Filamentous phage infection: required interactions with the TolA protein.

Infection of Escherichia coli by the filamentous phage f1 is initiated by binding of the phage to the tip of the F conjugative pilus via the gene III protein. Subsequent translocation of phage DNA requires the chromosomally encoded TolQ, TolR, and TolA proteins, after the pilus presumably has withdrawn, bringing the phage to the bacterial surface. Of these three proteins, TolA is proposed to span the periplasm, since it contains a long helical domain (domain II), which connects a cytoplasmic membrane anchor domain (domain I) to the carboxyl-terminal domain (domain III). By using a transducing phage, the requirement for TolA in an F+ strain was found to be absolute. The role of TolA domains II and III in the infective process was examined by analyzing the ability of various deletion mutants of tolA to facilitate infection. The C-terminal domain III was shown to be essential, whereas the polyglycine region separating domains I and II could be deleted with no effect. Deletion of helical domain II reduced the efficiency of infection, which could be restored to normal by retaining the C-terminal half of domain II. Soluble domain III, expressed in the periplasm but not in the cytoplasm or in the medium, interfered with infection of a tolA+ strain. The essential interaction of TolA domain III with phage via gene III protein appears to require interaction with a third component, either the pilus tip or a periplasmic entity.     

Interdomain interactions within the gene 3 protein of filamentous phage

A number of disorders related to cystic fibrosis have been described since the cloning of the cystic fibrosis gene, including infertility due to the congenital bilateral absence of the vas deferens. We have identified, in several patients, complex cystic fibrosis transmembrane conductance regulator genotypes like double-mutant alleles. We have now analyzed the structure-function relationships of one of these mutants, R74W-D1270N cystic fibrosis transmembrane conductance regulator, expressed in HeLa cells, to evaluate the contribution of each mutation in the phenotype. We found that R74W cystic fibrosis transmembrane conductance regulator appears to be a polymorphism, while D1270N cystic fibrosis transmembrane conductance regulator could be responsible for the congenital bilateral absence of the vas deferens phenotype. The combination of the two produced a more severe effect on the chloride conductance pathway as well as on the phenotype.     

Reprogramming the infection mechanism of a filamentous phage

When they infect Escherichia coli cells, the filamentous phages IF1 and fd first interact with a pilus and then target TolA as their common receptor. They use the domains N2 and N1 of their gene-3-proteins (G3P) for these interactions but differ in the mechanism of infection. In G3P of phage IF1, N1 and N2 are independent modules that are permanently binding-active. G3P of phage fd is usually in a closed state in which N1 and N2 are tightly associated. The TolA binding site is thus inaccessible and the phage incompetent for infection. Partial unfolding and prolyl isomerization must occur to abolish the domain interactions and expose the TolA binding site. This complex mechanism of phage fd could be changed to the simple infection mechanism of phage IF1 by reprogramming its G3P following physicochemical rules of protein stability. The redesigned phage fd was robust and as infectious as wild-type phage fd.     

A morphologic study of filamentous phage infection of Escherichia coli using biotinylated phages

Using biotinylated phage (BIO-phages), we observed the infection of filamentous phages into Escherichia coli JM109 morphologically. BIO-phages and BIO-phage-derived proteins, mainly pVIII, were detected in E. coli by using the avidin-biotin-peroxidase complex method with electron microscopy. Infected cells revealed positive staining on the outer and inner membranes and in the periplasmic space. Some cells showed specific or predominant staining of the outer membrane, whereas others showed predominant staining of the inner membrane or equivalent staining of the outer and inner membranes. The periplasmic spaces in some infected cells were expanded and filled with reaction products. Some cells showed wavy lines of positive staining in the periplasmic space. BIO-phages were detected as thick filaments or clusters covered with reaction products. The ends of the infecting phages were located on the surface of cells, in the periplasmic space, or on the inner membrane. These findings suggest that phage major coat proteins are integrated into the outer membrane and that phages cause periplasmic expansion during infection.     

细菌CRISPR-Cas 系统功能及其与噬菌体相互作用

摘要:近来研究发现,细菌CRISPR-Cas 系统在宿主菌抵抗可移动基因元件(mobile genetic elements,MGEs)的过程中发挥重要作用。CRISPR-Cas还参与宿主菌群体行为和毒力基因调控、DNA修复和基因组进化过程。本文着重综述细菌CRISPR-Cas系统的结构、类型、作用机制及其适应性免疫之外的其他功能(如对内源性基因表达的调控、促进基因组进化、DNA修复等);概述噬菌体抵抗CRISPR-Cas系统的机制,并对噬菌体-宿主菌相互作用进行探讨和展望。     

A visualization method of filamentous phage infection and phage-derived proteins in Escherichia coli using biotinylated phages.

Direct visualization of filamentous phage infection in Escherichia coli (E. coli) was attempted using biotinylated phages (BIO-phages). The biotinylation of the phages did not influence their infectivity into E. coli. E. coli infected with BIO-phages could be detected by using fluorescein-conjugated avidin with confocal laser scanning microscopy, and BIO-phages and BIO-phage-derived proteins in E. coli could be directly observed by using the avidin-biotin-peroxidase complex method with electron microscopy. This is the first report of direct visualization of phage infection and phage-derived proteins in the host cell using a biotin-avidin interaction. This simple and powerful method is applicable to the study of infection by various viruses.     

细菌群体感应系统与噬菌体的相互作用机制研究进展

细菌在与噬菌体的长期共进化过程中形成多种抵抗噬菌体侵染的机制,其中群体感应参与的细菌抵御噬菌体侵染机制成为近年来的研究热点。群体感应与噬菌体之间的相互作用是复杂和多样的,本文将重点综述群体感应在噬菌体侵染中的作用、调控在噬菌体裂解-溶源转变的作用,以及群体感应与噬菌体的其他相互影响等内容,为噬菌体在细菌性疾病的治疗提供理论依据。     

The use of filamentous phage M13 in protein engineering

M13B1 vector based on the filamentous phage M13 has been constructed. M13B1 phage carries the gene of resistance to ampicillin and contains the unique site of recognition for BamHI restriction endonuclease in gene VIII coding for the major coat protein. BamHI restriction site has been inserted into the gene of the major coat protein by means of oligonucleotide directed mutagenesis. The synthetic DNA fragment coding for the model peptides has been inserted through BamHI site into the M13B1 DNA. The possibility of inserting foreign peptides into the N-terminus at maintaining the viability of hybrid phages has been shown. The differences in specificity of the recombinant phage maturation have been determined by analysing the amino acid sequence of B-protein.     

Homodimeric peptides displayed by the major coat protein of filamentous phage

Peptide libraries displayed by filamentous bacteriophage have proven a powerful tool for the discovery of novel peptide agonists, antagonists and epitope mimics. Most phage-displayed peptides are fused to the N terminus of either the minor coat protein, pIII, or the major coat protein, pVIII. We report here that peptides containing cysteine residues, displayed as N-terminal fusions to pVIII, can form disulfide-bridged homodimers on the phage coat. Phage clones were randomly selected from libraries containing one or two fixed Cys residues, and surveyed for the presence of peptide-pVIII homodimers by SDS-PAGE analysis that involved pretreatment of the phage with reducing or thiol-modifying agents. For all phage whose recombinant peptide contained a single Cys residue, a significant fraction of the peptide-pVIII molecules were displayed as dimers on the phage coat. The dimeric form was in greater abundance than the monomer in almost all cases in which both forms could be reliably observed. Occasionally, peptides containing two Cys residues also formed dimers. These results indicate that, for a given pVIII-displayed peptide bearing a single Cys residue, a significant fraction of the peptide (>40 %) will dimerize regardless of its sequence; however, sequence constraints probably determine whether all of the peptide will dimerize. Similarly, only occasionally do peptides bearing two Cys residues form intermolecular disulfide bridges instead of intramolecular ones; this indicates that sequence constraints may also determine dimerization versus cyclization. Sucrose-gradient analysis of membranes from cells expressing pVIII fused to a peptide containing a single Cys residue showed that dimeric pVIII is present in the cell prior to its assembly onto phage. A model of the peptide-pVIII homodimer is discussed in light of existing models of the structure and assembly of the phage coat. The unique secondary structures created by the covalent association of peptides on the phage surface suggest a role for homo- and heterodimeric peptide libraries as novel sources of bioactive peptides.     

Functionally important correlated motions in the single-stranded DNA-binding protein encoded by filamentous phage Pf3

To elucidate the interplay between different parts of dimeric single-stranded DNA-binding proteins we have studied the correlated motions in the protein encoded by filamentous phage Pf3 via the combined use of 15N-NMR relaxation experiments, molecular dynamics simulations and essential dynamics calculations. These studies provide insight into the mechanism underlying the protein-DNA binding reaction. The most important motions can be described by a few essential modes. Most outstanding is the correlated symmetric motion of the DNA-binding wings, which are far apart in the structure. This motion determines the access of DNA to the DNA-binding domain. A correlation between the motion of the DNA-binding wing and the complex loop is indicated to play a role in the cooperative binding of the protein to DNA. These motions are in the nanosecond regime in correspondence with the 15N-NMR relaxation experiments.     

Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage

Background

Phage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display.

Methodology/Principal Findings

Here we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient.

Conclusions/Significance

Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before.     

Functional interactions of the genomes of Shigella sonnei phages and Escherichia coli phage T4 in mixed infection]

A comparative study of Shigella sonnei phages U and G and Escherichia coli phage T4 has shown that enzymes coded for by the Sh. sonnei phages can functionally substitute for some T4-coded products. This finding in indicative of an evolutionary relationship between T-even phages and disenteric phages U and G. The U phage is uncapable to compensate amber mutants for the genes that control the conversion of cytosine into 5-hydroxymethyl cytosine (5-HMC) and the glucosylation of the latter, which agrees with our earlier finding that the U phage DNA contains no 5-HMC. U and G phages are also found to exclude the T4 phage in the course of mixed infection.     

Regulation of filamentous bacteriophage length by modification of electrostatic interactions between coat protein and DNA

Bacteriophage fd gene VIII, which encodes the major capsid protein, was mutated to convert the serine residue at position 47 to a lysine residue (S47K), thereby increasing the number of positively charged residues in the C-terminal region of the protein from four to five. The S47K coat protein underwent correct membrane insertion and processing but could not encapsidate the viral DNA, nor was it incorporated detectably with wild-type coat proteins into hybrid bacteriophage particles. However, hybrid virions could be constructed from the S47K coat protein and a second mutant coat protein, K48Q, the latter containing only three lysine residues in its C-terminal region. K48Q phage particles are approximately 35% longer than wild-type. Introducing the S47K protein shortened these particles, the S47K/K48Q hybrids exhibiting a range of lengths between those of K48Q and wild-type. These results indicate that filamentous bacteriophage length (and the DNA packaging underlying it) are regulated by unusually flexible electrostatic interactions between the C-terminal domain of the coat protein and the DNA. They strongly suggest that wild-type bacteriophage fd makes optimal use of the minimum number of coat protein subunits to package the DNA compactly.     

fii, a bacterial locus required for filamentous phage infection and its relation to colicin-tolerant tolA and tolB.

We describe mutations in a new bacterial locus, designated fii, which do not allow the filamentous bacteriophage f1 to infect bacteria harboring the F plasmid. Mutations at this locus do not affect the ability of F plasmid-containing bacteria to undergo conjugation or be infected by the F plasmid-specific RNA phage f2. The filamentous phage can still adsorb to the F sex pilus, but the DNA is unable to enter the bacteria. All fii mutants become tolerant to colicins E1, E2, and E3. Strains with amber mutations in fii also are unable to plaque P1, even though they can be infected with this phage. Mutations in fii also prevent infection of bacteria harboring the N plasmid by the filamentous bacteriophage IKe. The fii locus maps adjacent to tolA, mutants of which demonstrate tolerance to high levels of the E and K colicins. The three genes tolA, tolB, and fii are shown to reside on a 4.3-kilobase fragment of the Escherichia coli chromosome. Each gene has been cloned into a chimeric plasmid and shown to complement, in trans, mutations at the corresponding chromosomal locus. Studies in maxicells show that the product of fii appears to be a 24-kilodalton protein which copurifies with the cell envelope. The product of tolA has been identified tentatively as a 51-kilodalton protein. Data from cloning, Tn5 mutagenesis, and P1 transduction studies are consistent with the gene order sucA-fii-tolA-tolB-aroG near 17 min on the E. coli map.     

A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein

Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets. This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency. Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII. Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection. Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage. The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target. We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.     

Relation between Px phages and generalised transducing particles of phage P22

Summary P22 mutants with an altered ability to form generalised transducing particles were tested for their ability to form the hybrid Px phages during growth on Py-lysogenic strains of Salmonella typhimurium. The mutant which produces more (less) transducing particles produces also more (less) Px phages. It is suggested that Px particles might be regarded as self-replicating generalised transducing particles.     

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.     

A proline switch controls folding and domain interactions in the gene-3-protein of the filamentous phage fd

The amino-terminal domains N1 and N2 of the gene-3-protein of phage fd form a bilobal structural and functional entity that protrudes from the phage tip. Domain N2 initiates the infection of Escherichia coli by binding to the F pilus. This binding results in the dissociation of the two domains and allows N1 to interact with the TolA receptor at the cell surface. The refolding of the N1-N2 fragment begins with the folding of domain N1, which takes a few milliseconds, followed by the folding of domain N2, which is complete within five minutes. The subsequent domain assembly is unusually slow and shows a time-constant of 6200 s at 25 degrees C. We found that the rate of this reaction is controlled by the trans to cis isomerization of the Gln212-Pro213 bond in the hinge subdomain of N2, a region that provides many interactions between N1 and N2 in the gene-3-protein. The substitution of Pro213 by Gly accelerated domain association 30-fold and revealed that the folding of the two individual domains and their assembly are indeed sequential steps in the refolding of the gene-3-protein. In the course of infection, the domains must separate to expose the binding site for TolA on domain N1. The kinetic block of domain reassembly caused by Pro213 isomerization could ensure that after the initial binding of N2 to the F pilus the open state persists until N1 and TolA are close enough for their mutual interaction. Pro213 isomerization might thus serve as a slow conformational switch in the function of the gene-3-protein.