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A S Lynch

Publications and source records attributed to A S Lynch.

17 recordsLinked to original sources
PubMed1999

A mutational study of the ArcA-P binding sequences in the aldA promoter of Escherichia coli.

The aldA gene (encoding aldehyde dehydrogenase) of Escherichia coli is anaerobically repressed by ArcA-P, the phosphorylated response regulator of the ArcB/A two-component signal transduction system. The promoter region of aldA contains two 10-bp sequences (5'-TGTTAATTAA-3') that perfectly match the proposed ArcA-P binding consensus (5'-[A/T]GTTAATTA[A/T]-3'). One consensus sequence is on the coding strand (-13 to -4 from the transcriptional start point), whereas the other is on the template strand (position -2 to -11). In this study we used the aldA promoter to test the validity of the proposed consensus sequence. DNase I protection experiments confirmed the 10-bp sequence to be a strong ArcA-P binding site. Alteration of the wild-type sequence from 5'-TGTTAATTAAC-3' to 5'-TCTTAATTAAG-3' or 5'-TATTAATTAAT-3' by site-directed mutagenesis markedly decreased the in vitro affinity of the promoter region for ArcA-P, and abolished the anaerobic repression of mutant att lambda::phi (aldA'-lacZ) transcriptional reporter constructs. Both the in vitro and in vivo results therefore support the proposed consensus sequence.

Aldehyde Dehydrogenase↗
PubMed1997

Regulation of Escherichia coli cell envelope proteins involved in protein folding and degradation by the Cpx two-component system.

We show that the two-component signal transduction system of Escherichia coli, CpxA-CpxR, controls the expression of genes encoding cell envelope proteins involved in protein folding and degradation. These findings are based on three lines of evidence. First, activation of the Cpx pathway induces 5- to 10-fold the synthesis of DsbA, required for disulfide bond formation, and DegP, a major periplasmic protease. Second, using electrophoretic mobility shift and DNase I protection assays, we have shown that phosphorylated CpxR binds to elements upstream of the transcription start sites of dsbA, degP, and ppiA (rotA), the latter coding for a peptidyl-prolyl cis/trans isomerase. Third, we have demonstrated increased in vivo transcription of all three genes, dsbA, degP, and ppiA, when the Cpx pathway is activated. We have identified a putative CpxR consensus binding site that is found upstream of a number of other E. coli genes. These findings suggest a potentially extensive Cpx regulon including genes transcribed by sigma70 and sigma(E), which encode factors involved in protein folding as well as other cellular functions.

Amino Acid Isomerases↗
PubMed1997

In vitro phosphorylation study of the arc two-component signal transduction system of Escherichia coli.

The ArcB and ArcA proteins constitute a two-component signal transduction system that plays a broad role in transcriptional regulation. Under anoxic or environmentally reducing conditions, the sensor kinase (ArcB) is stimulated to autophosphorylate at the expense of ATP and subsequently transphosphorylates the response regulator (ArcA). ArcB is a complex, membrane-bound protein comprising at least three cytoplasmic domains, an N-terminal transmitter domain with a conserved His292 residue (H1), a central receiver domain with a conserved Asp576 residue (D1), and a C-terminal alternative transmitter domain with a conserved His717 residue (H2). To study the phosphoryl transfer pathways of the Arc system, we prepared the following His-tagged proteins: H1, D1, H2, H1-D1, D1-H2, H1-D1-H2, and ArcA. Incubations of various combinations of Arc proteins with [gamma-32P]ATP indicated that H1, but not D1 or H2, catalyzes autophosphorylation; that H1-P transfers the phosphoryl group to D1 much more rapidly than to ArcA; and that D1 accelerates the transphosphorylation of H2. Finally, ArcA is phosphorylated much more rapidly by H2-P than by H1-P. Available data are consistent with a signal transduction model in which (i) reception of a membrane signal(s) triggers autophosphorylation of H1 at His292, (ii) the phosphoryl group can migrate to D1 at Asp576 and subsequently to H2 at His717, and (iii) ArcA receives the phosphoryl group from either His292 or His717, the relative contribution of which is regulated by cytosolic effectors.

Bacterial Outer Membrane Proteins↗
PubMed1996

Molecular dissection of a protein SopB essential for Escherichia coli F plasmid partition.

Biochemical and genetic experiments were carried out to deduce the structural and functional domains of SopB protein involved in the equipartition of F plasmid. The protein is dimeric. Proteolytic and chemical footprinting studies support earlier genetic analyses that the binding of SopB to specific sites within the F plasmid sopC locus involves mainly the C-terminal region. In vivo, the expression of a high level of SopB protein is known to repress sopC-linked genes. This silencing activity is shown to be unaffected by the deletion of 35 N-terminal residues, but abolished when 71 or more were removed from the N terminus. An excess of SopB protein does not extend its in vitro binding outside sopC, implicating participation of a host factor(s) in SopB-mediated gene silencing. A data base search identified a number of SopB homologues, including both chromosomally encoded bacterial proteins and phage- and plasmid-encoded proteins known to be involved in partition. Sequence homology is limited to the N-terminal half, suggesting that the N-terminal regions of these proteins are conserved to interact with a conserved cellular structure(s), whereas the C-terminal regions have diverged to bind different nucleotide sequences.

Amino Acid Sequence↗
PubMed1996

Transcriptional control mediated by the ArcA two-component response regulator protein of Escherichia coli: characterization of DNA binding at target promoters.

ArcA protein bearing an amino-terminal, oligohistidine extension has been purified, and its DNA binding activity has been characterized with or without prior incubation with carbamoyl phosphate. Electrophoretic mobility shift assays and DNase I protection assays indicate that where the phosphorylated form of the ArcA protein (ArcA-P) is expected to act as a transcriptional repressor (e.g., of lctPRD and gltA-sdhCDAB), the effect is likely to be mediated by sequestration of cis-controlling transcriptional regulatory elements. In contrast, in the case of cydAB, for which ArcA-P is expected to function as a transcriptional activator, two discrete binding sites have been identified upstream of a known promoter, and activation from these sites is likely to be mediated by a mechanism typical of the type I class of prokaryotic transcriptional activators. An additional ArcA-P binding site has also been located downstream of the known promoter, and a distinct role for this site in the regulation of the cydAB operon during anoxic growth transitions is suggested. These results are discussed within the framework of an overall model of signaling by the Arc two-component signal transduction system in response to changes in aerobiosis.

Bacterial Outer Membrane Proteins↗
PubMed1995

SopB protein-mediated silencing of genes linked to the sopC locus of Escherichia coli F plasmid.

Expression of a high level of F-plasmid-encoded SopB protein in Escherichia coli is found to repress genes linked to sopC, a sequence element of F consisting of 12 tandemly joined imperfect repeats of a 43-bp motif. Repression of a gene can occur over a distance of at least 10 kb from the sopC element and is not affected by the relative orientation of sopC. In the repressed state, accessibility of intracellular DNA to cellular proteins is greatly reduced in the region containing sopC, as monitored by the trapping of the covalent intermediate between DNA and DNA gyrase and by Dam methylase-catalyzed DNA methylation. These results signify the formation of a nucleoprotein structure emanating from sopC and are discussed in terms of position-dependent silencing of genes in general and the IncG type of plasmid incompatibility in particular.

Ampicillin↗
PubMed1995

Developmental expression pattern screen for genes predicted in the C. elegans genome sequencing project.

Maximum use should be made of information generated in the genome sequencing projects. Toward this end, we have initiated a genome sequence-based, expression pattern screen of genes predicted from the Caenorhabditis elegans genome sequence data. We examined beta-galactosidase expression patterns in C. elegans lines transformed with lacZ reporter gene fusions constructed using predicted C. elegans gene promoter regions. Of the predicted genes in the cosmids analysed so far, 67% are amenable to the approach and 54% of examined genes yielded a developmental expression pattern. Expression pattern information is being made generally available using computer databases.

Animals↗
PubMed1994

Characterization of the CysB protein of Klebsiella aerogenes: direct evidence that N-acetylserine rather than O-acetylserine serves as the inducer of the cysteine regulon.

The cysB gene of Klebsiella aerogenes has been cloned, sequenced and shown to complement the cysteine auxotrophic phenotype of Escherichia coli cysB mutants. The K. aerogenes cysB gene is predicted to encode a protein of 324 amino acid residues that shares approx. 95% sequence similarity with the Salmonella typhimurium and E. coli CysB proteins. Gel-retardation assays demonstrate that the purified protein binds to DNA fragments containing either the K. aerogenes cysb promoter or the S. typhimurium cysJIH promoter. Acetylserine enhances CysB binding to the cysJIH promoter fragment while diminishing its binding to the cysB promoter fragment. Fluorescence-emission-spectroscopy measurements suggest strongly that N-acetylserine binds to CysB apoprotein but that O-acetylserine does not, and support the notion that N-acetylserine is the physiological inducer of cysteine biosynthesis.

Amino Acid Sequence↗
PubMed1994

Use of an inducible site-specific recombinase to probe the structure of protein-DNA complexes involved in F plasmid partition in Escherichia coli.

The induced expression of a tightly regulated site-specific recombinase is shown to efficiently form intracellular DNA rings of well-defined nucleotide sequences in Escherichia coli. To provide information on the organization of an intracellular protein-DNA complex, the linking number distributions of excised DNA rings containing cognate binding sites for the protein can be measured after their isolation. Application of this approach to the partition system of the E. coli F plasmid suggests that the SopB protein and the sopC locus, the latter being composed of 12 tandemly joined imperfect repeats of a 43 base-pair motif, form a complex in which the DNA is wrapped right-handedly around a multimeric protein core; the presence of a single copy of a 43 base-pair motif on a DNA appears to be sufficient to nucleate the formation of this nucleoprotein complex.

Bacterial Proteins↗
PubMed1993

Transcription and DNA supercoiling.

Transcription and supercoiling of the DNA template are interrelated. This review summarizes recent progress in the study of how template topology affects transcription, and how transcription affects template topology inside wild-type and DNA topoisomerase mutant cells. The interplay between DNA supercoiling and transcription raises interesting questions on the regulation of adjacent genes, the organization of intracellular DNA, and the coupling between transcription and other cellular processes involving DNA.

Animals↗
PubMed1993

Anchoring of DNA to the bacterial cytoplasmic membrane through cotranscriptional synthesis of polypeptides encoding membrane proteins or proteins for export: a mechanism of plasmid hypernegative supercoiling in mutants deficient in DNA topoisomerase I.

A homologous set of plasmids expressing tet, lacY, and melB, genes encoding integral cytoplasmic membrane proteins, and tolC and ampC, genes encoding proteins for export through the cytoplasmic membrane, was constructed for studying the effects of transcription and translation of such genes on the hypernegative supercoiling of plasmids in Escherichia coli cells deficient in DNA topoisomerase I. The results support the view that intracellular bacterial DNA is anchored to the cytoplasmic membrane at many points through cotranscriptional synthesis of membrane proteins or proteins designated for export across the cytoplasmic membrane; in the latter case, the presence of the signal peptide appears to be unnecessary for cotranscriptional membrane association.

Base Sequence↗
PubMed1989

DNA supercoiling in Escherichia coli: topA mutations can be suppressed by DNA amplifications involving the tolC locus.

The level of DNA supercoiling is crucial for many cellular processes, including gene expression, and is determined, primarily, by the opposing actions of two enzymes: topoisomerase I and DNA gyrase. Escherichia coli strains lacking topoisomerase I (topA mutants) normally fail to grow in the absence of compensatory mutations which are presumed to relax DNA. We have found that, in media of low osmolarity, topA mutants are viable in the absence of any compensatory mutation, consistent with the view that decreased extracellular osmolarity causes a relaxation of cellular DNA. At higher osmolarity most compensatory mutations, as expected, are in the gyrA and gyrB genes. The only other locus at which compensatory mutations arise, designated toc, is shown to involve the amplification of a region of chromosomal DNA which includes the tolC gene. However, amplification of tolC alone is insufficient to explain the phenotypes of toc mutants. tolC insertion mutations alter the distribution of plasmid topoisomers in vivo. This effect is probably indirect, possibly a result of altered membrane structure and an alteration in the cell's osmotic barrier. As tolC is a highly pleiotropic locus, affecting the expression of many genes, it is possible that some of the TolC phenotypes are a direct result of this topological change. The possible relationship between toc and tolC mutations, and the means by which tolC mutations might affect DNA supercoiling, are discussed.

Blotting, Southern↗