Identification of the receptor-binding protein in 936-species lactococcal bacteriophages

Abstract

The aim of this work was to identify genes responsible for host recognition in the lactococcal phages sk1 and bIL170 belonging to species 936. These phages have a high level of DNA identity but different host ranges. Bioinformatic analysis indicated that homologous genes, orf18 in sk1 and orf20 in bIL170, could be the receptor-binding protein (RBP) genes, since the resulting proteins were unrelated in the C-terminal part and showed homology to different groups of proteins hypothetically involved in host recognition. Consequently, chimeric bIL170 phages carrying orf18 from sk1 were generated. The recombinant phages were able to form plaques on the sk1 host Lactococcus lactis MG1614, and recombination was verified by PCR analysis directly with the plaques. A polyclonal antiserum raised against the C-terminal part of phage sk1 ORF18 was used in immunogold electron microscopy to demonstrate that ORF18 is located at the tip of the tail. Sequence analysis of corresponding proteins from other lactococcal phages belonging to species 936 showed that the N-terminal parts of the RBPs were very similar, while the C-terminal parts varied, suggesting that the C-terminal part plays a role in receptor binding. The phages investigated could be grouped into sk1-like phages (p2, fd13, jj50, and phi 7) and bIL170-like phages (P008, P113G, P272, and bIL66) on the basis of the homology of their RBPs to the C-terminal part of ORF18 in sk1 and ORF20 in bIL170, respectively. Interestingly, sk1-like phages bind to and infect a defined group of L. lactis subsp. cremoris strains, while bIL170-like phages bind to and infect a defined group of L. lactis subsp. lactis strains.

FIG. 1.

Gene organization of the genome regions in sk1 and bIL170 encoding tail proteins. Homologous genes in phages sk1 and bIL170 are connected by dashed lines. The grey boxes indicate DNA sequences with homology in sk1 and bIL170. The open boxes indicate DNA sequences with no homology in sk1 and bIL170. NPS, putative neck passage structure; TMP, putative tape measure protein.

FIG. 2.

PCR assay for confirmation of homologous recombination between orf18 from sk1 and the bIL170 genome. (A) Combined gene map for phages sk1 and bIL170, showing the putative RBP gene and surrounding genes. Areas of homology and nonhomology are indicated by solid and dotted lines, respectively. The arrows represent primers. Primers sk1orf16F and sk1orf20R (Table 2) bind to both sk1 and bIL170 DNA. Primers sk1orf18F and bIL170orf22R (Table 2) bind exclusively to sk1 and bIL170 DNA, respectively. The lines below the gene map indicate the DNA distances between the primers. (B) PCR analysis with all four primers used in the reaction. Lane 1, control PCR performed directly with a lysate from L. lactis IL1403(pKDU1) infected with phage bIL170; lanes 2 to 5, PCR performed directly with single plaques; lane L, 1-kb DNA ladder (Life Technologies Inc., Rockville, Md.).

FIG. 3.

Western (A) and dot blot (B) analyses of sk1 and bIL170 phages performed with antibodies raised against the C-terminal part of ORF18 from sk1. Lane M contained a protein marker (BioWhittaker Molecular Applications, Rockland, Maine).

FIG. 4.

Immunogold electron micrograph of phage sk1. The primary antibodies were those described in the legend to Fig. 3. Secondary antibodies conjugated to gold particles that were 5 nm in diameter were used.

FIG. 5.

Alignment of the deduced amino acid sequences of the RBPs in lytic lactococcal phages. The stars above the sequences indicate positions at which there is a single, fully conserved residue. The alignment scores are shown below the ruler.