Biodiversity and classification of lactococcal phages

Abstract

For this study, an in-depth review of the classification of Lactococcus lactis phages was performed. Reference phages as well as unclassified phages from international collections were analyzed by stringent DNA-DNA hybridization studies, electron microscopy observations, and sequence analyses. A new classification scheme for lactococcal phages is proposed that reduces the current 12 groups to 8. However, two new phages (Q54 and 1706), which are unrelated to known lactococcal phages, may belong to new emerging groups. The multiplex PCR method currently used for the rapid identification of phages from the three main lactococcal groups (936, c2, and P335) was improved and tested against the other groups, none of which gave a PCR product, confirming the specificity of this detection tool. However, this method does not detect all members of the highly diverse P335 group. The lactococcal phages characterized here were deposited in the Félix d'Hérelle Reference Center for Bacterial Viruses and represent a highly diverse viral community from the dairy environment.

FIG. 1.

(A) Restriction profiles of lactococcal phages analyzed in this study. (B to G) Analysis of phage genomic DNAs by Southern hybridization, using the complete genomes of the following phages as probes: panel B, r1t; panel C, c2; panel D, P369; panel E, 949; panel F, Q54; panel G, 1706. Lanes 1 and 19, 1-kb DNA ladder (Invitrogen); lanes 2, phage bIL170 (genome digested with EcoRV); lanes 3, c2 (EcoRI); lanes 4, Q54 (EcoRI); lanes 5, GR6 (EcoRI); lanes 6, CB17 (EcoRI); lanes 7, ul36 (EcoRV); lanes 8, r1t (EcoRV); lanes 9, 1483 (EcoRV); lanes 10, T189 (EcoRV); lanes 11, P087 (EcoRV); lanes 12, 949 (EcoRV); lanes 13, bIL168 (EcoRV); lanes 14, 1706 (EcoRV); lanes 15, 1358 (EcoRI); lanes 16, KSY1 (AsnI); lanes 17, P369 (EcoRV); lanes 18, 1138 (EcoRV); lanes 20, BK5-T (EcoRV).

FIG. 2.

Identification of lactococcal phage species by multiplex PCR. (A) Three pairs of previously described primers (29) were used for PCR. (B) The same three sets of primers were used, except that the c2B primer was replaced by the c2C primer. Lanes 1 and 20, 100-bp DNA ladder (Invitrogen); lanes 2, negative control; lanes 3, bIL170; lanes 4, c2; lanes 5, GR6; lanes 6, CB17; lanes 7, Q54; lanes 8, r1t; lanes 9, 1483; lanes 10, ul36; lanes 11, T189; lanes 12, P087; lanes 13, 949; lanes 14, bIL168; lanes 15, 1706; lanes 16, 1358; lanes 17, KSY1; lanes 18, P369; lanes 19, 1138; lanes 21, BK5-T. (C) Analysis of the region covered by primers c2B and c2C in c2-like phages for which the gene coding for the major capsid protein is available in GenBank.

FIG. 3.

Alignment of the genetic maps of 10 completely sequenced P335-like phages (updated from reference 30). Deduced proteins sharing >60% amino acid identity are represented using the same colors and linked with gray shading when possible. Open reading frames with unique sequences are displayed in white. The dUTPase genes are identified by thick lines.