Phage T5 Straight Tail Fiber Is a Multifunctional Protein Acting as a Tape Measure and Carrying Fusogenic and Muralytic Activities

Abstract

We report a bioinformatic and functional characterization of Pb2, a 121-kDa multimeric protein that forms phage T5 straight fiber and is implicated in DNA transfer into the host. Pb2 was predicted to consist of three domains. Region I (residues 1-1030) was mainly organized in coiled coil and shared features of tape measure proteins. Region II (residues 1030 -1076) contained two ␣-helical transmembrane segments. Region III (residues 1135-1148) included a metallopeptidase motif. A truncated version of Pb2 (Pb2-Cterm, residues 964 -1148) was expressed and purified. Pb2-Cterm shared common features with fusogenic membrane polypeptides. It formed oligomeric structures and inserted into liposomes triggering their fusion. Pb2-Cterm caused ␤-galactosidase release from Escherichia coli cells and in vitro peptidoglycan hydrolysis. Based on these multifunctional properties, we propose that binding of phage T5 to its receptor triggers large conformational changes in Pb2. The coiled coil region would serve as a sensor for triggering the opening of the head-tail connector. The C-terminal region would gain access to the host envelope, permitting the local degradation of the peptidoglycan and the formation of the DNA pore by fusion of the two membranes.The mechanism by which the double-stranded DNA of tailed phages is transported through the envelope of Gram-negative bacteria is a complex process for which phages have developed diverse strategies. Transport may depend on transcription (T7) (1), on phage-encoded proteins (T7, T5, phi29) (1-3), and on host membrane potential (T4) (4, 5). The rate of DNA transport may also vary from phage to phage, reaching values as high as 3000 -4000 bp/s (T4) or as low as 800 bp/s (T7) (reviewed in Refs. 6 -8). Renewed attention was recently brought to these processes with studies focusing on the role of the capsid internal pressure as a trigger of DNA ejection (9) and on the structure of tail proteins participating in DNA ejection (10, 11) and with the development of single phage particles studies (12, 13). However, information on the in vivo DNA transport mechanism and on the phage/bacterial partners involved is still scarce.The Syphoviridae coliphage T5 is an interesting example of the complex strategies developed by phages to invade bacteria. It consists of a 90-nm large icosahedral capsid containing a 121,750-bp double-stranded DNA and a 250-nm-long flexible tail. The high resolution structure of the T5 particle was recently solved from cryoelectron microscopy images highlighting two unusual characteristics of this phage: the triangulation number of its capsid (t ϭ 13) and the 3-fold symmetry of its tail tube (14). Several other features make T5 an atypical phage (15). Its recently sequenced genome (GenBank TM accession numbers AY587007, AY692264, and AY543070 and Ref. 16) which is the largest among the T-odd viruses carries singlestranded interruptions at genetically defined positions on one of the DNA strands as well as large terminal redundancies in the form of 10,160-bp direct repeats ...

Insights into Bacteriophage T5 Structure from Analysis of Its Morphogenesis Genes and Protein Components

“…We did not observe any cross-linking or significant gold labeling with anti-pb2 IgG, arguing that pb2 is not accessible to antibodies in T5 particles. These observations challenge the previous assumption that pb2 is the major component of the central tail fiber (17) but rather support the idea that pb2 fills the tail tube with its long coiled-coil TMP domain and has its C-terminal hydrophobic do-main sequestered within the tail tip and not exposed to the external medium (16).…”

“…The tail tube contains in its central core the multifunctional and oligomeric protein pb2, whose long N-terminal coiled-coil domain plays the role of tape measure protein (TMP). In vitro, the C-terminal end of pb2 has the dual role of pore-forming protein and peptidoglycan hydrolase and most likely participates in the formation of the channel through which the phage genome crosses the host envelope (15,16). On the basis of biochemical studies, pb2 was designated the major component of the central fiber (17).…”

Structure, Adsorption to Host, and Infection Mechanism of Virulent Lactococcal Phage p2

“…Phages that attach to host protein receptors display a long tail fiber (26). In Gram-negative bacteria such as E. coli, this is illustrated by phages T5 and lambda binding to porins (54,55). In Gram-positive bacteria, this tail fiber is observed in Bacillus phage SPP1 (26,56) and lactococcal phage c2 (57), both binding to a host membrane protein from the type 7 secretion system, YueB (56) and Pip (58), respectively.…”

“…This large conformational change is probably somehow coupled to a signal to the stopper to open (26), releasing the dsDNA from the phage capsid. The dsDNA would in turn push out the TMP, which is suspected to guide delivery to the cytoplasm (54,63,64). In a majority of phages, the Tal protein harbors at its C terminus endolysin activity to digest the peptidoglycan and form a hole permitting the passage of the MTP and dsDNA at the onset of infection.…”

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

“…Phages, which attach to host's protein often, display a long, straight, element called "tail fiber" (45). In Gram-negative (Gram Ϫ ) bacteria (e.g., Escherichia coli), this is observed with phages binding to porins, such as phage T5 (68,69) or phage . In Gram ϩ bacteria, this tail fiber is observed in phage SPP1 (45,66) and lactococcal phage c2 (70), which bind to extracellular components of the type 7 secretion system (T7SS), called, respectively, YueB (66) and PIP (70).…”