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

Abstract

The Gram-positive bacterium Lactococcus lactis is used for the production of cheeses and other fermented dairy products. Accidental infection of L. lactis cells by virulent lactococcal tailed phages is one of the major risks of fermentation failures in industrial dairy factories. Lactococcal phage 1358 possesses a host range limited to a few L. lactis strains and strong genomic similarities to Listeria phages. We report here the X-ray structures of phage 1358 receptor binding protein (RBP) in complex with monosaccharides. Each monomer of its trimeric RBP is formed of two domains: a "shoulder" domain linking the RBP to the rest of the phage and a jelly roll fold "head/host recognition" domain. This domain harbors a saccharide binding crevice located in the middle of a monomer. Crystal structures identified two sites at the RBP surface, ϳ8 Å from each other, one accommodating a GlcNAc monosaccharide and the other accommodating a GlcNAc or a glucose 1-phosphate (Glc1P) monosaccharide. GlcNAc and GlcNAc1P are components of the polysaccharide pellicle that we identified at the cell surface of L. lactis SMQ-388, the host of phage 1358. We therefore modeled a galactofuranose (Galf) sugar bridging the two GlcNAc saccharides, suggesting that the trisaccharidic motif GlcNAc-Galf-GlcNAc (or Glc1P) might be common to receptors of genetically distinct lactococcal phages p2, TP091-1, and 1358. Strain specificity might therefore be elicited by steric clashes induced by the remaining components of the pellicle hexasaccharide. Taken together, these results provide a first insight into the molecular mechanism of host receptor recognition by lactococcal phages. IMPORTANCESiphophages infecting the Gram-positive bacterium Lactococcus lactis are sources of milk fermentation failures in the dairy industry. We report here the structure of the pellicle polysaccharide from L. lactis SMQ-388, the specific host strain of phage 1358. We determined the X-ray structures of the lytic lactococcal phage 1358 receptor binding protein (RBP) in complex with monosaccharides. The positions and nature of monosaccharides bound to the RBP are in agreement with the pellicle structure and suggest a general binding mode of lactococcal phages to their pellicle saccharidic receptor.

Cryo-Electron Microscopy Structure of Lactococcal Siphophage 1358 Virion

“…Indeed, ORF19 is similar to a short Tal protein, such as that of lactococcal phage p2 or of coliphage T4 (gp27), and differs from the long Tal proteins of lactococcal phage TP901-1 or Tuc2009 (40). As indicated above, we recently reported the X-ray structure of ORF20, the RBP of phage 1358 (22). HHpred analysis of ORF20 also revealed a striking similarity between its ϳ170 first residues and the N-terminal region of the RBP of the lactococcal p2 (936 group) (10)(11)(12)14).…”

“…We were able to fit the trimeric RBP structure (ORF20) of phage 1358 (22) into the baseplate EM map of the closed conformation. We successively assigned six RBPs trimers (18ϫ ORF20) to their electron densities ( Fig.…”

“…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).…”

“…Moreover, the tail of 1358 is significantly shorter than those of other lactococcal phages, and it is decorated by hairy appendages. We also recently reported the Xray structure of its RBP (22). Each monomer of its trimeric RBP is formed of two domains: a "shoulder" domain linking the RBP to the rest of the phage and a jelly-roll fold "head/host recognition" domain.…”

Genetically Engineered Phages: a Review of Advances over the Last Decade

“…By genetically engineering phages, it may be possible to overcome many of these limitations (44). The engineering of specific phages and components has been facilitated by the ever-growing abundance of fully sequenced phage genomes in public databases (45,46) and by research into elucidating the structures of phage components (47)(48)(49)(50)(51) and the interactions between phages and their host bacteria (52)(53)(54). This review focuses on advances made in phage engineering techniques and applications in the past decade.…”