Stress response and intraspecific diversity in Oenococcus oeni

Oenococcus oeni is a lactic acid bacterium with an applied importance because it is the main microorganism responsible for the malolactic fermentation of wine (Lonvaud-Funel, 1999). Since wine is a peculiar niche in which many factors, such as low pH and ethanol, act as stresses on bacterial cells, the study of stress response in O. oeni is of particular interest, especially for developing novel starter cultures (Beltramo et al., 2006). Furthermore, O. oeni has a small genome size (about 1.8 Mbp), as shown by the 14 available sequenced genomes (Mills et al., 2005; Borneman et al., 2010; Borneman et al., 2012a), and another genomic peculiarity, i.e., the lack of the mismatch repair pathway, which is probably responsible for the high diversity within this species (Makarova et al., 2006; Marcobal et al., 2008). Therefore O. oeni is an interesting species both for applied purposes and for basic research. In the literature, the response to the stress factors of wine has been mainly addressed with the analysis of the expression of only few genes (about 30). On the other hand, the availability of genome sequences for some strains provides a valuable source of information about the distribution of specific genetic traits that could be linked to the different behaviour of diverse strains. In the present thesis, stress response and its correlation with intraspecific diversity were investigated in O. oeni. Different techniques were applied and integrated: whole-genome transcriptomics with DNA microarray, single gene expression analysis, comparative genomics, genetic screenings on a collection of strains presenting different oenological properties, and, moreover, some of these strains were genetically and physiologically characterized for the first time. The first chapter regarded a global transcription analysis of strain O. oeni PSU-1, which offered a wider picture on stress response compared to the previous literature. This experiment delineated a pan transcriptome of 766 genes, differentially expressed in at least one condition of the four tested (pH 3.5, 10% EtOH, pH+EtOH, and 42°C applied for 6 hours), and a core transcriptome of 28 genes, modulated in all the four stresses. In particular, among these latter genes a transcriptional regulator (OEOE_0412) and a thioredoxin (OEOE_1702) were included. The presence of a regulator differentially expressed in all the applied conditions motivated the further analysis of the transcriptional regulators, key elements in the stress response, within O. oeni species. Indeed, in the second chapter, all the predicted regulators in PSU-1 genome were identified, and about half of them resulted modulated in at least one stress condition. Besides the known ctsR, and OEOE_0412, the unique present in the core transcriptome, other two interesting regulators emerged: OEOE_0878, and OEOE_1398, which were part of an operon and a regulon, respectively. All these genes were described for the first time as involved in the stress response of O. oeni. Since the expression in response to stress depends on the presence of a specific genetic background, and, given the high diversity within O. oeni (de las Rivas et al., 2004; Bilhère et al., 2009; Bridier et al., 2010), one strain cannot be considered representative of the whole species. Therefore, in the third chapter, the study focused on a collection of 38 strains, constituted by the 14 sequenced strains, and 24 isolates from Italian wines showing different behaviours in microvinification experiments (complete malolactic fermentation, growth but incomplete fermentation, or reduction of vitality). In this collection, different aspects of genetic diversity were investigated, and the correlation with stress response was evaluated. Besides the identification of two new flexibility regions, the results showed that the different oenological properties of strains could not be clearly linked to presence/absence of specific genes, but derive from a more complex genetic background. However, it was possible to observe that the majority of the analyzed strains from Amarone wine (with about 15% of ethanol) presented island or plasmid-related genes. Therefore, these elements seemed to be important, not only for strain diversity, but also for adaptation and resistance to the ecological niche, especially for strains from this peculiar wine. Interest in thioredoxin genes derived from both the analyses of the core transcriptome of O. oeni PSU-1 (OEOE_1702) and of genetic diversity (OEOE_0350), therefore these genetic traits, together with thioredoxin reductases, were investigated in the fourth chapter. The distribution of those genes was assessed in the whole collection of strains, and, moreover, their expression was analyzed during the malolactic fermentation of six strains with different characteristics. The trxA OEOE_1702 showed a marked upregulation in most of the strains considered, thus it seemed to have a crucial function during malolactic fermentation, especially in its final phase. The strain with the worst oenological properties (Rz81) presented a completely different expression profile for the trx genes, suggesting that the Trx system plays an important role in stress response. Among the 24 strains from Italian wines considered in this study, a sub-collection was represented by 13 isolates deriving from a single spontaneous malolactic fermentation in Amarone wine. In the fifth chapter these strains were genetically and phenotipically characterized taking into account enzymatic activity and growth assays. A high intraspecific biodiversity emerged from the analysis of this O. oeni natural population. In particular, variability appeared also in the distribution of genes related to the exopolysaccharide production, which may favor the bacterial survival in a stressful environment such as Amarone wine. In conclusion, the investigation of stress response in the species O. oeni, carried out in this thesis project, used an integration of transcriptomics, genomics, and strain collection analysis. This approach allowed to achieve an improved knowledge of the stress response machinery of this species and more information on its biodiversity.