Search tips
Search criteria

Results 1-14 (14)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
more »
Document Types
1.  Autogenous control of PspF, a constitutively active enhancer-binding protein of Escherichia coli. 
Journal of Bacteriology  1997;179(16):5232-5237.
Escherichia coli sigma54-dependent phage shock protein operon (pspA to -E) transcription is under the control of PspF, a constitutively active activator. Sigma70-dependent transcription of pspF is under autogenous control by wild-type PspF but not by a DNA-binding mutant, PspF deltaHTH. Negative autoregulation of PspF is continual and not affected by stimuli, like f1 pIV, that induce the pspA to -E operon. PspF production is independent of PspA (the negative regulator of the pspA to -E operon) and of PspB and -C (positive regulators).
PMCID: PMC179386  PMID: 9260970
2.  The RIB element in the goaG-pspF intergenic region of Escherichia coli. 
Journal of Bacteriology  1997;179(10):3095-3102.
The sequence (2,700 bp) between the aldH and pspF genes of Escherichia coli was determined. The pspF gene encodes a sigma54 transcriptional activator of the phage shock protein (psp) operon (pspA to pspE). Downstream of the pspF transcribed region are two open reading frames (ORFs), ordL and goaG, convergently oriented with respect to pspF. These two ORFs, together with the adjacent aldH gene, may constitute a novel operon (aldH-ordL-goaG). The goaG-pspF intergenic region contains a complex extragenic mosaic element, RIB. The structure of this RIB element, which belongs to the BIME-1 family, is Y(REP1) > 16 < Z1(REP2), where Y and Z1 are palindromic units and the central 16 bases contain an L motif with an ihf consensus sequence. DNA fragments containing the L motif of the psp RIB element effectively bind integration host factor (IHF), while the Y palindromic unit (REP1) of the same RIB element binds DNA gyrase weakly. Computer prediction of the pspF mRNA secondary structure suggested that the transcribed stem-loop structures formed by the 3'-flanking region of the pspF transcript containing the RIB element can stabilize and protect pspF mRNA. Analysis of pspF steady-state mRNA levels showed that transcripts with an intact RIB element are much more abundant than those truncated at the 3' end by deletion of either the entire RIB element or a single Z1 sequence (REP2). Thus, the pspF 3'-flanking region containing the RIB element has an important role in the stabilization of the pspF transcript.
PMCID: PMC179083  PMID: 9150200
3.  Essential role of a sodium dodecyl sulfate-resistant protein IV multimer in assembly-export of filamentous phage. 
Journal of Bacteriology  1996;178(7):1962-1970.
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.
PMCID: PMC177892  PMID: 8606171
4.  Identification, nucleotide sequence, and characterization of PspF, the transcriptional activator of the Escherichia coli stress-induced psp operon. 
Journal of Bacteriology  1996;178(7):1936-1945.
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.
PMCID: PMC177889  PMID: 8606168
5.  Thioredoxin or glutaredoxin in Escherichia coli is essential for sulfate reduction but not for deoxyribonucleotide synthesis. 
Journal of Bacteriology  1990;172(4):1923-1929.
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.
PMCID: PMC208687  PMID: 2180911
6.  Genetic analysis of the filamentous bacteriophage packaging signal and of the proteins that interact with it. 
Journal of Virology  1989;63(8):3284-3295.
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.
PMCID: PMC250900  PMID: 2746731
7.  Site-specific methylases induce the SOS DNA repair response in Escherichia coli. 
Journal of Bacteriology  1987;169(7):3243-3250.
Expression of the site-specific adenine methylase HhaII (GmeANTC, where me is methyl) or PstI (CTGCmeAG) induced the SOS DNA repair response in Escherichia coli. In contrast, expression of methylases indigenous to E. coli either did not induce SOS (EcoRI [GAmeATTC] or induced SOS to a lesser extent (dam [GmeATC]). Recognition of adenine-methylated DNA required the product of a previously undescribed gene, which we named mrr (methylated adenine recognition and restriction). We suggest that mrr encodes an endonuclease that cleaves DNA containing N6-methyladenine and that DNA double-strand breaks induce the SOS response. Cytosine methylases foreign to E. coli (MspI [meCCGG], HaeIII [GGmeCC], BamHI [GGATmeCC], HhaI [GmeCGC], BsuRI [GGmeCC], and M.Spr) also induced SOS, whereas one indigenous to E. coli (EcoRII [CmeCA/TGG]) did not. SOS induction by cytosine methylation required the rglB locus, which encodes an endonuclease that cleaves DNA containing 5-hydroxymethyl- or 5-methylcytosine (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986).
PMCID: PMC212376  PMID: 3036779
8.  Direct cloning of the trxB gene that encodes thioredoxin reductase. 
Journal of Bacteriology  1985;163(1):238-242.
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.
PMCID: PMC219103  PMID: 2989245
9.  Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. 
Journal of Bacteriology  1984;159(3):1034-1039.
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.
PMCID: PMC215764  PMID: 6384177
10.  Characterization of the cloned fip gene and its product. 
Journal of Bacteriology  1984;157(2):526-532.
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.
PMCID: PMC215278  PMID: 6319363
11.  A bacterial gene, fip, required for filamentous bacteriophage fl assembly. 
Journal of Bacteriology  1983;154(3):1064-1076.
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.
PMCID: PMC217576  PMID: 6343342
12.  Processing of alkaline phosphatase precursor to the mature enzyme by an Escherichia coli inner membrane preparation. 
Journal of Bacteriology  1980;142(2):726-728.
An inner membrane preparation co-translationally cleaved both the alkaline phosphatase and bacteriophage f1 coat protein precursors to the mature proteins. Post-translational outer membrane proteolysis of pre-alkaline phosphatase generated a protein smaller than the authentic monomer.
PMCID: PMC294062  PMID: 6991486
13.  Mechanism of export of colicin E1 and colicin E3. 
Journal of Bacteriology  1979;138(3):770-778.
The mechanism of export of colicins E1 and E3 was examined. Neither colicin E1, colicin E3, Nor colicin E3 immunity protein appears to be synthesized as a precursor protein with an amino-terminal extension. Instead, the colicins, as well as the colicin E3 immunity protein, appear to leave the cells where they are made, long after their synthesis, by a nonspecific mechanism which results in increased permeability of the producing cells. Induction of ColE3-containing cells with mitomycin C leads to actual lysis of those cells, as some time after synthesis of the colicin E3 and its immunity protein has been completed. Induction of ColE1-containing cells results in increased permeability of the cells, but not in actual lysis, and most of the colicin E1 produced never leaves the producing cells. Intracellular proteins such as elongation factor G can be found outside of colicinogenic cells after mitomycin C induction, along with the colicin. Until substantial increases in permeability occur, most of the colicin remains cell associated, in the soluble cytosol, rather than in a membrane-associated form.
PMCID: PMC218103  PMID: 378936
14.  Analysis of the proteins and cis-acting elements regulating the stress-induced phage shock protein operon. 
Nucleic Acids Research  1995;23(11):2030-2036.
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.
PMCID: PMC306980  PMID: 7596833

Results 1-14 (14)