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The Siphoviridae family of bacteriophages is the largest viral family on earth and comprises members infecting both bacteria and archaea. Lactococcal siphophages infect the Gram-positive bacterium
Hundreds of
Phages from the c2 group have been shown to recognize and infect
This review focuses on the structure of lactococcal phages p2, a lytic phage, and TP901-1, a lysogenic phage, belonging to the predominant 936 and P335 groups, respectively. Their complete structures have been tackled using electron microscopy (EM), and the structure of their components involved in adhesion was determined by X-ray crystallography. These structural data, together with functional studies, made it possible to reveal striking features of lactococcal phages concerning their baseplate activation, and the specificity of their receptor binding proteins (RBPs). Since the recent discovery of the
Knowledge on phage structures has to date primarily relied on the structures of Myoviridae or Podoviridae, since the flexible tail of Siphoviridae has prevented the application of single-particle reconstruction in a straightforward manner (
Phages p2 and TP901-1 have similar structural genomic modules resembling that of phage SPP1 (
While no peptidoglycan-digesting enzyme could be identified within the p2 structural cassette, the C-terminal moiety of the TP901-1 Tal has been shown to possess such an activity. Recent data demonstrated that TP901-1 mutated virions with Tal depleted of the peptidoglycan digesting enzyme domain could still infect their host during the
Llama immunization with p2 virions allowed to isolate single-domain llama antibody fragments (named VHH or nanobodies;
With in view to determine the receptor binding site of the p2 RBP, its crystal structure was determined alone and in complex with VHH5 (
The phage TP901-1 RBP trimer structure was determined by X-ray diffraction (
Following the helix bundle domain, a short linker structure (residues 31–39) connects the α-helical domain (17–30) and the β-prism (40–63;
The RBP head domain of TP901-1 (residues 64–163;
The high-resolution structure of the p2 RBP (1.7 Å resolution) revealed the presence of three glycerol molecules (originating from the cryoprotectant liquor) bound at the interface between the head domains (
The structure of the complex between p2 RBP and VHH5 was also determined by X-ray crystallography and revealed that the nanobody covers a large area of the head domain (
Glycerol molecules were also observed bound at the interface between head domains in the crystal structure of the phage TP901-1 RBP at 1.6 Å resolution (
The superposition of the binding network of glycerol in both RBPs shows their striking similarity (
The structures of the TP901-1 RBP in complex with nanobodies (
The affinity of glycerol and phospho-glycerol for the RBPs suggested that lipoteichoic acids (LTA) could act as receptors for lactococcal phages. Conversely, the observation that many sugars bind equally well to the RBPs and the fact that the structure of LTAs is too simple to explain the different specificities of hundreds of lactoccocal phages constituted arguments against this hypothesis. A recent report by
Preliminary experiments using surface plasmon resonance (SPR) explained the specificity mechanism of phage p2 RBP for
Based on their genomic location, we hypothesized that
List of the components of the baseplates from lactococcal phages mentioned in this review.
Protein\phage | Abbreviations | p2 | TP901-1 | Tuc2009 |
---|---|---|---|---|
Tape measure protein | TMP | ORF14 | ORF45 | ORF48 |
Distal tail protein | Dit | ORF15 | ORF46 | ORF49 |
Tail associated lysozyme | Tal | ORF16 | ORF47 | ORF50 |
Baseplate protein (upper) | BppU | n/a | ORF48 | ORF51 |
Baseplate protein A | BppA | n/a | n/a | ORF52 |
Receptor binding protein Baseplate protein (lower) | RBP (BppL) | ORF18 | ORF49 | ORF53 |
ORF15 (Dit) is composed of two domains. The N-terminal domain (“ring domain” 1–132) shows a split barrel-like fold similar to that found in phage Lambda gpV (
ORF16 (Tal) is a 398 residue-long protein harboring four domains (
The structure of ORF18 (RBP) is similar to that of ORF18 crystallized alone, with one exception: the N-terminal residues 2–17 of ORF18 in the baseplate structure are ordered and visible in the electron density. This is due to a tight interaction with the three-digit hand from the ORF15 galectin domain (
The structure of the baseplate reported above exhibited an unexpected conformation. Indeed, one would have expected the head domains of the RBPs (ORF18), which harbor the receptor-binding sites, to point “downward,” i.e., in the direction of the host cell surface. Instead, the RBPs were observed in a “heads-up” conformation, a position not compatible with optimal adhesion. However, the baseplate crystal structure fitted without rearrangement in the baseplate region of the p2 virion reconstruction (
Since it was noticed that in some cases lactococcal phages infection required Ca2+, attempts were made to obtain crystals in the presence of Ca2+ or Sr2+ without VHH5. New crystal forms were obtained readily with both cations, and their structure determined. Both structures were found to be identical, although the Sr2+ complex diffracted to higher resolution (
Following the same strategy employed for phage p2, attempts were made to express the phage TP901-1 baseplate by cloning a segment encompassing the
The Dit forms a hexameric circular-shaped core with a 80 Å diameter, which delineates a 37 Å wide central channel (
The 18 BppU assemble as six asymmetric trimers connecting the Dit central core and the RBPs (
The RBP structure is identical to the structure of its isolated form, with the three domains forming a trimer (
This baseplate structure is most probably shared, more or less closely, by several phages from the P355 group and beyond. EM studies have demonstrated the structural resemblance of the P335 phage Tuc2009 baseplate with that of TP901-1 (
Comparison of the structures of the p2 and TP901-1 baseplates revealed that the latter is already in a “ready to adsorb” conformation without requiring any conformational change. This observation could be correlated with functional data as p2-like phages are non-infectious in the absence of Ca2+ whereas TP901-1-like phages do not require Ca2+ for infection (
The fine specificity of lactococcal phages for their
According to the literature, a large number of phages exhibit a baseplate, beyond those infecting
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported, in part, by grants from the Marseille-Nice Génopole, the CNRS and the Agence Nationale de la Recherche (grants ANR-07-BLAN-0095, “Siphophages” and ANR-11-BSV8-004-01 “Lactophages”).