Structural Characterization and Assembly of the Distal Tail Structure of the Temperate Lactococcal Bacteriophage TP901-1

Abstract

The tail structures of bacteriophages infecting gram-positive bacteria are largely unexplored, although the phage tail mediates the initial interaction with the host cell. The temperate Lactococcus lactis phage TP901-1 of the Siphoviridae family has a long noncontractile tail with a distal baseplate. In the present study, we investigated the distal tail structures and tail assembly of phage TP901-1 by introducing nonsense mutations into the late transcribed genes dit (orf46), tal TP901-1 (orf47), bppU (orf48), bppL (orf49), and orf50. Transmission electron microscopy examination of mutant and wild-type TP901-1 phages showed that the baseplate consisted of two different disks and that a central tail fiber is protruding below the baseplate. Evaluation of the mutant tail morphologies with protein profiles and Western blots revealed that the upper and lower baseplate disks consist of the proteins BppU and BppL, respectively. Likewise, Dit and Tal TP901-1 were shown to be structural tail proteins essential for tail formation, and Tal TP901-1 was furthermore identified as the tail fiber protein by immunogold labeling experiments. Determination of infection efficiencies of the mutant phages showed that the baseplate is fundamental for host infection and the lower disk protein, BppL, is suggested to interact with the host receptor. In contrast, ORF50 was found to be nonessential for tail assembly and host infection. A model for TP901-1 tail assembly, in which the function of eight specific proteins is considered, is presented.

Structure and Molecular Assignment of Lactococcal Phage TP901-1 Baseplate

“…It was demonstrated that the Tal C termini of Tuc2009 (and most likely of TP901-1) has a proteolytic activity, probably used to cleave the peptidoglycan and allow DNA transit into the host cytoplasm (36). Tal also exhibits an autocatalytic activity, and a consensus self-cleav- age site was identified in Tuc2009 (GGSSG2GGD) as well as in TP901-1 (14,36,37). In contrast, the C-terminal domain of SPP1 gp21 appears to be dedicated to receptor recognition (38).…”

“…Native and Mutant Phage Production and PurificationThe preparation of phage mutants has been described previously (14). Native and mutant phages were induced with 3 g/ml mitomycin C from their lysogenic L. lactis 901-1 strains grown at 30°C in GM17 broth.…”

Crystal Structure of Bacteriophage SPP1 Distal Tail Protein (gp19.1)

“…This is the case for the TMP (gp18), Dit (gp19.1), and tail-spike (gp21). However, these two lactococcal phages possess a large and bulky baseplate of 1 to 2 MDa anchored at the end of the tail-tube (3,4,(7)(8)(9), whereas SPP1 displays only an elongated tail-tip (10). Therefore, despite conserved tail architecture among these viruses, the host-adsorption apparatus distinguishes them.…”

“…phage p2 (7)) or for baseplate complexes that attach to the RBPs (e.g. P335 phages (3,4,8,9,51)). In phages lacking a baseplate, like SPP1, the Dit role is limited to connect the tail-tube and tail-tip, this latter component recognizing the YueB receptor (47).…”

“…Their vast majority is Siphoviridae, characterized by the presence of a long non-contractile tail. The assembly pathway of this structure essential for infection was genetically dissected for Escherichia coli phage (1,2) and for the host-adsorption apparatus of phages TP901-1 and Tuc2009 that infect the Gram-positive bacterium Lactococcus lactis (3,4). However, the molecular mechanisms underlying tail assembly remain poorly understood.…”