Phage T5 Straight Tail Fiber Is a Multifunctional Protein Acting as a Tape Measure and Carrying Fusogenic and Muralytic Activities
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 α-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 β-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. 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 α-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 β-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) (1Molineux I.J. Mol. Microbiol. 2001; 40: 1-8Crossref PubMed Scopus (149) Google Scholar), on phage-encoded proteins (T7, T5, phi29) (1Molineux I.J. Mol. Microbiol. 2001; 40: 1-8Crossref PubMed Scopus (149) Google Scholar, 2McCorquodale D.J. Shaw A.R. Shaw P.K. Chinnadurai G. J. Virol. 1977; 22: 480-488Crossref PubMed Google Scholar, 3Gonzalez-Huici V. Salas M. Hermoso J. Mol. Microbiol. 2004; 52: 529-540Crossref PubMed Scopus (79) Google Scholar), and on host membrane potential (T4) (4Labedan B. Goldberg E.B. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4669-4673Crossref PubMed Scopus (61) Google Scholar, 5Goldberg E. Grinius L. Letellier L. Karam J. D Molecular Biology of Bacteriophage T4. American Society of Microbiology, Washington, DC1994: 347-356Google Scholar). 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. 6Letellier L. Boulanger P. De Frutos M. Jacquot J.P. Res. Microbiol. 2003; 154: 283-287Crossref PubMed Scopus (33) Google Scholar, 7Letellier L. Boulanger P. Plancon L. Jacquot P. Santamaria M. Front. Biosci. 2004; 9: 1228-1339Crossref PubMed Scopus (52) Google Scholar, 8Letellier L. Plancon L. Boulanger P. McGrath S Bacteriophage: Genetics and Molecular Biology. Caster Academic Press, Norfolk, UK2007: 209-228Google Scholar). 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 (9Inamdar M.M. Gelbart W.M. Phillips R. Biophys. J. 2006; 91: 411-420Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and on the structure of tail proteins participating in DNA ejection (10Plisson C. White H.E. Auzat I. Zafarani A. Sao-Jose C. Lhuillier S. Tavares P. Orlova E. EMBO J. 2007; 26: 3720-3728Crossref PubMed Scopus (106) Google Scholar, 11Olia A. Casjens S. Cingolani G. Nat. Struct. Biol. 2007; 14: 1221-1226Crossref Scopus (58) Google Scholar) and with the development of single phage particles studies (12Mangenot S. Hochrein M. Radler J. Letellier L. Curr. Biol. 2005; 15: 1-20Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 13Grayson P. Han L. Winther T. Phillips R. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 14652-14657Crossref PubMed Scopus (98) Google Scholar). 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 = 13) and the 3-fold symmetry of its tail tube (14Effantin G. Boulanger P. Neumann E. Letellier L. Conway J. J. Mol. Biol. 2006; 361: 993-1002Crossref PubMed Scopus (94) Google Scholar). Several other features make T5 an atypical phage (15McCorquodale J.D. Warner H.R. Calendar R The Viruses. 1. Plenum Press, New York1988: 439-476Google Scholar). Its recently sequenced genome (GenBank® accession numbers AY587007, AY692264, and AY543070 and Ref. 16Wang J. Jiang Y. Vincent M. Sun Y. Yu H. Bao Q. Kong H. Hu S. Virology. 2005; 332: 45-65Crossref PubMed Scopus (82) Google Scholar) which is the largest among the T-odd viruses carries single-stranded 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 (17Rhoades M. Rhoades E.A. J. Mol. Biol. 1972; 69: 187-200Crossref PubMed Scopus (23) Google Scholar). Another distinctive feature of T5 is the two-step transport of its genome (18Lanni Y.T. Bacteriol. Rev. 1968; 32: 227-242Crossref PubMed Google Scholar). 8% of the DNA (FST 2The abbreviations used are: FST, first step transfer; SST, second step transfer; LDAO, N,N-dimethyldodecylamine-N-oxide; MM, molecular mass; DDM, n-dodecyl-β-d-maltoside; TMP, tape measure protein; BN-PAGE, blue native polyacrylamide gel electrophoresis; LUV, large unilamellar vesicle(s); ONPG, o-nitrophenyl-β-d-galactopyranose; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Pb2-Cterm, truncated version of Pb2; HPLC, high pressure liquid chromatography. 2The abbreviations used are: FST, first step transfer; SST, second step transfer; LDAO, N,N-dimethyldodecylamine-N-oxide; MM, molecular mass; DDM, n-dodecyl-β-d-maltoside; TMP, tape measure protein; BN-PAGE, blue native polyacrylamide gel electrophoresis; LUV, large unilamellar vesicle(s); ONPG, o-nitrophenyl-β-d-galactopyranose; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Pb2-Cterm, truncated version of Pb2; HPLC, high pressure liquid chromatography. DNA) enters first the cytoplasm. A 4-min pause follows during which proteins encoded by this fragment are synthesized. Two of them (A1 and A2) then contribute to the transfer of the rest of the DNA (SST DNA) (2McCorquodale D.J. Shaw A.R. Shaw P.K. Chinnadurai G. J. Virol. 1977; 22: 480-488Crossref PubMed Google Scholar, 19Snyder Jr., C.E. Benzinger R.H. J. Virol. 1981; 40: 248-257Crossref PubMed Google Scholar). Numerous data have been collected on the interactions between the phage and its receptor (20Feucht A. Heinzelmann G. Heller K.J. FEBS Lett. 1989; 255: 435-440Crossref PubMed Scopus (7) Google Scholar, 21Plançon L. Janmot C. le Maire M. Desmadril M. Bonhivers M. Letellier L. Boulanger P. J. Mol. Biol. 2002; 318: 557-569Crossref PubMed Scopus (40) Google Scholar) but only few on the transport mechanism. T5 DNA transfer is initiated by reversible binding of the three L-shaped fibers to the O-antigen of the lipopolysaccharide, followed by irreversible binding of pb5, the receptor-binding protein, located at the distal end of the base plate to the iron-siderophore receptor FhuA (reviewed in Ref. 22Heller K.J. Arch. Microbiol. 1992; 158: 235-248Crossref PubMed Scopus (82) Google Scholar). Then uncharacterized events lead to the opening of the capsid and the release of the DNA, which is transferred base pair after base pair through the host envelope. A transient efflux of cytoplasmic potassium accompanies DNA transport. We proposed that it corresponded to the insertion and transient opening in the cytoplasmic membrane of a pore through which the DNA was transferred (23Boulanger P. Letellier L. J. Biol. Chem. 1992; 267: 3168-3172Abstract Full Text PDF PubMed Google Scholar). 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The with the was used for of Pb2-Cterm in E. coli The was used for as bacteria at in was by from DNA containing the and The was and into the to that was in E. coli The fragment Pb2-Cterm was by and inserted into the The encoded Pb2-Cterm to the by with and DNA and of coli the was to an = in with Pb2-Cterm was then with for to an = and by The was at protein as and at and in the of in of by three through a at and The containing Pb2-Cterm was in of for and The was a with of with and to Pb2-Cterm was at and by on which also for Pb2-Cterm was at and in DDM, The of Pb2-Cterm was of Pb2-Cterm from the from the low of bacteria was The was for at in and at to the The was in two as for cytoplasmic structure was to J. B. PubMed Google Scholar). coil was to Ref. A. M. J. PubMed Scopus Google and the predicted T. S. S. 14: PubMed Scopus Google Scholar). J. D.J. Res. PubMed Scopus Google Scholar). was used to A. V. L. De E. M. 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The was by a of into of Pb2-Cterm to of the in of of the was G. A. J. Struct. Biol. PubMed Scopus Google Scholar). the of of Pb2-Cterm or with the liposomes or of Pb2-Cterm into the liposomes to by with a in at the of a and with to with Pb2-Cterm the contained collected from the for and the protein was by of a The changes of the by the the of of Pb2-Cterm from the of the at in at a of of their by with Pb2-Cterm for with a particle of from E. coli and Pb2-Cterm E. coli cells and to an = in Pb2-Cterm was to of the for at and at β-galactosidase was by of the with an of of was from the at The β-galactosidase was with that of the of an of bacteria by of Pb2-Cterm E. coli cells as cells with of Pb2-Cterm, for at and for of their Pb2-Cterm on was from E. coli cells and as B. PubMed Scopus Google Scholar). of of Pb2-Cterm was with of peptidoglycan in of for at or with Pb2-Cterm then by for at in of potassium and by on a was with a of to in from the at by and of and of to the of and is located the region of the genome of the and tail Pb2 for of the of T5 proteins M. Virology. PubMed Scopus Google Scholar), to an of in a phage particle (15McCorquodale J.D. Warner H.R. Calendar R The Viruses. 1. Plenum Press, New York1988: 439-476Google Scholar). Pb2 is the T5 tail protein that is by during M. J. Mol. Biol. PubMed Scopus Google Scholar). and of Pb2 from T5 led us to conclude that was at the C-terminal in a of after A bioinformatic three in Pb2. Region I (residues 1–1030) was mainly organized in coiled with the for (residues and high coiled coil is of I. Virology. Scholar). studies have that the of phages are located and the tail and base plate Curr. Microbiol. 2003; PubMed Scopus Google Scholar, S. H. R. L. Heller K.J. J. 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However, the brought to the we to a and in to its Pb2 in E. coli led to that in a and to we at and Pb2 with the E. coli envelope, we on the of a truncated version of Pb2 (Pb2-Cterm, one of the predicted coiled coil the and the metallopeptidase in E. coli of Pb2-Cterm a in the caused that the protein was Pb2-Cterm was to the and the cytoplasm. The cytoplasmic formed that by also the protein from the membranes. two-step of Pb2-Cterm from led to a an of with from the with the protein from the cytoplasmic its was that of the membrane Pb2-Cterm as a in from the or from the The of the for to but was the this that formed The at the for the three and was the protein to microscopy this corresponded to a of the of the protein and that of the binding was from the le Maire M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, P. le Maire M. Bonhivers M. S. Desmadril M. Letellier L. PubMed Scopus Google Scholar). The of was that of Pb2-Cterm an of the of the to the the we a to protein of to a of a Pb2-Cterm with its of the at the this that Pb2-Cterm is mainly However, the of the that this with other was by a Pb2-Cterm at positions that the of used between and an that formed R.H. R.H. B. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). was after for corresponded to from the to the microscopy images of Pb2-Cterm was at a of The the largest The to of The of a of of the particles on the The base to The form of Pb2-Cterm by was by an of the particles of them formed large was with an in but their and the resolution a of their particles that to of but their a the data that Pb2-Cterm, from the or the in of from other and in Pb2-Cterm a and first the of Pb2-Cterm to with by the of the protein on a Pb2-Cterm at the of the in the of but with the with we a that is used for of membrane proteins into liposomes G. A. J. Struct. Biol. PubMed Scopus Google Scholar), Pb2-Cterm and an that the protein was into the the of Pb2-Cterm to in an of the by the Its with the Pb2-Cterm and a in which was with the liposomes or the protein was by an the of the from of to of for with Pb2-Cterm at a of is to fusion of the liposomes to their the of particles was these that Pb2-Cterm to fusogenic triggers an of the by Pb2-Cterm was to the at the and the was at The by the liposomes was from the base The of with the protein was of an to Pb2-Cterm in vivo we functional E. coli cells with Pb2-Cterm cytoplasmic as by the of in the a β-galactosidase of the with bacteria. efflux was to DDM, its in large with the with the protein, release of β-galactosidase the efflux of large from the that Pb2-Cterm and the We Pb2-Cterm a peptidoglycan of peptidoglycan by three a a and the of the was peptidoglycan was first with Pb2-Cterm and then with Pb2-Cterm by on the by this that the protein is to at the Pb2-Cterm a on E. coli was by bacteria with the protein as of the bacteria with Pb2-Cterm at a of on the mechanism the of phage T5 straight tail fiber Pb2 from a protein to a The first of this on the of Pb2 in is a of among phage it to a protein from Curr. Microbiol. 2003; PubMed Scopus Google Scholar, H. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Pb2 was an to this However, led us to conclude that Pb2 the The Pb2 on the tail well that of the from Pb2 is among the largest tail a protein, and a large of the Pb2 region is predicted to in coiled this this region of the protein, in an to the 250-nm-long tail tube I. Virology. Scholar, T. C. J. J. V. J. L. S. J. Jr., 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Pb2 common features with phage the number of Pb2 and proteins in the phage particle are E. Grinius L. Letellier L. Karam J. D Molecular Biology of Bacteriophage T4. American Society of Microbiology, Washington, DC1994: 347-356Google to 6Letellier L. Boulanger P. De Frutos M. Jacquot J.P. Res. Microbiol. 2003; 154: 283-287Crossref PubMed Scopus (33) Google Scholar), and are by in C-terminal during phage (reviewed in Ref. Curr. Microbiol. Google Scholar). this that Pb2 forms the straight fiber (24Heller K.J. H. J. Bacteriol. PubMed Google Scholar) and the phage tail. is from that in phage and straight fiber to two and I. Biophys. 26: PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Bacteriol. PubMed Google Scholar). is the T5 tail then We that Pb2, is of the complex for of the tail protein, followed by I. J. Mol. Biol. 91: PubMed Scopus Google Scholar). Pb2 to or other proteins in tail to The of T5 tail from that of a is in tail of double-stranded DNA for its phage this the of two proteins with and that are located from the tail protein and from the Y. 2001; PubMed Scopus Google Scholar). We to in the region of T5 tail T5 also from phages by the of the receptor binding protein, pb5, is located in the tail but the capsid the the T5 of which are is 3-fold (14Effantin G. Boulanger P. Neumann E. Letellier L. Conway J. J. Mol. Biol. 2006; 361: 993-1002Crossref PubMed Scopus (94) Google Scholar). this the of T5 and this phage an interesting for studies have that binding of T5 to its receptor FhuA in the insertion of Pb2 in the host G. Boulanger P. Letellier L. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar) and in liposomes with FhuA J. Letellier L. Curr. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the of Pb2 was the only region predicted to it was to its in membrane in E. coli of a truncated version of Pb2 (Pb2-Cterm, this region that the features of a membrane It was in the as and in membranes. forms by a or and The of of was with the of their le Maire M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and that Pb2-Cterm in to the from in data by microscopy that Pb2-Cterm formed large was with an in or the of the truncated is to that of Pb2 in the phage particle M. Virology. PubMed Scopus Google Scholar). Pb2-Cterm interesting features in It inserted into liposomes and into to its as a membrane Pb2-Cterm also an of the by the liposomes and of their that it a fusogenic fusogenic was at a as low as fusion the region predicted to the which a high of and two that are for triggering fusogenic processes J.P. I. Biophys. Res. 2002; PubMed Scopus Google Scholar). Pb2-Cterm was also in vivo triggering the release of cytoplasmic β-galactosidase and in The of Pb2-Cterm irreversible was Pb2-Cterm in it to which of the is and its is in the host envelope. It that this is a with L. and and Molecular New Scholar). that Pb2-Cterm and the The of the two transmembrane and of receptor Pb2-Cterm inserted into was by degradation of is to the metallopeptidase located at the end of the Pb2-Cterm have been or in phages proteins at the of and S. Scholar, E.A. J. Mol. Biol. 2007; PubMed Scopus Google Scholar, M. I. Mol. Microbiol. 2004; PubMed Scopus Google Scholar). It was that T5 phage contained a protein with M. I. Mol. Microbiol. 2004; PubMed Scopus Google Scholar). However, the of the T5 genome that of its proteins M. Virology. PubMed Scopus Google Scholar) us to this the from a Syphoviridae a that a is also in of phages a large of that a common for the M. Mol. Microbiol. 2006; PubMed Scopus Google Scholar). is among Syphoviridae phages from Gram-negative bacteria an that Pb2 in the phage and in its host. the phage Pb2 would in an with its coiled coil region the tail and its fusogenic C-terminal region the distal of the in the straight of T5 to FhuA its receptor-binding protein, pb5, would changes in Pb2; the coiled coil would serve as a sensor for triggering the opening of the head-tail and the release of the DNA from the capsid and the C-terminal region would gain access to the conformational changes are by images of T5 with liposomes J. Letellier L. Curr. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and by that Pb2 to T5 with FhuA A. A. R. H. Heller K.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). conformational changes would in local degradation of the peptidoglycan and formation of a pore by fusion of the and membranes. would serve two the between the two as to the DNA from and forming the DNA the number of Pb2 in the phage particle E. Grinius L. Letellier L. Karam J. D Molecular Biology of Bacteriophage T4. American Society of Microbiology, Washington, DC1994: 347-356Google to 6Letellier L. Boulanger P. De Frutos M. Jacquot J.P. Res. Microbiol. 2003; 154: 283-287Crossref PubMed Scopus (33) Google Scholar) this pore large for the DNA to Pb2 is into the host envelope or to the phage is vivo Pb2 is with the envelope DNA transfer G. Boulanger P. Letellier L. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). vitro the straight tail fiber the membrane DNA is but to the phage tail J. Letellier L. Curr. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Another is to the two-step mechanism of DNA transport. We that the efflux of cytoplasmic which with DNA after of DNA and after of proteins and transfer of the DNA (23Boulanger P. Letellier L. J. Biol. Chem. 1992; 267: 3168-3172Abstract Full Text PDF PubMed Google Scholar). that the pore would first DNA transfer and then during the of the proteins and during the transfer of the The trigger for pore the of the of Pb2 with the region of the DNA by potential structures and to repeats and M. V. S. J. 52: PubMed Scopus Google Scholar). The of a protein in vitro for of it is to DNA We C. and A. for and P. for protein