Table 1.
O. oeni strains used in this study.
Table 2.
Primers list.
Figure 1.
Genetic organization of pOENI-1 and comparison with related sequences.
A. Genetic organization of plasmid pOENI-1. ORFs are represented by numbered arrows and identified by corresponding protein tags (see also Table 3). B. Sequence comparison of pOENI-1 and related plasmids p1 (CP000424) and pH10 (CP002430). ORFs “c, d” (purple arrows) share 99% similarity with ORFs of pOENI-1v2. C. Portions of chromosomes in O. oeni ATCC BAA 1163 and O. oeni PSU1. The gene OEOE_0812 in O. oeni PSU1 (green arrow) is disrupted in O. oeni ATCC BAA 1163 by an 10 genes insert comprising four genes conserved in pOENI-1 (red arrows) and six genes unrelated to pOENI-1 (pink arrows). The insert is bordered by an 8-bp repeated sequence (dark triangles). D. Genetic organization of pOENI-1v2. ORFs numbered from 1 to 20 share more than 99% nucleotide sequence similarity with corresponding ORFs in pOENI-1. ORFs shaded in purple are not detected in pOENI-1 and code for transposases (a, e, f,), hypothetical proteins (b, c) and a recombinase (d). Pseudogenes are symbolized by arrowheads containing the symbol ψ. Regions of sequence similarity are indicated in percentages and shaded in blue. ori: putative origin of replication.
Table 3.
ORFs and predicted proteins of pOENI-1.
Figure 2.
Distribution of pOENI-1 genes in 44 O. oeni strains.
The dendrogram was constructed from DNA banding patterns obtained by NotI-PFGE analysis of 44 O. oeni strains. Oenococcus kitaharae was used as outgroup. Strain S11 was positioned on the basis of MLST data since no NotI-PFGE pattern was obtained for this strain. The presence (filled square) or absence (empty squares) of plasmid genes repA, tauE, oye and of the chromosomal gene OEOE_0812 were determined by PCR. The presence/absence of a region encompassing the oye and parB genes was also investigated. IOEB: Institute of oenology of Bordeaux, S: SARCO, ATCC: American type culture collection. Indutrial strains are marked with asterisks. Letters A and B in the dendrogram represent two phylogenetic groups of strains [36].
Table 4.
Plasmid/oye copy number.
Figure 3.
Control of plasmid-free strains.
A. Comparison of NotI-PFGE patterns of plasmid containing strains (C9+, C10+) and isogenic plasmid-less derivatives (C9− and C10−). Red arrows indicate bands corresponding to plasmids in strains C9+ and C10+. B. Absence of plasmids in strains C9− and C10− was confirmed by multiplex PCR targeting a plasmid gene (ORF 20, 821-bp PCR product) and a chromosomal gene (mleA, 430-bp PCR product).
Figure 4.
Comparison of MLF kinetics of isogenic strains with/without plasmids.
Kinetics of L-malate conversion (solid lines) and monitoring of cell population (dotted lines) were monitored following inoculation of bacteria to 107 cells ml−1 in a red wine containing 3 g l−1 L-malate. A control was performed without added bacteria. Values are means of two biological replicates.
Figure 5.
Comparison of growth in wine of isogenic strains with/without plasmids.
Kinetics of alcoholic fermentation (CO2 released, dark line), MLF (colored solid lines) and bacterial populations (colored dotted lines) were monitored in a sterile grape must inoculated with industrial wine yeasts and 103.ml−1 bacteria carrying pOENI-1 or pOENI-1v2 (red lines), bacteria without plasmids (blue lines) or a mixture of both (green lines). Kinetics of AF (dark symbols) is the mean of the three experiments.
Table 5.
Percentage of plasmid-carrying/plasmid-free cells at different times of winemaking.
Figure 6.
Frequency of tauE and oye genes during wine fermentations.
A. B. The oye and tauE gene were quantified by qPCR analysis of 95 samples of must or wine collected at different stages of winemaking. Data obtained from rpoB quantifications were plotted on the x-axis to appraise the O. oeni population and on the y-axis to make easier the comparison between the O. oeni population (rpoB, filled squares) and the tauE or oye copy number (empty squares). Data are means of two independent determinations. C. The average ratios of tauE/rpoB or oye/rpoB were calculated from samples collected in must or AF (10–105 cells.ml−1) and during MLF (105–109 cells.ml−1). The boxes and lines represent the means (small squares), standard errors (large squares) and standard deviations (lines).
Figure 7.
PCR detection of pOENI-1 and related plasmids in wines.
PCR assays were performed using DNA templates from O. oeni C9 (pOENI-1), O. oeni S11 (pOENI-1v2) and 30 samples of wine collected during MLF (A–E). The number of samples sharing the same PCR product is indicated in parentheses. The primers allowed detection of pOENI-1 repA (panel A), tauE (panel B) and a region extending from ORF11 (oye) to ORF 13 (parB) (panel C). M: DNA size markers.