Identification of Lactococcus lactis genes required for bacteriophage adsorption

Abstract

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and phi 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that phi645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that phi 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and phi 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and phi 645 that showed no homology in the C-terminal part.

FIG. 1.

Southern analyses of chromosomal DNA from selected integrants of L. lactis IL1403 with reduced binding of phage bIL170. Chromosomal DNA was digested with EcoRI, and pGh9:ISS1 was used as a probe. The two bands in each lane represent chromosomal DNA with a flanking pGh9:ISS1 sequence and a flanking ISS1 sequence (25). The numbers at the top indicate the integrants used (see Table 4).

FIG. 2.

Integration sites of pGh9:ISS1 in integrants of L. lactis IL1403 (A) and L. lactis Wg2 (B) resistant to phages bIL170 and φ645, respectively. The integration sites in the resulting proteins are indicated by open triangles and numbers that correspond to the integrants shown in Table 4 and in Fig. 1. For L. lactis Wg2 the approximate integration sites are indicated in the homologous L. lactis IL1403 proteins.

FIG. 3.

RT-PCR of phage-resistant integrants of L. lactis IL1403. (A) Integrants 5 and 8 (see Table 4 and Fig. 2) with pGh9:ISS1 integrated into the ycaG and rgpE genes, respectively. Primers annealed downstream from the integrated pGh9:ISS1 in rgpF and ycbA (nucleotides 6637 and 9866; accession no. AE006258) (Table 3). (B) Integrant 9 (see Table 4 and Fig. 2) with a solitary ISS1 sequence integrated into the rgpE gene. Lane 9A, primers annealing on each side of the ISS1 sequence in the rgpC and rgpF genes (nucleotides 2314 and 7835; accession no. AE006258) (Table 3); lane 9B, primers annealing in the ISS1 sequence (ISS1F [Table 2]) and in the rgpF gene (nucleotide 7855; accession no. AE006258) (Table 3); lane L, 1-kb DNA ladder. RT, reverse transcriptase.

FIG. 4.

Operon in L. lactis IL1403 containing the ycaG, rgpE, ycbB, and ycbC genes. The light grey arrows represent genes interrupted in integrants of L. lactis IL1403 (ycaG and rgpE) and L. lactis Wg2 (ycbB and ycbC) resistant to phages bIL170 and φ645, respectively. The displaced arrows indicate overlapping open reading frames. The bent arrow indicates the promoter, and the hairpin symbols indicate the terminators. LPS, lipopolysaccharide.

FIG. 5.

Alignment of amino acid sequences of the RBPs in φ645 and bIL170. The stars above the sequences indicate positions which have a single, fully conserved residue. The alignment scores are shown below the ruler.