Novel P335-like Phage Resistance Arises from Deletion within Putative Autolysin yccB in Lactococcus lactis

Abstract

Lactococcus lactis and Lactococcus cremoris are broadly utilized as starter cultures for fermented dairy products and are inherently impacted by bacteriophage (phage) attacks in the industrial environment. Consequently, the generation of bacteriophage-insensitive mutants (BIMs) is a standard approach for addressing phage susceptibility in dairy starter strains. In this study, we characterized spontaneous BIMs of L. lactis DGCC12699 that gained resistance against homologous P335-like phages. Phage resistance was found to result from mutations in the YjdB domain of yccB, a putative autolysin gene. We further observed that alteration of a fused tail-associated lysin-receptor binding protein (Tal-RBP) in the phage restored infectivity on the yccB BIMs. Additional investigation found yccB homologs to be widespread in L. lactis and L. cremoris and that different yccB homologs are highly correlated with cell wall polysaccharide (CWPS) type/subtype. CWPS are known lactococcal phage receptors, and we found that truncation of a glycosyltransferase in the cwps operon also resulted in resistance to these P335-like phages. However, characterization of the CWPS mutant identified notable differences from the yccB mutants, suggesting the two resistance mechanisms are distinct. As phage resistance correlated with yccB mutation has not been previously described in L. lactis, this study offers insight into a novel gene involved in lactococcal phage sensitivity.

Keywords: Lactococcus; autolysin; bacteriophage; resistance; yccB.

Figure 1

Predicted YccB protein translation comparison. Protein domains found in YccB are shown in different colors and labeled accordingly. Dashed lines indicate protein truncation due to mutation. An amino acid scale is included on top where the active site of the protein is also shown in light green.

Figure 2

Phage genome alignments. Regions of similarity exhibiting a minimum identity overlap of 200 bp are shown in varying scales of gray according to the percentage identity observed. Genes exhibiting globally related functions possess the same color scheme and are labeled accordingly (yellow = structural, dark blue = DNA packaging, purple = life cycle regulation, red = DNA replication or repair, brown = domain of unknown function (DUF), light blue = other function, gray = hypothetical protein).

Figure 3

Nucleotide alignment of the genes encoding the Tal-RBPs. In the consensus identity bar (top), green indicates identity, yellow indicates polymorphism, and red indicates low identity. Yellow arrows indicate CDS. Black areas in the grey bars indicate differences among the three phages.

Figure 4

Molecular modeling and comparisons. Structural alignment of the AlphaFold predicted Tal-RBP models for: (a) Phages D6867 (green) and D7138 (cyan). The region representing the deletion in D7138 is shown in red on D6867; (b) Phages D6867 (green) and D7893 (purple).

Figure 5

Comparison of yccB homologs and CWPS type. A phylogenetic tree of yccB homologs found in a subset of public lactococcal strains; branch color indicates CWPS type and text color/highlight indicates CWPS subtype. Like-colored strains clustering together indicate a strong relationship between yccB homolog and CWPS type/subtype.

Figure 6

CINAC milk acidification activity test comparison of DGCC12699, BIM2, and BIM21. Each strain was run in duplicate and the averages of the data are shown on the plot. The average time for each strain to reach target pH 5.2 (T5.2) is displayed in the upper right.