Evolution of Lactococcus lactis phages within a cheese factory

Abstract

We have sequenced the double-stranded DNA genomes of six lactococcal phages (SL4, CB13, CB14, CB19, CB20, and GR7) from the 936 group that were isolated over a 9-year period from whey samples obtained from a Canadian cheese factory. These six phages infected the same two industrial Lactococcus lactis strains out of 30 tested. The CB14 and GR7 genomes were found to be 100% identical even though they were isolated 14 months apart, indicating that a phage can survive in a cheese plant for more than a year. The other four genomes were related but notably different. The length of the genomes varied from 28,144 to 32,182 bp, and they coded for 51 to 55 open reading frames. All five genomes possessed a 3' overhang cos site that was 11 nucleotides long. Several structural proteins were also identified by nano-high-performance liquid chromatography-tandem mass spectrometry, confirming bioinformatic analyses. Comparative analyses suggested that the most recently isolated phages (CB19 and CB20) were derived, in part, from older phage isolates (CB13 and CB14/GR7). The organization of the five distinct genomes was similar to the previously sequenced lactococcal phage genomes of the 936 group, and from these sequences, a core genome was determined for lactococcal phages of the 936 group.

FIG. 1.

Schematic representation of the genomic organization of phages belonging to the 936 group. Each line represents a different phage genome and each arrow represents a putative protein. Each genome was compared only with the successive genome in this figure. ORFs of the same color represent those that share more than 80% amino acid identity. The percentages have been calculated for the smallest proteins. The white ORFs are unique. Gray shading connects genome regions conserved in all phages. Finally, arrows with thick outlines represent structural proteins observed on SDS-PAGE gels and identified by MS or by N-terminal sequencing (for the protein identification of phage bIL170, see references and 33).

FIG. 2.

Schematic representation of the genomic organization of lactococcal phages SL4, CB13, CB14/GR7, CB19, and CB20. Each line represents a different phage genome, and each arrow represents a putative protein. The ORFs in black or gray represent those that share 100% amino acid identity. Arrows in white indicate proteins that share less than 100% amino acid identity.

FIG. 3.

(A) Analysis of the structural proteins of phages SL4, CB13, CB14, CB19, and CB20 by SDS-PAGE. M is the broad-range protein marker (Bio-Rad). The numbers represent the proteins identified by MS. (B) Electron micrograph of phage CB19. (C) Electron micrograph of phage SL4. (D) Identification of the phage structural proteins by nano-HPLC coupled with MS/MS.