Modular Structure of the Receptor Binding Proteins of Lactococcus lactis Phages

Abstract

Lactococcus lactis is a Gram-positive bacterium widely used by the dairy industry. Several industrial L. lactis strains are sensitive to various distinct bacteriophages. Most of them belong to the Siphoviridae family and comprise several species, among which the 936 and P335 are prominent. Members of these two phage species recognize their hosts through the interaction of their receptor-binding protein (RBP) with external cell wall saccharidices of the host, the “receptors.” We report here the 1.65 Å resolution crystal structure of the RBP from phage TP901-1, a member of the P335 species. This RBP of 163 amino acids is a homotrimer comprising three domains: a helical N terminus, an interlaced β-prism, and a β-barrel, the head domain (residues 64-163), which binds a glycerol molecule. Fluorescence quenching experiments indicated that the RBP exhibits high affinity for glycerol, muramyl-dipeptide, and other saccharides in solution. The structural comparison of this RBP with that of lactococcal phage p2 RBP, a member of the 936 species (Spinelli, S., Desmyter, A., Verrips, C. T., de Haard, J. W., Moineau, S., and Cambillau, C. (2006) Nat. Struct. Mol. Biol. 13, 85-89) suggests a large extent of modularity in RBPs of lactococcal phages. Lactococcus lactis is a Gram-positive bacterium widely used by the dairy industry. Several industrial L. lactis strains are sensitive to various distinct bacteriophages. Most of them belong to the Siphoviridae family and comprise several species, among which the 936 and P335 are prominent. Members of these two phage species recognize their hosts through the interaction of their receptor-binding protein (RBP) with external cell wall saccharidices of the host, the “receptors.” We report here the 1.65 Å resolution crystal structure of the RBP from phage TP901-1, a member of the P335 species. This RBP of 163 amino acids is a homotrimer comprising three domains: a helical N terminus, an interlaced β-prism, and a β-barrel, the head domain (residues 64-163), which binds a glycerol molecule. Fluorescence quenching experiments indicated that the RBP exhibits high affinity for glycerol, muramyl-dipeptide, and other saccharides in solution. The structural comparison of this RBP with that of lactococcal phage p2 RBP, a member of the 936 species (Spinelli, S., Desmyter, A., Verrips, C. T., de Haard, J. W., Moineau, S., and Cambillau, C. (2006) Nat. Struct. Mol. Biol. 13, 85-89) suggests a large extent of modularity in RBPs of lactococcal phages. Phages of Lactococcus lactis are a major problem in industrial milk fermentation, because they are ubiquitous within their process environments as well as within pasteurized milk (1Moineau S. Tremblay D. Labrie S. ASM News. 2002; 68: 388-393Google Scholar). They belong to several different species of the Siphoviridae family (small isometric capsid and long noncontractile tail), among which the genetically distinct species 936, P335, and c2 are the three prominent (2Moineau S. Fortier J. Ackermann H.-W. Can. J. Microbiol. 1992; 38: 875-882Crossref Scopus (88) Google Scholar, 3Moineau S.M. Borkaev B.J. Holler S.A. Walker J.K. Kondo E.R. Vedamuthu Vandenbergh P.A. J. Dairy Sci. 1996; 79: 2104-2111Abstract Full Text PDF Scopus (95) Google Scholar, 4Bissonnette F. Labrie S. Deveau H. Lamoureux M. Moineau S. J. Dairy Sci. 2000; 83: 620-627Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 5Josephsen J. Nielsen E.W. Milchwissenschaft. 1988; 43: 219-223Google Scholar, 6Josephsen J. Neve H. Salminen S. von Wright A. Lactic Acid Bacteria: Microbiology and Functional Aspects. Marcel Dekker, Inc., New York1998: 385-436Google Scholar, 7Moineau S. Pandian S. Klaenhammer T.R. Appl. Environ. Microbiol. 1993; 59: 197-202Crossref PubMed Google Scholar). The first steps of phage infection require interactions between the phage receptor-binding proteins (RBPs) 3The abbreviations used are: RBP, receptor binding protein; ORF, open reading frame; r.m.s., root mean square; EM, electro