Detecting Lactococcus lactis Prophages by Mitomycin C-Mediated Induction Coupled to Flow Cytometry Analysis

Abstract

Most analyzed Lactococcus lactis strains are predicted to harbor one or more prophage genomes within their chromosome; however, the true extent of the inducibility and functionality of such prophages cannot easily be deduced from sequence analysis alone. Chemical treatment of lysogenic strains with Mitomycin C is known to cause induction of temperate phages, though it is not always easy to clearly identify a lysogenic strain or to measure the number of released phage particles. Here, we report the application of flow cytometry as a reliable tool for the detection and enumeration of released lactococcal prophages using the green dye SYTO-9.

Characterization and induction of prophages in human gut-associated Bifidobacterium hosts

“…The extent of lysis and phage release can be assessed with flow cytometry. Flow cytometry has been used to identify stained, PEG precipitated Lactococcus lactis phages in culture supernatants after mitomycin C induction 41 . Here, a modified strategy was used to quantify mitomycin C-induced changes in supernatant composition, since phage release is expected to increase both the abundance and fluorescence of flow cytometric events (see Materials and Methods).…”

“…Sample preparation and processing for flow cytometric analysis was performed similarly to previously described methods 41 . Strains were grown in RCM to early log phase, treated with mitomycin C (or were left untreated), and filtered (described above).…”

“…Forward scatter (FSC-H), side scatter (SSC-H), and fluorescence (FL1-H) parameters were measured using instrument settings that were calibrated to reproduce mitomycin C-treated Lactococcus lactis UC509.9 and NZ9000(TP901-1) induction results reported previously using a different flow cytometer (Supplementary Fig. S3a ) 41 . Several types of controls were used for downstream analysis, including distilled H 2 O, ¼ strength Ringer’s solution, and ¼ strength Ringer’s solution with beads, with and without mitomycin C added.…”

“…Also, fluorescence intensity of stained particles does not correlate with genome size 66 . Therefore, flow cytometry events due to debris or bead standards were gated and removed similarly to previously described methods 41 . To define the gates, the signal distribution of each parameter (FSC-H, SSC-H, FL1-H) was analyzed in several control samples to identify the signal range associated with each event type (Supplementary Fig.…”

Lysogenization of a Lactococcal Host with Three Distinct Temperate Phages Provides Homologous and Heterologous Phage Resistance

“…Strains of Lactococcus lactis are among the most intensely applied starter cultures in mesophilic fermentations. Most genomes of lactococcal strains are reported to harbor one or more prophages [1][2][3][4]. Because phages pose a threat to consistent fermentation regimes, they have traditionally been viewed as a problematic entity.…”