Escherichia coli

About: Escherichia coli is a research topic. Over the lifetime, 59041 publications have been published within this topic receiving 2050337 citations. The topic is also known as: E. coli & E coli jdj.

Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: strategy for targeting and cloning of genes from gram-positive bacteria.

Abstract: The conjugative transposon Tn916 (15 kilobases), originally identified in Streptococcus faecalis DS16, has been cloned as an intact element on the pBR322-derived vector pGL101 in Escherichia coli. The EcoRI F' (EcoRI F::Tn916) fragment of pAM211 (pAD1::Tn916) was cloned into the single EcoRI site of pGL101 to form the chimera, pAM120, by selecting for the expression of Tn916-encoded tetracycline resistance (Tcr). Interestingly, in the absence of continued selection for Tcr, Tn916 excised from pAM120 at high frequency. This excision event resulted in a plasmid species consisting of the pGL101 vector and a 2.7-kilobase restriction fragment comigrating with the EcoRI F fragment of pAD1 during agarose gel electrophoresis. Filter blot hybridization experiments showed the 2.7-kilobase fragment generated as a result of Tn916 excision to be homologous with the EcoRI F fragment of pAD1. Analogous results were obtained with another chimera, pAM170, generated by ligating the EcoRI D' (EcoRI D::Tn916) fragment of pAM210 (pAD1::Tn916) to EcoRI-digested pGL101. Comparison of the AluI and RsaI cleavage patterns of the EcoRI F fragment isolated after Tn916 excision with those from an EcoRI F fragment derived from pAD1 failed to detect any difference in the two fragments: data in support of a precise Tn916 excision event in E. coli. Subcloning experiments showed that an intact transposon was required for Tn916 excision and located the Tcr determinant near the single HindIII site on Tn916. Although excision occurred with high frequency in E. coli, Tn916 insertion into the E. coli chromosome was a much rarer event. Tcr transformants were not obtained when pAM120 DNA was used to transform a polA1 strain, E. coli C2368.

Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli.

Abstract: Background: The overproduction of recombinant proteins in host cells often leads to their misfolding and aggregation. Previous attempts to increase the solubility of recombinant proteins by co-overproduction of individual chaperones were only partially successful. We now assessed the effects of combined overproduction of the functionally cooperating chaperone network of the E. coli cytosol on the solubility of recombinant proteins. Results: A two-step procedure was found to show the strongest enhancement of solubility. In a first step, the four chaperone systems GroEL/GroES, DnaK/DnaJ/GrpE, ClpB and the small HSPs IbpA/IbpB, were coordinately co-overproduced with recombinant proteins to optimize de novo folding. In a second step, protein biosynthesis was inhibited to permit chaperone mediated refolding of misfolded and aggregated proteins in vivo. This novel strategy increased the solubility of 70% of 64 different heterologous proteins tested up to 42-fold. Conclusion: The engineered E. coli strains and the two-step procedure presented here led to a remarkable increase in the solubility of a various recombinant proteins and should be applicable to a wide range of target proteins produced in biotechnology.

Substrate specificity of the Escherichia coli endonuclease III : excision of thymine- and cytosine-derived lesions in DNA produced by radiation-generated free radicals

Abstract: The excision of modified bases from DNA by Escherichia coli endonuclease III was investigated. Modified bases were produced in DNA by exposure of dilute buffered solutions of DNA to ionizing radiation under oxic or anoxic conditions. The technique of gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify 16 pyrimidine- and purine-derived DNA lesions. DNA substrates were incubated either with the native enzyme or with the heat-inactivated enzyme. Subsequently, DNA was precipitated. Pellets were analyzed by GC/MS after hydrolysis and derivatization. Supernatant fractions were analyzed after derivatization without hydrolysis. The results provided unequivocal evidence for the excision by E. coli endonuclease III of a number of thymine- and cytosine-derived lesions from DNA. These were 5,6-dihydrothymine, 5-hydroxy-5-methylhydantoin, thymine glycol, 5-hydroxy-6-hydrothymine, 5,6-dihydrouracil, alloxan, uracil glycol, and 5-hydroxy-6-hydrouracil. None of the purine-derived lesions was excised by endonuclease III. The present work extends the substrate specificity of E. coli endonuclease III to another thymine-derived and four cytosine-derived lesions. It is the first investigation of the substrate specificity of this repair enzyme in the context of a large number of pyrimidine- and purine-derived lesions in DNA.

Emergence of human pandemic O25:H4-ST131 CTX-M-15 extended-spectrum-β-lactamase-producing Escherichia coli among companion animals

Abstract: Objectives: In view of the intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15 extended-spectrum b-lactamase (ESBL) in human clinical settings it would be of great interest to explore itsexistence in animals to unravel a possible reservoir function and the origin and transmission of this group ofmultiresistant strains.Methods: A total of 177 clinical phenotypically ESBL-producing E. coli isolates, mainly obtained from companionanimals with urinary tract infections, wound infections and diarrhoea, were collected in a veterinary diagnosticlaboratory covering a European-wide service area. They were screened for molecular subtype O25b and multilocussequence type 131. O25b-ST131 isolates were subsequently tested for ESBL types, and phenotypic andgenotypic resistance determinants. Further characterization of the strains was performed by PFGE and virulencegene typing.Results: Ten (5.6%) of 177 phenotypically ESBL-producing E. coli isolates, nine strains from dogs and one strainfrom a horse, were allocated to the B2-O25b-ST131 lineage. Nine of these isolates harboured a CTX-M-15-typeb-lactamase enzyme while one strain possessed an SHV-12-type ESBL. Macrorestriction analysis revealed acluster formation of six of the animal CTX-M-15-type ESBL-producing strains from five different Europeancountries together with a human control strain constituting a group of clonally related strains at a similarityvalue of 87.0%.Conclusions: Our findings demonstrate that the group of clonally related human B2-O25:H4-ST131 CTX-M-15-type ESBL-producing E. coli strains is present in companion animals from various European countries. This highlightsthe possibility of inter-species transmission of these multiresistant strains from human to animal and viceversa.