scholar]
  32. Sambrook J., Fritsch E. F., Maniatis T. ( 1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  33. Snoep J. L., de Graef M. R., Teixeira de Mattos M. J., Neijssel O. M. ( 1992a). Pyruvate catabolism during transient state conditions in chemostat cultures of Enterococcus faecalis NCTC 775: importance of internal pyruvate concentrations and NADH/NAD+ ratios. J Gen Microbiol 138:2015–2020[PubMed] [CrossRef]
     [Google Scholar]
  34. Snoep J. L., Westphal A. H., Benen J. A., Teixeira de Mattos M. J., Neijssel O. M., de Kok A. ( 1992b). Isolation and characterisation of the pyruvate dehydrogenase complex of anaerobically grown Enterococcus faecalis NCTC 775. Eur J Biochem 203:245–250 [View Article][PubMed]
     [Google Scholar]
  35. Tsau J. L., Guffanti A. A., Montville T. J. ( 1992). Conversion of pyruvate to acetoin helps to maintain pH homeostasis in Lactobacillus plantarum . Appl Environ Microbiol 58:891–894
     [Google Scholar]
  36. Turinsky A. J., Moir-Blais T. R., Grundy F. J., Henkin T. M. ( 2000). Bacillus subtilis ccpA gene mutants specifically defective in activation of acetoin biosynthesis. J Bacteriol 182:5611–5614 [View Article][PubMed]
     [Google Scholar]
  37. Weinrick B., Dunman P. M., McAleese F., Murphy E., Projan S. J., Fang Y., Novick R. P. ( 2004). Effect of mild acid on gene expression in Staphylococcus aureus . J Bacteriol 186:8407–8423 [View Article][PubMed]
     [Google Scholar]
  38. Wilks J. C., Kitko R. D., Cleeton S. H., Lee G. E., Ugwu C. S., Jones B. D., BonDurant S. S., Slonczewski J. L. ( 2009). Acid and base stress and transcriptomic responses in Bacillus subtilis . Appl Environ Microbiol 75:981–990 [View Article][PubMed]
     [Google Scholar]
  39. Xiao Z., Xu P. ( 2007). Acetoin metabolism in bacteria. Crit Rev Microbiol 33:127–140 [View Article][PubMed]
     [Google Scholar]
  40. Yoon S. S., Mekalanos J. J. ( 2006). 2,3-Butanediol synthesis and the emergence of the Vibrio cholerae El Tor biotype. Infect Immun 74:6547–6556 [View Article][PubMed]
     [Google Scholar]
  41. Zuker M. ( 2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415 [View Article][PubMed]
     [Google Scholar]
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Disruption of the alsSD operon of Enterococcus faecalis impairs growth on pyruvate at low pH
Microbiology 157, 2708 (2011); https://doi.org/10.1099/mic.0.047662-0
/content/journal/micro/10.1099/mic.0.047662-0
/content/journal/micro/10.1099/mic.0.047662-0
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