Structural basis for host recognition and superinfection exclusion by bacteriophage T5.

A key but poorly understood stage of the bacteriophage life cycle is the binding of phage receptor-binding proteins (RBPs) to receptors on the host cell surface, leading to injection of the phage genome and, for lytic phages, host cell lysis. To prevent secondary infection by the same or a closely related phage and nonproductive phage adsorption to lysed cell fragments, superinfection exclusion (SE) proteins can prevent the binding of RBPs via modulation of the host receptor structure in ways that are also unclear. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the phage T5 outer membrane (OM) receptor FhuA in complex with the T5 RBP pb5, and the crystal structure of FhuA complexed to the OM SE lipoprotein Llp. Pb5 inserts four loops deeply into the extracellular lumen of FhuA and contacts the plug but does not cause any conformational changes in the receptor, supporting the view that DNA translocation does not occur through the lumen of OM channels. The FhuA-Llp structure reveals that Llp is periplasmic and binds to a nonnative conformation of the plug of FhuA, causing the inward folding of two extracellular loops via "reverse" allostery. The inward-folded loops of FhuA overlap with the pb5 binding site, explaining how Llp binding to FhuA abolishes further infection of Escherichia coli by phage T5 and suggesting a mechanism for SE via the jamming of TonB-dependent transporters by small phage lipoproteins.

Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR-Cas effector complexes.

Prokaryotes encode various host defense systems that provide protection against mobile genetic elements. Restriction-modification (R-M) and CRISPR-Cas systems mediate host defense by sequence specific targeting of invasive DNA. T-even bacteriophages employ covalent modifications of nucleobases to avoid binding and therefore cleavage of their DNA by restriction endonucleases. Here, we describe that DNA glucosylation of bacteriophage genomes affects interference of some but not all CRISPR-Cas systems. We show that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR-Cas systems by lowering the affinity of the Cascade and Cas9-crRNA complexes for their target DNA. On the contrary, the type V-A nuclease Cas12a (also known as Cpf1) is not impaired in binding and cleavage of glucosylated target DNA, likely due to a more open structural architecture of the protein. Our results suggest that CRISPR-Cas systems have contributed to the selective pressure on phages to develop more generic solutions to escape sequence specific host defense systems.

Long tail fibres of the novel broad-host-range T-even bacteriophage S16 specifically recognize Salmonella OmpC.

We report isolation and characterization of the novel T4-like Salmonella bacteriophage vB_SenM-S16. S16 features a T-even morphology and a highly modified 160 kbp dsDNA genome with 36.9 mol % G+C, containing 269 putative coding sequences and three tRNA genes. S16 is a virulent phage, and exhibits a maximally broad host range within the genus Salmonella, but does not infect other bacteria. Synthesis of functional S16 full-length long tail fibre (LTF) in Escherichia coli was possible by coexpression of gp37 and gp38. Surface plasmon resonance analysis revealed nanomolar equilibrium affinity of the LTF to its receptor on Salmonella cells. We show that OmpC serves as primary binding ligand, and that S16 adsorption can be transferred to E. coli by substitution of ompC with the Salmonella homologue. S16 also infects 'rough' Salmonella strains which are defective in lipopolysaccharide synthesis and/or its carbohydrate substitution, indicating that this interaction does not require an intact LPS structure. Altogether, its virulent nature, broad host range and apparent lack of host DNA transduction render S16 highly suitable for biocontrol of Salmonella in foods and animal production. The S16 LTF represents a highly specific affinity reagent useful for cell decoration and labelling, as well as bacterial immobilization and separation.

Investigation of the calcium-induced activation of the bacteriophage T5 peptidoglycan hydrolase promoting host cell lysis.

Peptidoglycan hydrolase of bacteriophage T5 (EndoT5) is a Ca2+-dependent l-alanyl-d-glutamate peptidase, although the mode of Ca2+ binding and its physiological significance remain obscure. Site-directed mutagenesis was used to elucidate the role of the polar amino acids of the mobile loop of EndoT5 (111-130) in Ca2+ binding. The mutant proteins were purified to electrophoretic homogeneity, the overall structures were characterized by circular dichroism, and the calcium dissociation constants were determined via NMR spectroscopy. The data suggest that polar amino acids D113, N115, and S117 of EndoT5 are involved in the coordination of calcium ions by forming the core of the EF-like Ca2+-binding loop while the charged residues D122 and E123 of EndoT5 contribute to maintaining the loop net charge density. The results suggest that Ca2+ binding to the EndoT5 molecule could be essential for the stabilization of the long mobile loop in the catalytically active "open" conformation. The possible mechanism of Ca2+ regulation of EndoT5 activity during bacteriophage T5's life cycle through the Ca2+ concentration difference between the cytoplasm and the periplasm of the host bacteria cell has been discussed. The study reveals valuable insight into the role of calcium in the regulation of phage-induced bacterial lysis.

Phage T4 endonuclease SegD that is similar to group I intron endonucleases does not initiate homing of its own gene.

Homing endonucleases are a group of site-specific endonucleases that initiate homing, a nonreciprocal transfer of its own gene into a new allele lacking this gene. This work describes a novel phage T4 endonuclease, SegD, which is homologous to the GIY-YIG family of homing endonucleases. Like other T4 homing endonucleases SegD recognizes an extended, 16bp long, site, cleaves it asymmetrically to form 3'-protruding ends and digests both unmodified DNA and modified T-even phage DNA with similar efficiencies. Surprisingly, we revealed that SegD cleavage site was identical in the genomes of segD- and segD+ phages. We found that segD gene was expressed during the T4 developmental cycle. Nevertheless, endonuclease SegD was not able to initiate homing of its own gene as well as genetic recombination between phages in its site inserted into the rII locus.

A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1.

Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown.

Accurate enzymatic cleavage in vitro of a 2'-deoxyribose-substituted ribonuclease III processing signal.

To assess the involvement of the RNA cleavage site-proximal 2' hydroxyl group in the RNase III catalytic mechanism, a specific processing substrate was chemically synthesized to contain a 2'-deoxyribose residue at the scissile phosphodiester bond. The RNA substrate, corresponding to the phage T7 R1.1 primary processing signal, can be accurately cleaved in vitro by RNase III. A fully deoxyribose-substituted R1.1 processing signal is not cleaved by RNase III, nor does it in excess inhibit cleavage of unmodified substrate. These results show that the 2' hydroxyl group proximal to the scissile bond is not an essential participant in the RNase III processing reaction; however, other 2' hydroxyl groups are important for substrate reactivity, and may be involved in establishing proper double helical conformation, and/or specific substrate contacts with RNase III.

Reaction of T7 DNA with a polycyclic aromatic hydrocarbon. Lack of structural perturbation.

Bacteriophage T7 DNA reacts uniformly with trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene(anti-BPDE). The reaction product retains the native configuration so that only one site sensitive to S1 nuclease is produced for every 70 anti-BPDE adducts. DNA treated with anti-BPDE is retained on benzoylated naphthoylated DEAE-cellulose even after washing with 1.0 M salt solutions. About 100 adducts per T7 molecule are required for adherence which is not due to breaks or single-stranded regions since adherence is not affected by S1 nuclease treatment. The binding of anti-BPDE reacted DNA to benzoylated naphthoylated DEAE-cellulose is cooperative and requires many residues per bound fragment. Treatment of T7 DNA treated with anti-BPDE with restriction endonuclease yields smaller molecules, still containing adducts, which do not adhere. We interpret these results to mean that reaction with BPDE does not involve deformation of the DNA structure and that the adducts lie in a position which they are readily accessible for interaction with aromatic groups on the column resin.

DNA-RNA polymerase complexes associated with the membrane from bacteriophage T2- or T4-infected Escherichia coli. I. General properties.

Properties of DNA-RNA polymerase complexes, apparently bound to a fraction of the cell membrane of bacteriophage T2- or T4-infected Escherichia coli, are described. Evidence is presented to show that the complexes initiate the asymmetric synthesis of RNA, and release the finished product. The transcription capacity per unit of beta' + beta was 10 times higher at 6 min than at 30 min after infection.

DNA-RNA polymerase complexes associated with the membrane from bacteriophage T2- or T4-infected Escherichia coli. II. A comparison of in vitro RNA with the in vivo RNA products.

Membrane preparations, containing DNA-RNA polymerase from bacteriophage T2- or T4-infected Escherichia coli, caused the asymmetric synthesis of RNA classes similar to those produced sequentially in vivo: 1, Pre-early RNA was formed when a membrane preparation was isolated 1 min after infection. 2, Pre-early and delayed early RNA, in roughly the same proportion as that obtained in vivo, was synthesized by a preparation obtained 6 min after infection. 3, Pre-early, delayed early and late genes were transcribed by preparations isolated from cells during the late period of infection. In late preparations, however, less late and more pre-early RNA was usually synthesized in vitro than that obtained in vivo. Late RNA was not synthesized when a phage T4 mutant defective in either gene 33 or gene 55 was used. Evidence is presented to show that late RNA synthesis cannot solely be accounted for from the completion of already initiated chains.

Defective DNA injection by alkylated and nonalkylated bacteriophage T7.

DNA injection by alkylated and nonalkylated bacteriophage T7 has been analyzed by a physical method which involved Southern hybridization to identify noninjected regions of DNA. Treatment of phage with methyl methanesulfonate reduced the amount of DNA injected into wild-type Escherichia coli cells. This reduction was correlated with a decreased injection of DNA segments located on the right-hand third of the T7 genome. An essentially identical injection defect was observed when alkylated phage infected E. coli mutant cells unable to repair 3-methyladenine. Furthermore, untreated phage particles were discovered to be naturally injection-defective. Some injected all their DNA except those segments located in the rightmost 15% of the T7 genome, while other injected no DNA at all. In the presence of rifampicin, untreated phages injected only segments from the left end of the genome. These results provide direct physical evidence that T7 DNA injection is strictly unidirectional, starting from the left end of the T7 genome. The injection defect quantified here for alkylated phage is probably partially, if not totally, responsible for phage inactivation, when that inactivation is measured in wild-type E. coli cells. Since alkylated phage injected the same DNA sequences into both wild-type and repair-deficient cells, we conclude that DNA injection is independent of the host-cell's capacity for repair of 3-methyladenine residues.

The ferrichrome-iron receptor of Escherichia coli K-12. Antigenicity of the fhuA protein.

The ferrichrome-iron receptor in the outer membrane of Escherichia coli K-12 was isolated by preparative SDS-polyacrylamide gel electrophoresis and electroelution of the protein from the gel into solution. This protein, called the fhuA (=tonA) gene product, was biologically active in non-ionic detergent solutions because it was able to inactivate T5 phages. Antibodies were raised against fhuA protein by injecting rabbits with isolated material in polyacrylamide chips. Titers of specific immunoglobulin were confirmed by microenzyme-linked immunosorbent assay. The gamma globulin fraction of anti-fhuA protein completely blocked the adsorption of T5 phage, and partially inhibited ferrichrome-promoted iron uptake.

Host-cell reactivation of alkylated T7 bacteriophage.

Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (AB1157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.

Synthesis of the capsid protein inhibits development of bacteriophage T3 mutants that abortively infect F plasmid-containing cells.

Mutants of bacteriophage T3 that lack gene 1.2 resemble wild-type phage T7 in that they are unable productively to infect F plasmid-containing cells of Escherichia coli. Pseudorevertants of a T3 gene 1.2 deletion mutant that have regained the ability to plate efficiently on male cells have been isolated and characterized. At least two mutations in the gene for the major capsid protein are necessary for these phages to bypass F-mediated restriction. One mutation serves to reduce the rate of synthesis of the capsid protein; a second mutation apparently alters an unknown property that is intrinsic to the free, or unassembled form of the protein. During the abortive infection of an F-containing host, synthesis of the wild-type capsid protein directly inhibits further phage development.

Recombination between bacteriophage T4 and plasmid pBR322 molecules containing cloned T4 DNA.

Reciprocal recombination between T4 DNA cloned in plasmid pBR322 and homologous sequences in bacteriophage T4 genomes leads to integration of complete plasmid molecules into phage genomes. Indirect evidence of this integration comes from two kinds of experiments. Packaging of pBR322 DNA into mature phage particles can be detected by a DNA--DNA hybridization assay only when a T4 restriction fragment is cloned in the plasmid. The density of the pBR322 DNA synthesized after phage infection is also consistent with integration of plasmid vector DNA into vegetative phage genomes. Direct evidence of plasmid integration into phage genomes in the region of DNA homology comes from genetic and biochemical analysis of cytosine-containing DNA isolated from mature phage particles. Agarose gel electrophoresis of restriction endonuclease-digested DNA, followed by Southern blot analysis with nick-translated probes, shows that entire plasmid molecules become integrated into phage genomes in the region of T4 DNA homology. In addition, this analysis shows that genomes containing multiple copies of complete plasmid molecules are also formed. Among phage particles containing at least one integrated copy, the average number of integrated plasmid molecules is almost ten. A cloning experiment done with restricted DNA confirms these conclusions and illustrates a method for walking along the T4 genome.

Genome plasticity in the distal tail fiber locus of the T-even bacteriophage: recombination between conserved motifs swaps adhesin specificity.

The adsorption specificity of the T-even phages is determined by the protein sequence near the tip of the long tail fibers. These adhesin sequences are highly variable in both their sequence and specificity for bacterial receptors. The tail fiber adhesin domains are located in different genes in closely related phages of the T-even type. In phage T4, the adhesin sequence is encoded by the C-terminal domain of the large tail fiber gene (gene 37), but in T2, the adhesin is a separate gene product (gene 38) that binds to the tip of T2 tail fibers. Analysis of phage T6 and Ac3 sequences reveals additional variant forms of this locus. The tail fiber host specificity determinants can be exchanged, although the different loci have only limited homology. Chimeric fibers can be created by crossovers either between small homologies within the structural part of the fiber gene or in conserved motifs of the adhesin domain. For example, the T2 adhesin determinants are flanked by G-rich DNA motifs and exchanges involving these sequences can replace the specificity determinants. These features of the distal tail fiber loci genetically link their different forms and can mediate acquisition of diverse host range determinants, including those that allow it to cross species boundaries and infect taxonomically distant hosts.

Phage and host genetic determinants of the specific anticodon loop cleavages in bacteriophage T4-infected Escherichia coli CTr5X.

Anticodon loop cleavages of two host tRNA species occur in bacteriophage T4-infected Escherichia coli CTr5X, a host strain restricting phage mutants deficient in polynucleotide kinase (pnk) or RNA ligase (rli). The cleavage products accumulate with the mutants but are further processed in wt infection through polynucleotide kinase and RNA ligase reactions. Inactivating mutations in stp suppress pnk- or rli- mutations in E. coli CTr5X and, as shown here, also abolish the anticodon nuclease, implicating the stp product with this activity. We show also that there exist other suppressing mutations of a pnk- (pseT2) mutation that appear not to affect the anticodon nuclease and are not in stp. It has been shown that a single locus in E. coli CTr5X, termed prr, determines the restriction of pnk- or rli- mutants. A transductant carrying prr featured upon infection the anticodon nuclease reaction products, suggesting that prr determines the specific manifestation of this activity. However, prr does not encode the tRNA species that are vulnerable to the anticodon nuclease.

Substitution of a single bacteriophage T3 residue in bacteriophage T7 RNA polymerase at position 748 results in a switch in promoter specificity.

The bacteriophage T3 and T7 RNA polymerases (RNAP) are closely related, yet exhibit high specificity for their own promoter sequences. In this work the primary determinant of T7 versus T3 promoter specificity has been localized to a single amino acid residue at position 748 in the T7 RNAP. Substitution of this residue (Asn) with the corresponding residue found in T3 RNAP (Asp) results in a switch in promoter specificity, and specifically alters recognition of the base pairs (bp) at positions -11 and, possibly, -10 in the promoter. A complementary mutation in T3 RNAP (T3-D749N) results in a similar switch in promoter preference for that enzyme. The hierarchy of bp preference by the mutant and wild-type enzymes for bp at -10 and -11, and the results of previous experiments, lead to a model for specificity in which it is proposed that N748 in T7 RNAP (and D749 in T3 RNAP) make specific hydrogen bonds with bases at -11 and -10 on the non-template strand in the major groove. The specificity determining region of T7 RNAP does not appear to exhibit homology to any known sequence-dependent DNA binding motif.

Self cleavage of a precursor RNA from bacteriophage T4.

We found that a precursor of an RNA molecule from T4-infected Escherichia coli cells (p2Spl; precursor of species 1) has the capacity to cleave itself in a specific position. This cleavage is similar to a cleavage carried out by the aid of a protein, RNase F, that has been previously identified. This cleavage could lead to the maturation of an RNA (species 1) found in T4-infected E. coli cells. The reaction is time and temperature-dependent and is relatively slow as compared to the protein-dependent reaction. It requires at least a monovalent cation and is aided by non-ionic detergents. In the absence of detergent the cleavage can occur but at a reduced rate. The substrate does not contain hidden nicks and a variety of experiments suggest that it does not contain a protein. Moreover, we found no indication that the cleavage is due to contaminating nucleases in the substrate or in the reagents. The intact secondary and tertiary structures of the molecule are necessary for the cleavage to occur. The finding of a self cleaving RNA molecule has interesting evolutionary implications.

Single mutations in a gene for a tail fiber component of an Escherichia coli phage can cause an extension from a protein to a carbohydrate as a receptor.

The T-even type Escherichia coli phage Ox2 recognizes the outer membrane protein OmpA as a receptor. This recognition is accomplished by the 266 residue protein 38, which is located at the free ends of the virion's long tail fibers. Host-range mutants had been isolated in three consecutive steps: Ox2----Ox2h5----Ox2h10----Ox2h12, with Ox2h12 recognizing the outer membrane protein OmpC efficiently and having lost some affinity for OmpA. Protein 38 consists, in comparison with these proteins of other phages, of two constant and one contiguous array of four hypervariable regions; the alterations leading to Ox2h12 were all found within the latter area. Starting with Ox2h12, further host-range mutants could be isolated on strains resistant to the respective phage: Ox2h12----h12h1----h12h1.1----h12h1.11----h12 h1.111. It was found that Ox2h12h1.1 (and a derivative of Ox2h10, h10h4) probably uses, instead of OmpA or OmpC, yet another outer membrane protein, designated OmpX. Ox2h12h1.11 was obtained on a strain lacking OmpA, -C and -X. This phage could not grow on a mutant of E. coli B, possessing a lipopolysaccharide (LPS) with a defective core oligosaccharide; Ox2h12h1.111 was obtained from this strain. It turned out that the latter two mutants used LPS as a receptor, most likely via its glucose residues. Selection for resistance to them in E. coli B (ompA+, ompC-, ompX-) yielded exclusively LPS mutants, and in another strain, possessing OmpA, C and X, the majority of resistant mutants were of this type. Isolated LPS inactivated the mutant phages very well and was inactive towards Ox2h12. By recombining the genes of mutant phages into the genome of parental phages it could be shown that the phenotypes were associated with gene 38. All mutant alterations (mostly single amino acid substitutions) were found within the hypervariable regions of protein 38. In particular, a substitution leading to Ox2h12h1.11 (Arg170----Ser) had occurred at the same site that led to Ox2h10 (His170----Arg), which binds to OmpC in addition to OmpA. It is concluded that not only can protein 38 gain the ability to switch from a protein to a carbohydrate as a receptor but can do so using the same domain of the polypeptide.