A phage display system designed to detect and study protein–protein interactions

Analysing protein–protein interactions is critical in proteomics and drug discovery. The usage of 2-Hybrid (2λ) systems is limited to an in vivo environment. We describe a bacteriophage 2-Hybrid system for studying protein interactions in vitro . Bait and prey are displayed as fusions to the surface of phage λ that are marked with different selectable drug-resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug-resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9-CUE, a Ubiquitin binding domain, and by the interaction of (Gly-Glu)₄ and (Gly-Arg)₄ peptides. Interruptions of the phage interactions by non-fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a λ-display library. The unique usefulness to 2λ is also described.     

Combining phage display and screening of cDNA expression libraries: a new approach for identifying the target antigen of an scFv preselected by phage display

A potential method for identifying new tumor-specific antibody structures as well as tumor-associated antigens is by selecting scFv phage libraries on tumor cells. This phage display technique involves multiple rounds of phage binding to target cells, washing to remove non-specific phage and elution to retrieve specific binding phage. Although the binding properties of an isolated tumor-specific scFv can be evaluated by ELISA, FACS and immunohistochemistry, it still remains a challenge to define the corresponding antigen. Here, we provide evidence that the target antigen of a given scFv displayed on phages can be detected in an immobilized lambda phage cDNA expression library containing thousands of irrelevant clones. The library contained CD30-negative breast-cancer specific cDNA as well as human CD30 receptor cDNA. The interaction of anti-CD30 scFv phages and their target antigen after blotting onto nitrocellulose filters was documented under defined conditions. Screening of different ratios between CD30 receptor and breast cancer specific clones (1:1 and 1:200) revealed that the CD30 antigen could be detected by anti-CD30 scFv phages using at least 5x10(12) plaque forming units of filamentous phages per blot. These investigations demonstrate that it is possible to detect the target antigen of a preselected scFv displayed on filamentous phages in lambda phage cDNA expression libraries.     

Specificity determinants in the attachment sites of bacteriophages HK022 and lambda.

The Int proteins of bacteriophages HK022 and lambda promote recombination between phage and bacterial attachment sites. Although the proteins and attachment sites of the two phages are similar, neither protein promotes efficient recombination between the pair of attachment sites used by the other phage. To analyze this difference in specificity, we constructed and characterized chimeric attachment sites in which segments of one site were replaced with corresponding segments of the other. Most such chimeras recombined with appropriate partner sites in vivo and in vitro, and their differential responses to the Int proteins of the two phages allowed us to locate determinants of the specificity difference in the bacterial attachment sites and a central segment of the phage attachment sites. The location of these determinants encompasses three of the four core-type binding sites for lambda Int: C, B, and most importantly, B'. The regions corresponding to the C' core binding site and the arm-type binding sites of lambda Int play no role in the specificity difference and, indeed, are well conserved in the two phages. We found, unexpectedly, that the effect of replacement of an Int-binding region on the recombinational potency of one chimeric site was reversed by a change of partner. This novel context effect suggests that postsynaptic interactions affect the specificity of recognition of attachment sites by Int.     

Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.     

Modelling the stability of Stx lysogens

Shiga-toxin-converting bacteriophages (Stx phages) are temperate phages of Escherichia coli, and can cause severe human disease. The spread of shiga toxins by Stx phages is directly linked to lysogen stability because toxins are only synthesized and released once the lytic cycle is initiated. Lysogens of Stx phages are known to be less stable than those of the related lambda phage; this is often described in terms of a 'hair-trigger' molecular switch from lysogeny to lysis. We have developed a mathematical model to examine whether known differences in operator regions and binding affinities between Stx phages and lambda phage can account for the lower stability of Stx lysogens. The Stx phage 933W has only two binding sites in its left operator region (compared to three in phage lambda), but this has a minimal effect on 933W lysogen stability. However, the relatively weak binding affinity between repressor molecules and the second binding site in the right operator is found to significantly reduce the stability of its lysogens, and may account for the hair-trigger nature of the switch. Reduced lysogen stability can lead to increased frequency of genetic recombination in bacterial genomes. The development of the mathematical model has considerable utility in understanding the behaviour and evolution of the molecular switch, with implications for phage-related diseases.     

Improved in vitro packaging of coliphage lambda DNA: a one-strain system free from endogenous phage

In previous systems for in vitro packaging of lambda DNA, phages are produced from the packaging components as well as from added DNA. We have developed a new genetic strategy for in vitro packaging that bypasses this endogenous phage problem. Our system employs a single bacterial strain whose lambda prophage codes for all of the packaging proteins but is deleted for cos, the packaging origin. Crude extracts of the single lysogen: (i) are virtually free from endogenous phages, (ii) package added lambda DNA efficiently and (iii) are easy to prepare. Using the cos- in vitro packaging system we show that packaging of lambda linear monomers is a second-order reaction, but that packaging from concatemers prepared by annealing or ligation is first order. We conclude that in our cos- system, linear monomers are a poor substrate for in vitro packaging but that packaging from concatemers works well.     

大分子量重组蛋白在丝状噬菌体表面的展示表达及其与小分子相互作用的初步研究

利用pHEN1KM13噬菌粒系统表达融合蛋白,进而确定大分子量重组蛋白在丝状噬菌体表达的部位及其表达后的生物活性。通过蛋白酶切处理前后噬菌体侵染细菌能力的变化快速地检测大分子蛋白质能否在噬菌体表面展示表达;比较了谷胱甘肽S转移酶及其与三个不同长度连接臂融合的外源蛋白在噬菌体表面的表达和组装,确定了不大于40kD的重组蛋白分子能展示表达在丝状噬菌体表面;并利用已知的小分子化合物与蛋白质的相互作用证明了组装在噬菌体表面的谷胱甘肽S转移酶重组蛋白仍保持其天然的结合活性,为利用噬菌体展示系统研究蛋白质与小分子化合物的相互作用建立了基础。     

Large variabilities in host strain susceptibility and phage host range govern interactions between lytic marine phages and their Flavobacterium hosts

Phages are a main mortality factor for marine bacterioplankton and are thought to regulate bacterial community composition through host-specific infection and lysis. In the present study we demonstrate for a marine phage-host assemblage that interactions are complex and that specificity and efficiency of infection and lysis are highly variable among phages infectious to strains of the same bacterial species. Twenty-three Bacteroidetes strains and 46 phages from Swedish and Danish coastal waters were analyzed. Based on genotypic and phenotypic analyses, 21 of the isolates could be considered strains of Cellulophaga baltica (Flavobacteriaceae). Nevertheless, all bacterial strains showed unique phage susceptibility patterns and differed by up to 6 orders of magnitude in sensitivity to the same titer of phage. The isolated phages showed pronounced variations in genome size (8 to >242 kb) and host range (infecting 1 to 20 bacterial strains). Our data indicate that marine bacterioplankton are susceptible to multiple co-occurring phages and that sensitivity towards phage infection is strain specific and exists as a continuum between highly sensitive and resistant, implying an extremely complex web of phage-host interactions. Hence, effects of phages on bacterioplankton community composition and dynamics may go undetected in studies where strain identity is not resolvable, i.e., in studies based on the phylogenetic resolution provided by 16S rRNA gene or internal transcribed spacer sequences.     

Efficient excision of phage lambda from the Escherichia coli chromosome requires the Fis protein.

The Escherichia coli protein Fis has been shown to bind a single site in the recombination region of phage lambda and to stimulate excisive recombination in vitro (J. F. Thompson, L. Moitoso de Vargas, C. Koch, R. Kahmann, and A. Landy, Cell 50:901-908, 1987). We demonstrate that mutant strains deficient in fis expression show dramatically reduced rates of lambda excision in vivo. Phage yields after induction of a stable lysogen are reduced more than 200-fold in fis cells. The defect observed in phage yield is not due to inefficient phage replication or lytic growth. Direct examination of excisive recombination products reveals a severe defect in the rate of recombination in the absence of Fis. The excision defect observed in fis cells can be fully reproduced in fis+ cells by using phages that lack the Fis binding site on attR, indicating that the entire stimulatory effect of Fis on excisive recombination is due to binding at that site.     

Evidence of bar minigene expression and tRNA2Ile sequestration as peptidyl-tRNA2Ile during lambda bacteriophage development

Lambda bacteriophage development is impaired in Escherichia coli cells defective for peptidyl (pep)-tRNA hydrolase (Pth). Single-base-pair mutations (bar(-)) that affect translatable two-codon open reading frames named bar minigenes (barI or barII) in the lambda phage genome promote the development of this phage in Pth-defective cells (rap cells). When the barI minigene is cloned and overexpressed from a plasmid, it inhibits protein synthesis and cell growth in rap cells by sequestering tRNA(2)(Ile) as pep-tRNA(2)(Ile). Either tRNA(2)(Ile) or Pth may reverse these effects. In this paper we present evidence that both barI and barII minigenes are translatable elements that sequester tRNA(2)(Ile) as pep-tRNA(2)(Ile). In addition, overexpression of the barI minigene impairs the development even of bar(-) phages in rap cells. Interestingly, tRNA or Pth may reestablish lambda phage development. These results suggest that lambda bar minigenes are expressed and tRNA(2)(Ile) is sequestered as pep-tRNA(2)(Ile) during lambda phage development.     

Ni2+高效结合肽的筛选与作用研究

利用噬菌体随机十二肽库和金属亲和层析对重金属Ni2 进行结合肽筛选。经4轮生物淘洗、噬菌体扩增和DNA测序,获得一组多肽序列。GenBank Blast分析未发现同源序列,Clustal W多重序列比对也未找到Ni2 金属结合肽结合基序,但可能含有多聚组氨酸(His)2-5。噬菌体单克隆金属离子螯合树脂的亲和力测定和反筛、抑菌解毒试验表明:展示有金属结合肽的噬菌体不仅对Ni2 具有高亲和力,而且对其它金属离子也有作用,Cu2 、Ni2 、Co2 、Zn2 等金属离子对金属结合肽的亲和力显著高于Cd2 和Cr2 ,展示金属结合肽的噬菌体对重金属Ni2 具有一定的耐受和解毒作用。显微形态学观察也显示金属结合肽与金属螯合树脂的作用。对于了解重金属与多肽的相互作用机理以及环境重金属修复等均具有重要意义和价值。     

Large Variabilities in Host Strain Susceptibility and Phage Host Range Govern Interactions between Lytic Marine Phages and Their Flavobacterium Hosts

Phages are a main mortality factor for marine bacterioplankton and are thought to regulate bacterial community composition through host-specific infection and lysis. In the present study we demonstrate for a marine phage-host assemblage that interactions are complex and that specificity and efficiency of infection and lysis are highly variable among phages infectious to strains of the same bacterial species. Twenty-three Bacteroidetes strains and 46 phages from Swedish and Danish coastal waters were analyzed. Based on genotypic and phenotypic analyses, 21 of the isolates could be considered strains of Cellulophaga baltica (Flavobacteriaceae). Nevertheless, all bacterial strains showed unique phage susceptibility patterns and differed by up to 6 orders of magnitude in sensitivity to the same titer of phage. The isolated phages showed pronounced variations in genome size (8 to >242 kb) and host range (infecting 1 to 20 bacterial strains). Our data indicate that marine bacterioplankton are susceptible to multiple co-occurring phages and that sensitivity towards phage infection is strain specific and exists as a continuum between highly sensitive and resistant, implying an extremely complex web of phage-host interactions. Hence, effects of phages on bacterioplankton community composition and dynamics may go undetected in studies where strain identity is not resolvable, i.e., in studies based on the phylogenetic resolution provided by 16S rRNA gene or internal transcribed spacer sequences.     

Cd2+结合肽的筛选及与重金属离子的亲和作用

利用噬菌体随机十二肽库和亲和层析技术对重金属Cd进行亲和筛选,共获得两条Cd结合肽序列。将展示有Cd2 结合肽的噬菌体单克隆扩增物对不同重金属离子(Cd2 、Cr2 、Cu2 、Co2 、Zn2 、Ni2 )螯合的树脂进行亲和测定,结果表明Cu2 、Co2 、Zn2 、Ni2 对结合肽的亲和力高于Cd2 和Cr2 。抑菌解毒试验进一步确认了Cd2 结合肽对大肠杆菌重金属的解毒作用。显微观察可见金属结合肽与金属螯合树脂混合后分散度发生改变。     

Detection and identification of methicillin resistant and sensitive strains of Staphylococcus aureus using tandem measurements

Discrimination of methicillin resistant (MRSA) and sensitive (MSSA) strains of Staphylococcus aureus, was achieved by the specially selected lytic bacteriophage with a wide host range of S. aureus strains and a penicillin-binding protein (PBP 2a) specific antibody. A quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to analyze bacteria-phage interactions. The lytic phages were transformed into phage spheroids by exposure to water-chloroform interface. Phage spheroid monolayers were transferred onto QCM-D sensors by Langmuir-Blodgett (LB) technique. Biosensors were tested in the flow mode with bacterial water suspensions, while collecting frequency and energy dissipation changes. Bacteria-spheroid interactions resulted in decreased resonance frequency and an increase in dissipation energy for both MRSA and MSSA strains. Following the bacterial binding, these sensors were further exposed to a flow of the penicillin-binding protein (PBP 2a) specific antibody conjugated latex beads. Sensors tested with MRSA responded to PBP 2a antibody beads; while sensors examined with MSSA gave no response. This experimental difference establishes an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Both free and immobilized bacteriophages strongly inhibit bacterial growth on solid/air interfaces and in water suspensions. After lytic phages are transformed into spheroids, they retain their strong lytic activity and demonstrate high bacterial capture efficiency. The phage and phage spheroids can be used for screening and disinfection of antibiotic resistant bacteria. Other applications may include use on biosensors, bacteriophage therapy, and antimicrobial surfaces.     

The terminase of bacteriophage lambda. Functional domains for cosB binding and multimer assembly

Terminase is a protein complex involved in lambda DNA packaging. The subunits of terminase, gpNul and gpA, are the products of genes Nul and A. The actions of terminase include DNA binding, prohead binding and DNA nicking. Phage 21 is a lambdoid phage that also makes a terminase, encoded by genes 1 and 2. The terminases of 21 and lambda are not interchangeable. This specificity involves two actions of terminase; DNA binding and prohead binding. In addition, the subunits of lambda terminase will not form functional multimers with the subunits of 21 terminase. lambda-21 hybrid phages can be produced as a result of recombination. We describe here lambda-21 hybrid phages that have hybrid terminase genes. The packaging specificities of the hybrids and the structure of their genes were compared in order to identify functional domains of terminase. The packaging specificities were determined in vivo by complementation tests and helper packaging experiments. Restriction enzyme site mapping and sequencing located the sites at which recombination occurred to produce the hybrid phages. lambda-21 hybrid 51 carries the lambda A gene, and a hybrid 1/Nul gene. The crossover that produced this phage occurred near the middle of the 1 and Nul genes. The amino-terminal portion of the hybrid protein is homologous to gp1 and the carboxy-terminal portion is homologous to gpNul. It binds to 21 DNA and forms functional multimers with gpA, providing evidence that the amino-terminal portion of gpNul is involved in DNA binding and the carboxy-terminal portion of gpNul is involved in the interaction with gpA. lambda-21 hybrid 54 has a hybrid 2/A gene. The amino terminus of the hybrid protein of lambda-21 hybrid 54 is homologous with gp2. This protein forms functional multimers only with gp1, providing evidence that the amino terminus of gpA is involved in the interaction with gpNul. These studies identify three functional domains of terminase.     

Rule-based simulation of temperate bacteriophage infection: Restriction–modification as a limiter to infection in bacterial populations

An individual-based model (IbM) for bacterial adaptation and evolution, COSMIC-Rules, has been employed to simulate interactions of virtual temperate bacteriophages (phages) and their bacterial hosts. Outcomes of infection mimic those of a phage such as lambda, which can enter either the lytic or lysogenic cycle, depending on the nutritional status of the host. Infection of different hosts possessing differing restriction and modification systems is also simulated. Phages restricted upon infection of one restricting host can be adapted (by host-controlled modification of the phage genome) and subsequently propagate with full efficiency on this host. However, such ability is lost if the progeny phages are passaged through a new host with a different restriction and modification system before attempted re-infection of the original restrictive host. The simulations show that adaptation and re-adaptation to a particular host-controlled restriction and modification system result in lower efficiency and delayed lysis of bacterial cells compared with infection of non-restricting host bacteria.     

Structural insights into the inhibition mechanism of bacterial toxin LsoA by bacteriophage antitoxin Dmd

Bacteria have obtained a variety of resistance mechanisms including toxin‐antitoxin (TA) systems against bacteriophages (phages), whereas phages have also evolved to overcome bacterial anti‐phage mechanisms. Dmd from T4 phage can suppress the toxicities of homologous toxins LsoA and RnlA from Escherichia coli, representing the first example of a phage antitoxin against multiple bacterial toxins in known TA systems. Here, the crystal structure of LsoA‐Dmd complex showed Dmd is inserted into the deep groove between the N‐terminal repeated domain (NRD) and the Dmd‐binding domain (DBD) of LsoA. The NRD shifts significantly from a ‘closed’ to an ‘open’ conformation upon Dmd binding. Site‐directed mutagenesis of Dmd revealed the conserved residues (W31 and N40) are necessary for LsoA binding and the toxicity suppression as determined by pull‐down and cell toxicity assays. Further mutagenesis identified the conserved Dmd‐binding residues (R243, E246 and R305) of LsoA are vital for its toxicity, and suggested Dmd and LsoB may possess different inhibitory mechanisms against LsoA toxicity. Our structure‐function studies demonstrate Dmd can recognize LsoA and inhibit its toxicity by occupying the active site possibly via substrate mimicry. These findings have provided unique insights into the defense and counter‐defense mechanisms between bacteria and phages in their co‐evolution.