Faculty Opinions recommendation of Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research.

ABSTRACT Pseudomonas aeruginosa, a γ-proteobacterium, is motile by means of a single polar flagellum and is chemotactic to a variety of organic compounds and phosphate. P. aeruginosa has multiple homologues of Escherichia coli chemotaxis genes that are organized into five gene clusters. Previously, it was demonstrated that genes in cluster I and cluster V are essential for chemotaxis. A third cluster (cluster II) contains a complete set of che genes, as well as two genes, mcpA and mcpB, encoding methyl-accepting chemotaxis proteins. Mutations were constructed in several of the cluster II che genes and in the mcp genes to examine their possible contributions to P. aeruginosa chemotaxis. A cheB2 mutant was partially impaired in chemotaxis in soft-agar swarm plate assays. Providing cheB2 in trans complemented this defect. Further, overexpression of CheB2 restored chemotaxis to a completely nonchemotactic, cluster I, cheB-deficient strain to near wild-type levels. An mcpA mutant was defective in chemotaxis in media that were low in magnesium. The defect could be relieved by the addition of magnesium to the swarm plate medium. An mcpB mutant was defective in chemotaxis when assayed in dilute rich soft-agar swarm medium or in minimal-medium swarm plates containing any 1 of 60 chemoattractants. The mutant phenotype could be complemented by the addition of mcpB in trans. Overexpression of either McpA or McpB in P. aeruginosa or Escherichia coli resulted in impairment of chemotaxis, and these cells had smooth-swimming phenotypes when observed under the microscope. Expression of P. aeruginosa cheA2, cheB2, or cheW2 in E. coli K-12 completely disrupted wild-type chemotaxis, while expression of cheY2 had no effect. These results indicate that che cluster II genes are expressed in P. aeruginosa and are required for an optimal chemotactic response.

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Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽

10.1093/genetics/125.4.691 ◽

1990 ◽

Vol 125 (4) ◽

pp. 691-702 ◽

Cited By ~ 6

Author(s):

B L Berg ◽

V Stewart

Keyword(s):

Escherichia Coli ◽

Nitrate Reductase ◽

Formate Dehydrogenase ◽

Recombinant Dna ◽

Structural Genes ◽

Respiratory Pathway ◽

E Coli ◽

Formate Oxidation ◽

Operon Expression ◽

K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

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Microbial computational genomics of gene regulation

Pure and Applied Chemistry ◽

10.1351/pac200274060899 ◽

2002 ◽

Vol 74 (6) ◽

pp. 899-905 ◽

Cited By ~ 3

Author(s):

Julio Collado-Vides ◽

Gabriel Moreno-Hagelsieb ◽

Arturo Medrano-Soto

Keyword(s):

Transcription Initiation ◽

Transcription Unit ◽

Microbial Genomes ◽

E Coli ◽

Regulatory Interactions ◽

Living Bacterium ◽

Complete Set ◽

Operon Organization ◽

Unit Organization ◽

K 12

Escherichia coli is a free-living bacterium that condensates a large legacy of knowledge as a result of years of experimental work in molecular biology. It represents a point of departure for analyses and comparisons with the ever-increasing number of finished microbial genomes. For years, we have been gathering knowledge from the literature on transcriptional regulation and operon organization in E. coli K-12, and organizing it in a relational database, RegulonDB. RegulonDB contains information of 2025 % of the expected total sets of regulatory interactions at the level of transcription initiation. We have used this knowledge to generate computational methods to predict the missing sets in the genome of E. coli, focusing on prediction of promoters, regulatory sites, regulatory proteins, operons, and transcription units. These predictions constitute separate pieces of a single puzzle. By putting them all together, we shall be able to predict the complete set of regulatory interactions and transcription unit organization of E. coli. Orthologous genes in other genomes of known co-regulated sets of genes in E. coli, along with their corresponding predicted operons, and their predicted transcriptional regulators, shall permit the extension of the previous goal to many more microbial genomes.

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Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.152.1.81-88.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 81-88

Author(s):

E H Berglin ◽

M B Edlund ◽

G K Nyberg ◽

J Carlsson

Keyword(s):

Escherichia Coli ◽

Hydrogen Peroxide ◽

Metal Ion ◽

Chelating Agent ◽

Fold Increase ◽

Bactericidal Effect ◽

Anaerobic Conditions ◽

Dna Breakage ◽

E Coli ◽

K 12

Under anaerobic conditions an exponentially growing culture of Escherichia coli K-12 was exposed to hydrogen peroxide in the presence of various compounds. Hydrogen peroxide (0.1 mM) together with 0.1 mM L-cysteine or L-cystine killed the organisms more rapidly than 10 mM hydrogen peroxide alone. The exposure of E. coli to hydrogen peroxide in the presence of L-cysteine inhibited some of the catalase. This inhibition, however, could not fully explain the 100-fold increase in hydrogen peroxide sensitivity of the organism in the presence of L-cysteine. Of other compounds tested only some thiols potentiated the bactericidal effect of hydrogen peroxide. These thiols were effective, however, only at concentrations significantly higher than 0.1 mM. The effect of L-cysteine and L-cystine could be annihilated by the metal ion chelating agent 2,2'-bipyridyl. DNA breakage in E. coli K-12 was demonstrated under conditions where the organisms were killed by hydrogen peroxide.

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pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.186.1.192-199.2004 ◽

2004 ◽

Vol 186 (1) ◽

pp. 192-199 ◽

Cited By ~ 65

Author(s):

Elizabeth Yohannes ◽

D. Michael Barnhart ◽

Joan L. Slonczewski

Keyword(s):

Escherichia Coli ◽

Ph Regulation ◽

Metabolic Enzymes ◽

Anaerobic Growth ◽

High Ph ◽

E Coli ◽

Catabolic Enzymes ◽

Periplasmic Proteins ◽

Ph Dependent ◽

K 12

ABSTRACT During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.

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Escherichia coli as a genetic tool

Journal of Hygiene ◽

10.1017/s0022172400060708 ◽

1985 ◽

Vol 95 (3) ◽

pp. 611-618

Author(s):

Naomi Datta

Keyword(s):

Escherichia Coli ◽

Academic Research ◽

Genetically Engineered ◽

Cloning And Expression ◽

Biological Research ◽

New Techniques ◽

E Coli ◽

Genetic Tool ◽

The Past ◽

Made In

SUMMARYThe study of Escherichia coli and its plasmids and bacteriophages has provided a vast body of genetical information, much of it relevant to the whole of biology. This was true even before the development of the new techniques, for cloning and analysing DNA, that have revolutionized biological research during the past decade. Thousands of millions of dollars are now invested in industrial uses of these techniques, which all depend on discoveries made in the course of academic research on E. coli. Much of the background of knowledge necessary for the cloning and expression of genetically engineered information, as well as the techniques themselves, came from work with this organism.

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The Small Noncoding DsrA RNA Is an Acid Resistance Regulator in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.186.18.6179-6185.2004 ◽

2004 ◽

Vol 186 (18) ◽

pp. 6179-6185 ◽

Cited By ~ 65

Author(s):

Richard A. Lease ◽

Dorie Smith ◽

Kathleen McDonough ◽

Marlene Belfort

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Resistance Genes ◽

Acid Resistance ◽

Dna Arrays ◽

E Coli ◽

Specific Mrnas ◽

Steady State Levels ◽

Control Translation ◽

K 12

ABSTRACT DsrA RNA is a small (87-nucleotide) regulatory RNA of Escherichia coli that acts by RNA-RNA interactions to control translation and turnover of specific mRNAs. Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (σs), and H-NS, a histone-like nucleoid protein and global transcription repressor. Genes regulated globally by RpoS and H-NS include stress response proteins and virulence factors for pathogenic E. coli. Here, by using transcription profiling via DNA arrays, we have identified genes induced by DsrA. Steady-state levels of mRNAs from many genes increased with DsrA overproduction, including multiple acid resistance genes of E. coli. Quantitative primer extension analysis verified the induction of individual acid resistance genes in the hdeAB, gadAX, and gadBC operons. E. coli K-12 strains, as well as pathogenic E. coli O157:H7, exhibited compromised acid resistance in dsrA mutants. Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant. Thus, DsrA RNA plays a regulatory role in acid resistance. Whether DsrA targets acid resistance genes directly by base pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in either event, our results suggest that DsrA RNA may enhance the virulence of pathogenic E. coli.

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afa-8 Gene Cluster Is Carried by a Pathogenicity Island Inserted into the tRNAPhe of Human and Bovine Pathogenic Escherichia coli Isolates

Infection and Immunity ◽

10.1128/iai.69.2.937-948.2001 ◽

2001 ◽

Vol 69 (2) ◽

pp. 937-948 ◽

Cited By ~ 48

Author(s):

Lila Lalioui ◽

Chantal Le Bouguénec

Keyword(s):

Escherichia Coli ◽

Direct Repeat ◽

Pathogenicity Island ◽

Genomic Region ◽

Open Reading Frames ◽

Blood Isolate ◽

Distinctive Features ◽

E Coli ◽

Pathogenic Escherichia Coli ◽

K 12

ABSTRACT We recently described a new afimbrial adhesin, AfaE-VIII, produced by animal strains associated with diarrhea and septicemia and by human isolates associated with extraintestinal infections. Here, we report that the afa-8 operon, encoding AfaE-VIII adhesin, from the human blood isolate Escherichia coli AL862 is carried by a 61-kb genomic region with characteristics typical of a pathogenicity island (PAI), including a size larger than 10 kb, the presence of an integrase-encoding gene, the insertion into a tRNA locus (pheR), and the presence of a small direct repeat at each extremity. Moreover, the G+C content of the afa-8 operon (46.4%) is lower than that of the E. coli K-12/MG1655 chromosome (50.8%). Within this PAI, designated PAI IAL862, we identified open reading frames able to code for products similar to proteins involved in sugar utilization. Four probes spanning these sequences hybridized with 74.3% of pathogenicafa-8-positive E. coli strains isolated from humans and animals, 25% of human pathogenic afa-8-negativeE. coli strains, and only 8% of fecal strains (P = 0.05), indicating that these sequences are strongly associated with the afa-8 operon and that this genetic association may define a PAI widely distributed among human and animal afa-8-positive strains. One of the distinctive features of this study is that E. coli AL862 also carries another afa-8-containing PAI (PAI IIAL862), which appeared to be similar in size and genetic organization to PAI IAL862 and was inserted into the pheV gene. We investigated the insertion sites of afa-8-containing PAI in human and bovine pathogenic E. coli strains and found that this PAI preferentially inserted into the pheV gene.

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Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.180.7.1814-1821.1998 ◽

1998 ◽

Vol 180 (7) ◽

pp. 1814-1821 ◽

Cited By ~ 52

Author(s):

Yong Yang ◽

Ho-Ching Tiffany Tsui ◽

Tsz-Kwong Man ◽

Malcolm E. Winkler

Keyword(s):

Escherichia Coli ◽

De Novo ◽

Specific Activity ◽

Salvage Pathway ◽

Pyridoxal Kinase ◽

Triple Mutant ◽

Reading Frame ◽

E Coli ◽

Specific Activities ◽

K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

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