Characterization of the First Virulent Phage Infecting Oenococcus oeni, the Queen of the Cellars

Abstract

There has been little exploration of how phages contribute to the diversity of the bacterial community associated with winemaking and may impact fermentations and product quality. Prophages of Oenococcus oeni, the most common species of lactic acid bacteria (LAB) associated with malolactic fermentation of wine, have been described, but no data is available regarding phages of O. oeni with true virulent lifestyles. The current study reports on the incidence and characterization of the first group of virulent oenophages named Vinitor, isolated from the enological environment. Vinitor phages are morphologically very similar to siphoviruses infecting other LAB. Although widespread during winemaking, they are more abundant in musts than temperate oenophages. We obtained the complete genomic sequences of phages Vinitor162 and Vinitor27, isolated from white and red wines, respectively. The assembled genomes shared 97.6% nucleotide identity and belong to the same species. Coupled with phylogenetic analysis, our study revealed that the genomes of Vinitor phages are architecturally mosaics and represent unique combinations of modules amongst LAB infecting-phages. Our data also provide some clues to possible evolutionary connections between Vinitor and (pro)phages associated to epiphytic and insect-related bacteria.

Keywords: Oenococcus; fermentation; grapes; lactic acid bacteria; lytic phage; phage (bacteriophage); phage phylogenetics; wine.

FIGURE 1

Plaque morphology of phage Vinitor162 on its O. oeni host.

FIGURE 2

Uranyl acetate-stained transmission electron micrographs of phage Vinitor162. The white arrows show the extended unique thin tail fiber.

FIGURE 3

Architecture of the oenophages Vinitor162 and Vinitor27 genomes. The predicted functions of the Vinitor genes are indicated below the maps. The presumptive modules are colored. The gray vertical blocks between phage sequences indicate regions of shared homology according to BLASTn, and the degree of nucleotide identity is indicated by the intensity of gray. ORFs in white characters have small differences in their nucleic sequences and those in red are specific to each Vinitor phage. The figure was generated with Easyfig.

FIGURE 4

Phylogenetic relationships of Vinitor phages and related phages. Phylogenetic trees using the TerL (A), MCP (B) proteins and whole genome nucleotide sequences (C) are shown. A and B; Maximum likelihood phylogenetic trees were computed using the best-fit substitution models for given alignments as determined by PhyML with Bayesian Information Criterion with a LRT SH-like method for branch support as reported above. The values are shown for nodes with ≥ 50% support. The scale bar represents the number of amino acid substitutions per site. Asterisks represent pac-containing phages. Red and green arrows represent Vinitor162 and other oenophages, respectively. C. Phylogenetic position of Vinitor162 and Vinitor27 using VICTOR. Current virus taxon nomenclature by ICTV is given with a color code for the different branches as follows: sub-family Mccleskeyvirinae Genus Limdunavirus (purple); sub-family Mccleyskyvirinae Genus Unaquatrovirus (blue); Genus Cequinquevirus (orange); Genus Coetzeevirus turquoise blue); Genus Maenadvirus (bright green); Genus Lenusvirus (fuschia); sub-family Tybeckvirinae Genus Lidleunavirus (yellow); Genus Brüssowvirus (brown); Genus Moineauvirus (light blue); Genus Ceduovirus (pink); Unclassified virus (black); Vinitor phage (bold red); other Oenophages (bold green).

FIGURE 5

The proposed adhesion apparatus of Vinitor phages is based upon HHpred analysis. The classical Dit structural ring is colored in red. The Tal structural domains are colored in light green. The atypical CBM insertions (5 types) are colored in dark green. The putative location of the structural domain is shown by a red a circle and the 2–5 CBMs by red arrows on the EM micrographs. The Protein Data Bank identification numbers are displayed below each CBM label and identify the best HHpred hit.