Identification of the Receptor-Binding Protein in 936-Species Lactococcal Bacteriophages

Phages infecting Lactococcus lactis, a Gram-positive bacterium, are a recurrent problem in the dairy industry. Despite their economical importance, the knowledge on these phages, belonging mostly to Siphoviridae, lags behind that accumulated for members of Myoviridae. The three-dimensional structures of the receptor-binding proteins (RBP) of three lactococcal phages have been determined recently, illustrating their modular assembly and assigning the nature of their bacterial receptor. These RBPs are attached to the baseplate, a large phage organelle, located at the tip of the tail. Tuc2009 baseplate is formed by the products of 6 open read frames, including the RBP. Because phage binding to its receptor induces DNA release, it has been postulated that the baseplate might be the trigger for DNA injection. We embarked on a structural study of the lactococcal phages baseplate, ultimately to gain insight into the triggering mechanism following receptor binding. Structural features of the Tuc2009 baseplate were established using size exclusion chromatography coupled to on-line UVvisible absorbance, light scattering, and refractive index detection (MALS/UV/RI). Combining the results of this approach with literature data led us to propose a "low resolution" model of Tuc2009 baseplate. This model will serve as a knowledge base to submit relevant complexes to crystallization trials.The very first event in viral infection is the attachment of the virion particle to its host. This occurs through the recognition of a host cell wall receptor, which can be a polysaccharide or a protein (or both), by the viral receptor-binding protein (RBP) 3 (1-3). In tailed Siphoviridae bacteriophages (Caudovirales order), which infect Lactococcus lactis and belong to the P335 group (4), the RBP is positioned in an organelle at the tip of the tail, the baseplate (5-7). Baseplates of phages TP901-1 and Tuc2009 have been the subject of thorough investigations (5-7), which have led to a consensus model for baseplate assembly in these two phages. Recently, the x-ray structure of the RBP from phage TP901-1 was determined (8). Comparisons of RBP structures from phages p2 and bIL170, which belong to the so-called 936 group, led us to propose that lactococcal phages RBPs consist of three domains or modules, the shoulders, neck, and head. The modules can be exchanged among phages, which may lead to an alteration in host specificity (9, 10).Phages TP901-1 and Tuc2009 share ϳ78 -97% amino acid identity in the individual protein components that make up their baseplate, which is formed by 5 and 6 proteins, respectively (Table 1): the tape measure protein (TMP), the distal tail protein (Dit), the tail-associated lysozyme (Tal), the upper baseplate protein (BppU), and the lower baseplate protein, which acts as the RBP (BppL). An additional protein is present in the base plate of Tuc2009, which was designated the baseplate-associated protein (BppA). TMPs are generally the longest ORF products observed in bacteriophages. According to secondary structure pred...

Section: Discussionmentioning

confidence: 56%

Section: Discussionmentioning

confidence: 94%

A Topological Model of the Baseplate of Lactococcal Phage Tuc2009

Sciara

Blangy

Siponen

et al. 2008

“…The first steps of phage infection require interactions between the phage receptor-binding proteins (RBPs) 2 (1, 2) and the receptors at the host cell surface. Although some RBPs are located at the tip of fibers (3), others belong to an elongated structure, the tail spike (4,5).…”

mentioning

confidence: 99%

Structure and Molecular Assignment of Lactococcal Phage TP901-1 Baseplate

Bebeacua

Bron

Lai

et al. 2010

P335 lactococcal phages infect the Gram؉ bacterium Lactococcus lactis using a large multiprotein complex located at the distal part of the tail and termed baseplate (BP). The BP harbors the receptor-binding proteins (RBPs), which allow the specific recognition of saccharidic receptors localized on the host cell surface. We report here the electron microscopic structure of the phage TP901-1 wild-type BP as well as those of two mutants bppL ؊ and bppU ؊ , lacking BppL (the RBPs) or both peripheral BP components (BppL and BppU), respectively. We also achieved an electron microscopic reconstruction of a partial BP complex, formed by BppU and BppL. This complex exhibits a tripod shape and is composed of nine BppLs and three BppUs. These structures, combined with lightscattering measurements, led us to propose that the TP901-1 BP harbors six tripods at its periphery, located around the central tube formed by ORF46 (Dit) hexamers, at its proximal end, and a ORF47 (Tal) trimer at its distal extremity. A total of 54 BppLs (18 RBPs) are thus available to mediate host anchoring with a large apparent avidity. TP901-1 BP exhibits an infection-ready conformation and differs strikingly from the lactococcal phage p2 BP, bearing only 6 RBPs, and which needs a conformational change to reach its activated state. The comparison of several Siphoviridae structures uncovers a close organization of their central BP core whereas striking differences occur at the periphery, leading to diverse mechanisms of host recognition.The first steps of phage infection require interactions between the phage receptor-binding proteins (RBPs) 2 (1, 2) and the receptors at the host cell surface. Although some RBPs are located at the tip of fibers (3), others belong to an elongated structure, the tail spike (4, 5). In bacteriophages infecting the Gram ϩ bacterium Lactococcus lactis such as p2 (936 group), TP901-1, and Tuc2009 (P335 group), RBPs are part of a large organelle (1-2 MDa) termed the baseplate (BP). We previously solved RBP structures of phages p2 (6, 7) and TP901-1 (8) as well as the RBP C-terminal domain ("head domain") of phage bIL170 (936 group) (9). It appeared that the RBP of phage TP901-1 (termed BppL, lower baseplate protein) was cleaved during crystallization, and the polypeptidic chain in the crystal structure starts either at residue 16 or at residue 32 (10). Proteolytic cleavage was also observed for the homologous RBP from Tuc2009 (11) as well as in the structure of a chimeric RBP comprising the N-terminal and linker domains of phage TP901-1 RBP fused to the C-terminal domain of phage p2 RBP (12). We demonstrated that individually expressed Tuc2009 BppU (upper baseplate protein) and BppL did not interact when mixed, and we attributed this to the proteolytic cleavage of the BppL N terminus that should normally plug into BppU (13). In contrast, co-expression of BppU and BppL yielded a well defined 3:9 complex (13).The BP architecture of the P335 phages TP901-1 and Tuc2009 has been investigated thoroughly using mutagenesis and immuno...

“…The first steps of phage infection require interactions between the phage receptor-binding proteins (RBPs) 3 (8,9) and the receptors at the host cell surface. These mediating RBPs are located at the distal structure of their long tail (150 -200 nm).…”

mentioning

confidence: 99%

Modular Structure of the Receptor Binding Proteins of Lactococcus lactis Phages

Spinelli

Campanacci

Blangy

et al. 2006

105

127

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 ␤-prism, and a ␤-barrel, the head domain (residues 64 -163), which binds a glycerol molecule. 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 (1). 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 (2-7).The first steps of phage infection require interactions between the phage receptor-binding proteins (RBPs) 3 (8, 9) and the receptors at the host cell surface. These mediating RBPs are located at the distal structure of their long tail (150 -200 nm). Lactococcal phages from species 936 or P335 bind to carbohydrate receptors at the surface of the cell wall, the exact nature of them being still unknown (10 -15). A better understanding at a molecular level of this recognition mechanism would increase the possibility of designing novel tools to inactivate the RBPs, thereby preventing phage infection.In this context, solving the structure of lactococcal phage RBPs is a significant step in understanding the phage-host interactions. To this end, we have previously determined the first crystal structure of a RBP from a lactococcal phage, namely from the lytic phage p2 (936 species) (16). This RBP is formed of three monomers related by a 3-fold noncrystallographic axis, each assembling three domains, from N to C terminus: the shoulders, the interlaced neck, and the heads. 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).The lactococcal temperate phage TP901-1 belongs to the P335 species. It has a genome size of 37,667 bp with 56 open reading frames (ORFs) (18). Phage TP901-1 has a long noncontractile tail with a distal baseplate (19). Recently, one of its ORFs (ORF49 or BppL) has been identified as being the phage RBP (11,20). The mechanism of assembly of the baseplate of phage TP901-1 was also recently deciphered, indicating that BppL forms the lower baseplate a...