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

Abstract

Oenococcus oeni is the lactic acid bacterium (LAB) that most commonly drives malolactic fermentation in wine. Although oenococcal prophages are highly prevalent, their implications on bacterial fitness have remained unexplored and more research is required in this field. An important step toward achieving this goal is the ability to produce isogenic pairs of strains that differ only by the lysogenic presence of a given prophage, allowing further comparisons of different phenotypic traits. A novel protocol for the rapid isolation of lysogens is presented. Bacteria were first picked from the center of turbid plaques produced by temperate oenophages on a sensitive nonlysogenic host. When streaked onto an agar medium containing red grape juice (RGJ), cells segregated into white and red colonies. PCR amplifications with phage-specific primers demonstrated that only lysogens underwent white-red morphotypic switching. The method proved successful for various oenophages irrespective of their genomic content and attachment site used for site-specific recombination in the bacterial chromosome. The color switch was also observed when a sensitive nonlysogenic strain was infected with an exogenously provided lytic phage, suggesting that intracolonial lysis triggers the change. Last, lysogens also produced red colonies on white grape juice agar supplemented with polyphenolic compounds. We posit that spontaneous prophage excision produces cell lysis events in lysogenic colonies growing on RGJ agar, which, in turn, foster interactions between lysed materials and polyphenolic compounds to yield colonies easily distinguishable by their red color. Furthermore, the technique was used successfully with other species of LAB. IMPORTANCE The presence of white and red colonies on red grape juice (RGJ) agar during enumeration of Oenococcus oeni in wine samples is frequently observed by stakeholders in the wine industry. Our study brings an explanation for this intriguing phenomenon and establishes a link between the white-red color switch and the lysogenic state of O. oeni. It also provides a simple and inexpensive method to distinguish between lysogenic and nonlysogenic derivatives in O. oeni with a minimum of expended time and effort. Noteworthy, the protocol could be adapted to two other species of LAB, namely, Leuconostoc citreum and Lactobacillus plantarum. It could be an effective tool to provide genetic, ecological, and functional insights into lysogeny and aid in improving biotechnological processes involving members of the lactic acid bacterium (LAB) family.

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

“…The phage did not encode recognizable integrase, excisionase or repressor genes confirming that Vinitor phages have an exclusively lytic life cycle. Despite the peculiarity of its genome sequence and high number of orphans, phage Vinitor162 shared synteny with other reported temperate/ex-temperate oenophages in terms of genome organization, with the DNA packaging module followed by the structural module, the lysis module and the replication module ( Jaomanjaka et al, 2013 ; Chaïb et al, 2019 ).…”

“…Lysates of Vinitor162 or Vinitor27 were produced on O. oeni IOEB-SARCO 277 which does not contain endogenous phage and is sensitive to all oenophages so far isolated in our laboratory ( Chaïb et al, 2019 ). Phage amplification was carried out in MRS broth supplemented with MgSO 4 (3.75 g/L) and CaCl 2 (2.375 g/L) (MRS Φ ) with a multiplicity of infection of 0.03.…”

“…Noteworthy, lysogeny was shown to be prevalent among MLF commercial cultures and further studies are now needed to better undertand lysis-lysogeny decision strategies in wine. Intringuinly, a small set of oenophages collected in 2015 drew our attention as they produced clear plaques and did not exhibit several key genomic features which are globally applicable to previously described temperate oenophages ( Philippe et al, 2017 ; Chaïb et al, 2019 ). In the current study, whole-genome sequencing of two phages of the group, further named Vinitor, confirmed their virulent nature, reinforcing the notion that phage predation of O. oeni occurs during wine making.…”

Draft Genome Sequence of Oenococcus kitaharae CRBO2176, Isolated from Homemade Water Kefir

“…A homemade kefir prepared from figs (Bordeaux, France) was plated onto red grape juice agar ( 9 ), and the plates were incubated at 25°C for 5 days under anaerobic conditions. Slow-growing isolates were purified three times by single-colony isolation.…”

“French Phage Network” Annual Conference—Fifth Meeting Report

“…Red grape polyphenolic compounds were shown to interact with dead cell material. The method was further adapted to other LAB species [34].…”