Differences in Lactococcal Cell Wall Polysaccharide Structure Are Major Determining Factors in Bacteriophage Sensitivity

Abstract

Analysis of the genetic locus encompassing a cell wall polysaccharide (CWPS) biosynthesis operon of eight strains of Lactococcus lactis, identified as belonging to the same CWPS type C genotype, revealed the presence of a variable region among the strains examined. The results allowed the identification of five subgroups of the C type named subtypes C1 to C5. This variable region contains genes encoding glycosyltransferases that display low or no sequence homology between the subgroups. In this study, we purified an acidic polysaccharide from the cell wall of L. lactis 3107 (subtype C2) and confirmed that it is structurally different from the previously established CWPS of subtype C1 L. lactis MG1363. The CWPS of L. lactis 3107 is composed of pentasaccharide repeating units linked by phosphodiester bonds with the structure 6-α-Glc-3-β-Galf-3-β-GlcNAc-2-β-Galf-6-α-GlcNAc-1-P. Combinations of genes from the variable region of subtype C2 were introduced into a mutant of subtype C1 L. lactis NZ9000 deficient in CWPS biosynthesis. The resulting recombinant mutant synthesized a polysaccharide with a composition characteristic of that of subtype C2 L. lactis 3107 and not wild-type C1 L. lactis NZ9000. By challenging the recombinant mutant with various lactococcal phages, we demonstrated that CWPS is the host cell surface receptor of tested bacteriophages of both the P335 and 936 groups and that differences between the CWPS structures play a crucial role in determining phage host range.

Cryo-Electron Microscopy Structure of Lactococcal Siphophage 1358 Virion

“…It is tempting to speculate that this viral adaptation is an evolutionary response to the industrial practice of rotating numerous L. lactis strains. Another significant outcome of these studies was the discovery of the binding partner of the RBPs, a polysaccharide pellicle at the surface of the lactococcal cells (20)(21)(22). The diversity of the pellicle composition between L. lactis strains explains, at least in part, the strain specificity of most lactococcal phages (21).…”

Molecular Insights on the Recognition of a Lactococcus lactis Cell Wall Pellicle by the Phage 1358 Receptor Binding Protein

“…A comparative analysis of the gene cluster of the pellicle revealed diversity among L. lactis strains but also an apparent correlation between the lactococcal pellicle genotype of a given strain and the host range of tested 936-type phages (29). This correlation was recently confirmed through the characterization of the pellicle of L. lactis 3107, the host of phage TP091-1 (28).…”

“…Amino group location was concluded from the high field signal position of aminated carbons (CH signals at 45 to 60 ppm). ␤-Galf was identified by 13 C chemical shifts in comparison with published values (28,47,48), based on the known monosaccharide composition. Complete assignment of 1 H and 13 C spectra showed two ␣-GlcNAc saccharides (A and E), one ␤-GlcNAc saccharide (F), two ␤-Galf saccharides (B and C), and one ␣-Glc saccharide (D) ( Table 3).…”

“…These surface polysaccharides were shown to be composed of repeating units of hexasaccharide phosphate that are distinct from other bacterial polysaccharides (11,28). A comparative analysis of the gene cluster of the pellicle revealed diversity among L. lactis strains but also an apparent correlation between the lactococcal pellicle genotype of a given strain and the host range of tested 936-type phages (29).…”

“…We analyzed the pellicle of its host L. lactis SMQ-388 and determined that it possesses a phospho-hexasaccharidic repeat, differing from the L. lactis hosts of phage p2 (11) and TP091-1 (28). These data led us to model a trisaccharidic moiety, GlcNAc-galactofuranose (Galf)-GlcNAc/glucose 1-phosphate (Glc1P), that is surprisingly common to the receptors of phages p2, TP091-1, and 1358.…”