Skip to main content

Advertisement

SpringerLink
Log in

Validation of the use of multiple internal control genes, and the application of real-time quantitative PCR, to study esterase gene expression in Oenococcus oeni

  • Methods and protocols
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The study of gene expression and accurate quantitation of target genes in any organism depends on correct normalisation. Due to the increase in studies on Oenococcus oeni gene expression, there is a clear need for alternative reference genes in order to reliably measure expression levels. In this manuscript, we propose the approach of using multiple reference genes to provide a more robust basis for establishing a reference gene set. The identification and evaluation of a panel of nine reference genes, including the commonly used ldhD, for real-time PCR normalisation was performed in O. oeni. Expression levels of these reference genes were then measured by real-time qPCR in an independent set of O. oeni samples (n = 30). The nine genes were ranked according to their stability of gene expression measure (M) using geNorm to identify the most consistently expressed reference genes. This approach resulted in the identification of multiple reference genes that may be used for a screening and more robust normalisation of target gene expression measured by real-time RT-qPCR. Expression of esterase genes was then measured in these O. oeni samples in the presence of known esterase substrates. The results give an indication of how these genes may be involved in ester synthesis and hydrolysis in O. oeni.

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
Fig. 3

Similar content being viewed by others

References

  • Beltramo C, Desroche N, Tourdot-Maréchal R, Grandvalet C, Guzzo J (2006) Real-time PCR for characterizing the stress response of Oenococcus oeni in a wine-like medium. Res Microbiol 157(3):267–274

    Article  CAS  Google Scholar 

  • Bendtsen JD, Kiemer L, Fausbøll A, Brunak S (2005) Non-classical protein secretion in bacteria. BMC Microbiology 5(58):doi:10.1186/1471-2180-1185-1158

    Google Scholar 

  • Bourdineaud JP, Nehme B, Tesse S, Lonvaud-Funel A (2003) The ftsH gene of the wine bacterium Oenococcus oeni is involved in protection against environmental stress. Appl Environ Microbiol 69(5):2512–2520

    Article  CAS  Google Scholar 

  • Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  CAS  Google Scholar 

  • Carr FJ, Chill D, Maida N (2002) The lactic acid bacteria: a literature survey. Crit Rev Microbiol 28(4):281–370

    Article  CAS  Google Scholar 

  • Chou K, Shen H (2010) Cell-PLoc 2.0: an improved package of web-servers for predicting subcellular localization of proteins in various organisms. Natural Science 2(10):1090–1103

    Article  CAS  Google Scholar 

  • Costantini A, Vaudano E, Rantsiou K, Cocolin L, Garcia-Moruno E (2011) Quantitative expression analysis of mleP gene and two genes involved in the ABC transport system in Oenococcus oeni during rehydration. Appl Microbiol Biotechnol 91(6):1601–1609

    Article  CAS  Google Scholar 

  • Davis CR, Wibowo D, Eschenbruch R, Lee TH, Fleet GH (1985) Practical implications of malolactic fermentation: a review. Am J Enol Vitic 36(4):290–301

    CAS  Google Scholar 

  • de Revel G, Martin N, Pripis-Nicolau L, Lonvaud-Funel A, Bertrand A (1999) Contribution to the knowledge of malolactic fermentation influence on wine aroma. J Agric Food Chem 47(10):4003–4008

    Article  Google Scholar 

  • Delaquis P, Cliff M, King M, Girard B, Hall J, Reynolds A (2000) Effect of two commercial malolactic cultures on the chemical and sensory properties of Chancellor wines vinified with different yeasts and fermentation temperatures. Am J Enol Vitic 51(1):42–48

    CAS  Google Scholar 

  • Desroche N, Beltramo C, Guzzo J (2005) Determination of an internal control to apply reverse transcription quantitative PCR to study stress response in the lactic acid bacterium Oenococcus oeni. J Microbiol Methods 60(3):325–333

    Article  CAS  Google Scholar 

  • Duary RK, Batish VK, Grover S (2010) Expression of the atpD gene in probiotic Lactobacillus plantarum strains under in vitro acidic conditions using RT-qPCR. Res Microbiol 161(5):399–405

    Article  CAS  Google Scholar 

  • Duquenne M, Fleurot I, Aigle M, Darrigo C, Borezee-Durant E, Derzelle S, Bouix M, Deperrois-Lafarge V, Delacroix-Buchet A (2010) Tool for quantification of staphylococcal enterotoxin gene expression in cheese. Appl Environ Microbiol 76(5):1367–1374

    Article  CAS  Google Scholar 

  • Fiocco D, Crisetti E, Capozzi V, Spano G (2008) Validation of an internal control gene to apply reverse transcription quantitative PCR to study heat, cold and ethanol stresses in Lactobacillus plantarum. World J Microb Biot 24(6):899–902

    Article  CAS  Google Scholar 

  • Grandvalet C, Coucheney F, Beltramo C, Guzzo J (2005) CtsR is the master regulator of stress response gene expression in Oenococcus oeni. J Bacteriol 187:5614–5623

    Article  CAS  Google Scholar 

  • Guzzo J (2011) Stress Responses of Oenococcus oeni. In: Tsakalidou E, Papadimitriou K (eds) Stress responses of lactic acid bacteria. Springer, New York, pp 349–365

    Chapter  Google Scholar 

  • Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biology, 8(2:R19), doi:10.1186/gb-2007-1188-1182-r1119

  • Kim OB, Richter H, Zaunmüller T, Graf S, Unden G (2011) Glucose transport by Oenococcus oeni: role of secondary transporters and phosphotransferase systems. J Bacteriol. doi:10.1128/JB.06038-11

  • Kumar P, Henikoff S, Ng PC (2009) Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 4(7):1073–1081

    Article  CAS  Google Scholar 

  • Maicas S, Gil J-V, Pardo I, Ferrer S (1999) Improvement of volatile composition of wines by controlled addition of malolactic bacteria. Food Res Int 32:491–496

    Article  CAS  Google Scholar 

  • Matthews A, Grbin P, Jiranek V (2006) A survey of lactic acid bacteria for enzymes of interest to oenology. Aust J Grape Wine Res 12:235–244

    Article  CAS  Google Scholar 

  • Mills DA, Rawsthorne H, Parker C, Tamir D, Makarova K (2005) Genomic analysis of Oenococcus oeni PSU-1 and its relevance to winemaking. FEMS Microbiol Rev 29:465–475

    CAS  Google Scholar 

  • Olguín N, Bordons A, Reguant C (2009) Influence of ethanol and pH on the gene expression of the citrate pathway in Oenococcus oeni. Food Microbiol 26(2):197–203

    Article  Google Scholar 

  • Olguín N, Bordons A, Reguant C (2010) Multigenic expression analysis as an approach to understanding the behaviour of Oenococcus oeni in wine-like conditions. Int J Food Microbiol 144(1):88–95

    Article  Google Scholar 

  • Olguín N, Alegret JO, Bordons A, Reguant C (2011) β-Glucosidase activity and bgl gene expression of Oenococcus oeni strains in model media and Cabernet Sauvignon wine. Wine Am J Enol Vitic 62(1):99–105

    Article  Google Scholar 

  • Pozo-Bayón MA, Alegría EG, Polo MC, Tenorio C, Martín-Álvarez PJ, Calvo de la Banda MT, Ruiz-Larrea F, Moreno-Arribas MV (2005) Wine volatile and amino acid composition after malolactic fermentation: effect of Oenococcus oeni and Lactobacillus plantarum starter cultures. J Agric Food Chem 53:8729–8735

    Article  Google Scholar 

  • Ritt JF, Remize F, Grandvalet C, Guzzo J, Atlan D, Alexandre H (2009) Peptidases specific for proline-containing peptides and their unusual peptide-dependent regulation in Oenococcus oeni. J Appl Microbiol 106(3):801–813

    Article  CAS  Google Scholar 

  • Ritz M, Garenaux A, Berge M, Federighi M (2009) Determination of rpoA as the most suitable internal control to study stress response in C. jejuni by RT-qPCR and application to oxidative stress. J Microbiol Methods 76(2):196–200

    Article  CAS  Google Scholar 

  • Savli H, Karadenizli A, Kolayli F, Gundes S, Ozbek U, Vahaboglu H (2003) Expression stability of six housekeeping genes: a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J Med Microbiol 52(5):403–408

    Article  CAS  Google Scholar 

  • Seta FD, Boschi-Muller S, Vignais ML, Branlant G (1997) Characterization of Escherichia coli strains with gapA and gapB genes deleted. J Bacteriol 179(16):5218–5221

    CAS  Google Scholar 

  • Sumby KM, Matthews AH, Grbin PR, Jiranek V (2009) Cloning and characterization of an intracellular esterase from the wine-associated lactic acid bacterium Oenococcus oeni. Appl Environ Microbiol 75(21):6729–6735

    Article  CAS  Google Scholar 

  • Sumby KM, Grbin PR, Jiranek V (2010) Microbial modulation of aromatic esters in wine: current knowledge and future prospects. Food Chem 121(1):1–16

    Article  CAS  Google Scholar 

  • Sumby KM, Grbin PR, Jiranek V (2012a) Characterisation of EstCOo8 and EstC34, intracellular esterases, from the wine associated lactic acid bacterium Oenococcus oeni and Lactobacillus hilgardii. (in press)

  • Sumby KM, Grbin PR, Jiranek V (2012b) Ester synthesis and hydrolysis in an aqueous environment, and strain specific changes during malolactic fermentation in wine with Oenococcus oeni. (in press)

  • Turroni S, Bendazzoli C, Dipalo SCF, Candela M, Vitali B, Gotti R, Brigidi P (2010) Oxalate-degrading activity in Bifidobacterium animalis subsp. lactis: impact of acidic conditions on the transcriptional levels of the oxalyl coenzyme A (CoA) decarboxylase and formyl-CoA transferase genes. Appl Environ Microbiol 76(16):5609–5620

    Article  CAS  Google Scholar 

  • Ugliano M, Moio L (2005) Changes in the concentration of yeast-derived volatile compounds of red wine during malolactic fermentation with four commercial starter cultures of Oenococcus oeni. J Agric Food Chem 53:10134–10139

    Article  CAS  Google Scholar 

  • Vandecasteele SJ, Peetermans WE, Merckx R, Van Eldere J (2001) Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under different conditions. J Bacteriol 183(24):7094–7101

    Article  CAS  Google Scholar 

  • Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):research0034–research0034.0011

    Article  Google Scholar 

  • Vaudano E, Noti O, Costantini A, Garcia-Moruno E (2011) Identification of reference genes suitable for normalization of RT-qPCR expression data in Saccharomyces cerevisiae during alcoholic fermentation. Biotechnol Lett 33(8):1593–1599

    Article  CAS  Google Scholar 

  • Yu C-S, Chen Y-C, Lu C-H, Hwang J-K (2006) Prediction of protein subcellular localization. Protein Struct Funct Bioinforma 64(3):643–651

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The research in this paper was undertaken as part of project UA 05/01 supported by Australia’s grape growers and winemakers through their investment body, the Grape and Wine Research Development Corporation, with matching funds from the Australian Government. K.S. is supported by a Grape and Wine Research Development Corporation scholarship. The University of Adelaide is a member of the Wine Innovation Cluster in Adelaide (wineinnovationcluster.com).

Author information

Authors and Affiliations

  1. School of Agriculture, Food and Wine, The University of Adelaide, PMB 1, Glen Osmond, Adelaide, SA, 5064, Australia

    Krista M. Sumby, Paul R. Grbin & Vladimir Jiranek

Authors
  1. Krista M. Sumby
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paul R. Grbin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vladimir Jiranek
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vladimir Jiranek.

Additional information

Adelaide University is a member of the Wine Innovation Cluster

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sumby, K.M., Grbin, P.R. & Jiranek, V. Validation of the use of multiple internal control genes, and the application of real-time quantitative PCR, to study esterase gene expression in Oenococcus oeni . Appl Microbiol Biotechnol 96, 1039–1047 (2012). https://doi.org/10.1007/s00253-012-4409-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-012-4409-1

Keywords

Search

Navigation