T4 Infection Research Articles

A four-dimensional structure of T4 infection

Biochemical and genetic data defining the assembly pathway and structural biology of the T4 tail apparatus are merging to create a four-dimensional image reconstruction. Human inventions seem to be large-scale replicas of molecular devices honed by evolution.

Adenylate Kinase of Escherichia coli, a Component of the Phage T4 dNTP Synthetase Complex

Adenylate kinase, which catalyzes the reversible ATP-dependent phosphorylation of AMP to ADP and dAMP to dADP, can also catalyze the conversion of nucleoside diphosphates to the corresponding triphosphates. Lu and Inouye (Lu, Q., and Inouye, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5720-5725) showed that an Escherichia coli ndk mutant, lacking nucleoside diphosphate kinase, can use adenylate kinase as an alternative source of nucleoside triphosphates. Bacteriophage T4 can reproduce in an Escherichia coli ndk mutant, implying that adenylate kinase can meet a demand for deoxyribonucleoside triphosphates that increases by up to 10-fold as a result of T4 infection. In terms of kinetic linkage and specific protein-protein associations, NDP kinase is an integral component of T4 dNTP synthetase, a multienzyme complex containing phage-coded enzymes, which facilitates the synthesis of dNTPs and their flow into DNA. Here we asked whether, by similar criteria, adenylate kinase of the host cell is also a specific component of the complex. Experiments involving protein affinity chromatography, immunoprecipitation, optical biosensor measurements, and glutathione S-transferase pulldowns demonstrated direct interactions between adenylate kinase and several phage-coded enzymes, as well as E. coli nucleoside diphosphate kinase. These results identify adenylate kinase as a specific component of the complex. The rate of DNA synthesis after infection of an ndk mutant was found to be about 40% of the rate seen in wild-type infection, implying that complementation of the missing NDP kinase function by adenylate kinase is fairly efficient, but that adenylate kinase becomes rate-limiting for DNA synthesis when it is the sole source of dNTPs.

The iron–siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11–Pro16 β-hairpin region in the recognition mechanism

The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA(-) strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a K(d) of 1.2 microM. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand-receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val11-Pro16 beta-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.

Phage-induced change in the stability of mRNAs

The stability of mRNA in Escherichia coli cells changed after phage T4 infection. Stable E. coli mRNAs such as lpp and ompA were drastically destabilized immediately after infection. In contrast, T4 phage soc mRNA that had been unstable before infection became stabilized after infection. The host RNases E and G both contributed to the destabilization of these mRNAs. Accordingly, these RNases may alter their target RNAs before and after infection. An RNA chaperon, Hfq, and polyadenylation at 3′ ends of mRNA are known key factors for destabilization of ompA and lpp mRNAs in uninfected cells. However, they had no effect on the destabilization of E. coli mRNAs after infection. On the other hand, T4 infection in the presence of rifampicin or infection of a deletion mutant, Δ tk2, did not destabilize the host mRNAs. These results strongly suggest that a phage-encoded factor is responsible for the destabilization of host mRNAs. Destabilization of host mRNAs was also observed after infection by phages T2 and T7.

Multiple Roles of T7 RNA Polymerase and T7 Lysozyme During Bacteriophage T7 Infection

T7 RNA polymerase selectively transcribes T7 genes during infection but is also involved in DNA replication, maturation and packaging. T7 lysozyme is an amidase that cuts a bond in the peptidoglycan layer of the cell wall, but it also binds T7 RNA polymerase and inhibits transcription, and it stimulates replication and packaging of T7 DNA. To better understand the roles of these two proteins during T7 infection, mutants of each were constructed or selected and their biochemical and physiological behavior analyzed. The amidase activity of lysozyme is needed for abrupt lysis and release of phage particles but appears to have no role in replication and packaging. The interaction between polymerase and lysozyme stimulates both replication and packaging. Polymerase mutants that gain the ability to grow normally in the absence of an interaction with lysozyme still fail to shut down late transcription and, remarkably, have become hypersensitive to inhibition when lysozyme is able to bind. These lysozyme-hypersensitive polymerases behave without lysozyme similarly to wild-type polymerase with lysozyme: both remain longer at the promoter before establishing a lysozyme-resistant elongation complex and both increase the length of pausing when elongation complexes encounter an eight-base recognition sequence involved in DNA packaging. Replication origins contain T7 promoters, but the role of T7 RNA polymerase in initiating replication is not understood well enough to more than speculate how the lysozyme–polymerase interaction stimulates replication. Maturation and packaging is apparently initiated through interaction between prohead-terminase complexes and transcription elongation complexes paused at the sequence TATCTGT(T/A), well conserved at the right-end of the concatemer junction of T7-like phages. A model that is consistent with the structure of an elongation complex and a large body of mutational and biochemical data is proposed to explain sequence-specific pausing and potential termination at the consensus recognition sequence (C/T)ATCTGT(T/A).

Blocking the T4 lysis inhibition phenotype

Nonlysogenic Escherichia coli K cells exhibit a delay in lysis when infected by T4 rII phage termed lysis inhibition (LIN). E. coli K cells expressing λ rexB from either a prophage defective for rexA, or a multicopy plasmid supported T4 rII infection, but prevented the establishment of LIN. In addition, E. coli null mutations in either the periplasmic “tail-specific protease” tsp, or the 10Sa RNA ssrA, completely blocked the establishment of LIN following T4 infections. The expression of rexB in the absence of rexA resulted in several cellular phenotypes, including aberrant cell surface morphology, the partial to near complete suppression of mutations of λ S and T4 t holin genes, and lysis by cells aging on plates or growing with high rexB expression at elevated temperatures. These activities of RexB were impeded in the presence of RexA.

Bacteriophage T4-encoded Stp can be replaced as activator of anticodon nuclease by a normal host cell metabolite.

The bacterial tRNALys-specific anticodon nuclease is known as a phage T4 exclusion system. In the uninfected host cell anticodon nuclease is kept latent due to the association of its core protein PrrC with the DNA restriction-modification endonuclease EcoprrI. Stp, the T4-encoded peptide inhibitor of EcoprrI activates the latent enzyme. Previous in vitro work indicated that the activation by Stp is sensitive to DNase and requires added nucleotides. Biochemical and mutational data reported here suggest that Stp activates the latent holoenzyme when its EcoprrI component is tethered to a cognate DNA substrate. Moreover, the activation is driven by GTP hydrolysis, possibly mediated by the NTPase domain of PrrC. The data also reveal that Stp can be replaced as the activator of latent anticodon nuclease by certain pyrimidine nucleotides, the most potent of which is dTTP. The activation by dTTP likewise requires an EcoprrI DNA substrate and GTP hydrolysis but involves a different form of the latent holoenzyme/DNA complex. Moreover, whereas Stp relays its activating effect through EcoprrI, dTTP targets PrrC. The activation of the latent enzyme by a normal cell constituent hints that anticodon nuclease plays additional roles, other than warding off phage T4 infection.

Three-dimensional structure of bacteriophage T4 baseplate.

The baseplate of bacteriophage T4 is a multiprotein molecular machine that controls host cell recognition, attachment, tail sheath contraction and viral DNA ejection. We report here the three-dimensional structure of the baseplate-tail tube complex determined to a resolution of 12 A by cryoelectron microscopy. The baseplate has a six-fold symmetric, dome-like structure approximately 520 A in diameter and approximately 270 A long, assembled around a central hub. A 940 A-long and 96 A-diameter tail tube, coaxial with the hub, is connected to the top of the baseplate. At the center of the dome is a needle-like structure that was previously identified as a cell puncturing device. We have identified the locations of six proteins with known atomic structures, and established the position and shape of several other baseplate proteins. The baseplate structure suggests a mechanism of baseplate triggering and structural transition during the initial stages of T4 infection.

Architecture of the Replication Complex and DNA Loops at the Fork Generated by the Bacteriophage T4 Proteins

Rolling circle replication has previously been reconstituted in vitro using M13 duplex circles containing preformed forks and the 10 purified T4 bacteriophage replication proteins. Leading and lagging strand synthesis in these reactions is coupled and the size of the Okazaki fragments produced is typical of those generated in T4 infections. In this study the structure of the DNAs and DNA-protein complexes engaged in these in vitro reactions has been examined by electron microscopy. Following deproteinization, circular duplex templates with linear tails as great as 100 kb are observed. The tails are fully duplex except for one to three single-stranded DNA segments close to the fork. This pattern reflects Okazaki fragments stopped at different stages in their synthesis. Examination of the DNA-protein complexes in these reactions reveals M13 duplex circles in which 64% contain a single large protein mass (replication complex) and a linear duplex tail. In 56% of the replicating molecules with a tail there is at least one fully duplex loop at the replication complex resulting from the portion of the lagging strand engaged in Okazaki fragment synthesis folding back to the replisome. The single-stranded DNA segments at the fork bound by gene 32 and 59 proteins are not extended but rather appear organized into highly compact structures ("bobbins"). These bobbins constitute a major portion of the mass of the full replication complex.

バクテリオファージT4のテイルリゾチーム(gp5)の切断部位近傍の構造と切断の特異性の解析

バクテリオファージT4のテイルリゾチーム(gp5)の切断部位近傍の構造と切断の特異性の解析

Microarray Analysis of Gene Expression during Bacteriophage T4 Infection

Genomic microarrays were used to examine the complex temporal program of gene expression exhibited by bacteriophage T4 during the course of development. The microarray data confirm the existence of distinct early, middle, and late transcriptional classes during the bacteriophage replicative cycle. This approach allows assignment of previously uncharacterized genes to specific temporal classes. The genomic expression data verify many promoter assignments and predict the existence of previously unidentified promoters.

A Bipartite Bacteriophage T4 SOC and HOC Randomized Peptide Display Library: Detection and Analysis of Phage T4 Terminase (gp17) and Late σ Factor (gp55) Interaction

HOC and SOC are dispensable T4 capsid proteins that can be used for phage display of multiple copies of peptides and proteins. A bipartite phage T4 peptide library was created by displaying on tetra-alanine linker peptides five randomized amino acids from the carboxyl-terminus of SOC and five randomized amino acids from the amino terminus of HOC. The bipartite library was biopanned against the phage T4 terminase large subunit gp17 to identify T4 gene products that may interact with the terminase. The sequences of selected phages displayed matches to those T4 gene products previously known by genetic and biochemical criteria to interact with gp17: gp20 (portal protein), gp32 (single-stranded DNA binding protein), gp16 (terminase small subunit), and gp17 (self). In addition, matches were found to gp55 (T4 late σ factor), gp45 (sliding clamp), gp44 (clamp loader), gp2 (DNA end protein), and gp23 (major capsid protein). Abundant amino acid sequence matches were found to aa region 118–134 of gp55. Immunoprecipitation and affinity column chromatography demonstrated direct binding of gp17 and gp55; moreover, gp17 bound specifically to a column-coupled peptide corresponding to gp55 residues 111–136. Measurements of gene 17 and other mRNA levels in mutant-infected bacteria did not support a role of gp17–gp55 interaction in regulation of terminase or other late gene transcription. However, whereas DNA concatemers that accumulate in prohead and terminase defective phage T4 infections could be packaged in vitro to ∼10% wild-type efficiency, 55am33am defective concatemeric DNA was packaged at least 100-fold less efficiently. Moreover, gp55 residues 111–136 peptide specifically blocked DNA packaging in vitro. These results suggest that the T4 terminase interaction with T4 late σ factor gp55 plays a role in DNA packaging in vivo. The gp55 interaction may function to load the terminase onto DNA for packaging.

Bacteriophage T4 multiplication in a glucose-limited Escherichia coli biofilm

An Escherichia coli K-12 biofilm was grown at a dilution rate of 0.028 h-1 for 48 h in a glucose-limited chemostat coupled to a modified Robbins' device to determine its susceptibility to infection by bacteriophage T4. Bacteriophage T4 at a multiplicity of infection (MOI) of 10 caused a log reduction in biofilm density (expressed as colony forming units (CFU) per cm2) at 90 min postinfection. After 6 h, a net decrease and equilibrium in viral titer was seen. When biofilms were exposed to T4 phage at a MOI of 100, viral titer doubled after 90 min. After 6 h, viral titers (expressed as plaque forming units (PFU) per cm2) stabilized at levels approximately one order of magnitude higher than seen at a MOI of 10. Scanning confocal laser microscopy images also indicated disruption of biofilm morphology following T4 infection with the effects being more pronounced at a MOI of 100 than at a MOI of 10. These results imply that biofilms under carbon limitation can act as natural reservoirs for bacteriophage and that bacteriophage can have some influence on biofilm morphology.Key words: bacteriophage T4, biofilm, biofilm morphology, bacteriophage ecology, carbon limitation.

Two types of recombination hotspots in bacteriophage T4: one requires DNA damage and a replication origin and the other does not.

Recombination hotspots have previously been discovered in bacteriophage T4 by two different approaches, marker rescue recombination from heavily damaged phage genomes and recombination during co-infection by two undamaged phage genomes. The phage replication origin ori(34) is located in a region that has a hotspot in both assays. To determine the relationship between the origin and the two kinds of hotspots, we generated phage carrying point mutations that should inactivate ori(34) but not affect the gene 34 reading frame (within which ori(34) is located). The mutations eliminated the function of the origin, as judged by both autonomous replication of plasmids during T4 infection and two-dimensional gel analysis of phage genomic replication intermediates. As expected from past studies, the ori(34) mutations also eliminated the hotspot for marker rescue recombination from UV-irradiated genomes. However, the origin mutations had no effect on the recombination hotspot that is observed with co-infecting undamaged phage genomes, demonstrating that some DNA sequence other than the origin is responsible for inflated recombination between undamaged genomes. The hotspots for marker rescue recombination may result from a replication fork restart process that acts upon origin-initiated replication forks that become blocked at nearby DNA damage. The two-dimensional gel analysis also revealed phage T4 replication intermediates not previously detected by this method, including origin theta forms.

Bacteriophage T4 multiplication in a glucose-limited <i>Escherichia coli</i> biofilm

An Escherichia coli K-12 biofilm was grown at a dilution rate of 0.028 h(-1) for 48 h in a glucose-limited chemostat coupled to a modified Robbins' device to determine its susceptibility to infection by bacteriophage T4. Bacteriophage T4 at a multiplicity of infection (MOI) of 10 caused a log reduction in biofilm density (expressed as colony forming units (CFU) per cm2) at 90 min postinfection. After 6 h, a net decrease and equilibrium in viral titer was seen. When biofilms were exposed to T4 phage at a MOI of 100, viral titer doubled after 90 min. After 6 h, viral titers (expressed as plaque forming units (PFU) per cm2) stabilized at levels approximately one order of magnitude higher than seen at a MOI of 10. Scanning confocal laser microscopy images also indicated disruption of biofilm morphology following T4 infection with the effects being more pronounced at a MOI of 100 than at a MOI of 10. These results imply that biofilms under carbon limitation can act as natural reservoirs for bacteriophage and that bacteriophage can have some influence on biofilm morphology.

Interaction of the Bacteriophage T4 Gene 59 Helicase Loading Protein and Gene 41 Helicase with Each Other and with Fork, Flap, and Cruciform DNA

Interaction of the Bacteriophage T4 Gene 59 Helicase Loading Protein and Gene 41 Helicase with Each Other and with Fork, Flap, and Cruciform DNA

Double-strand break repair in tandem repeats during bacteriophage T4 infection.

Recombinational repair of double-strand breaks in tandemly repeated sequences often results in the loss of one or more copies of the repeat. The single-strand annealing (SSA) model for repair has been proposed to account for this nonconservative recombination. In this study we present a plasmid-based physical assay that measures SSA during bacteriophage T4 infection and apply this assay to the genetic analysis of break repair. SSA occurs readily in broken plasmid DNA and is independent of the strand exchange protein UvsX and its accessory factor UvsY. We use the unique features of T4 DNA metabolism to examine the link between SSA repair and DNA replication and demonstrate directly that the DNA polymerase and the major replicative helicase of the phage are not required for SSA repair. We also show that the Escherichia coli RecBCD enzyme can mediate the degradation of broken DNA during early, but not late, times of infection. Finally, we consider the status of broken ends during the course of the infection and propose a model for SSA during T4 infections.

The Major Head Protein of Bacteriophage T4 Binds Specifically to Elongation Factor Tu

The Lit protease in Escherichia coli K-12 strains induces cell death in response to bacteriophage T4 infection by cleaving translation elongation factor (EF) Tu and shutting down translation. Suicide of the cell is timed to the appearance late in the maturation of the phage of a short peptide sequence in the major head protein, the Gol peptide, which activates proteolysis. In the present work we demonstrate that the Gol peptide binds specifically to domains II and III of EF-Tu, creating the unique substrate for the Lit protease, which then cleaves domain I, the guanine nucleotide binding domain. The conformation of EF-Tu is important for binding and Lit cleavage, because both are sensitive to the identity of the bound nucleotide, with GDP being preferred over GTP. We propose that association of the T4 coat protein with EF-Tu plays a role in phage head assembly but that this association marks infected cells for suicide when Lit is present. Based on these data and recent observations on human immunodeficiency virus type 1 maturation, we speculate that associations between host translation factors and coat proteins may be integral to viral assembly in both prokaryotes and eukaryotes.

Endoribonuclease RegB from bacteriophage T4 is necessary for the degradation of early but not middle or late mRNAs

The RegB endoribonuclease from bacteriophage T4 cleaves early mRNAs specifically in the middle of the sequence GGAG. We show here that RegB is required for the degradation of bulk T4 early mRNA. In the absence of RegB, the chemical half-life of early transcripts is increased nearly fourfold, whereas their functional half-life is increased twofold. RegB also regulates the translation of several prereplicative genes. The synthesis of several early proteins is down-regulated, probably as a consequence of RegB cleavages in the Shine-Dalgarno sequence of these genes. The synthesis of several other proteins is up-regulated, suggesting that processing by RegB might improve translation by changing the conformation of a transcript. In contrast, RegB does not affect the average half-life of middle and late mRNA. An analysis of the susceptibility to RegB of many GGAG motifs carried by these mRNA species showed that most middle and all late GGAG-carrying mRNAs escape RegB processing in spite of the fact that the enzyme is acting at least until ten minutes post-infection. The sensitivity or resistance to RegB observed during phage infection could be reproduced in uninfected Escherichia coli cells and in vitro. This shows that the GGAG-carrying RNAs that are uncut during T4 infection are not substrates, whatever the period of the T4 cycle when the transcripts are made.

Bacteriophage T4 gene 59 helicase assembly protein binds replication fork DNA. The 1.45 Å resolution crystal structure reveals a novel α-helical two-domain fold

The bacteriophage T4 gene 59 helicase assembly protein is required for recombination-dependent DNA replication, which is the predominant mode of DNA replication in the late stage of T4 infection. T4 gene 59 helicase assembly protein accelerates the loading of the T4 gene 41 helicase during DNA synthesis by the T4 replication system in vitro. T4 gene 59 helicase assembly protein binds to both T4 gene 41 helicase and T4 gene 32 single-stranded DNA binding protein, and to single and double-stranded DNA. We show here that T4 gene 59 helicase assembly protein binds most tightly to fork DNA substrates, with either single or almost entirely double-stranded arms. Our studies suggest that the helicase assembly protein is responsible for loading T4 gene 41 helicase specifically at replication forks, and that its binding sites for each arm must hold more than six, but not more than 12 nucleotides. The 1.45 Å resolution crystal structure of the full-length 217-residue monomeric T4 gene 59 helicase assembly protein reveals a novel α-helical bundle fold with two domains of similar size. Surface residues are predominantly basic (pI 9.37) with clusters of acidic residues but exposed hydrophobic residues suggest sites for potential contact with DNA and with other protein molecules. The N-terminal domain has structural similarity to the double-stranded DNA binding domain of rat HMG1A. We propose a speculative model of how the T4 gene 59 helicase assembly protein might bind to fork DNA based on the similarity to HMG1, the location of the basic and hydrophobic regions, and the site size of the fork arms needed for tight fork DNA binding. The fork-binding model suggests putative binding sites for the T4 gene 32 single-stranded DNA binding protein and for the hexameric T4 gene 41 helicase assembly.