Phage Encounters Recorded in CRISPR Arrays in the Genus Oenococcus

Abstract

The Oenococcus genus comprises four recognized species, and members have been found in different types of beverages, including wine, kefir, cider and kombucha. In this work, we implemented two complementary strategies to assess whether oenococcal hosts of different species and habitats were connected through their bacteriophages. First, we investigated the diversity of CRISPR-Cas systems using a genome-mining approach, and CRISPR-endowed strains were identified in three species. A census of the spacers from the four identified CRISPR-Cas loci showed that each spacer space was mostly dominated by species-specific sequences. Yet, we characterized a limited records of potentially recent and also ancient infections between O. kitaharae and O. sicerae and phages of O. oeni, suggesting that some related phages have interacted in diverse ways with their Oenococcus hosts over evolutionary time. Second, phage-host interaction analyses were performed experimentally with a diversified panel of phages and strains. None of the tested phages could infect strains across the species barrier. Yet, some infections occurred between phages and hosts from distinct beverages in the O. oeni species.

Keywords: CRISPR; Oenococcus; bacteriophage; fermented beverage; host spectra; spacers.

Figure 1

Distribution and architecture of CRISPR loci in the Oenococcus genus. Direct repeats (DR) and spacers are represented by rectangles and lozenges. Genes coding for hypothetical proteins (HP) are in grey. Double slash marks represent DNA regions that are not shown. Sequence ID for the restriction endonuclease R and S subunits and putative Ig-like proteins in O. kitaharae CRBO2176 are MCV3295975.1, MCV3295974.1 and MCV3295973.1, respectively.

Figure 2

Scheme for the classification and rapid genotyping of oenophages. LM, lysogeny maintenance; Rep, replication-associated protein; TerS, small unit of the terminase; TMP, tape measure protein; Doc, toxin–antitoxin system. Representative phages containing the genes encoding the distinct proteins are indicated. Pro indicates that the phage is a prophage. Because some strains are poly-lysogens, the integrase type (A to F) is also indicated.

Figure 3

Distribution of the protospacers in oenophages. (A) Phylogenetic tree of the 34 phages of O. oeni, including OE333PA (ex-temperate), OE33PA6 (temperate) and Vinitor (strictly lytic), which were isolated as free replicating phage particles from must or wine samples. Other genomes correspond to prophages, and reference to their cognate integrase group was also added, since many lysogens harbour two or three prophages belonging to distinct integrase groups. In the leftward column, dots were used to summarize the presence of protospacers targeted by the CRISPR of O. kitaharae (purple) or O. sicerae (blue and/or green); (B) presence of spacers in plasmids of O. oeni; (C) presence of spacers in the genome of other LAB. Lb. plantarum, Lactiplantibacillus plantarum; Liq. mali, Liquorilactobacillus mali.

Figure 4

Auto-immunity and location of STS in O. sicerae UCMA15228. The genomic organization of the prophage genome targeted by the type I CRISPR system is shown. The location of the protospacers incorporated into the CRISPR locus as STS (SP_OSI30, SP_OSI36, SP_OSI86, SP_OSI87 and SP_OSI98) are indicated above the map. The four putative acr genes are in green. Tgase: Transglycosylase.

Figure 5

Variability of the protospacer sequences incorporated as SP_OSI78, SP_OSI89 (A) and SP_OSII1 (B) in oenophages. Analysis of the Rep5 proteins in Vinitor 23 and its homologue in Vinitor 162 (C). The variable region marked in red results from the deletion of the protospacer matching SP_OSII1 in Vinitor 162.