Use of bioinformatics to predict a function for the GS element in Mycobacterium avium subspecies paratuberculosis

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.     

Effect of DNA copy number on genetic stability of phage-displayed peptides

A small model peptide, the FLAG epitope, was cloned into two filamentous phage display vectors, f88-4 and fd88-4, creating phages f88-FLAG and fd88-FLAG, respectively. Both vectors have a gene VIII display cassette (in addition to their normal phage gene VIII) and display the cloned peptide on a few percent of the virion's 3000-4000 pVIII (major coat protein) subunits. Vector f88-4 has a replication defect and attains low DNA copy number in infected cells, while vector fd88-4 has no replication defect and attains the normal, high DNA copy number characteristic of wild-type filamentous phage. Almost no loss of displayed peptide was observed during six rounds of propagation of low copy number f88-FLAG phage. In contrast, when high copy number fd88-FLAG phage was similarly propagated, variant clones that did not display the FLAG epitope accumulated gradually. The loss of displayed peptide from the high copy number vector is undoubtedly slow enough to be overcome by even weak affinity selection, and high copy number vectors have important advantages that make their use worth considering, at least when the displayed peptides are small.     

A general insert label for peptide display on chimeric filamentous bacteriophages

The foreign insert intended to be displayed via recombinant phage proteins can have a negative effect on protein expression and phage assembly. A typical example is the case of display of peptides longer than 6 amino acid residues on the major coat protein, protein VIII of the filamentous bacteriophages M13 and fd. A solution to this problem has been the use of "two-gene systems" generating chimeric phages that concomitantly express wild-type protein VIII along with recombinant protein VIII. Although the two-gene systems are much more permissive in regard to insert length and composition, some cases can still adversely affect phage assembly. Although these phages genotypically contain the desired DNA of the insert, they appear to be phenotypically wild type. To avoid false-negative results when using chimeric phages in binding studies, it is necessary to confirm that the observed lack of phage recognition is not due to faulty assembly and display of the intended insert. Here we describe a strategy for generating antibodies that specifically recognize recombinant protein VIII regardless of the nature of its foreign insert. These antibodies can be used as a general monitor of the display of recombinant protein VIII into phage particles.     

Construction and exploitation in model experiments of functional selection of a landscape library expressed from a phagemid

Phage display has evolved during the past 15 years as a powerful technique to select, from libraries of peptides or proteins, binders for various targets or to evolve new functions in proteins. In recent years, the knowledge acquired in phage display technology was exploited to engineer phages as vehicles for receptor-mediated gene delivery. The first vectors generated provided the proof of the concept that development of gene delivery vehicles based on phages was feasible. Results obtained showed that the level of receptor ligand display was an essential factor that determines the efficiency of transduction and suggested that phagemids might be more appropriate than phages for gene delivery. However, due to the limitations of the existing display systems, vectors constructed up to now allowed only relatively low levels of ligand display. The transduction efficiency of these vectors was relatively poor. Here, we describe the construction and optimization of a new phagemid display system that was designed to allow the functional selection of peptides that promote gene delivery from phagemids in a high display format. Peptides are displayed on every copy of the major coat protein pVIII and are expressed from the phagemid itself. The phagemid is rescued as particles by a modified R408 helper phage, deficient in pVIII production. Besides an expression cassette for pVIII, the phagemid also contains the SV40 origin of replication, the GFP gene and the neomycin resistance marker. As a model we constructed a library of octapeptides and showed that the library is amenable to selection on cos-7 cells. Several selection approaches were investigated and a preliminary analysis of the peptides selected was carried out.     

Protection against Influenza A Virus Challenge with M2e-Displaying Filamentous Escherichia coli Phages

Human influenza viruses are responsible for annual epidemics and occasional pandemics that cause severe illness and mortality in all age groups worldwide. Matrix protein 2 (M2) of influenza A virus is a tetrameric type III membrane protein that functions as a proton-selective channel. The extracellular domain of M2 (M2e) is conserved in human and avian influenza A viruses and is being pursued as a component for a universal influenza A vaccine. To develop a M2e vaccine that is economical and easy to purify, we genetically fused M2e amino acids 2–16 to the N-terminus of pVIII, the major coat protein of filamentous bacteriophage f88. We show that the resulting recombinant f88−M2e2-16 phages are replication competent and display the introduced part of M2e on the phage surface. Immunization of mice with purified f88−M2e2-16 phages in the presence of incomplete Freund’s adjuvant, induced robust M2e-specific serum IgG and protected BALB/c mice against challenge with human and avian influenza A viruses. Thus, replication competent filamentous bacteriophages can be used as efficient and economical carriers to display conserved B cell epitopes of influenza A.     

Adapter-Directed Display: A Modular Design for Shuttling Display on Phage Surfaces

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems.     

Construction and characterization of a 9-mer phage display pVIII-library with regulated peptide density

The construction of a new phagemid vector for display of peptides on the pVIII major coat protein of filamentous bacteriophage is described, in which expression of pVIII-peptide fusions was placed under the control of the arabinose-inducible P_BAD promoter. The new phagemid showed excellent capacity for the regulation of peptide expression, as judged by enzyme-linked immunosorbent assay (ELISA) and electron microscopy of immunogold-labeled FLAG peptides displayed on phages. Regulation of the density of peptide fusions displayed on phages may offer advantages in the search for new peptide ligands due to the possibility of regulating the stringency of binding, reducing selection based on avidity effects during biopanning. Furthermore, the peptide expression in the absence of inducer was effectively shut off, minimizing growth bias of individual clones. A 9-mer phage display library prepared using the constructed phagemid was generated by insertion of randomly synthesized oligonucleotides close to the N-terminal of the pVIII protein. The library comprised a total of 9.4 × 10⁹ unique transformants, and was confirmed to show high diversity. The functional utility of the library was confirmed by the successful affinity selection of peptides binding to matrix metalloproteinase-9 (MMP-9). The majority of selected peptides shared the consensus motif R(D/N)XXG(M/L)(V/I)XQ, not previously selected during biopanning against MMP-9.     

Phenogenetic characterization of a group of giant Phi KZ-like bacteriophages of Pseudomonas aeruginosa

A comparative study was made of a group of Pseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage phi KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage phi KZ genome (288,334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1-19]. By DNA homology, the phages were assigned to three species (represented by phage phi KZ, Lin68, and EL, respectively) and two new genera (phi KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the phi KZ species (phi KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the phi KZ genus. The origin and evolution of the phi KZ-like phages are discussed. Analysis of protein sequences encoded by the phage phi KZ genome made it possible to assume wide migration of the phi KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage phi KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.     

Site-specific recombination of T2 phage using IP008 long tail fiber genes provides a targeted method for expanding host range while retaining lytic activity

The application of bacteriophages (phages) in therapy urgently requires the production of wide-host-range recombinant phages that possess strong lytic activity. The wide-host-range IP008 phage was classified by transmission electron microscopy analysis as an A2 morphotype member of the Myoviridae family of the order Caudovirales . IP008 showed a high homology (99.4% similarity in the amino acid alignment of the major capsid protein Gp 23) with KEP10, another wide-host-range phage. The long tail fiber genes (genes 37 and 38 ) from the genome of T2 were replaced with those of the IP008 phage by homologous recombination. The host range of the recombinant phages was identical to that of IP008. Furthermore, the recombinant phage bacterial lytic activity was restored. Future analyses of host-range mutants of the closely related phages T2 and IP008 could lead to a more precise localization of the genetic factors responsible for receptor specificity.     

Interspecies migration and evolution of bacteriophages of the genus phiKZ: The purpose and criteria of the search for new phiKZ-like bacteriophages

Some properties of bacteriophages with large (200 kb and more) sequenced genomes have been compared. In contrast to other large bacteriophages from different families, bacteriophages active on pseudomonads of various species (phiKZ-like bacterio phages) have some common features, which suggests their phylogenetic relationship and independence of their evolution as a result of migration among bacteria of this family. Among such common features are the absence in the genomes of these phages of sites sensitive to endonuclease PstI, the absence of genes encoding DNA polymerases that are similar to the known enzymes of this type, possible dependence of replication of the phage genome on bacterial DNA polymerase, and a considerably larger average gene size as compared to that for other phages. Criteria are suggested for searching for novel phiKZ-like bacteriophages: the size of a phag e particle, production by bacteria infected with such phages of a large amount of highly viscous mucus. Taking into account the use of these bacteriophages in therapeutic preparations (due to a broad spectrum of lytic activity) and a poor knowledge of a majority of their gene products, it seems necessary to perform a more comprehensive genetic analysis of phages of this genus or their mutants for selecting those adequate for phage therapy.     

Inversion and deletion mutants in Bacillus subtilis bacteriophage SPP1 as a consequence of cloning

Summary Properties of an inversion and a deletion mutant of B. subtilis phage SPP1 which arose during cloning are described. The results are related to the biology of this bacteriophage.In preceding communications from our laboratories (Heilmann and Reeve 1982, Behrens et al. 1983) we reported the properties of genetically engineered SPP1 bacteriophages, which could be used as cloning vehicles in B. subtilis. These phages contain a unique restriction site within a dispensable region of their genomes. In the course of cloning experiments using these phage vectors, we have occasionally observed the appearance of not only the original vector and desired hybrid phages, but also of SPP1 phages which had undergone extensive genomic rearrangements. Properties of two such phages, SPP1 inv1, which was found to contain a large inversion and of SPP1 delV, a deletion mutant, which defines an additional dispensable region of the SPP1 genome, are described in this communication.     

Genomic characterization of two Staphylococcus epidermidis bacteriophages with anti-biofilm potential

ABSTRACT: BACKGROUND: Staphylococcus epidermidis is a commensal bacterium but can colonize the hospital environment due to its ability to form biofilms favouring adhesion to host tissues, medical devices and increasing resistance to antibiotics. In this context, the use of phages to destroy biofilms is an interesting alternative. RESULTS: The complete genomes of two Staphylococcus epidermidis bacteriophages, vB_SepiSphiIPLA5 and vB_SepiS-phiIPLA7, have been analyzed. Their genomes are 43,581 bp and 42,123 bp, and contain 67 and 59 orfs. Bioinformatic analyses enabled the assignment of putative functions to 36 and 29 gene products, respectively, including DNA packaging and morphogenetic proteins, lysis components, and proteins necessary for DNA recombination, regulation, modification and replication. A point mutation in vB_SepiS-phiIPLA5 lysogeny control-associated genes explained its strictly lytic behaviour. Comparative analysis of phi- IPLA5 and phi-IPLA7 genome structure resembled those of S. epidermidis phiPH15 and phiCNPH82 phages. A mosaic structure of S. epidermidis prophage genomes was revealed by PCR analysis of three marker genes (integrase, major head protein and holin). Using these genes, high prevalence (73%) of phage DNA in a representative S. epidermidis strain collection consisting of 60 isolates from women with mastitis and healthy women was determined. Putative pectin lyase-like domains detected in virion-associated proteins of both phages could be involved in exopolysaccharide (EPS) depolymerization, as evidenced by both the presence of a clear halo surrounding the phage lysis zone and the phage-mediated biofilm degradation. CONCLUSIONS: Staphylococcus epidermidis bacteriophages, vB_SepiS-phiIPLA5 and vB_SepiS-phiIPLA7, have a mosaic structure similar to other widespread S. epidermidis prophages. Virions of these phages are provided of pectin lyase-like domains, which may be regarded as promising anti-biofilm tools.     

Genetic studies of lactococcal bacteriophages—taxonomic differentiations and DNA analysis: evidence for 3' cohesive ends

Twenty-four bacteriophages of Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris were classified. Two groups of bacteriophages morphologically defined as prolate or isometric types by electron microscopy were examined for their genome sizes, protein patterns and DNA homologies. These criteria showed that prolate phages are quite homogeneous. In contrast, isometric phages exhibit more differences, particularly in particle sizes and protein compositions. Analysis of DNA hybridizations confirmed that prolate phages can be grouped together as can be isometric phages but for one exception, phage I52. These two families were clearly defined. The unique phage which does not fit in either group probably belongs to a third one which is much less represented. No obvious relationships between these criteria and the lytic spectra were detected. Evidence of the presence of cohesive ends in phage genomes is also presented in this study. A more detailed analysis performed on one member of the prolate group revealed 3' protruding ends made up of around 13 nucleotides on complementary single strands.     

Recombinant derivatives of Bacillus subtilis phage Z containing the DNA methyltransferase genes of related methylation-proficient phages

The DNA methyltransferase (Mtase) genes of temperate Bacillus subtilis phages SPR, phi 3T, SP beta and rho 11 can be transferred by transfection and recombination to the genome of the related non-modifying phage Z. Integration of the Mtase genes occurs in phage Z DNA at a unique location which is homologous with the flanking regions of the Mtase genes of the related phages. In lysogenic cells carrying recombinant phages, expression of the Mtase genes is repressed, irrespective of whether the Mtase genes were derived from phage donors which were homo- or heteroimmune to phage Z.     

噬菌体展示技术系统发展进展

噬菌体展示技术(Phage display technology,PDT)是一种特殊的基因工程重组表达技术,噬菌体展示技术系统(Phage display system,PDS)是指包括经过遗传改造后的系列噬菌体、辅助噬菌体、宿主细菌等集成平台(含试剂盒)。文章从噬菌体分子遗传学及其基因(基因组)遗传工程改良角度,基于噬菌体M13、λ、T4和T7等4大类典型噬菌体展示技术系统的发展进展进行了综述。重点强调不同展示系统中的核心部件及其基因工程改造的分子遗传学原理、不同展示锚定位点的技术特征、相关试剂盒的研制状况及选择依据。     

Rethinking the Evolution of Single-Stranded RNA (ssRNA) Bacteriophages Based on Genomic Sequences and Characterizations of Two R-Plasmid-Dependent ssRNA Phages, C-1 and Hgal1

We have sequenced and characterized two R-plasmid-dependent single-stranded RNA bacteriophages (RPD ssRNA phages), C-1 and Hagl1. Phage C-1 requires a conjugative plasmid of the IncC group, while Hgal1 requires the IncH group. Both the adsorption rate constants and one-step growth curves are determined for both phages. We also empirically confirmed the lysis function of the predicted lysis genes. Genomic sequencing and phylogenetic analyses showed that both phages belong to the Levivirus group and are most closely related to another IncP-plasmid-dependent ssRNA phage, PRR1. Furthermore, our result strongly suggests that the stereotypical bauplans of genome organization found in Levivirus and Allolevivirus predate phage specialization for conjugative plasmids, suggesting that the utilization of conjugative plasmids for cell attachment and entry comprises independent evolutionary events for these two main clades of ssRNA phages. Our result is also consistent with findings of a previous study, making the Levivirus-like genome organization ancestral and the Allolevivirus-like genome derived. To obtain a deeper insight into the evolution of ssRNA phages, more phages specializing for various conjugative plasmids and infecting different bacterial species would be needed.     

New temperate Pseudomonas aeruginosa phage, phi297: Specific features of genome structure

The genome structure and some specific features of temperate Pseudomonas aeruginosa phage phi297 are considered. Analysis of sequencing data and genome annotation suggest that the phi297 genome displays a mosaic structure, which has formed through combining gene blocks from bacteria of taxonomically remote groups and/or their phages. The results of a comparison of the phi297 DNA homology level and pattern with the genome sequences of the currently known related P. aeruginosa bacteriophages are interpreted from the perspective of assumed active migration of these phages between different bacterial species.     

Phage display as a novel screening method to identify extracellular proteins

Extracellular proteins are involved in many diverse and essential cell functions and in pathogenic bacteria, and they may also serve as virulence factors. Therefore, there is a need for methods that identify the genes encoding this group of proteins in a bacterial genome. Here, we present such a method based on the phage display technology. A novel gene III-based phagemid vector, pG3DSS, was constructed that lacks the signal sequence which normally orientates the encoded fusion protein to the Escherichia coli cell membrane, where it is assembled into the phage particle. When randomly fragmented DNA is inserted into this vector, only phagemids containing an insert encoding a signal sequence will give rise to phage particles displaying a fusion protein. These phages also display an E-tag epitope in fusion with protein III, which enables isolation of phages displaying a fusion protein, using antibodies against the epitope. From a library constructed from Staphylococcus aureus chromosomal DNA, genes encoding secreted as well as transmembrane proteins were isolated, including adhesins, enzymes and transport proteins.