Identification and physical characterization of a Col E1 hybrid plasmid containing a catalase gene of Escherichia coli

1983 ◽  
Vol 61 (12) ◽  
pp. 1315-1321 ◽  
Author(s):  
Peter C. Loewen ◽  
Barbara L. Triggs ◽  
Glen R. Klassen ◽  
Joel H. Weiner
Keyword(s):  
Escherichia Coli ◽  
Catalase Activity ◽  
Physical Map ◽  
Polyacrylamide Gel Electrophoresis ◽  
Hybrid Plasmid ◽  
E Coli ◽  
Catalase Gene ◽  
Restriction Sites ◽  
Catalase Peroxidase

A hybrid Escherichia coli: Col E1 plasmid, pLC36-19, containing a catalase gene has been identified in the Clarke and Carbon colony bank. Catalase activity was amplified two- to three-fold in the pLC36-19-containing strain relative to other hybrid-plasmid-containing strains and this activity could be induced three- or four-fold by hydrogen peroxide or ascorbic acid. The plasmid was transferred to a strain chromosomally deficient in catalase synthesis, resulting in a strain with high and inducible levels of catalase. The plasmid was also transferred to a minicell-producing strain and minicells harbouring the plasmid were found to synthesize a labelled protein with a molecular weight of 84 000 characteristic of catalase from E. coli. A catalase activity was also synthesized by the plasmid-containing minicells. Two catalase activities with associated peroxidase activities coded for by the plasmid were separable by polyacrylamide gel electrophoresis and migrated coincident with chromosomally encoded catalase–peroxidase activities. A third catalase activity which did not have an associated peroxidase activity was not coded for by the plasmid. A physical map of the 25.5-kilobase pair plasmid was constructed by restriction nuclease analysis and the relative positions of 38 restriction sites were defined.

Characterization of novel ybjG and dacC variants in Escherichia coli

2013 ◽  
Vol 62 (11) ◽  
pp. 1728-1734 ◽  
Author(s):  
Dongguo Wang ◽  
Enping Hu ◽  
Jiayu Chen ◽  
Xiulin Tao ◽  
Katelyn Gutierrez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multiple Drug Resistance ◽  
Multiple Drug ◽  
The Novel ◽  
Conventional Pcr ◽  
E Coli ◽  
Novel Variants ◽  
Restriction Sites ◽  
Genetic Structures

A total of 69 strains of Escherichia coli from patients in the Taizhou Municipal Hospital, China, were isolated, and 11 strains were identified that were resistant to bacitracin, chloramphenicol, tetracycline and erythromycin. These strains were PCR positive for at least two out of three genes, ybjG, dacC and mdfA, by gene mapping with conventional PCR detection. Conjugation experiments demonstrated that these genes existed in plasmids that conferred resistance. Novel ybjG and dacC variants were isolated from E. coli strains EC2163 and EC2347, which were obtained from the sputum of intensive care unit patients. Genetic mapping showed that the genes were located on 8200 kb plasmid regions flanked by EcoRI restriction sites. Three distinct genetic structures were identified among the 11 PCR-positive strains of E. coli, and two contained the novel ybjG and dacC variants. The putative amino acid differences in the ybjG and dacC gene variants were characterized. These results provide evidence for novel variants of ybjG and dacC, and suggest that multiple drug resistance in hospital strains of E. coli depends on the synergistic function of ybjG, dacC and mdfA within three distinct genetic structures in conjugative plasmids.

scholarly journals Integrated Genomic Map from Uropathogenic Escherichia coli J96

2000 ◽  
Vol 68 (10) ◽  
pp. 5933-5942 ◽  
Author(s):  
Lyla J. Melkerson-Watson ◽  
Christopher K. Rode ◽  
Lixin Zhang ◽  
Betsy Foxman ◽  
Craig A. Bloch
Keyword(s):  
Escherichia Coli ◽  
Virulence Genes ◽  
Housekeeping Genes ◽  
E Coli ◽  
Restriction Sites ◽  
Physical And Genetic Map ◽  
K 12 ◽  
Genomic Map ◽  
Insertion Mutants

ABSTRACT Escherichia coli J96 is a uropathogen having both broad similarities to and striking differences from nonpathogenic, laboratoryE. coli K-12. Strain J96 contains three large (>100-kb) unique genomic segments integrated on the chromosome; two are recognized as pathogenicity islands containing urovirulence genes. Additionally, the strain possesses a fourth smaller accessory segment of 28 kb and two deletions relative to strain K-12. We report an integrated physical and genetic map of the 5,120-kb J96 genome. The chromosome contains 26 NotI, 13 BlnI, and 7 I-CeuI macrorestriction sites. Macrorestriction mapping was rapidly accomplished by a novel transposon-based procedure: analysis of modified minitransposon insertions served to align the overlapping macrorestriction fragments generated by three different enzymes (each sharing a common cleavage site within the insert), thus integrating the three different digestion patterns and ordering the fragments. The resulting map, generated from a total of 54 mini-Tn10insertions, was supplemented with auxanography and Southern analysis to indicate the positions of insertionally disrupted aminosynthetic genes and cloned virulence genes, respectively. Thus, it contains not only physical, macrorestriction landmarks but also the loci for eight housekeeping genes shared with strain K-12 and eight acknowledged urovirulence genes; the latter confirmed clustering of virulence genes at the large unique accessory chromosomal segments. The 115-kb J96 plasmid was resolved by pulsed-field gel electrophoresis inNotI digests. However, because the plasmid lacks restriction sites for the enzymes BlnI and I-CeuI, it was visualized in BlnI and I-CeuI digests only of derivatives carrying plasmid inserts artificially introducing these sites. Owing to an I-SceI site on the transposon, the plasmid could also be visualized and sized from plasmid insertion mutants after digestion with this enzyme. The insertional strains generated in construction of the integrated genomic map provide useful physical and genetic markers for further characterization of the J96 genome.

scholarly journals Expression and Characterization of Streptococcal rgp Genes Required for Rhamnan Synthesis in Escherichia coli

2002 ◽  
Vol 70 (6) ◽  
pp. 2891-2898 ◽  
Author(s):  
Yukie Shibata ◽  
Yoshihisa Yamashita ◽  
Kazuhisa Ozaki ◽  
Yoshio Nakano ◽  
Toshihiko Koga
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polymerization Process ◽  
Streptococcus Sobrinus ◽  
E Coli ◽  
Genes Encoding ◽  
Group A ◽  
Specific Antigens

ABSTRACT Six genes (rgpA through rgpF) that were involved in assembling the rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans were previously identified (Y. Yamashita, Y. Tsukioka, K. Tomihisa, Y. Nakano, and T. Koga, J. Bacteriol. 180:5803-5807, 1998). The group-specific antigens of Lancefield group A, C, and E streptococci and the polysaccharide antigen of Streptococcus sobrinus have the same rhamnan backbone as the RGP of S. mutans. Escherichia coli harboring plasmid pRGP1 containing all six rgp genes did not synthesize complete RGP. However, E. coli carrying a plasmid with all of the rgp genes except for rgpE synthesized the rhamnan backbone of RGP without glucose side chains, suggesting that in addition to rgpE, another gene is required for glucose side-chain formation. Synthesis of the rhamnan backbone in E. coli required the initiation of transfer of N-acetylglucosamine to a lipid carrier and the expression of the rgpC and rgpD genes encoding the putative ABC transporter specific for RGP. The similarities in RGP synthesis between E. coli and S. mutans suggest common pathways for rhamnan synthesis. Therefore, we evaluated the rhamnosyl polymerization process in E. coli by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the lipooligosaccharide (LOS). An E. coli transformant harboring rgpA produced the LOS modified by the addition of a single rhamnose residue. Furthermore, the rgpA, rgpB, and rgpF genes of pRGP1 were independently mutated by an internal deletion, and the LOS chemotypes of their transformants were examined. The transformant with an rgpA deletion showed the same LOS profile as E. coli without a plasmid. The transformant with an rgpB deletion showed the same LOS profile as E. coli harboring rgpA alone. The transformant with an rgpF deletion showed the LOS band with the most retarded migration. On the basis of these results, we speculated that RgpA, RgpB, and RgpF, in that order, function in rhamnan polymerization.

scholarly journals Construction and Characterization of a 1,3-Propanediol Operon

1998 ◽  
Vol 64 (1) ◽  
pp. 98-105 ◽  
Author(s):  
Frank A. Skraly ◽  
Betsy L. Lytle ◽  
Douglas C. Cameron
Keyword(s):  
Escherichia Coli ◽  
Second Phase ◽  
Fed Batch ◽  
Glycerol Dehydratase ◽  
E Coli ◽  
Restriction Sites ◽  
Unique Restriction ◽  
Coenzyme B ◽  
Single Promoter

ABSTRACT The genes for the production of 1,3-propanediol (1,3-PD) inKlebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, are naturally under the control of two different promoters and are transcribed in different directions. These genes were reconfigured into an operon containing dhaB followed bydhaT under the control of a single promoter. The operon contains unique restriction sites to facilitate replacement of the promoter and other modifications. In a fed-batch cofermentation of glycerol and glucose, Escherichia coli containing the operon consumed 9.3 g of glycerol per liter and produced 6.3 g of 1,3-PD per liter. The fermentation had two distinct phases. In the first phase, significant cell growth occurred and the products were mainly 1,3-PD and acetate. In the second phase, very little growth occurred and the main products were 1,3-PD and pyruvate. The first enzyme in the 1,3-PD pathway, glycerol dehydratase, requires coenzyme B12, which must be provided in E. colifermentations. However, the amount of coenzyme B12 needed was quite small, with 10 nM sufficient for good 1,3-PD production in batch cofermentations. 1,3-PD is a useful intermediate in the production of polyesters. The 1,3-PD operon was designed so that it can be readily modified for expression in other prokaryotic hosts; therefore, it is useful for metabolic engineering of 1,3-PD pathways from glycerol and other substrates such as glucose.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

scholarly journals The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli

1996 ◽  
Vol 316 (1) ◽  
pp. 251-257 ◽  
Author(s):  
Michinori MUTSUDA ◽  
Takahiro ISHIKAWA ◽  
Toru TAKEDA ◽  
Shigeru SHIGEOKA
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Molecular Mass ◽  
Peroxidase Activity ◽  
Catalase Activity ◽  
Native Enzyme ◽  
Nucleotide Sequence Analysis ◽  
Synechococcus Pcc 7942 ◽  
Catalase Peroxidase ◽  
Pcc 7942

Synechococcus PCC 7942, a cyanobacterium, possesses catalase–peroxidase as the sole hydrogen peroxide-scavenging system. The enzyme has been purified to electrophoretic homogenenity from the cells. The native enzyme had a molecular mass of 150 kDa and was composed of two identical subunits of molecular mass 79 kDa. The apparent Km value of the catalase activity for H2O2 was 4.2±0.27 mM and the kcat value was 2.6×104 s-1. The enzyme contained high catalase activity and an appreciable peroxidase activity with o-dianisidine and pyrogallol. The catalase activity was not inhibited by 3-amino-1,2,4-triazole but by KCN and NaN3 (apparent Ki values 19.3±0.84 and 20.2±0.95 μM respectively). The enzyme showed an absorption spectrum of typical protohaem and contained one protohaem molecule per dimer. The gene encoding catalase–peroxidase was cloned from the chromosomal DNA of Synechococcus PCC 7942. A 2160 bp open reading frame (ORF), coding a catalase–peroxidase of 720 amino acid residues (approx. 79.9 kDa), was observed. The deduced amino acid sequence coincided with that of the N-terminus of the purified enzyme and showed a remarkable similarity to those of a family of catalase–peroxidases of prokaryotic cells. Escherichia coli BL21(DE3)plysS, harbouring a recombinant plasmid containing the catalase–peroxidase gene, produced a large amount of proteins that co-migrated on SDS/PAGE with the native enzyme. The recombinant enzyme showed the same ratio of catalase activity to peroxidase activity with o-dianisidine and the same Km for H2O2 as the native enzyme.

scholarly journals Isolation and characterization of escherichia coli in ready-to-eat foods vended in Islamic University, Kushtia

1970 ◽  
Vol 18 ◽  
pp. 99-103 ◽  
Author(s):  
S Biswas ◽  
MAK Parvez ◽  
M Shafiquzzaman ◽  
S Nahar ◽  
MN Rahman
Keyword(s):  
Escherichia Coli ◽  
Pattern Analysis ◽  
University Campus ◽  
Rflp Pattern ◽  
E Coli ◽  
Fish Meat ◽  
Isolation And Characterization ◽  
Food Borne ◽  
Identification And Characterization

Context: Escherichia coli is shed in the feces of warm blooded animals and humans and thus potential for public health. Detection and characterization of E. coli in the ready-to-eat (RTE) foods concerns due to their presence indicates fecal contamination of the food.   Objective: To identify, characterize and RFLP pattern analysis of E. coli isolated from RTE foods vended in Islamic University campus, Kushtia.   Materials and Methods: Fifty samples from four types of consumed foods in six student halls of residence, some temporary restaurants of Islamic University, Kushtia were assessed for bacterial contamination by standard methods. Identification and characterization of E. coli isolates were performed using IMViC tests. Genomic DNA was used to perform RFLP pattern analysis.   Results: Thirty seven out of 50 (74%) examined samples of RTE foods had E. coli contamination. The highest number of E. coli was isolated from vegetable oriented RTE foods (90.90%) and fish, meat and cereals samples were also significantly E. coli positive. RFLP profiling of two E. coli isolates were observed.   Conclusion: The results of this study provide evidence that some RTE foods had unsatisfactory levels of contamination with E. coli. Thus street vended RTE food could be important potential vehicles for food-borne diseases. Molecular characterization may be exploited to identify food borne pathogen among different species.  Keywords: Ready-to-eat foods; Escherichia coli; RFLP pattern DOI: http://dx.doi.org/10.3329/jbs.v18i0.8783 JBS 2010; 18(0): 99-103

scholarly journals Evaluation of antipyretic activity of hydroalcoholic extract of Corchorus depressus Linn. in Escherichia coli–induced pyretic rabbits

2018 ◽  
Vol 16 ◽  
pp. 205873921879295
Author(s):  
Saeed Ahmad ◽  
Muhammad Akram ◽  
Syed Muhammad Ali Shah ◽  
Sabira Sultana
Keyword(s):  
Escherichia Coli ◽  
Positive Control ◽  
Negative Control ◽  
The Body ◽  
Hydroalcoholic Extract ◽  
Antipyretic Effect ◽  
E Coli ◽  
Antipyretic Activity ◽  
Experimental Treatments

This study was conducted to investigate the antipyretic effect of the hydroalcoholic extract of Corchorus depressus Linn. against Escherichia coli ( E. coli)-induced pyrexia in rabbits. Hydroalcohalic extracts of C. depressus were given orally at 25, 50, and 100 mg/kg for antipyretic affect in E. coli-induced fever in rabbits. The animals were divided into five groups of five each. Among these five groups, three received various doses of experimental treatments, whereas the fourth one served as positive control and received paracetamol. The fifth group of animals served as negative control and received no treatment. The body temperature of the rabbits was measured rectally over a period of 5 h. C. depressus exhibited better effects at dose rate of 25, 50, and 100 mg/kg. The hydroalcoholic extract of C. depressus has significant antipyretic effect. These results lend support to the popular use of C. depressus in traditional medicine as a remedy for pyrexia and suggest that the characterization of the principles for such activity deserves further investigation.

scholarly journals Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

2001 ◽  
Vol 183 (21) ◽  
pp. 6466-6477 ◽  
Author(s):  
Christopher Kirkpatrick ◽  
Lisa M. Maurer ◽  
Nikki E. Oyelakin ◽  
Yuliya N. Yoncheva ◽  
Russell Maurer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Opposite Effect ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Resistance ◽  
Luria Broth ◽  
Acetyl Coa ◽  
Fermentation Products ◽  
E Coli

ABSTRACT Acetate and formate are major fermentation products ofEscherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-ptastrain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of theackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.

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