Mutant Phage Research Articles

The Cotransduction of pET System Plasmids by Mutants of T4 and RB43 Bacteriophages

The study of the cotransduction of the plasmid pairs pET-3a-pLysE and pET-3a-pLysS by the mutant phage T4alc7 showed that the antibiotic resistance markers of the plasmids were cotransduced with a high frequency. The analysis of the plasmid DNA of cotransductants and cotransformants showed that the mutant phage T4alc7 can be used for obtaining the monomeric and oligomeric forms of plasmids and for the cotransduction of two-plasmid overproduction systems into E. coli strains. The plaque mutants RB43-03 and RB43-13 derived from bacteriophage RB43 were found to be able to cotransduce the antibiotic resistance markers of pET-3a and pLysE plasmids.

In Vivo Assembly of Phage ϕ29 Replication Protein p1 into Membrane-associated Multimeric Structures

The mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown. In the case of the Bacillus subtilis phage 29, the viral protein p1 enhances the rate of in vivo viral DNA replication. Previous work showed that p1 generates highly ordered structures in vitro. We now show that protein p1, like integral membrane proteins, has an amphiphilic nature. Furthermore, immunoelectron microscopy studies reveal that p1 has a peripheral subcellular location. By combining in vivo chemical cross-linking and cell fractionation techniques, we also demonstrate that p1 assembles in infected cells into multimeric structures that are associated with the bacterial membrane. These structures exist both during viral DNA replication and when 29 DNA synthesis is blocked due to the lack of viral replisome components. In addition, protein p1 encoded by plasmid generates membrane-associated multimers and supports DNA replication of a p1-lacking mutant phage, suggesting that the pre-assembled structures are functional. We propose that a phage structure assembled on the cell membrane provides a specific site for 29 DNA replication.

A Consensus Tetrapeptide Selected by Phage Display Adopts the Conformation of a Dominant Discontinuous Epitope of a Monoclonal Anti-VWF Antibody That Inhibits the von Willebrand Factor-Collagen Interaction

Monoclonal antibody (mAb) 82D6A3 is an anti-von Willebrand factor (VWF) mAb directed against the A3-domain of VWF that inhibits the VWF binding to fibrillar collagens type I and III in vitro and in vivo. To identify the discontinuous epitope of this mAb, we used phage display, mutant analysis, and peptide modeling. All 82D6A3-binding phages displayed peptides containing the consensus sequence SPWR that could be aligned with P981W982 in the VWF A3-domain. Next, the binding of mAb 82D6A3 to 27 Ala mutants with mutations in the A3-domain of VWF revealed that amino acids Arg963, Pro981, Asp1009, Arg1016, Ser1020, Met1022, and His1023 are part of the epitope of mAb 82D6A3. Inspection of residues Ser1020, Arg1016, Pro981, and Trp982 in the three-dimensional structure of the A3-domain demonstrated that these residues are close together in space, pointing out that the structure of the SPWR consensus sequence might mimic this discontinuous epitope. Modeling of a cyclic 6-mer peptide containing the consensus sequence and superposition of its three-dimensional structure onto the VWF A3-domain demonstrated that the Ser and Arg in the peptide matched the Ser1020 and Arg1016 in the A3-domain. The Pro residue of the peptide served as a spacer, and the side chain of the Trp pointed in the direction of Trp982. In conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.

Sequence Analysis of the Genome of the Temperate Yersinia enterocolitica Phage PY54

The temperate Yersinia phage PY54 belongs to the unusual group of phages that replicate as linear plasmids with covalently closed ends. Besides Escherichia coli phage N15, PY54 is the only member of this group to be identified. We have determined the complete sequence (46,339 bp) of the PY54 genome. Bioinformatic analyses revealed 67 open reading frames (ORFs) with good coding potential located on both DNA strands. The comparison of the deduced PY54 gene products with known proteins encoded by other phages and bacteria along with functional studies have enabled us to assign the possible functions of 25 ORFs. In the left arm of the PY54 genome, we identified a number of ORFs that obviously code for head and tail proteins. Furthermore, this part of the phage genome contains genes probably involved in plasmid partitioning. Regarding the predicted gene functions and gene order, the PY54 and N15 left arms are similar. However, there are only weak DNA homologies and, in contrast to N15, the Yersinia phage harbours only a few ORFs related to genes found in lambdoid phages. The PY54 right arm comprises mainly regulatory genes as well as genes important for plasmid replication, DNA methylation, and host cell lysis. Out of 36 deduced products of the right arm, 13 revealed strongest database homologies to N15 proteins, of which the protelomerase and the Rep protein are exclusively homologous to their N15 counterparts. A number of PY54 genes essential for the lytic or lysogenic cycle were identified by functional analysis and characterization of phage mutants. In order to study transcription during the lytic and lysogenic stage, we analysed 34 PY54 ORFs by reverse transcriptase (RT)-PCR. The phage transcription patterns in lysogenic bacteria and at the late lytic stage of infection are nearly identical. The reasons for this finding are spontaneous release of phages during lysogeny and a high rate of phages that lysogenize their Yersinia host upon infection.

Development of a novel method of lytic phage delivery by use of a bacteriophage P22 site-specific recombination system

Bacteriophage therapy represents a potential alternative to the use of antibiotics to control proliferation of pathogenic bacteria. As an alternative to the strategy where a limited number of doses of large numbers of lytic bacteriophages are administered, a novel method delivery system was developed so that phages are continually released into the culture. Specifically, a non-pathogenic Escherichia coli strain was constructed that was lysogenic for a lytic mutant of bacteriophage λ. This lysogen was shown to be effective at decreasing the number of λ-sensitive E. coli in vitro. Construction of this E. coli strain was accomplished by development of a plasmid-based system utilizing the site-specific recombination machinery of bacteriophage P22 to integrate DNA constructs into the host chromosome. This recombination system is useful for strain construction and other genetic manipulations in both E. coli and Salmonella enterica serovars.

Identification of crucial residues of conformational epitopes on VP2 protein of infectious bursal disease virus by phage display

A new method for identifying epitopes in viral proteins expressed by filamentous phage has been developed. Filamentous phage fUSE 1 containing the variable region of the VP2 gene of infectious bursal disease virus (IBDV) strain 002–73 was constructed. Neutralizing monoclonal antibodies 17–82 and 33–10 raised against VP2 protein were used to bind phage containing the original variable region of VP2. The phage bound to monoclonal antibodies, were removed by protein G Sepharose and the unbound phage (escape mutants) were isolated for sequencing to locate the mutations. The crucial amino acid residues for conformational neutralizing epitopes recognized by the monoclonal antibodies were located in the first main hydrophilic region (amino acids from 210 to 225) and the central region of the variable region of VP2. The amino acid residues on both ends of the variable region of VP2 affected considerably the binding of monoclonal antibodies. This technique might be useful for selecting escape mutants of phage displaying the original antigenic regions of other viruses to define the crucial amino acid residues of their conformational epitopes, especially viruses that cannot be grown in cell cultures.

Analyzing Transcription Antitermination in Lambdoid Phages Encoding Toxin Genes

Publisher Summary This chapter analyzes the transcription of antitermination in lambdoid phages encoding toxin genes. Lambdoid phages carry the genes encoding Shiga toxin (Stx A and B): most commonly, the genes encoding the two subunits of either the stx1 or stx2 genes. The two best-studied stx-carrying phages are Stx1 phage H-19B and Stx2 phage 933W. Four methods of analysis were used to determine whether H-19B has an antitermination system similar to that of λ and assess its specific mechanism of action: DNA sequence determination of regions of the H-19B genome that may play a role in antitermination based on studies of λ N-mediated antitermination; growth of H-19B in E. coli mutants defective for λ antitermination; construction and analysis of H-19B phage mutants; and reporter constructs designed to study antitermination mediated by the H-19B N protein. Analysis of H-19B Q-mediated antitermination involved similar methods as those employed in analysis of N-mediated antitermination: sequence determination of the late regulatory region of H-19B, which includes the Q gene, the Prʹ promoter with its associated qut site, and the t R ʹ terminator, as well as stx and lysis genes; phage mutants; and reporter–terminator constructs to measure factors required for antitermination at downstream terminators.

Coevolution of bacteriophage PP01 and Escherichia coli O157:H7 in continuous culture.

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h(-1) and by 3 orders of magnitude at a lower dilution rate (0.327 h(-1)). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h(-1) and persisted until the end of the experiment (approximately 200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.

Mutations in the N-Terminus of the Major Coat Protein (pVIII, gp8) of Filamentous Bacteriophage Affect Infectivity

Filamentous bacteriophages are nonlytic, male-specific bacteriophages which infect Escherichia coli carrying an F-episome. The molecular mechanism of infection remains elusive, including the role of the major coat protein pVIII. In order to evaluate the contributions of major coat protein pVIII in the process of infection, two phage display libraries were generated. One library consisted of random amino acids at positions 2, 4, 5, 8, 11 and 12 of the N-terminus of major coat protein pVIII. The second library was generated by randomizing these positions as well as position 1. All these residues were previously shown to be exposed at the surface of the virions by being accessible to ligands. The infectivity of randomly selected mutant phages was analyzed. The present results demonstrate that phages modified at these positions can be correctly assembled and secreted into the exoplasm, although the efficiency was slightly lower than that of wild-type phage. Their infectivity varied greatly, and a general structural pattern underlying infectivity did not emerge. However, residual differences were observed between infectious and defective phage; in general, uncharged polar amino acids present at positions 5 and 11 of the N-terminus of pVIII reduced phage infectivity, whereas polar residues at position 8 facilitated infection. The first position of pVIII is remarkably critical for infection; when this alanine was substituted with other residues, most of the phages lost their infectivity. These results shed new light on the true complexity of random peptide pVIII phage display libraries.

Characterization of the two-component abortive phage infection mechanism AbiT from Lactococcus lactis.

During the production of fermented dairy products, virulent bacteriophages infecting Lactococcus lactis can delay or stop the milk acidification process. A solution to this biological problem consists of introducing natural phage barriers into the strains used by the dairy industry. One such hurdle is called abortive infection (Abi) and causes premature cell death with no or little phage progeny. Here, we describe the isolation and characterization of a novel Abi mechanism encoded by plasmid pED1 from L. lactis. The system is composed of two constitutively cotranscribed genes encoding putative proteins of 127 and 213 amino acids, named AbiTi and AbiTii, respectively. Site-directed mutagenesis indicated that a hydrophobic region at the C-terminal extremity of AbiTi is essential to the antiphage phenotype. The AbiT system is effective against phages of the 936 and P335 species (efficiency of plaquing between 10(-5) and 10(-7)) and causes a 20-fold reduction in the efficiency to form centers of infection as well as a 10- to 12-fold reduction in the burst size. Its efficacy could be improved by raising the plasmid copy number, but changing the intrinsic ratio of AbiTi and AbiTii did not greatly affect the antiphage activity. The monitoring of the intracellular phage infection process by DNA replication, gene expression, and electron microscopy as well as the study of phage mutants by genome mapping indicated that AbiT is likely to act at a later stage of the phage lytic cycle.

Amino acid alterations in Gp38 of host range mutants of PP01 and evidence for their infection of an ompC null mutant of Escherichia coli O157:H7.

The previously isolated T-even type coliphage PP01, specifically infective to Escherichia coli O157:H7, uses the outer membrane protein OmpC as a receptor. The characterization of a spontaneous PP01-resistant strain indicated that it had lost ompC due to the deletion of a 14-kbp region upstream of and partially including ompC. Two host range mutants, able to infect an ompC null mutant, were isolated. Sequencing of gene 38, which codes for the receptor recognition protein Gp38, indicated three mutations in one mutant and two in the other. Both mutant proteins had a Gly208Arg, a Gly161Arg or Gly101His replacement, respectively, and the one mutant phage in addition a Trp189Arg replacement. These alterations suggest that the host range was mediated by a more positively charged Gp38.

Amino acid alterations in Gp38 of host range mutants of PP01 and evidence for their infection of an ompC null mutant of Escherichia coli O157:H7

The previously isolated T-even type coliphage PP01, specifically infective to Escherichia coli O157:H7, uses the outer membrane protein OmpC as a receptor. The characterization of a spontaneous PP01-resistant strain indicated that it had lost ompC due to the deletion of a 14-kbp region upstream of and partially including ompC. Two host range mutants, able to infect an ompC null mutant, were isolated. Sequencing of gene 38, which codes for the receptor recognition protein Gp38, indicated three mutations in one mutant and two in the other. Both mutant proteins had a Gly208Arg, a Gly161Arg or Gly101His replacement, respectively, and the one mutant phage in addition a Trp189Arg replacement. These alterations suggest that the host range was mediated by a more positively charged Gp38.

A P22 scaffold protein mutation increases the robustness of head assembly in the presence of excess portal protein.

Bacteriophage with linear, double-stranded DNA genomes package DNA into preassembled protein shells called procapsids. Located at one vertex in the procapsid is a portal complex composed of a ring of 12 subunits of portal protein. The portal complex serves as a docking site for the DNA packaging enzymes, a conduit for the passage of DNA, and a binding site for the phage tail. An excess of the P22 portal protein alters the assembly pathway of the procapsid, giving rise to defective procapsid-like particles and aberrant heads. In the present study, we report the isolation of escape mutant phage that are able to replicate more efficiently than wild-type phage in the presence of excess portal protein. The escape mutations all mapped to the same phage genome segment spanning the portal, scaffold, coat, and open reading frame 69 genes. The mutations present in five of the escape mutants were determined by DNA sequencing. Interestingly, each mutant contained the same mutation in the scaffold gene, which changes the glycine at position 287 to glutamate. This mutation alone conferred an escape phenotype, and the heads assembled by phage harboring only this mutation had reduced levels of portal protein and exhibited increased head assembly fidelity in the presence of excess portal protein. Because this mutation resides in a region of scaffold protein necessary for coat protein binding, these findings suggest that the P22 scaffold protein may define the portal vertices in an indirect manner, possibly by regulating the fidelity of coat protein polymerization.

Using 2-Aminopurine Fluorescence to Measure Incorporation of Incorrect Nucleotides by Wild Type and Mutant Bacteriophage T4 DNA Polymerases

The ability of wild type and mutant T4 DNA polymerases to discriminate in the utilization of the base analog 2-aminopurine (2AP) and the fluorescence of 2AP were used to determine how DNA polymerases distinguish between correct and incorrect nucleotides. Because T4 DNA polymerase incorporates dTMP opposite 2AP under single-turnover conditions, it was possible to compare directly the kinetic parameters for incorporation of dTMP opposite template 2AP to the parameters for incorporation of dTMP opposite template A without the complication of enzyme dissociation. The most significant difference detected was in the K(d) for dTTP, which was 10-fold higher for incorporation of dTMP opposite template 2AP (approximately 367 microm) than for incorporation of dTMP opposite template A (approximately 31 microm). In contrast, the dTMP incorporation rate was reduced only about 2-fold from about 318 s(-1) with template A to about 165 s(-1) for template 2AP. Discrimination is due to the high selectivity in the initial nucleotide-binding step. T4 DNA polymerase binding to DNA with 2AP in the template position induces formation of a nucleotide binding pocket that is preshaped to bind dTTP and to exclude other nucleotides. If nucleotide binding is hindered, initiation of the proofreading pathway acts as an error avoidance mechanism to prevent incorporation of incorrect nucleotides.

Lactococcus lactis AbiD1 abortive infection efficiency is drastically increased by a phage protein

Sensitivity of phage bIL66 to the AbiD1 Lactococcus lactis abortive infection mechanism was previously shown to be determined by the phage middle-time-expressed operon composed of four orfs. Using spontaneous bIL66 mutants resistant to AbiD1, we established that this sensitivity is determined by the orf1 encoded protein. Overproduction of Orf1 in trans in AbiD1 + cells was shown to increase AbiD1 efficiency on both wild-type phage bIL66 and mutants resistant to AbiD1. Such an increase was not observed following overproduction of mutant Orf1. We propose that wild-type, but not a mutant Orf1, activates AbiD1 expression or activity.

Lactococcus lactis AbiD1 abortive infection efficiency is drastically increased by a phage protein.

Sensitivity of phage bIL66 to the AbiD1 Lactococcus lactis abortive infection mechanism was previously shown to be determined by the phage middle-time-expressed operon composed of four orfs. Using spontaneous bIL66 mutants resistant to AbiD1, we established that this sensitivity is determined by the orf1 encoded protein. Overproduction of Orf1 in trans in AbiD1(+) cells was shown to increase AbiD1 efficiency on both wild-type phage bIL66 and mutants resistant to AbiD1. Such an increase was not observed following overproduction of mutant Orf1. We propose that wild-type, but not a mutant Orf1, activates AbiD1 expression or activity.

Single-chain variable fragment antibodies against the neural adhesion molecule CHL1 (close homolog of L1) enhance neurite outgrowth.

The neural cell adhesion molecule CHL1 (close homolog of L1) plays important roles in neurite outgrowth and neuronal survival in vitro. Reproducible and functionally active CHL1 antibodies are critical for a better understanding of the functional properties of CHL1 in vitro and in vivo. We have isolated human single-chain variable fragment (scFv) antibodies against mouse CHL1 from a human synthetic phage display library. To improve the binding activity of such antibodies, a clone (C12) was selected for affinity maturation by combined random mutagenesis of the V(H) gene and site-directed cassette mutagenesis to introduce random mutations in the complementarity determining region 3 (CDR3) of the V(L) gene. From the mutant phage display library, we selected a clone (6C2) that gave the strongest signal as determined by ELISA. The dissociation constant of 6C2 (Kd 2.28 x 10(-8) M) was increased approximately 85-fold compared with the wild-type clone C12 (Kd 1.93 x 10(-6) M). 6C2 detected CHL1 by Western blot analysis in mouse brain homogenates and detected CHL1 in CHL1-transfected cells by immunofluorescence. Furthermore, the wild-type and affinity-matured antibodies promoted neurite outgrowth of hippocampal and cerebellar neurons in vitro. Our results suggest that the affinity-matured CHL1 scFv antibody will serve a range of applications in vitro and in vivo.

Evolution of bacteriophage in continuous culture: a model system to test antiviral gene therapies for the emergence of phage escape mutants.

The emergence of viral escape mutants is usually a highly undesirable phenomenon. This phenomenon is frequently observed in antiviral drug applications for the treatment of viral infections and can undermine long-term therapeutic success. Here, we propose a strategy for evaluating a given antiviral approach in terms of its potential to provoke the appearance of resistant virus mutants. By use of Q beta RNA phage as a model system, the effect of an antiviral gene therapy, i.e., a virus-specific repressor protein expressed by a recombinant Escherichia coli host, was studied over the course of more than 100 generations. In 13 experiments carried out in parallel, 12 phage populations became resistant and 1 became extinct. Sequence analysis revealed that only two distinct phage mutants emerged in the 12 surviving phage populations. For both escape mutants, sequence variations located in the repressor binding site of the viral genomic RNA, which decrease affinity for the repressor protein, conferred resistance to translational repression. The results clearly suggest the feasibility of the proposed strategy for the evaluation of antiviral approaches in terms of their potential to allow resistant mutants to appear. In addition, the strategy proved to be a valuable tool for observing virus-specific molecular targets under the impact of antiviral drugs.

Delineating the Site of Interaction on the pIII Protein of Filamentous Bacteriophage fd with the F-pilus of Escherichia coli

The minor coat protein pIII at one end of the filamentous bacteriophage fd, mediates the infection of Escherichia coli cells displaying an F-pilus. pIII has three domains (D1, D2 and D3), terminating with a short hydrophobic segment at the C-terminal end. Domain D2 binds to the tip of F-pilus, which is followed by retraction of the pilus and penetration of the E. coli cell membrane, the latter involving an interaction between domain D1 and the TolA protein in the membrane. Surface residues on the D2 domain of pIII were replaced systematically with alanine. Mutant virions were screened for D2-pilus interaction in vivo by measuring the release of infectious virions from E. coli F + cells infected with the mutants. A competitive ELISA was developed to measure in vitro the ability of mutant phages to bind to purified pili. This allowed the identification of amino acid residues involved in binding to F and to EDP208 pili. These residues were found to cluster on the outer rim of the 3D structure of the D2 domain, unexpectedly identifying this as the F-pilus binding region on the pIII protein.

Knotting probability of DNA molecules confined in restricted volumes: DNA knotting in phage capsids.

When linear double-stranded DNA is packed inside bacteriophage capsids, it becomes highly compacted. However, the phage is believed to be fully effective only if the DNA is not entangled. Nevertheless, when DNA is extracted from a tailless mutant of the P4 phage, DNA is found to be cyclic and knotted (probability of 0.95). The knot spectrum is very complex, and most of the knots have a large number of crossings. We quantified the frequency and crossing numbers of these knots and concluded that, for the P4 tailless mutant, at least half the knotted molecules are formed while the DNA is still inside the viral capsid rather than during extraction. To analyze the origin of the knots formed inside the capsid, we compared our experimental results to Monte Carlo simulations of random knotting of equilateral polygons in confined volumes. These simulations showed that confinement of closed chains to tightly restricted volumes results in high knotting probabilities and the formation of knots with large crossing numbers. We conclude that the formation of the knots inside the viral capsid is driven mainly by the effects of confinement.