Development of a New Method for Detection and Identification of Oenococcus oeni Bacteriophages Based on Endolysin Gene Sequence and Randomly Amplified Polymorphic DNA

Abstract

M alolactic fermentation (MLF) is a secondary fermentation that occurs in wine after the alcoholic fermentation. This fermentation can take place spontaneously through the action of the population of lactic acid bacteria (LAB) present in the wine or by the use of commercial starters, mainly belonging to the species Oenococcus oeni, the principal LAB species responsible for MLF in wine (1, 2).As in other fermented foods, like dairy products, in which bacteriophages represent a potential risk for the fermentative process (3, 4), bacteriophages have also been reported to be a possible cause of unsuccessful MLF in wine (5, 6). Therefore, preparation of commercial starters that take into account the different sensitivities of O. oeni strains to different phages has been proposed (5, 7). However, under laboratory conditions, O. oeni has a very low growth rate, and its poorly visible colonies are hardly produced in agar medium, hampering the accurate detection of O. oeni phages, which has contributed to the low number of studies conducted until now on this topic. For instance, while PCR-based approaches to detect bacteriophages have been used in bacteria of the genera Lactobacillus and Lactococcus present in dairy products (8-11), such methods have never been applied to detect phages infecting O. oeni. In the present study, a new PCR method for the detection of bacteriophages of O. oeni without the need for a sensitive indicator strain and for a growth step on solid medium to confirm the presence of prophages was developed.Until now, typing of O. oeni bacteriophages has been conducted by morphological characterization, structural-protein composition, host range analysis, and/or restriction enzyme analysis (5, 12-15). The last approach (15) was the first DNA-based technique that allowed the grouping of different O. oeni phage types.Bacteriophages of O. oeni present in wines were first observed by Sozzi et al. (12) by electron microscopy (EM) and were subsequently described as being different in size (5). Later, EM analyses of four O. oeni phages showed that they were very similar, exhibiting a Bradley type B morphology, with the presence of a head, a tail, and a base plate (13). Similar results were reported after EM analysis of 10 phages isolated from wines (14), which also showed similar morphology with slight differences in the head size, tail length, and presence of a base plate. These phages were divided into four groups based on structural-protein composition, host range, and restriction enzyme analysis. Likewise, comparable morphologies and structural protein profiles have also been reported in 17 O. oeni mitomycin C (mit C)-induced phages from different O. oeni strains, which were further divided into six groups based on their enzymatic restriction patterns (15).Endolysins, also termed lysins, are enzymes encoded by most double-stranded DNA phage genomes that lyse bacterial cell walls and cause host death (16,17). Detection of the lys gene has recently been used to evaluate phages infecting Lactobacillus helve-

Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes

“…As regards the protection against phage attacks, opposite effects have been described: certain EPS are specifically recognized by certain phages and predispose bacteria to the attack by these phages, while others would be a protective barrier [57], [67]. It might be interesting in the future to connect the diversification of eps genes with the high variability in Oenophages recently described [12], [68], [69].…”

Complete Genome Sequence of Lytic Oenococcus oeni Bacteriophage OE33PA

“…After alcoholic fermentation, most wines undergo malolactic fermentation (MLF), driven by Oenococcus oeni , to improve their organoleptic properties and microbiological stability ( 1 ). Phages infecting O. oeni have been examined, as they could delay MLF ( 2 – 6 ) and open the way for less-desired indigenous populations to dominate in wines, impairing quality ( 1 , 7 ). However, so far, no free phage infecting O. oeni has been sequenced, and our current knowledge about phage genomes is derived from the sequencing of prophage loci recognized in prokaryotic genome sequencing projects ( 8 , 9 ).…”

Lysogeny in the Lactic Acid Bacterium Oenococcus oeni Is Responsible for Modified Colony Morphology on Red Grape Juice Agar

“…ORF1 and ORF4 correspond to the integrase and endolysin sequences, respectively. Both sequences have been previously reported to share significant overall nucleotide and deduced amino acid similarities, even though polymorphic zones allowed the design of specific primer couples (20,21).…”

“…The pervasiveness of prophages has been long recognized in the O. oeni species (17)(18)(19)(20)(21)(22). Prophages belong to the Siphoviridae family and are typically mosaic (19).…”