Expression analysis of genes related to citrate metabolism in Oenococcus oeni PSU-1 after stress exposure by DNA microarray

Oenococcus oeni is a bacterial species of great interest for some winemaking processes as it is the principal agent of malolactic fermentation, which could confer positive sensory traits and improve microbial stability. Moreover, besides malic acid, O. oeni can also metabolise citric acid leading to the production of flavoring compounds, such as diacetyl, acetoin, butanediol and acetate, through 3 pathways (citrate, pyruvate and butanediol), involving 15 genes. In literature only some of these genes were analysed for their expression by RT-qPCR. The aim of the present study was to obtain a complete view on transcriptional variation of all the genes related to citrate metabolism in the reference strain PSU-1 under stress conditions using DNA microarray. Low pH (3.5), low pH and ethanol (10% v/v), and heat shock at 42°C were applied to cultures in mid-exponential phase for half the replication time in modified MRS. The results showed that the regulator of the citrate lyase gene cluster (citR) increased its transcription in presence of low pH and ethanol, whereas citrate lyase genes were up-regulated at pH 3.5 and down-regulated at 42°C. Expression data concerning genes involved in butanediol pathway revealed that alsS and alsD exhibited up-regulation at pH 3.5 and both the genes annotated as acetoin reductase (butA) were down-regulated in all tested conditions. The transcription of ldhD was repressed under all stresses applied, whereas the genes responsible for the conversion of pyruvate in acetate (pdh and ackA) were differentially expressed in low pH and ethanol, and at 42°C. This study demonstrates an overall response of the citrate metabolism genes in O. oeni after 6 hours of stress application. Furthermore it is possible to conclude that: (i) at low pH O. oeni induces the cit operon, as previously described in Lactococcus lactis, (ii) the additional presence of ethanol does not affect most of the genes, (iii) heat shock elicits a general repression of transcription