3.A.17 The Phage T7 Injectisome (T7 Injectisome) Family
Bacteriophage such as T7 and λ inject their DNA into the E. coli cell using specific membrane proteins. For example, λ uses the LamB maltoporin of the sugar porin (SP) family (TC #1.B.3) and the mannose Enzyme II complex of the PTS mannose (Man) family (TC #4.A.6) to serve as receptor and allow transport across the outer and inner membranes of the Gram-negative bacterial envelope, respectively. The route of entry of T7 phage DNA is not known. However, considerable evidence has suggested that RNA polymerase and the type I restriction enzyme ATP hydrolysis via the HsdR helicase ATP-binding domain of EcoK1 can provide the energy for DNA translocation. EcoK1 specifies (1) DNA methyl transfer activity, endonuclease activity, and ATPase translocation activity. DNA translocation and cleavage are ATP dependent. One subunit of this oligomeric complex, HsdR, contributes to these activities. Mutations in the seven motifs characteristic of the DEAD-box family of proteins that comprise known or putative helicases severely impair the ATPase activity in vitro and DNA translocase activity in vivo. It is believed that these motifs are relevant to the coupling of ATP hydrolysis to DNA translocation. Other type I restriction/modification (R-M) systems can apparently promote translocation of DNA. They probably energize translocation by moving the DNA towards the bound enzyme in an ATP-dependent process.
Key enzymes or protein complexes involved in DNA replication, recombination, repair and transcription may all have the ability to energize DNA translocation. RNA polymerase can perform this function, translocating the entire T7 genome from its capsid into the bacterial cell. Thus, when T7 infects the cell, about 850 bp of the 40 kd genome are ejected from the phage particle into the host. Entry of the remaining DNA is probably coupled to transcription. RNA polymerase recognizes strong promoters in the first DNA segment. As RNA ploymerase transcribes the viral genome, it pulls the downstream DNA into the cell. The first gene to be transcribed, 0.3, the product of which inactivates any resident type I R-M system, protects unmodified DNA from restriction. Another early T7 gene encodes the T7 RNA polymerase which normally completes transfer of the genome from the phage head to the cell cytoplasm, again as a result of transcription. When transcription is blocked, (e.g., using rifampin), the normal translocation process is blocked. However the leading 850 bp contains a target restriction site for EcoKI, and this restriction enzyme can complete transfer of the entire genome. Thus, in the absence of transcription, entry of the T7 genome can be used to assay the translocation activity of EcoKI. EcoKI consists of three subunits: HsdR, HsdM and HsdS (R2M2S). HsdR is required for restriction and translocation. SM2 alone can catalyze modification. Translocation is believed to precede cleavage. It is therefore clear that several different enzyme complexes, all which apparently use nucleotide hydrolysis to energize translocation, can provide the same function of energizing DNA translocation. This is an unusual case of energization promiscuity.
A channel in the phage particle extends through the tail, the head-tail connector and the internal core. Phage proteins probably form an extensible channel across the cell envelope to achieve DNA transport. Two of the phage proteins ejected from the phage head may establish a molecular motor that ratchets the phage genome into the cell. Internal phage core proteins ejected from T7 that may form the channel for DNA transport into the cell include gp14 (21 kDa; 18 molecules per genome), gp15 (84 kDa; 12 molecules) and gp16 (144 kDa; 3 molecules) (Hu et al. 2013). After ejection, gp14 resides in the outer membrane while gp15 and gp16 reside in the inner membrane and cytoplasm. gp16 (1318 aas) possesses transglycosidase activity for peptidoglycan hydrolysis, and this activity is required for efficient infection. It also exhibits hydrophobic regions that might span the cytoplasmic membrane. Both gp15 and gp16 have been implicated in enzymatic translocation of T7 DNA, possibly as an initial molecular motor, but RNA polymerase may complete the job.
T7 DNA entry into the E. coli cell depends both on the pmf and ATP hydrolysis. However, both phage lambda and T5 DNA can enter energy poisoned cells, and they can enter liposomes.
The T7 pore is probably formed from three internal core proteins of the virus, gp14, gp15 and gp16. These proteins are ejected from the virion. They insert into the envelope, probably forming a channel across the cell envelope for uptake of the viral DNA (Molineux, 2001). A speculative model has been proposed suggesting that normally, only part of the phage genome enters the cytoplasm by phage ejection, and that the remainder enters due to transcription-coupled DNA translocation. A 'molecular ratchet' mechanism has been proposed (Molineux, 2001).
gp14 (196 aas) exhibits three peaks of hydrophobicity (residue positions 1-20, 70-90 and 155-175). Only the last of these is predicted to be a TMS using the WHAT program. gp15 (747 aas) has 0 or 1 peak of hydrophobicity and is largely hydrophilic. gp16 (1318 aas) has 5 or 6 putative TMSs, 2 (or possibly 3) at about residue positions 350-420, and three more in the region of positions 1100-1250.
Homologues of gp14, gp15 and gp16 are found in other phage such as Yersinia phage phiA1122 and phiYe03-12, E. coli phage T3, and Pseudomonas phage gh-1.
Many tailed bacteriophages assemble ejection proteins and a portal-tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel extending the portal-tail channel for the delivery of genomic DNA in cell infection. Chen et al. 2021 reported the structure of the mature bacteriophage T7, including the ejection proteins, as well as the structures of the full and empty T7 particles in complex with their cell receptor lipopolysaccharide. The near-atomic-resolution reconstruction showed that the ejection proteins in the mature T7 assemble into a core, which comprises a fourfold gene product 16 (gp16) ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The gp15 and gp16 are mainly composed of helix bundles, and gp16, with two 3 TMS bundles at residues 310 - 420 and 1130 - 1270, respectively, harbors a lytic transglycosylase domain for degrading the bacterial peptidoglycan layer. When interacting with the lipopolysaccharide, the T7 tail nozzle opens. Six copies of gp14 anchor to the tail nozzle, extending the nozzle across the lipopolysaccharide lipid bilayer. The structures of gp15 and gp16 in the mature T7 suggest that they should undergo remarkable conformational changes to form the transenvelope channel. Hydrophobic α-helices were observed in gp16 but not in gp15, suggesting that gp15 forms the channel in the hydrophilic periplasm and gp16 forms the channel in the cytoplasmic membrane (Chen et al. 2021).
The reaction catalyzed by the phage DNA translocase is:
DNA (out) + nucleotide triphosphate(s) (NTP) (in) → DNA (in) + [RNA (in) + P2 (in)] or [NDP (in) + Pi (in)]