Control of plasmid-free strains.

<p>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 (<i>mleA</i>, 430-bp PCR product).</p

Percentage of plasmid-carrying/plasmid-free cells at different times of winemaking.

a<p>strains inoculated in must.</p

Primers list.

a<p>Primers used in qPCR assays.</p>b<p>Product sizes obtained for pOENI-1 and pOENI-1v2.</p

Distribution of pOENI-1 genes in 44 <i>O. oeni</i> strains.

<p>The dendrogram was constructed from DNA banding patterns obtained by NotI-PFGE analysis of 44 <i>O. oeni</i> strains. <i>Oenococcus kitaharae</i> 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 <i>repA</i>, <i>tauE</i>, <i>oye</i> and of the chromosomal gene OEOE_0812 were determined by PCR. The presence/absence of a region encompassing the <i>oye</i> and <i>parB</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049082#pone.0049082-Bilhere1" target="_blank">[36]</a>.</p

Comparison of growth in wine of isogenic strains with/without plasmids.

<p>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.</p

<i>O. oeni</i> strains used in this study.

a<p>IOEB: Institute of oenology of Bordeaux, S: SARCO, ATCC: American type culture collection.</p

Genetic organization of pOENI-1 and comparison with related sequences.

<p>A. Genetic organization of plasmid pOENI-1. ORFs are represented by numbered arrows and identified by corresponding protein tags (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049082#pone-0049082-t003" target="_blank">Table 3</a>). 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 <i>O. oeni</i> ATCC BAA 1163 and <i>O. oeni</i> PSU1. The gene OEOE_0812 in <i>O. oeni</i> PSU1 (green arrow) is disrupted in <i>O. oeni</i> 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.</p

Comparison of MLF kinetics of isogenic strains with/without plasmids.

<p>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.</p

PCR detection of pOENI-1 and related plasmids in wines.

<p>PCR assays were performed using DNA templates from <i>O. oeni</i> C9 (pOENI-1), <i>O. oeni</i> 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 <i>repA</i> (panel A), <i>tauE</i> (panel B) and a region extending from ORF11 (<i>oye</i>) to ORF 13 (<i>parB</i>) (panel C). M: DNA size markers.</p

Frequency of <i>tauE</i> and <i>oye</i> genes during wine fermentations.

<p>A. B. The <i>oye</i> and <i>tauE</i> gene were quantified by qPCR analysis of 95 samples of must or wine collected at different stages of winemaking. Data obtained from <i>rpoB</i> quantifications were plotted on the x-axis to appraise the <i>O. oeni</i> population and on the y-axis to make easier the comparison between the <i>O. oeni</i> population (<i>rpoB</i>, filled squares) and the <i>tauE</i> or <i>oye</i> copy number (empty squares). Data are means of two independent determinations. C. The average ratios of <i>tauE/rpoB</i> or <i>oye/rpoB</i> 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).</p