The genetic and physical basis of variability in Escherichia coli strains carrying a reference Inc N group plasmid

1981 ◽  
Vol 27 (6) ◽  
pp. 616-626 ◽  
Author(s):  
M. Konarska-Kozlowska ◽  
V. N. Iyer
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Sequence Homology ◽  
Dna Hybridization ◽  
Tetracycline Resistance ◽  
Resistance Marker ◽  
E Coli ◽  
Reference Plasmid ◽  
Dna Fragment ◽  
K 12

The nature and basis of variability in the conjugational behaviour of RM98+ (RM98-carrying) strains of Escherichia coli K-12 that are otherwise similar in phenotype was studied. An explanation for such variability is provided.Some RM98+ strains of E. coli have a plasmid aggregate, which upon conjugation yields two different conjugative plasmids. The first (pCU1) is an N conjugative group plasmid by all available criteria. The second (pCU2) could not be placed in any conjugative group known among the Enterobacteriaceae. Reciprocal DNA hybridization experiments and the gel patterns displayed by the two plasmid DNAs upon digestion with different restriction endonucleases indicate no extensive sequence homology between pCU1 and pCU2. pCU2 DNA is much longer than pCU1 DNA.Despite the absence of extensive homology, the DNA of pCU1 and pCU2 can interact. Derivatives can be selected that have all the antibiotic markers of the aggregate plasmid but that neither contain nor segregate pCU2. It is shown that in such strains a DNA fragment of molecular weight 7.9 × 106 has been added to pCU1 concurrently with a tetracycline resistance marker originally present in pCU2 and absent in pCU1. These observations suggest that tetracycline resistance in pCU2 may be part of a large translocatable element.RM98 has been used to designate a reference Inc N group plasmid. The results presented indicate that this can lead to ambiguity. pCU1 would now be the appropriate reference plasmid.

scholarly journals Evidence of Horizontal Transfer of theEcoO109I Restriction-Modification Gene to Escherichia coli Chromosomal DNA

1999 ◽  
Vol 181 (21) ◽  
pp. 6822-6827 ◽  
Author(s):  
Keiko Kita ◽  
Junko Tsuda ◽  
Toshinobu Kato ◽  
Kenji Okamoto ◽  
Hideshi Yanase ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Horizontal Transfer ◽  
Attachment Site ◽  
Chromosomal Dna ◽  
E Coli ◽  
Bacteriophage P4 ◽  
Dna Fragment ◽  
Stable Maintenance ◽  
K 12 ◽  
Restriction Modification

ABSTRACT A DNA fragment carrying the genes coding for EcoO109I endonuclease and EcoO109I methylase, which recognize the nucleotide sequence 5′-(A/G)GGNCC(C/T)-3′, was cloned from the chromosomal DNA of Escherichia coli H709c. TheEcoO109I restriction-modification (R-M) system was found to be inserted between the int and psu genes from satellite bacteriophage P4, which were lysogenized in the chromosome at the P4 phage attachment site of the corresponding leuX gene observed in E. coli K-12 chromosomal DNA. Thesid gene of the prophage was inactivated by insertion of one copy of IS21. These findings may shed light on the horizontal transfer and stable maintenance of the R-M system.

scholarly journals Use of Cibracon Blue-Sepharose 6B to Detect Modifications of the Non-allosteric 6-Phosphofructokinase from Escherichia Coli K-12

1981 ◽  
Vol 34 (1) ◽  
pp. 125 ◽  
Author(s):  
Ken N Ewings ◽  
Horst W Doelle
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
E Coli ◽  
Blue Dextran ◽  
Strong Affinity ◽  
K 12 ◽  
Blue Sepharose

In crude cell-free extracts of aerobically grown E. coli K-12, the non-allosteric form of 6-phosphofructokinase has a tetrameric molecular weight of 140000 with a low affinity � 5%) for the blue dextran chromophore-Cibracon Blue. The allosteric form has the same tetrameric molecular weight, but possesses a strong affinity for the blue dextran chromophore.

scholarly journals Molecular cloning of pheR in Escherichia coli K-12

1982 ◽  
Vol 152 (1) ◽  
pp. 1-6
Author(s):  
J Gowrishankar ◽  
J Pittard
Keyword(s):  
Escherichia Coli ◽  
Tetracycline Resistance ◽  
Plasmid Vector ◽  
Chorismate Mutase ◽  
Gamma Delta ◽  
E Coli ◽  
Subunit Molecular Weight ◽  
K 12 ◽  
Vector Pbr322 ◽  
Pair Fragment

The regulator gene pheR, which in Escherichia coli controls the expression of pheA, the structural gene for chorismate mutase P-prephenate dehydratase, was cloned on to multicopy plasmids directly from the E. coli chromosome; this was achieved with the aid of the tetracycline resistance transposon, Tn10, that had been inserted very close to the pheR gene. Subsequently, pheR was subcloned on a 1.1-kilobase-pair fragment on the plasmid vector pBR322; its position on the plasmid was localized by the method of gamma delta-mediated transpositional inactivation. The pheR gene product was identified in maxicells and found to be a protein of subunit molecular weight 19,000, suggesting that the coding segment of the gene is about 500 nucleotide pairs long.

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

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 691-702 ◽  
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.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

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

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.

scholarly journals Molecular types, virulence profiles and antimicrobial resistance of Escherichia coli causing bovine mastitis

2019 ◽  
Vol 6 (1) ◽  
pp. e000369 ◽  
Author(s):  
Magdalena Nüesch-Inderbinen ◽  
Nadine Käppeli ◽  
Marina Morach ◽  
Corinne Eicher ◽  
Sabrina Corti ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bovine Mastitis ◽  
Tetracycline Resistance ◽  
Diffusion Method ◽  
Sample Population ◽  
Disk Diffusion Method ◽  
E Coli ◽  
Phylogenetic Grouping ◽  
Resistance Rates ◽  
Susceptibility Profiles

BackgroundEscherichia coli is an important aetiological agent of bovine mastitis worldwide.MethodsIn this study, 82 E. coli from bovine mastitis milk samples from 49 farms were analysed for their genetic diversity using phylogenetic grouping and multilocus sequence typing. The isolates were examined by PCR for a selection of virulence factors (VFs). Antimicrobial susceptibility profiles were assessed using the disk diffusion method.ResultsThe most prevalent phylogroups were group B1 (41.5 per cent of the isolates) and group A (30.5 per cent). A variety of 35 different sequence types (STs) were identified, including ST1125 (11 per cent), ST58 (9.8 per cent), ST10 (8.5 per cent) and ST88 (7.3 per cent). Aggregate VF scores (the number of unique VFs detected for each isolate) ranged from 1 to 3 for 63.4 per cent of the isolates and were at least 4 for 12.2 per cent. For 24.4 per cent of the isolates, the score was 0. The three most frequent VFs were traT, fyuA and iutA. The majority (72 per cent) of the isolates harboured traT. The majority (68.3 per cent) of the isolates were fully susceptible to all antimicrobials tested, with 22 per cent resistant to ampicillin and 14.6 per cent to tetracycline. Resistance rates were low for gentamicin (3.7 per cent), amoxicillin/clavulanic acid (2.4 per cent) and ceftiofur (1.2 per cent), respectively.ConclusionAmong the study’s sample population, E. coli strains were genotypically diverse, even in cows from the same farm, although some STs occurred more frequently than others. Susceptibility to clinically relevant compounds remained high.

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

2004 ◽  
Vol 186 (1) ◽  
pp. 192-199 ◽  
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.

Cloning and expression of Xylella fastidiosa antigens in Escherichia coli and Erwinia stewartii

1989 ◽  
Vol 35 (4) ◽  
pp. 487-491 ◽  
Author(s):  
Paul H. Goodwin
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Genomic Dna ◽  
Xylella Fastidiosa ◽  
Gene Bank ◽  
Cloning And Expression ◽  
Western Blots ◽  
E Coli ◽  
Cosmid Vector ◽  
Erwinia Stewartii

Xylella fastidiosa DNA, partially digested with Sau3A, was ligated into the cosmid vector, pUCD615. Approximately 4500 ampicillin-resistant Escherichia coli colonies were obtained. The frequency of complementation of leucine auxotrophy in transfected E. coli indicated that the cosmid gene bank was representative of X. fastidiosa genomic DNA. Colonies were lysed directly onto nitrocellulose membranes using a thermo-inducible λ lysogen and screened for expression of X. fastidiosa antigens. Approximately 16.5% of a random sample of clones were found to express X. fastidiosa antigens as determined by Western blots. These proteins comigrated with proteins of X. fastidiosa and ranged in molecular weight from 10 000 to 160 000. Conjugation of several of the plasmids into Erwinia stewartii resulted in expression of the similar molecular weight cloned proteins with similar levels of expression as in E. coli.Key words: Xylella fastidiosa, Pierce's disease, immunological clone screening, thermo-inducible lysogeny.

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