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M Russel

Publications and source records attributed to M Russel.

At least 37 records · Page 2Linked to original sources
PubMed1996

Essential role of a sodium dodecyl sulfate-resistant protein IV multimer in assembly-export of filamentous phage.

Filamentous phage f1 encodes protein IV (pIV), a protein essential for phage morphogenesis that localizes to the outer membrane of Escherichia coli, where it is found as a multimer of 10 to 12 subunits. Introduction of internal His or Strep affinity tags at different sites in pIV interfered with its function to a variable extent. A spontaneous second-site suppressor mutation in gene IV allowed several different insertion mutants to function. The identical mutation was also isolated as a suppressor of a multimerization-defective missense mutation. A high-molecular-mass pIV species is the predominant form of pIV present in cells. This species is stable in 4% sodium dodecyl sulfate at temperatures up to 65 degrees C and is largely preserved at 100 degrees C in Laemmli protein sample buffer containing 4% sodium dodecyl sulfate. The suppressor mutation makes the high-molecular-mass form of wild-type pIV extremely resistant to dissociation, and it stabilizes the high-molecular-mass form of several mutant pIV proteins to extents that correlate with their level of function. Mixed multimers of pIV(f1) and pIV(Ike) also remain associated during heating in sodium dodecyl sulfate-containing buffers. Thus, sodium dodecyl sulfate- and heat-resistant high-molecular-mass pIV is derived from pIV multimer and reflects the physiologically relevant form of the protein essential for assembly-export.

Amino Acid Sequence↗
PubMed1995

Moving through the membrane with filamentous phages.

Filamentous phages are small, highly evolved parasites that can reproduce and disseminate without killing their host. During assembly, virion proteins are transferred from the host membrane to the single-stranded DNA phase genome and simultaneously secreted from the cell. Filamentous phage assembly shares certain features with bacterial processes responsible for the assembly of cell-surface structures and for extracellular protein secretion.

Coliphages↗
PubMed1994

pIV, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer.

Filamentous phage pIV is an outer membrane protein required for phage assembly and secretion. Chemical cross-linking and sedimentation experiments have been used to demonstrate that pIV from f1-infected Escherichia coli exists as a homo-multimer, probably composed of 10 to 12 subunits. pIV secreted from spheroplasts remains soluble and does not form multimers. Synthesis of pIV from distantly related filamentous phages or from a bacterial homolog that participates in a specialized form of extra-cellular protein secretion in the same cell with pIVf1 resulted in the formation of mixed multimers. This suggests that the homologous proteins themselves form homo-multimers. These structures could form gated channels that conduct assembling phage or specific substrate proteins across the outer membrane to the extracellular milieu.

Bacterial Proteins↗
PubMed1994

Mutants at conserved positions in gene IV, a gene required for assembly and secretion of filamentous phages.

The filamentous phage protein pIV is required for assembly and secretion of the virus and possesses regions homologous to those found in a number of Gram-negative bacterial proteins that are essential components of a widely distributed extracellular protein-export system. These proteins form multimers that may constitute an outer membrane channel that allows phage/protein egress. Three sets of f1 gene IV mutants were isolated at positions that are absolutely (G355 and P375) or largely (F381) conserved amongst the 16 currently known family members. The G355 mutants were non-functional, interfered with assembly of pIV+ phage, and made Escherichia coli highly sensitive to deoxycholate. The P375 mutants were non-functional and defective in multimerization. Many of the F381 mutants retained substantial function, and even those in which charged residues had been introduced supported some phage assembly. Some inferences about the roles of these conserved amino acids are made from the mutant phenotypes.

Amino Acid Sequence↗
PubMed1993

Protein-protein interactions during filamentous phage assembly.

Filamentous phage proteins pI and pIV are morphogenetic proteins required for phage assembly but not part of the virion. Neither pI nor pIV from the related phages f1 and IKe can substitute for its equivalent in the other phage. When the two proteins are supplied as pairs, however, partial restoration of heterologous phage assembly occurs. This observation strongly suggests that the two proteins interact. A selection for revertants of a temperature sensitive mutant of f1 gene IV resulted in the isolation of a suppressor mutation in gene I. This suppressor is allele specific, and thus supports the hypothesis that pI and pIV interact. A selection for IKe phage that can efficiently utilize paired pI and pIV from from f1 led to the isolation of a phage with a mutation in gene VIII, which encodes the major coat protein of the virus. Analysis of the system suggests that it is pI that interacts with both pIV and pVIII. Thus the process by which filamentous phage are concomitantly assembled and secreted across the cell membranes is likely to involve a series of protein-protein interactions that are accessible to genetic analysis.

Alleles↗
PubMed1993

Analysis of the structure and subcellular location of filamentous phage pIV.

The gene IV protein of filamentous bacteriophages is an integral membrane protein required for phage assembly and export. A series of gene IV::phoA fusion, gene IV deletion, and gene IV missense mutations have been isolated and characterized. The alkaline phosphatase activity of the fusion proteins suggests that pIV lacks a cytoplasmic domain. Cell fractionation studies indicate that the carboxy-terminal half of pIV mediates its assembly into the membrane, although there is no single, discrete membrane localization domain. The properties of gene IV missense and deletion mutants, combined with an analysis of the similarities between pIVs from various filamentous phage and related bacterial export-mediating proteins, suggest that the amino-terminal half of pIV consists of a periplasmic substrate-binding domain that confers specificity to the assembly-export system.

Alkaline Phosphatase↗
PubMed1992

Construction of a microphage variant of filamentous bacteriophage.

The intergenic region in the genome of the Ff class of filamentous phage (comprising strains fl, fd and M13) genome constitutes 8% of the viral genome, and has essential functions in DNA replication and phage morphogenesis. The functional domains of this region may be inserted into separate sites of a plasmid to function independently. Here, we demonstrate the construction of a plasmid containing, sequentially, the origin of (+)-strand synthesis, the packaging signal and a terminator of (+)-strand synthesis. When host cells harboring this plasmid (pLS7) are infected with helper phage they produce a microphage particle containing all the structural elements of the mature, native phage. The microphage is 65 A in diameter and about 500 A long. It contains a 221-base single-stranded circle of DNA coated by about 95 copies of the major coat protein (gene 8 protein).

Bacteriophage M13↗
PubMed1992

Interchangeability of related proteins and autonomy of function. The morphogenetic proteins of filamentous phage f1 and IKe cannot replace one another.

The filamentous phage f1 and IKe infect a common host, are structurally highly similar and exhibit 55% identity at the DNA sequence level. Based on the idea that proteins that function autonomously will be more tolerant of multiple amino acid differences than proteins that must interact with other proteins to function, the ability of four individual proteins from f1 to substitute for their IKe equivalents to promote virus assembly in vivo has been examined. The reciprocal replacements were also examined. Only the single-strand DNA binding proteins (pV) were fully interchangeable. A minor capsid protein, pIX, was unable to substitute in assembly of the heterologous phage. Two proteins required for particle assembly that are not part of the phage particle, pI and pIV, were not interchangeable, although pIVf1 stimulated formation of a very small number of IKe particles in the absence of pIVIKe. The lack of interchangeability suggests that these morphogenetic proteins do not function autonomously, but rather interact with one or more phage proteins. The ability of certain overproduced proteins to interfere with assembly of wild-type f1 or IKe forms the basis for a model that suggests that phage assembly requires an interaction between pI and pIV.

Cloning, Molecular↗
PubMed1992

A modified TnphoA useful for single-stranded DNA sequencing.

The TnphoA transposon constructed by Manoil and Beckwith [Proc. Natl. Acad. Sci. USA 82 (1985) 8129-8133] has been modified to permit easy isolation of single-stranded (ss) DNA of target plasmids. The intergenic region (IG) of filamentous phage f1, which consists of the phage origin of replication and packaging signal, was inserted into a nonessential region of TnphoA. This modified transposon should be useful for the analysis of genes cloned in plasmids that lack a filamentous phage IG. Transposition of TnphoA-IG into a plasmid carries the IG with it; subsequently, after infection with a filamentous helper phage, ss plasmid DNA suitable for sequence analysis and useful for oligodeoxyribonucleotide-mediated mutagenesis of TnphoA-generated fusions can be isolated. The utility of TnphoA-IG was confirmed by analysis of 'blue hops' into the bla (encoding beta-lactamase) and pspE (encoding phage shock protein) genes whose products are secreted into the Escherichia coli periplasm.

Alkaline Phosphatase↗
PubMed1991

Filamentous phage assembly.

Filamentous phages present a genetically well-defined system for studying the ordered membrane assembly of five different phage-encoded proteins around the circular single-stranded DNA phage genome. Assembly occurs at high efficiency in vivo, catalysed by two phage-encoded membrane proteins and at least one host protein, thioredoxin. This review presents a description of the virion and its cytoplasmic precursor and summarizes the results of genetic and biochemical experiments that are beginning to elucidate the role of the three morphogenetic proteins. The recent discovery of bacterial transport proteins with homology to a phage morphogenetic protein located in the outer membrane suggests the existence of a common mechanism for moving complex macromolecules across bacterial membranes.

Amino Acid Sequence↗
PubMed1990

Prokaryotic secretion.

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Bacterial Outer Membrane Proteins↗
PubMed1990

Secretion and membrane integration of a filamentous phage-encoded morphogenetic protein.

The filamentous phage-encoded gene IV protein is required at high levels for virus assembly, although it is not a constituent of the virion. It is an integral membrane protein that does not contain an extended hydrophobic region of the kind often required for stable integration in the inner membrane. Rather, like a number of Escherichia coli outer membrane proteins, pIV is rich in charged amino acid residues and is predicted to consist of extensive beta-sheet structures. In phage-producing cells, pIV is primarily detected in the outer membrane, while in cells that produce it from the cloned gene, pIV is found in both the inner and outer membranes. The protein is synthesized as a precursor. Following cleavage of the signal sequence and translocation into the periplasm, the mature form is initially found as a soluble species. Soluble pIV then integrates into the membrane with a half-time of one to two minutes. Neither phage assembly nor other phage proteins are needed for this membrane integration, and phage assembly does not require the presence of the soluble form. The gene IV protein may be part of the structure through which the assembling phage is extruded.

Amino Acid Sequence↗
PubMed1990

Phage shock protein, a stress protein of Escherichia coli.

Filamentous phage infection induces the synthesis of large amounts of an Escherichia coli protein, phage shock protein (Psp), the product of a previously undescribed gene. This induction is due to the phage gene IV protein, pIV, an integral membrane protein. The uninduced level of Psp is undetectable, but when induced by prolonged synthesis of pIV, it can become one of the most abundant proteins in the cell. Psp is also synthesized transiently in response to several stresses (heat, ethanol, and osmotic shock). High-level synthesis occurs only after extreme treatment. Unlike the members of the heat shock regulon, Psp induction does not require the heat shock sigma factor, sigma 32; some stimuli that elicit sigma 32-dependent heat shock proteins do not induce Psp synthesis. The level of Psp induction after extreme stress is even higher in sigma 32 mutant cells, which are unable to mount a normal heat shock response, suggesting that these parallel stress responses are interrelated.

Bacterial Proteins↗
PubMed1990

Thioredoxin or glutaredoxin in Escherichia coli is essential for sulfate reduction but not for deoxyribonucleotide synthesis.

We have shown previously that Escherichia coli cells constructed to lack both thioredoxin and glutaredoxin are not viable unless they also acquire an additional mutation, which we called X. Here we show that X is a cysA mutation. Our data suggest that the inviability of a trxA grx double mutant is due to the accumulation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), an intermediate in the sulfate assimilation pathway. The presence of excess cystine at a concentration sufficient to repress the sulfate assimilation pathway obviates the need for an X mutation and prevents the lethality of a novel cys+ trxA grx double mutant designated strain A522. Mutations in genes required for PAPS synthesis (cysA or cysC) protect cells from the otherwise lethal effect of elimination of both thioredoxin and glutaredoxin even in the absence of excess cystine. Both thioredoxin and glutaredoxin have been shown to be hydrogen donors for PAPS reductase (cysH) in vitro (M. L.-S. Tsang, J. Bacteriol. 146:1059-1066, 1981), and one or the other of these compounds is presumably essential in vivo for growth on minimal medium containing sulfate as the sulfur source. The cells which lack both thioredoxin and glutaredoxin require cystine or glutathione for growth on minimal medium but maintain an active ribonucleotide reduction system. Thus, E. coli must contain a third hydrogen donor active with ribonucleotide reductase.

Adenine Nucleotides↗
PubMed1989

Crystallization and preliminary x-ray characterization of thioredoxin reductase from Escherichia coli.

Single crystals of thioredoxin reductase, suitable for x-ray diffraction studies, have been obtained at room temperature by vapor diffusion of 10-20 mg/ml protein solution against 35% polyethylene glycol containing 200 mM ammonium sulfate. Good quality crystals appear spontaneously only from a protein solution that had been stored for more than a year at 4 degrees C, although large single crystals are reproducibly obtained from fresh protein solutions by micro-seeding. The space group is P6(3)22 (a = b = 123.8 A, c = 81.6 A), with one monomer of the enzyme (34.5 kDa) in the crystallographic asymmetric unit. The crystals are well ordered and diffract to beyond 2 A resolution.

Crystallization↗