Cassettes of the f1 intergenic region.
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Cloning, Molecular↗
Biomedical subjects
M Russel
Publications and source records attributed to M Russel.
Genetic analysis of the filamentous bacteriophage packaging signal and of the proteins that interact with it.
The single-stranded DNA of filamentous phages (f1, fd, M13, Ike) contains a region that can fold into a hairpin structure that serves to earmark the DNA for encapsidation. Second-site suppressor mutants of f1 that can compensate for deletion of this packaging signal have been isolated and characterized. The mutations lie in three genes, two that encode virion proteins located at the end of the particle that is first to emerge from the cell, the end at which the packaging signal is located, and the third in a gene whose product is required for assembly but which is not itself a part of the virion. Analysis of base substitution and deletion mutations in the packaging signal suggests that both structural and sequence elements are important to its proper function.
Bacteriophages↗
Sequence of thioredoxin reductase from Escherichia coli. Relationship to other flavoprotein disulfide oxidoreductases.
The DNA sequence of the Escherichia coli gene encoding thioredoxin reductase has been determined. The predicted protein sequence agrees with an earlier determination of the 17 amino-terminal amino acids and with a fragment of the protein containing the redox-active half-cystines. Similarity between E. coli thioredoxin reductase and other flavoprotein disulfide oxidoreductases is quite limited, but three short segments, two of which are probably involved in FAD and NADPH binding, are highly conserved between thioredoxin reductase, glutathione reductase, dihydrolipoamide dehydrogenase, and mercuric reductase.
Amino Acid Sequence↗
Construction and characterization of glutaredoxin-negative mutants of Escherichia coli.
Deoxyribonucleotides, the precursors of DNA, are formed de novo by ribonucleotide reductase, and in vitro thioredoxin or glutathione plus glutaredoxin have been isolated as hydrogen donors. The in vivo hydrogen donor for ribonucleotide reductase is not known. To study this, the Escherichia coli glutaredoxin gene (255 base pairs) was inactivated by inserting a 2-kilobase kanamycin-resistance fragment into the coding sequence of the cloned gene. The inactivated gene was inserted into the E. coli chromosome and mapped to about 18.5 min. A gene replacement technique was used to obtain a strain, A407, that lacked glutaredoxin by radioimmunoassay and by enzymatic assay with ribonucleotide reductase. Glutaredoxin was found not to be essential for viability of E. coli. Thioredoxin is also not essential for viability, as had been shown earlier, but a double mutant lacking glutaredoxin and thioredoxin could not be obtained by P1 transduction on a defined medium, indicating that either thioredoxin or glutaredoxin is essential. In rich medium, very slowly growing, unstable transductants were obtained that at high frequency gave rise to better growing cells. One such isolate, A410, was shown to still lack glutaredoxin and thioredoxin.
Bacterial Proteins↗
Low-frequency infection of F- bacteria by transducing particles of filamentous bacteriophages.
Filamentous particles containing single-stranded plasmid and bacteriophage DNA are able to infect F- Escherichia coli at frequencies of approximately 10(-6). This infection is dependent on an intact particle and requires the products of the tolQ, tolR, and tolA genes of the bacteria. The addition of CaCl2 can increase the frequency about 100-fold, presumably by increasing the concentration of particles at the bacterial surface.
Coliphages↗
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.
Bacterial Proteins↗
Interaction of mutant thioredoxins of Escherichia coli with the gene 5 protein of phage T7. The redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity.
DNA polymerase activity in Escherichia coli cells infected with bacteriophage T7 resides in a protein complex consisting of the T7 gene 5 protein and E. coli thioredoxin in a 1 to 1 stoichiometry. We have analyzed nine mutant thioredoxins, both in vivo and in vitro, for their ability to interact with the T7 gene 5 protein and stimulate the DNA polymerase and exonuclease activities inherent in gene 5 protein. The efficiency of plating of T7 on E. coli thioredoxin mutants depends strongly on the copy number of the respective mutant thioredoxin allele. Plating efficiencies at a constant copy number correlate well with the affinity of the purified mutant proteins for T7 gene 5 protein. The observed dissociation constant, Kobs, is increased between 5 and several hundredfold at 42 degrees C compared to wild-type thioredoxin. The maximum polymerase activity of the reconstituted gene 5 protein-thioredoxin complex at saturating concentrations of mutant thioredoxins, however, is reduced by less than 20%. Consequently, none of the mutant thioredoxins acts as a competitive inhibitor of wild-type thioredoxin. The active-site disulfide of thioredoxin is not essential for the activities of the gene 5 protein-thioredoxin complex. Both cysteines can be replaced without significantly affecting the maximum polymerase or exonuclease activities. Substitution or alkylation of either cysteine, however, reduces the affinity for gene 5 protein drastically, indicating that the active site is part of the thioredoxin surface involved in the protein-protein interaction.
Bacterial Proteins↗
An improved filamentous helper phage for generating single-stranded plasmid DNA.
Gene cloning in plasmid vectors that contain a filamentous phage intergenic region presents several advantages. However, technical difficulties have been a problem, primarily low yields of packaged single stranded (ss) plasmid DNA from the rapid, small scale procedures usually employed, and ambiguities in sequencing reactions attributed to the contamination by helper phage ss DNA. We report here the construction and some properties of a new f1 helper phage. Using this phage, R408, plasmid ss DNA is packaged and exported preferentially over phage ss DNA, and the absolute yield of plasmid ss DNA is usually increased.
Base Sequence↗
Aspects of the growth and regulation of the filamentous phages.
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Coliphages↗
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.
Amino Acid Sequence↗
Modification of DNA by glucose 6-phosphate induces DNA rearrangements in an Escherichia coli plasmid.
Reducing sugars such as glucose or glucose 6-phosphate (Glc-6-P) have been shown previously to modify the amino groups of nucleotides and single-stranded DNA. We have examined the mutagenic effect of Glc-6-P-induced lesions in the double-stranded DNA plasmid pBR322. Seventeen mutants of the Ampr Tets phenotype were isolated from plasmid preparations whose transforming capacity had been decreased by incubation with Glc-6-P. A number of the mutant plasmids were found to have undergone gross DNA alterations, including insertions and deletions, as well as the development of multiple species originating from a single cell. The ability of an endogenous reducing sugar to induce extensive DNA rearrangements suggests that these lesions may be significant contributors to cellular mutation.
Chromosome Deletion↗
Direct cloning of the trxB gene that encodes thioredoxin reductase.
A strain was constructed which contains mutations in the genes encoding thioredoxin (trxA) and thioredoxin reductase (trxB) such that filamentous phage f1 cannot grow. The complementation of either mutation with its wild-type allele permits phage growth. We used this strain to select f1 phage which contain a cloned trxB gene. The location of the gene on the cloned fragment was determined, and its protein product was identified. Plasmid subclones that contain this gene overproduce thioredoxin reductase.
Cloning, Molecular↗
Characterization of the cloned fip gene and its product.
A DNA fragment encoding the fip (filamentous phage production) gene from Escherichia coli, when cloned in a filamentous phage vector, restored to the phage ability to assemble progeny in fip mutant hosts. The fip gene was located just upstream of and transcribed in the same direction as the rho gene. Minicells containing fip+ phage or plasmids synthesized a 12,500-dalton protein that was missing or truncated when the Fip+ phenotype was inactivated by insertion of Tn5. The fip protein was cytoplasmic and was partially purified.
Bacterial Proteins↗
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.
Alleles↗
Assessment and intervention issues with the nonspeaking child.
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Child↗
Insertion of proteins into bacterial membranes.
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Bacteria↗
A bacterial gene, fip, required for filamentous bacteriophage fl assembly.
An Escherichia coli mutant which does not support the growth of filamentous bacteriophage fl allows phage fl DNA synthesis and gene expression in mutant cells, but progeny particles are not assembled. The mutant cells have no other obvious phenotype. On the basis of experiments with phage containing nonlethal gene I mutations and with mutant fl selected for the ability to grow on mutant bacteria, we propose an interaction between the morphogenetic function encoded by gene I of the phage and the bacterial function altered in this mutant. The bacterial mutation defines a new gene, fip (for filamentous phage production), located near 84.2 min on the E coli chromosome.
Chromosome Mapping↗
Filamentous phage pre-coat is an integral membrane protein: analysis by a new method of membrane preparation.
We show, using a simple, rapid fractionation method, that the precursor to the filamentous phage major coat protein is an integral membrane protein. The method, which consists of treatment of Escherichia coli with 0.1 N NaOH followed by centrifugation, leaves a subset of inner and outer membrane proteins in the NaOH pellet. Most proteins partition into the NaOH pellet (membrane) or supernatant (cytoplasm and periplasm) in a manner consistent with their subcellular location as determined by more conventional techniques. We find no evidence for cytoplasmic filamentous phage pre-coat protein in either wild-type of mutant-infected cells. Our evidence suggests that a protein identified as "soluble procoat" by K. Ito, G. Mandell and W. Wickner may be the amber fragment of a different phage protein.
Bacterial Proteins↗