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. 2019 Sep 17;85(19):e00997-19.
doi: 10.1128/AEM.00997-19. Print 2019 Oct 1.

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

Affiliations

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

Amel Chaïb et al. Appl Environ Microbiol. .

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.

Keywords: bacteriophages; lactic acid bacteria; lysogeny.

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Figures

FIG 1
FIG 1
An individual plaque produced by the temperate phage CiNeA on its host O. oeni IOEBS277 on MRSϕ agar was picked and streaked on red grape juice agar. Both red and white colonies were observed after 5 days of incubation at 25°C.
FIG 2
FIG 2
Enumeration of a mixture 1:10 of O. oeni IOEBS277 and its lysogenic derivative IOEBS277 (CiNeA) on RGJ plates, showing different colony morphotypes (red/white).
FIG 3
FIG 3
Characteristics of the oenophages used in this study. Schematic representation of the organization of the 6 distinct oenophages used in the study, namely, 1491MCA, CiNeMCA, O608MCA, 10MCB, O33SAGD, and 9805MCD. Modules are indicated with the following color code: orange, lysogeny module; blue, replication-associated genes; green, DNA packaging; pink, morphogenesis module; red, lysis; black, distal modules; and purple, hypothetical genes. Information is taken from references , , , and . Conserved ORFs are numbered from 1 to 4.
FIG 4
FIG 4
Growth curves and spontaneous phage release of lysogenic derivatives of O. oeni IOES277 in red grape juice (RGJ) (A) or in MRS (B). Precultures were prepared in RGJ broth and MRS broth. Strains were IOEBS277 (open box), IOEBS277 (CiNeA) (green box), IOEBS277 (10MCB) (blue box), and IOEBS277 (OE33SAGD) (red box). The arrow represents the end of exponential growth.
FIG 5
FIG 5
Colonies produced by O. oeni IOEBS277 are white on RGJ agar (A) and turn to red following a lytic phage (B) or lysozyme spray application (C). A MRS culture was enumerated on RGJ agar in duplicate (control and assay) and plates were incubated at 25°C. After 3 days, the plate was air sprayed with the lytic phage OE33PA (lysate containing ∼109 PFU/ml) and further incubated for 3 days. Both control (A) and assay (B) were observed after 6 days of incubation. Alternately, a zone of the plate (represented by the circle) was sprayed with a freshly prepared lysozyme solution (10 mg/ml) in 10 mM Tris-HCl (pH 8.0) and 2 mM EDTA and observed after 15 min of incubation (C).
FIG 6
FIG 6
The white-to-red switch of lysogens is observed on white grape juice agar supplemented with an extract containing PC. A mixture of O. oeni IOEBS277 and its lysogenic derivative IOEB277 (CiNeA) (1:1) was enumerated on WGJ agar containing an extract containing PC (50 mg/ml) (A) or without PC (B). Red and white colonies were observed in the presence of PC only. PCR tests targeting phage sequences showed that they corresponded to lysogens and nonlysogens.
FIG 7
FIG 7
The test is applicable to other species of LAB. (A) A turbid plaque produced by phage A7MC on Leuconostoc citreum NRRL B-742 was streaked onto RGJ agar. Bacterial cells segregated into white (strain NRRL B-742) and red (lysogenized derivatives) colonies. (B) The method was adapted to RGJ broth. Cells were grown to stationary phase in RGJ broth with (+MC) or without mitomycin C (−MC). After 24 h (Lb. plantarum) or 72 h (O. oeni), the culture was centrifuged (2,000 × g for 2 min) and the color of the pellet was observed. The volumes of the cultures were 10 ml, except for those of IOEB277 (10MC), which were 5 ml.

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