Structure and Molecular Assignment of Lactococcal Phage TP901-1 Baseplate

Cecilia Bebeacua,Patrick Bron,Livia Lai,Christina Skovgaard Vegge,Lone Brøndsted,Silvia Spinelli,Valérie Campanacci,David Veesler,Marin Van Heel,Christian Cambillau

doi:10.1074/jbc.m110.175646

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 light-scattering 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.

Highlights

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
Proteolytic cleavage was 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 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]

Summary

EXPERIMENTAL PROCEDURES

Cloning and Protein Production—The bppU and bppL (NP_112712.1) genes as well as the whole DNA region containing the bppU and bppL genes with the intervening sequence were amplified from phage genomic DNA and cloned using the Gateway technology (Invitrogen) as described [13, 19]. The best-averaged class images were used as new references for a subsequent alignment cycle. At this stage, a C1 startup procedure was carried out to compute a first three-dimensional model. Heterogeneity was visible and revealed by comparison of class averages and projections of the three-dimensional model. This corresponded to arms opening movement in particles. An initial model was calculated from the aligned class averages, imposing 6-fold symmetry, and re-projected along the equator (IMAGIC Euler angle ␤ equal to 90°) with a difference of 20°.

RESULTS

Structures 3 and 4

DISCUSSION