Structure and Molecular Assignment of Lactococcal Phage TP901-1 Baseplate

Abstract

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...

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

“…4C). Our results also indicate that an equivalent protein is present in the highly elaborated TP901-1 baseplate (7,85). Considering that an identical Dit structure is present in phages as distant as SPP1 (bearing a simplified tail tip and interacting with a membrane protein receptor) and the P335 and 936 lactococcal phages (harboring large baseplates and adsorbing only to saccharidic components present in the host cell envelope) as well as the sequence similarity observed for this protein among several Gram-positive-bacterium-infecting phages (85), we postulate that a Dit-like protein with exactly the same architectural motif is found in all members of the Siphoviridae infecting Gram-positive bacteria.…”

Visualizing a Complete Siphoviridae Member by Single-Particle Electron Microscopy: the Structure of Lactococcal Phage TP901-1

“…Refinement was carried out for several rounds over 2°, imposing c12 symmetry for the connector and c6 symmetry for the baseplate. For further analysis and interpretation, however, we used the baseplate that we previously obtained (6). Fragments 2 to 6 (corresponding to the tail) were combined (ϳ4,000 particles) and submitted to helical processing.…”

“…Structural studies of the host adsorption apparatus of the Lactococcus lactis phages p2 and TP901-1 revealed distinct baseplate architectures and diverse strategies used by the two virions to initiate infection (6)(7)(8)(9)(10)(11). The phage p2 baseplate undergoes large conformational changes in the presence of Ca 2ϩ ions to appropriately orient its RBPs and establish multiple interactions with host saccharides at the onset of infection (8).…”

The First Structure of a Mycobacteriophage, the Mycobacterium abscessus subsp. bolletii Phage Araucaria

“…HHpred also predicted that the ORF71 protein shares structural similarity (98% similarity) with a prophage MUSO2 43-kDa tail protein (PDB 3CDD). This protein is structurally similar to phage T4 gp27 (50), to lactococcal phage p2 ORF16 protein (49), to SPP1 gp21 (51), and to TP901-1 Tal (ORF47 protein) (52). According to these predicted similarities, we then fit the phage p2 Dit-Tal complex (ORF15-ORF16) (49), a 1/1 assembly of Dit hexamer and Tal trimer, in the EM density of the Araucaria HAD ( Fig.…”