Determination of an internal control to apply reverse transcription quantitative PCR to study stress response in the lactic acid bacterium Oenococcus oeni

Abstract

The expression gene pattern reflects, in part, mechanisms involved in adaptation to environmental conditions. Thus, we established and validated a method that enables relative transcript quantification in different conditions in the lactic acid bacteria Oenococcus oeni, notably in a technological medium. First, we determined an internal control in our conditions by reverse transcription quantitative polymerase chain reaction (RT-qPCR) using the SYBR® Green I technology. Among the seven presumed housekeeping tested genes, the ldhD gene was retained for further experiments. Then, the PCR reproducibility was verified in our conditions and the comparative critical threshold (2^ΔΔC_T) method was applied to quantify the transcript level of genes. The quantification of transcript levels of several stress genes already studied in our laboratory by Northern blot after a heat shock and at the entry of stationary phase allowed us to validate this method. RT-qPCR appeared as a powerful tool to study O. oeni response in stress conditions and wine mimetic conditions.

Introduction

The expression pattern reflects, in part, mechanisms involved in adaptation to environmental conditions (Bustin, 2000). All adaptive responses imply several genetic switches that control metabolic changes that take place (Abee and Wouters, 1999). Thus, the ability to quantify transcriptional levels of specific genes could be useful to characterize the physiological state and stress response of bacteria. Current methods used to study gene expression, such as Northern hybridization, quantitative competitive polymerase chain reaction (PCR), and RNase protection assays, have a small dynamic range and a lack of sensitivity (Vandecasteele et al., 2001). The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful technique, which allows the quantification of steady-state mRNA levels in bacteria (Goerke et al., 2000, Corbella and Puyet, 2003, Vandecasteele et al., 2003, Devers et al., 2004).

Two quantifications could be used: relative quantification, or absolute quantification (Bustin, 2000, Bustin, 2002). Relative quantification is the analytic method of choice for many real-time PCR studies (Ginzinger, 2002) and it is best applied when there many genes to be tested in many conditions (Freeman et al., 1999). This method requires the use of internal control for data normalisation. Indeed, variations such as the amount of starting material, enzymatic efficiencies, and differences between cultures of overall transcriptional activity can interfere with the final quantification of cDNA levels (Vandesompele et al., 2002). Presumed housekeeping genes are used as internal controls in eukaryotic organisms. The expression of these genes is often considered to fluctuate very little in comparison to others genes. However, in given conditions, their expression can vary considerably (Thellin et al., 1999, Vandecasteele et al., 2001, Vandesompele et al., 2002). There is no consensus for internal control in bacteria. The most frequent strategy implies the determination of an internal control gene to normalize mRNA fractions for each study.

In this work, we presented a method to define an internal control for relative quantification of transcript levels in stress conditions and in wine mimetic conditions in the lactic acid bacterium Oenococcus oeni. This bacterium is mainly responsible for malolactic fermentation (MLF). During winemaking, the MLF follows alcoholic fermentation carried out by yeasts. This step decreases the total acidity of wine and also improves microbiological stability and organoleptic characteristics (Versari et al., 1999). Wine is a medium with very harsh environmental conditions: low pH, high alcohol content, and high concentrations of SO₂ (Versari et al., 1999). The capacity of O. oeni to survive and grow in wine is essential for MLF, and requires several mechanisms involving the generation of a proton motrice force (Salema et al., 1996), activation of membrane-bound H⁺-ATPases (Carreté et al., 2002), modification of membrane fluidity (Tourdot-Maréchal et al., 2000, da Silveira et al., 2003), and stress protein synthesis (Guzzo et al., 2000). Previous studies concerning O. oeni stress responses and MLF have used physiological and biochemical techniques. This approach consists in validation of a molecular method to characterize O. oeni transcripts levels in several stress conditions and technological medium.

The aim of this work is to explore transcription of presumed housekeeping genes to determine a reference for further studies. The validation of the RT-qPCR method using the SYBR® Green I technology to study relative transcript level after a heat shock and at the entry of stationary phase was performed.