Filamentous Phage Assembly Research Articles

THE OVER EXPRESSION OF THIOREDOXIN DURING MALIGNANCIES

Article type Review article Thioredoxin system comprised of thiorexin and NADPH dependent thiorexin reductase, is responsible for redox regulation of cells by controlling the apoptosis, proliferation and other vital processes of cells. The efficacy of thioredoxin system has been represented in a wide range of physiological and biological reactions in bacteria, yeast, plants, mammals and etc. including DNA synthesis, regulation of transcription factors, protein repairing, regulating the photosynthesis and controlling the apoptosis and preventing oxidative stresses, filamentous phage assembly, immune-modulating, neuronal survival, pregnancy and birth and many other physiological and biological functions. The up-regulation of thioredoxin has been observed in various malignancies, which was associated with tumor angiogenesis and development. In this regard, the thiordoxin system has become a putative target in new chemotherapeutic methods. In this study, we mentioned various features of thioredoxin system in malignant cells and reviewed the articles which have evaluated the expression rate of thioredoxin system in malignancies.

Filamentous Bacteriophage: Biology, Phage Display and Nanotechnology Applications

Filamentous bacteriophage, long and thin filaments that are secreted from the host cells without killing them, have been an antithesis to the standard view of head-and-tail bacterial killing machines. Episomally replicating filamentous phage Ff of Escherichia coli provide the majority of information about the principles and mechanisms of filamentous phage infection, episomal replication and assembly. Chromosomally- integrated "temperate" filamentous phage have complex replication and integration, which are currently under active investigation. The latter are directly or indirectly implicated in diseases caused by bacterial pathogens Vibrio cholerae, Pseudomonas aeruginosa and Neisseria meningitidis. In the first half of the review, both the Ff and temperate phage are described and compared. A large section of the review is devoted to an overview of phage display technology and its applications in nanotechnology.

Comprehensive Mutagenesis of the C-terminal Domain of the M13 Gene-3 Minor Coat Protein: The Requirements for Assembly into the Bacteriophage Particle

Filamentous bacteriophage assemble at the host membrane in a non-lytic process; the gene-3 minor coat protein (P3) is required for release from the membrane and subsequently, for recognition and infection of a new host. P3 contains at least three distinct domains: two N-terminal domains that mediate host recognition and infection, and a C-terminal domain (P3-C) that is required for release from the host cell following phage assembly and contributes to the structural stability of the phage particle. A comprehensive mutational analysis of the 150 residue P3-C revealed that only 24 side-chains, located within the last 70 residues of sequence, were necessary for efficient incorporation into a wild-type coat. The results reveal that the requirements for the assembly of P3 into the phage particle are quite lax and involve only a few key side-chains. These findings shed light on the functional and structural requirements for filamentous phage assembly, and they may provide guidelines for the engineering of improved coat proteins as scaffolds for phage display technology.

The pI and pXI assembly proteins serve separate and essential roles in filamentous phage assembly

Three non-capsid, phage-encoded proteins, pI, pIV and pXI, are required for assembly of the filamentous bacteriophage at the envelope of Escherichia coli. pIV forms the outer membrane component of the assembly site, and pI and pXI are predicted to form the cytoplasmic membrane component. pXI is the result of an in-frame internal translational initiation event in gene I and is identical with the carboxyl-terminal third of pI in amino acid sequence, membrane localization and topology. The two proteins share a cytoplasmic domain predicted to be an amphipathic helix, a transmembrane domain, and a periplasmic domain. By mutating the initiation site for pXI, a phage was made that produced only pI and was shown to absolutely require functional plasmid-encoded pXI for growth. Further mutational analysis was done to examine the functional determinants of the amphipathic helix and periplasmic domains of the pI and pXI proteins. The results show that the amphipathic helix region is very important for pI function but not for pXI function. Mutational analysis of the periplasmic domains of pI and pXI implies that these domains also perform separate functions, and suggests that the interaction between pI and pIV in the periplasm is critical for assembly. The results are discussed with regard to the separate roles that the pI and pXI proteins play in the overall process of phage assembly.

A trans-envelope protein complex needed for filamentous phage assembly and export.

Assembly and export of filamentous phage requires four non-capsid proteins: the outer membrane protein, pIV; the inner membrane proteins, pI and pXI; and a cytoplasmic host factor, thioredoxin. Chemical cross-linking of intact cells demonstrates a trans-membrane complex containing pI and pIV. Formation of the complex protects pI from proteolytic cleavage by an endogenous protease. This protection also requires pXI, which is identical to the C-terminal portion of pI. This indicates that pXI, which is required for phage assembly in its own right, is also part of the complex. This complex forms in the absence of any other phage proteins or the DNA substrate; hence, it represents the first preinitiation step of phage morphogenesis. On the basis of protease protection data, we propose that the preinitiation complex is converted to an initiation complex by binding phage DNA, thioredoxin and the initiating minor coat protein(s).

Roles of pIII in filamentous phage assembly

Filamentous phage protein III (pIII), located at one end of the phage, is required for infectivity and stability of the particle. Cells infected with phage from which gene III has been completely deleted produce particles that are not released into the medium but stay associated at the surface. These particles are much longer than normal phage. They can be released by subsequent expression of pIII. Viewed with the electron microscope, cells infected with gene III deletion phage are decorated with structures that resemble extremely long pili. Surprisingly, such cells are viable and can form colonies. The pIII deficiency can be complemented in trans, but there is a threshold concentration below which assembly does not occur. Above this threshold, pIII is used very efficiently and is incorporated into infectious but longer than unit length phage. As the concentration of pIII is increased, the number of infectious particles increases, and their average length decreases. pIII stabilizes pVI, a second phage protein found at the pIII end of the particle. In the absence of pIII, degradation of pVI is very rapid. pIII is thus not only required for infectivity and particle stability, but to terminate assembly and release the phage from its assembly site.

Filamentous phage structure, infection and assembly

The structural model of filamentous phage derived by X-ray fibre diffraction is supported by spectroscopic and genetic experiments. The structure of the receptor-binding domain at the end of the phage and the structure of the phage-coded intracellular DNA-binding protein have been determined at high resolution. The recent dissection of the virus life cycle by genetic and biochemical analyses, combined with structural information, suggests models for virus infection and assembly.

Filamentous phage assembly: variation on a protein export theme

Biogenesis of both filamentous phage and type-IV pili involves the assembly of many copies of a small, integral inner membrane protein (the phage major coat protein or pilin) into a helical, tubular array that passes through the outer membrane. The occurrence of related proteins required for assembly and export in both systems suggests that there may be similarities at the mechanistic level as well. This report summarizes the properties of filamentous phage and the proteins required for their assembly, with particular emphasis on features they may share with bacterial protein export and pilus biogenesis systems, and it presents evidence that supports the hypothesis that one of the phage proteins functions as an outer membrane export channel.

A permeabilized cell system that assembles filamentous bacteriophage.

A permeabilized cell system has been developed that is capable of assembling filamentous phage only upon addition of exogenous thioredoxin. The in vitro system exhibits the same component requirements seen in vivo: functional thioredoxin, an intact packaging signal in the substrate DNA, and the assembly protein, pIV. This crude in vitro system is insensitive to inhibitors of protein or DNA synthesis, demonstrating that particle assembly uses components that had accumulated before cell permeabilization. The temporal separation of the synthetic period, during which phage proteins and DNA accumulate, from the assembly period enabled us to examine the energy requirement for assembly. We show here that ATP hydrolysis is required for filamentous phage assembly and that the proton motive force is also important.

Module swaps between related translocator proteins pIVf1, pIVIKe and PulD: identification of a specificity domain

In Gram-negative bacteria, type II and type III secretion and filamentous phage assembly systems use related outer membrane proteins for substrate-specific transport across the outer membrane. We show here that the specificity domain of the phage f1 outer membrane protein pIV is contained within the 149 N-terminal amino acid residues. When the pIVf1 specificity domain is fused to the translocator domain of the related pIV of phage IKe, the chimeric construct supports f1 but not IKe assembly. Functional coupling between the two domains in this chimeric construct is poor and is improved by a single amino acid change in the translocator domain of the pIVIKe. In native pIVIKe, two amino acid changes within its specificity domain are both necessary and sufficient to change the specificity from IKe to f1 assembly. Analysis of 39 chimeric constructs between pIVf1 and the outer membrane protein PulD of the pullulanase secretion system failed to identify a comparable exchangeable specificity domain. These results indicate that the two domains may not function autonomously, and suggest that tertiary and quarternary changes of the entire translocator component rather than of an autonomous functional domain are required for specific translocation across the outer membrane.

The Products of Gene I and the Overlapping in-Frame Gene XI are Required for Filamentous Phage Assembly

The class I filamentous bacteriophage are non-lytic single-stranded DNA phage which are assembled at the cell envelope as they are extruded from the Gram-negative bacteria,Escherichia coli. The process requires the products of the phage genes I and IV, which reside in the inner and outer membrane, respectively, and are not present in the mature phage particle. Gene I encodes two proteins, the full length 348-residue pI and a smaller pI*, which this report shows is the result of an internal translation initiation event at methionine codon 241. Both pI and pI* are shown to be required for phage assembly. Therefore, pI* can be considered the product of an additional phage gene, XI, which is a separate in-frame gene that overlaps gene I. Both proteins contain a 13-residue region adjacent to the cytoplasmic face of the inner membrane that probably exists as a positively charged amphiphilic helix. Although this region is not required for membrane insertion of pI and pI*, it is shown to be required for phage assembly. Oligonucleotide-directed mutagenesis of this region, which removes positive charges or alters the hydrophobic face of the putative helix, renders pI and pI* unable to function in phage assembly. This region of pI and pI* is highly homologous in structure to the carboxyl-terminal 11 amino acids of pVIII, the main coat protein, which also reside adjacent to the cytoplasmic face of the inner membrane.

The role of the absorption complex in the termination of filamentous phage assembly

The adsorption complex of filamentous phage fd consists of two minor coat proteins, g3p and g6p, and is considered to be not only a structural entity, but also a functional unit to terminate phage assembly. Cells were infected with phage M13am8H1, which cannot assemble because it lacks the major coat protein g8p, although producting all of the other minor coat proteins. The membranes of infected cells were solubilized and analysed by non-denaturing PAGE and gel filtration. The data suggest the presence of the adsorption complex in these membranes. Furthermore, the non-polar gene 3 amber-mutant phage R171 was shown to lack g6p in the phage coat as well. The termination of assembly of this phage is disturbed, resulting in synthesis of polyphages. Electron micrographs and transient electrical birefringence show that these polyphages are eight times longer as compared to unit length phage. From these results, we conclude that the formation of the g3p-g6p complex is essential for correct termination of filamentous phage assembly.

Filamentous phage assembly: Morphogenetically defective mutants that do not kill the host

The filamentous phage virion is assembled without killing the host, by extrusion of the DNA through the envelope and concomitant acquisition of coat proteins from the inner membrane. When assembly is blocked, however, intracellular phage DNA and gene products accumulate and the host is killed. This “cell killing” is largely absent in phage fdtet, which carries a tetracycline-resistance determinant within the origin of minus-strand synthesis; as a result of the replication defect, phage DNA does not accumulate to high levels intracellularly when virion assembly is blocked. This allows morphogenetically defective mutants except those ablating gene V to be freely propagated in tetracycline-containing medium and studied in the absence of the confounding factor of cell morbidity. Because cultures can be initiated by transfection in the complete absence of input virions, extremely low levels of phage production can be assayed. Using this system, I show that genes III, VI, I, and IV are not required to form the complex between viral DNA and gene-V protein that is the intracellular precursor to mature virions; that genes I and/or IV are absolutely (or nearly absolutely) required for assembly; and that mos, a cis-acting sequence previously shown to enhance phage yield in some circumstances, is without such effect in others.

The role of thioredoxin in filamentous phage assembly. Construction, isolation, and characterization of mutant thioredoxins.

Filamentous phage assembly in vivo shows an absolute requirement for thioredoxin and a partial requirement for thioredoxin reductase. Mutants in which one or both of the active site cysteine residues of thioredoxin were changed to alanine or serine were constructed and shown to support filamentous phage assembly. Some of the mutants were almost as effective as wild-type thioredoxin, while others supported phage assembly only when high levels of the mutant protein were present in the infected cell. The mutant proteins were all inactive in an assay which couples oxidation of NADPH to reduction of 5,5'-dithiobis-2-nitrobenzoic acid) via thioredoxin reductase and thioredoxin. These active site mutants make phage assembly completely independent of thioredoxin reductase, which suggests that the phage needs, and the active site mutants provide, the proteins in the reduced conformation. Other mutants were isolated on the basis of their failure to support filamentous phage growth. These specified mutant thioredoxin proteins with varying levels of redox activity in vivo and in vitro. The locations of these mutations suggest that the surface of thioredoxin thought to interact with thioredoxin reductase also interacts with the filamentous phage assembly machinery. An in vivo assay for thioredoxin redox function, based on the ability of cells to utilize methionine sulfoxide, was developed. Met- cells containing mutant thioredoxins that are inactive in vitro do not form colonies on plates containing methionine sulfoxide as the sole methionine source.

Thioredoxin is required for filamentous phage assembly.

Sequence comparisons show that the fip gene product of Escherichia coli, which is required for filamentous phage assembly, is thioredoxin. Thioredoxin serves as a cofactor for reductive processes in many cell types and is a constituent of phage T7 DNA polymerase. The fip-1 mutation makes filamentous phage and T7 growth temperature sensitive in cells that carry it. The lesion lies within a highly conserved thioredoxin active site. Thioredoxin reductase (NADPH), as well as thioredoxin, is required for efficient filamentous phage production. Mutant phages defective in phage gene I are particularly sensitive to perturbations in the fip-thioredoxin system. A speculative model is presented in which thioredoxin reductase, thioredoxin, and the gene I protein interact to drive an engine for filamentous phage assembly.

Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid.

To determine whether the fip gene of Escherichia coli, which is required for filamentous phage assembly, is required for cell viability, we replaced the chromosomal copy of the gene with an inactive copy introduced on a plasmid. We found that the fip gene is dispensable. The method we devised, which should be generally useful, was also tested with an inactivated rho gene. As expected, the rho gene is essential.

F1, a Rod-shaped male-specific bacteriophage that contains DNA

Filamentous bacteriophages are interesting paradigms in structural molecular biology, in part because of the unusual mechanism of filamentous phage assembly. During assembly, several thousand copies of an intracellular DNA-binding protein bind to each copy of the replicating phage DNA, and are then displaced by membrane-spanning phage coat proteins as the nascent phage is extruded through the bacterial plasma membrane. This complicated process takes place without killing the host bacterium.The bacteriophage is a semi-flexible worm-like nucleoprotein filament. The virion comprises a tube of several thousand identical major coat protein subunits around a core of single-stranded circular DNA. Each protein subunit is a polymer of about 50 amino-acid residues, largely arranged in an α-helix. The subunits assemble into a helical sheath, with each subunit oriented at a small angle to the virion axis and interdigitated with neighbouring subunits. A few copies of “minor” phage proteins necessary for infection and/or extrusion of the virion are located at each end of the completed virion.Here we review both the structure of the virion and aspects of its function, such as the way the virion enters the host, multiplies, and exits to prey on further hosts. In particular we focus on our understanding of the way the components of the virion come together during assembly at the membrane. We try to follow a basic rule of empirical science, that one should chose the simplest theoretical explanation for experiments, but be prepared to modify or even abandon this explanation as new experiments add more detail.