Molecular cloning of Escherichia coli K-12 hexuronate system genes: exu region

Abstract

Lambda transducing bacteriophages carrying the exu region (min 66) of Escherichia coli K-12 (lambda pexu) were previously isolated. A restriction map of these phages is presented. Starting from the lambda pexu phage deoxyribonucleic acid, various endonuclease-generated exu fragments were subcloned into multicopy plasmid vectors, using in vitro recombination techniques. The precise location of the exu genes, relative to the endonuclease sites, was determined. Plasmids carrying uxaC and uxaA genes overproduced the corresponding enzymes 30- to 40-fold. When these plasmids were expressed in an in vitro protein-synthesizing system, two polypeptides of 50,500 and 53,000 molecular weights appeared and were identified as the uxaC and uxaA gene products. A 2.6-kilobase-pair deoxyribonucleic acid fragment was shown to code for a functional exuR repressor which controls the expression of the exu region. Plasmids containing this fragment overproduced the regulatory protein. It was possible to localize the operator region, uxaCo, which overlapped a PstI endonuclease site, and to confirm the transcriptional direction of the uxaC-uxaA operon from uxaC to uxaA.

Genetic and physical characterization of a plasmid carrying the E. coli kdgA gene

“…The isolation of plasmid pAL1 completes the molecular cloning of the genes involved in the hexuronate-catabolizing system [20][21][22]. This plasmid bears the kdgA and edd genes.…”

Cloning and endonuclease restriction analysis of uidA and uidR genes in Escherichia coli K-12: determination of transcription direction for the uidA gene.

“…Agarose and polyacrylamide gel electrophoresis. The conditions used for electrophoresis of DNA on agarose gels were identical to those described by Ritzenthaler et al (26). For polyacrylamide gel electrophoresis under nondenaturing conditions, samples were made 10o glycerol and 5% ,-mercaptoethanol and electrophoresed overnight at 4°C on 7.5% polyacrylamide gels by the methods of Laemmli (15) and Studier (29).…”

Physical mapping of the exuT and uxaC operators by use of exu plasmids and generation of deletion mutants in vitro

“…Tagaturonate and fructuronate enter cells by separate transport systems that are distinct from the system encoded by exuT. All of the genes belonging to the exu regulon have been cloned into plasmid vector pBR322 (5,39).…”

“…2). When this fragment containing a binding site for the exuR repressor is inserted into a multicopy plasmid, it titrates out the exuR repressor present in the cell and derepresses the uxaB operon, resulting in constitutive synthesis of the altronate oxidoreductase enzyme (39).…”

“…2) carries a functional exuT gene on a SaIl-EcoRI restriction fragment. The unique BamHI site of pRE6 is located within the exuT gene (39). Plasmid pMC874 also possesses a unique BamHl restriction site.…”