Phage-host interactions as a driver of population dynamics during wine fermentation: Betting on underdogs

Winemaking is a complex process in which numerous microorganisms, mainly yeasts and lactic acid bacteria (LAB), play important roles. After alcoholic fermentation (AF), most wines undergo malolactic fermentation (MLF) to improve their organoleptic properties and microbiological stability. Oenococcus oeni is mainly responsible for this crucial process where L-malic acid (MA) in wine converts to softer L-lactic acid. The bacterium is better adapted to the limiting conditions imposed by the wine matrix and performs MLF under regular winemaking conditions, especially in wines with a pH below 3.5. Traditionally, this process has been conducted by the natural microbiota present within the winery. However, the start, duration and qualitative impact of spontaneous MLF are unpredictable, which prompts winemakers to use pure starter cultures of selected bacteria to promote a more reliable, simple, fast and efficient fermentation. Yet, their use does not always ensure a problem-free fermentation. Spontaneous initiation of the process may prove very difficult or does not occur at all. Such difficulties arise from a combination of factors found in some wines upon the completion of AF (high ethanol concentration, low temperature and pH, low nutrient concentrations, presence of free and bound SO2). Alongside these well documented facts, research has also provided evidence that negative interactions between O. oeni and other biological entities such as yeasts may also impact MLF. Another insufficiently described, but highly significant factor inhibiting bacterial growth is connected to the presence of bacteriophages of O. oeni which are frequently associated to musts and wines. The purpose of this review is to summarize the current knowledge about the phage life cycles and possible impacts on the trajectory of the microbiota during winemaking

Appl. environ. microbiol.

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.[br/] 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 plantarurn. 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