Receptor-Binding Protein of Lactococcus lactis Phages: Identification and Characterization of the Saccharide Receptor-Binding Site

Abstract

Phage p2, a member of the lactococcal 936 phage species, infects Lactococcus lactis strains by binding initially to specific carbohydrate receptors using its receptor-binding protein (RBP). The structures of p2 RBP, a homotrimeric protein composed of three domains, and of its complex with a neutralizing llama VH domain (VHH5) have been determined (S. Spinelli, A. Desmyter, C. T. Verrips, H. J. de Haard, S. Moineau, and C. Cambillau, Nat. Struct. Mol. Biol. 13:85-89, 2006). Here, we show that VHH5 was able to neutralize 12 of 50 lactococcal phages belonging to the 936 species. Moreover, escape phage mutants no longer neutralized by VHH5 were isolated from 11 of these phages. All of the mutations (but one) cluster in the RBP/VHH5 interaction surface that delineates the receptor-binding area. A glycerol molecule, observed in the 1.7-Å resolution structure of RBP, was found to bind tightly (K d ‫؍‬ 0.26 M) in a crevice located in this area. Other saccharides bind RBP with comparable high affinity. These data prove the saccharidic nature of the bacterial receptor recognized by phage p2 and identify the position of its binding site in the RBP head domain.Bacteriophage infection is a major problem impairing any industrial fermentation that relies on bacteria to transform a substrate into fermented products or specific molecules. Several strains of the gram-positive bacterium Lactococcus lactis are used quite extensively worldwide for the manufacture of fermented milk products. For decades, the dairy industry has been dealing with phage infections of their L. lactis strains because virulent phages are ubiquitous within their environment (28).Phages of L. lactis are classified within several groups (21), although those commonly found in dairy plants belong to three different species, i.e., 936, P335, and c2 (18, 27, 29). From a taxonomical standpoint, these phages are all members of the Siphoviridae family (order Caudovirales) that also includes the coliphage lambda. Siphophages are characterized by a double-stranded DNA genome and a long noncontractile tail. The phages belonging to the species 936 and P335 have a small isometric capsid, while those from the c2 species have a prolate capsid.The bacteriophage infection process starts by the specific recognition between the phage receptor-binding protein (RBP) located at the tip of the tail and the receptor distributed over the host cell surface (14, 35). Bacterial receptors have been well studied in gram-negative bacteria, particularly in Escherichia coli, while similar information lags behind in grampositive bacteria (12). It has been previously shown that phages infecting some L. lactis strains adsorb initially to the cell wall surface and likely to various carbohydrates containing rhamnose, glucose, or galactose (for a review, see Forde and Fitzgerald [15]). For many phages, this binding step is reversible, and phages of the c2 species, for example, require a second irreversible binding step to a predicted membrane-attached protein (PIP) of 901 amino acids. However, ph...

Modular Structure of the Receptor Binding Proteins of Lactococcus lactis Phages

“…The central hydroxyl group is either free or substituted by a D-glucose or a D-alanine, a feature that might be involved in strain specificity. Interestingly, despite the different strains of L. lactis recognized by p2 and TP901-1 phages (L. lactis strains LM0230 and 3107, respectively), their glycerol binding sites are very similar (this study) (15). This suggests that secondary sites might be involved in the whole polysaccharide recognition or that the different decorations of the first phosphoglycerol moiety might both modulate the strain recognition and increase the affinity for TP901-1 RBP.…”

“…We have shown that this last domain harbors the putative saccharide-binding site, which can be blocked by a llama immunoglobulin VH domain of camelid antibody heavy chain (9,16,17). We recently showed that the binding of a glycerol molecule to the head domain led to the identification of the residues of the saccharide-binding site (15).…”

A Common Evolutionary Origin for Tailed-Bacteriophage Functional Modules and Bacterial Machineries

“…All dsDNA phage and prophage genomes seem therefore to be mosaic, with access by horizontal transfer to a large pool of genes, and the frequency of these events depends on the number of steps of genetic exchange required. The currently accumulated data validate this hypothesis, which is further illustrated by several cases such as (i) the conservation of a similar folding pattern in the head domain of lactococcal phage RBPs as well as in fiber proteins from reoviruses and adenoviruses, despite the absence of detectable sequence identity at the amino acid level (73,78,79,84); (ii) the production of a chimeric RBP between the lactococcal phages TP901-1 and p2, in which no major structural changes occurred to accommodate the domain grafting (77); (iii) the observation of similar overall folds and organizations in the tailspikes of bacteriophages P22 (infecting Salmonella), HK620 (infecting Escherichia coli H), and Sf6 (infecting Shigella) that exhibit a high sequence conservation in the N-terminal virion-binding domain and no sequence identity in the receptor-binding domain (6,51,62,80,81); (iv) evidence of repeated tail fiber horizontal gene transfer among unrelated phages belonging to both the Siphoviridae and Myoviridae families (30); and (v) the widespread occurrence of three distinct Ig-like domain types (I-set, FN3, and BIG-2) in the three main Caudovirales families among phages infecting both Gram-positive and Gram-negative hosts (28).…”

“…In addition to the horizontal transfer of genetic material, phage adaptation is also likely to occur by point mutations in key genes (for example, those encoding tail fibers) in order to guarantee the recognition of hosts throughout their own evolution (84). Besides, protein processing from larger precursors is a strategy used by phages to determine phage-specific properties such as host range (54).…”

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.…”