Structure, adsorption to host, and infection mechanism of virulent lactococcal phage p2.

Lactococcal siphophages from the 936 and P335 groups infect the Gram-positive bacterium Lactococcus lactis using receptor binding proteins (RBPs) attached to their baseplate, a large multiprotein complex at the distal part of the tail. We have previously reported the crystal and electron microscopy (EM) structures of the baseplates of phages p2 (936 group) and TP901-1 (P335 group) as well as the full EM structure of the TP901-1 virion. Here, we report the complete EM structure of siphophage p2, including its capsid, connector complex, tail, and baseplate. Furthermore, we show that the p2 tail is characterized by the presence of protruding decorations, which are related to adhesins and are likely contributed by the major tail protein C-terminal domains. This feature is reminiscent of the tail of Escherichia coli phage λ and Bacillus subtilis phage SPP1 and might point to a common mechanism for establishing initial interactions with their bacterial hosts. Comparative analyses showed that the architecture of the phage p2 baseplate differs largely from that of lactococcal phage TP901-1. We quantified the interaction of its RBP with the saccharidic receptor and determined that specificity is due to lower k(off) values of the RBP/saccharidic dissociation. Taken together, these results suggest that the infection of L. lactis strains by phage p2 is a multistep process that involves reversible attachment, followed by baseplate activation, specific attachment of the RBPs to the saccharidic receptor, and DNA ejection.

Functions involved in bacteriophage P2-induced host cell lysis and identification of a new tail gene.

Successful completion of the bacteriophage P2 lytic cycle requires phage-induced lysis of its Escherichia coli host, a process that is poorly understood. Genetic analysis of lysis-deficient mutants defined a single locus, gene K, which lies within the largest late transcription unit of P2 and maps between head gene L and tail gene R. We determined and analyzed the DNA sequence of a ca. 2.1-kb EcoRV fragment that spans the entire region from L to R, thus completing the sequence of this operon. This region contains all of the functions necessary for host cell lysis. Sequence analysis revealed five open reading frames, initially designated orf19 through orf23. All of the existing lysis mutants--ts60, am12, am76, and am218--were located in orf21, which must therefore correspond to gene K. The K gene product has extensive amino acid sequence similarity to the product of gene R of bacteriophage lambda, and its exhibits endolysin function. Site-directed mutagenesis and reverse genetics were used to create P2 amber mutants in each of the four other newly identified open reading frames. Both orf19 (gene X) and orf20 (gene Y) encode essential functions, whereas orf22 (lysA) and orf23 (lysB) are nonessential. Gene Y encodes a polypeptide with striking similarities to the family of holin proteins exemplified by gpS of phage lambda, and the Yam mutant displayed the expected properties of a holin mutant. The gene products of lysA and lysB, although nonessential, appear to play a role in the correct timing of lysis, since a lysA amber mutant caused slightly accelerated lysis and a lysB amber mutant slightly delayed lysis of nonpermissive strains. Gene X must encode a tail protein, since lysates from nonpermissive cells infected with the X amber mutant were complemented in vitro by similar lysates of cells infected with P2 head mutants but not with tail mutants.