Search PubMed⌕ Search

Biomedical subjects

P Model

Publications and source records attributed to P Model.

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

Identification, nucleotide sequence, and characterization of PspF, the transcriptional activator of the Escherichia coli stress-induced psp operon.

The phage shock protein (psp) operon (pspABCE) of Escherichia coli is strongly induced in response to a variety of stressful conditions or agents such as filamentous phage infection, ethanol treatment, osmotic shock, heat shock, and prolonged incubation in stationary phase. Transcription of the psp operon is driven from a sigma54 promoter and stimulated by integration host factor. We report here the identification of a transcriptional activator gene, designated pspF, which controls expression of the psp operon in E. coli. The pspF gene was identified by random miniTn10-tet transposon mutagenesis. Insertion of the transposon into the pspF gene abolished sigma54-dependent induction of the psp operon. The pspF gene is closely linked to the psp operon and is divergently transcribed from one major and two minor sigma 70 promoters, pspF encodes a 37-kDa protein which belongs to the enhancer-binding protein family of sigma54 transcriptional activators. PspF contains a catalytic domain, which in other sigma54 activators would be the central domain, and a C-terminal DNA-binding domain but entirely lacks an N-terminal regulatory domain and is constitutively active. The insertion mutant pspF::mTn10-tet (pspF877) encodes a truncated protein (PspF delta HTH) that lacks the DNA-binding helix-turn-helix (HTH) motif. Although the central catalytic domain is intact, PspF delta HTH at physiological concentration cannot activate psp expression. In the absence of inducing stimuli, multicopy-plasmid-borne PspF or PspF delta HTH overcomes repression of the psp operon mediated by the negative regulator PspA.

Amino Acid Sequence↗
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

Location of filamentous phage minor coat proteins in phage and in infected cells.

We show that all minor coat proteins of phage f1 are integral inner membrane proteins prior to assembly. Hence all phage structural and morphogenetic proteins are localized in the membrane of the infected cell, consistent with models of phage assembly in which morphogenesis is coincident with phage extrusion. Our data suggest that the minor coat proteins, pVI and pIII, are already associated with the major coat protein, pVIII, in the membrane. On the other hand pVI and pIII are not recovered as a complex from the membrane, even though experiments with dissociated phage show they are associated in phage. The minor coat proteins, pVII and pVIII, are also associated in phage. With the use of sera directed against the minor proteins, we show that the minor coat protein, pIX, is accessible in intact phage, but pVI and pVII are not. Consistent with earlier results, the attachment protein, pIII, clearly is exposed on the phage exterior. Infected cells contain about ten times more pVII and pIX than is incorporated into phage.

Base Sequence↗
PubMed1995

Analysis of the proteins and cis-acting elements regulating the stress-induced phage shock protein operon.

The phage shock protein operon (pspABCE) of Escherichia coli is strongly induced by adverse environmental conditions. Expression is controlled principally at the transcriptional level, and transcription is directed by the sigma factor sigma 54. PspB and PspC are required for high-level psp expression during osmotic shock, ethanol treatment and f1 infection, but heat-induced expression is independent of these proteins. We report here that the promoter region contains an upstream activation sequence (UAS) that is required for psp induction and has the enhancer-like ability to activate at a distance. A DNA-binding activity is detected in crude protein extracts that is dependent on the UAS and induced by heat shock. We further show that integration host factor (IHF) binds in vitro to a site between the UAS and sigma 54 recognition sequence. In bacteria lacking IHF, psp expression is substantially reduced in response to high temperature and ethanol. During osmotic shock in contrast, psp expression is only weakly stimulated by IHF, and IHF mutants can strongly induce the operon. The dependence of psp expression on IHF varies with the inducing condition, but does not correlate with dependence on PspB and PspC, indicating distinct, agent-specific activation mechanisms.

Bacterial Proteins↗
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

Role of an Escherichia coli stress-response operon in stationary-phase survival.

The phage shock protein operon (pspABCE) of Escherichia coli is strongly expressed in response to stressful environmental conditions, such as heat shock, ethanol treatment, osmotic shock, and filamentous phage infection. We show that bacteria lacking the pspABC genes exhibit a substantial decrease in the ability to survive prolonged incubation in stationary phase under alkaline conditions (pH 9). The psp mutant bacteria grow approximately as well as wild-type strains in the alkaline medium, and stationary-phase survival of the psp mutants improves substantially at pH values closer to the optimal growth range (pH 6-8). In late stationary-phase (1- to 2-day-old) cells, the operon can be strongly induced under certain conditions, and PspA can become one of the most highly expressed bacterial proteins. The combination of stationary-phase starvation and alkaline pH is likely to place a severe strain on the maintenance of endogenous energy sources, and, consistent with these effects, we find that psp expression is also induced by uncouplers of oxidative phosphorylation and other agents that interfere with energy production. The death rate of psp mutants in stationary phase is accelerated by the presence of wild-type bacteria in the same culture, suggesting that the psp operon may play a significant role in enabling E. coli to compete for survival under nutrient- or energy-limited conditions.

Bacterial Proteins↗
PubMed1994

Characterization by 1H NMR of a C32S,C35S double mutant of Escherichia coli thioredoxin confirms its resemblance to the reduced wild-type protein.

A mutant of Escherichia coli thioredoxin containing serine residues in place of the two active-site cysteines, termed C32S,C35S, previously shown to be partially able to substitute for reduced thioredoxin in certain phage systems, has been characterized by 1H NMR spectroscopy at pH values between 5.5 and 10. The 1H NMR spectrum of the mutant at pH 5.5 is very similar to that of the wild-type protein in either the reduced or oxidized state. Chemical shift changes in the vicinity of the active site serines indicate that the nearby hydrophobic pocket is somewhat changed, probably as a result of the replacement of the cysteine thiols with the smaller, more hydrophilic hydroxyl side chains and a change in the preferred chi 1 angles of the side chains. Although the pattern of amide protons persistent in 2H2O differs only slightly between the two forms of the wild-type protein, the pattern observed for the C32S,C35S mutant shows characteristic features that correspond closely with those of the reduced wild-type protein rather than with the oxidized form. The pH dependence of the mutant protein shows a single group titrating close to the active site with a pKa of 8.3, which we assign to the buried carboxyl group of Asp 26 by analogy with the behavior of wild-type thioredoxin. The pKa is significantly higher for the mutant protein, consistent with an increase in the hydrophobicity of the pocket where the carboxyl is buried, probably due to repacking caused by the removal of the cysteine thiols and the placement of the serine hydroxyls in positions where they interact better with solvent. The results demonstrate that the solution behavior of the mutant protein is similar in many ways to that of reduced wild-type thioredoxin, explaining its partial activity in the two essential roles of reduced thioredoxin as a subunit of phage T7 DNA polymerase and in the assembly of filamentous phage.

Amino Acid Sequence↗
PubMed1993

Cell wall sorting signals in surface proteins of gram-positive bacteria.

Staphylococcal protein A is anchored to the cell wall, a unique cellular compartment of Gram-positive bacteria. The sorting signal sufficient for cell wall anchoring consists of an LPXTG motif, a C-terminal hydrophobic domain and a charged tail. Homologous sequences are found in many surface proteins of Gram-positive bacteria and we explored the universality of these sequences to serve as cell wall sorting signals. We show that several signals are able to anchor fusion proteins to the staphylococcal cell wall. Some signals do not sort effectively, but acquire sorting activity once the spacing between the LPXTG motif and the charged tail has been increased to span the same length as in protein A. Thus, signals for cell wall anchoring in Gram-positive bacteria are as universal as signal (leader) sequences.

Amino Acid Sequence↗
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

Sorting of protein A to the staphylococcal cell wall.

The cell wall of gram-positive bacteria can be thought of as representing a unique cell compartment, which contains anchored surface proteins that require specific sorting signals. Some biologically important products are anchored in this way, including protein A and fibronectin binding protein of Staphylococcus aureus and streptococcal M protein. Studies of staphylococcal protein A and Escherichia coli alkaline phosphatase show that the signal both necessary and sufficient for cell wall anchoring consists of an LPXTGX motif, a C-terminal hydrophobic domain, and a charged tail. These sequence elements are conserved in many surface proteins from different gram-positive bacteria. We propose the existence of a hitherto undescribed sorting mechanism that positions proteins on the surface of gram-positive bacteria.

Amino Acid Sequence↗
PubMed1991

SOS induction as an in vivo assay of enzyme-DNA interactions.

We have constructed strains which are convenient and sensitive indicators of DNA damage and describe their use. These strains utilize an SOS::lac Z fusion constructed by Kenyon and Walker [Proc. Natl. Acad. Sci. USA 77 (1980) 2819-2823] and respond to DNA damage by producing beta-galactosidase. They can be used to characterize restriction systems and screen for restriction endonuclease mutants. Applications include the study of other enzymes involved in DNA metabolism, such as DNA methyltransferases, topoisomerases, recombinases, and DNA replication and repair enzymes.

DNA↗
PubMed1991

Convergent evolution of similar function in two structurally divergent enzymes.

An example of two related enzymes that catalyse similar reactions but possess different active sites is provided by comparing the structure of Escherichia coli thioredoxin reductase with glutathione reductase. Both are dimeric enzymes that catalyse the reduction of disulphides by pyridine nucleotides through an enzyme disulphide and a flavin. Human glutathione reductase contains four structural domains within each molecule: the flavin-adenine dinucleotide (FAD)- and nicotinamide-adenine dinucleotide phosphate (NADPH)-binding domains, the 'central' domain and the C-terminal domain that provides the dimer interface and part of the active site. Although both enzymes share the same catalytic mechanism and similar tertiary structures, their active sites do not resemble each other. We have determined the crystal structure of E. coli thioredoxin reductase at 2 A resolution, and show that thioredoxin reductase lacks the domain that provides the dimer interface in glutathione reductase, and forms a completely different dimeric structure. The catalytically active disulphides are located in different domains on opposite sides of the flavin ring system. This suggests that these enzymes diverged from an ancestral nucleotide-binding protein and acquired their disulphide reductase activities independently.

Amino Acid Sequence↗
PubMed1991

Characterization and sequence of the Escherichia coli stress-induced psp operon.

We describe a new Escherichia coli operon, the phage shock protein (psp) operon, which is induced in response to heat, ethanol, osmotic shock and infection by filamentous bacteriophages. The operon includes at least four genes: pspA, B, C and E. PspA associates with the inner membrane and has the heptad repeats characteristic of proteins that can form coiled coils. The operon encodes a factor that activates psp expression, and deletion analyses indicate that this protein is PspC; PspC is predicted to possess a leucine zipper, a motif present in many eukaryotic transcription factors. The pspE gene is expressed in response to stress as part of the operon, but is also transcribed from its own promoter under normal conditions. In vitro studies suggest that PspA and C are modified in vivo. Expression of the psp genes does not require the heat shock sigma factor, sigma32. The increased duration of psp induction in a sigma32 mutant suggests that a product (or products) of the heat shock response down-regulates expression of the operon.

Amino Acid Sequence↗
PubMed1991

Stress-induced expression of the Escherichia coli phage shock protein operon is dependent on sigma 54 and modulated by positive and negative feedback mechanisms.

The phage shock protein (psp) operon of Escherichia coli is strongly induced in response to heat, ethanol, osmotic shock, and infection by filamentous bacteriophages. The operon contains at least four genes--pspA, pspB, pspC, and pspE--and is regulated at the transcriptional level. We report here that psp expression is controlled by a network of positive and negative regulatory factors and that transcription in response to all inducing agents is directed by the sigma-factor sigma 54. Negative regulation is mediated by both PspA and the sigma 32-dependent heat shock proteins. The PspB and PspC proteins cooperatively activate expression, possibly by antagonizing the PspA-controlled repression. The strength of this activation is determined primarily by the concentration of PspC, whereas PspB enhances but is not absolutely essential for PspC-dependent expression. PspC is predicted to contain a leucine zipper, a motif responsible for the dimerization of many eukaryotic transcriptional activators. PspB and PspC, though not necessary for psp expression during heat shock, are required for the strong psp response to phage infection, osmotic shock, and ethanol treatment. The psp operon thus represents a third category of transcriptional control mechanisms, in addition to the sigma 32- and sigma E-dependent systems, for genes induced by heat and other stresses.

Bacterial Proteins↗
PubMed1990

Prokaryotic secretion.

Explore the source record for details and available documents.

Bacterial Outer Membrane Proteins↗
PubMed1990

Mutants of the EcoRI endonuclease with promiscuous substrate specificity implicate residues involved in substrate recognition.

The EcoRI restriction endonuclease cleaves DNA molecules at the sequence GAATTC. We devised a genetic screen to isolate EcoRI mutants with altered or broadened substrate specificity. In vitro, the purified mutant enzymes cleave both the wild-type substrate and sites which differ from this by one nucleotide (EcoRI star sites). These mutations identify four residues involved in substrate recognition and catalysis that are different from the amino acids proposed to recognize the substrate based on the EcoRI-DNA co-crystal structure. In fact, these mutations suppress EcoRI mutants altered at some of the proposed substrate binding residues (R145, R200). We argue that these mutations permit cleavage of additional DNA sequences either by perturbing or removing direct DNA-protein interactions or by facilitating conformational changes that allosterically couple substrate binding to DNA scission.

Base Sequence↗