Biomedical subjects

P Ritzenthaler

Publications and source records attributed to P Ritzenthaler.

43 records · Page 3Linked to original sources

PubMed1983

Use of uxu-lac fusion strains to study the regulation of the uxuAB operon in Escherichia coli K12.

In the hexuronate system of Escherichia coli, the uxuAB operon is negatively controlled by the UxuR and ExuR repressors. A Mudlac phage was used, by the method of Casadaban, to construct strains where a truncated lacZ was fused to uxuB. From these fusion strains, deletions of various lengths extending into the uxu region were created in vivo by selecting temperature-insensitive mutants. Operator-constitutive mutations were also selected for in such strains and their preliminary analysis is presented. Large amounts of either ExuR or UxuR repressor caused a strong decrease of the constitutive expression of the uxuAB operon in the operator mutants. The implications of this repression for the presence of one or two operator sites in the uxuAB operon are discussed.

Chromosome Deletion↗

PubMed1983

Construction of hybrid plasmids containing the Escherichia coli uxaB gene: analysis of its regulation and direction of transcription.

The uxaB gene of Escherichia coli, encoding for altronate oxidoreductase involved in the hexuronate degradative pathway, was isolated on a ColE1-uxaB hybrid plasmid from the Clarke and Carbon bank. The restriction map of this plasmid was established. The uxaB gene was mapped on a 1.5-megadalton HindIII-KpnI DNA fragment. Use of an in vitro gene fusion between uxaB and lacZ genes led to the determination that uxaB is transcribed from the KpnI towards the HindIII restriction sites. Gene amplification in cells containing various uxaB hybrid plasmids allowed us to show a gradation in the level of repression of exu operator sites by the exuR regulatory gene product.

Carbohydrate Dehydrogenases↗

PubMed1983

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

Operons uxaCA and exuT of the hexuronate system are very closely linked on the Escherichia coli genetic map. Using plasmid vectors constructed by Casadaban et al. (J. Bacteriol. 143:971-980, 1980), we formed exuT-lacZ and uxaA-lacZ fusions in vitro. The phenotypic properties of the new plasmids allowed us to confirm that the exuT and uxaCA operons are divergently transcribed. An analysis of these fusion plasmids and derivatives in the presence of multiple copies of the exuR regulatory gene demonstrated that the two operons possess separate control regions. The precise location of the operator site relative to endonuclease restriction sites was determined. In addition, deletions of different lengths were generated on exu plasmids by restriction enzymes and were recombined into the chromosome. The expression of the exu regulon genes in the resulting deletion mutants is in agreement with the postulated location of the exuT and uxaCA operators in the fusion plasmids.

Aldose-Ketose Isomerases↗

PubMed1982

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

The two genes of the Escherichia coli K-12 uid region (the structural gene uidA and the regulatory gene uidR) were isolated on a ColE1-uid hybrid plasmid from the bank of Clarke and Carbon. We made a restriction map of this region and correlated it with the genetic map by subcloning the uid restriction fragments into plasmids pBR322, pBR325, and pACYC177. In these plasmids, amplification of the products of the uidA and uidR genes occurred. The enzyme coded for by uidA was identified by polyacrylamide gel electrophoresis of crude extracts from strains containing the uidA plasmid. A 1-megadalton EcoRI-BamHI segment contained the uidAo operator, and the direction of transcription of the uidA gene was determined. The restriction analysis also suggested that the order of the loci in this region is manA, uidA, uidAo, uidR.

Cloning, Molecular↗

PubMed1982

Use of in vitro gene fusions to study the uxuR regulatory gene in Escherichia coli K-12: direction of transcription and regulation of its expression.

The uxuAB operon is composed of two genes coding for enzymes involved in hexuronate degradation. This operon is negatively controlled by the uxuR and exuR regulatory gene products. Fusions that brought lac gene expression under the control of transcriptional and translational signals within the uxuR gene were used to study uxuR regulation. These fusions were formed on plasmid cloning vectors constructed by Casadaban et al. (J. Bacteriol. 143:971-980, 1980). The transcriptional direction of the uxuR gene was deduced from the restriction pattern and the phenotypic properties of the new plasmids. The gene is transcribed counterclockwise on the standard Escherichia coli map, as is the uxuAB operon. Introduction in trans of a compatible plasmid carrying a wild-type uxuR gene in the lac fusion plasmid containing strain resulted in a decrease of beta-galactosidase synthesis. It was concluded that expression of the uxuR gene itself is repressed by its own product. The two other types of regulation found in the uxuAB operon, i.e., induction by fructuronate and catabolite control, also apply to the uxuR gene, whereas repression by the exuR repressor does not seem to occur for the uxuR gene.

Cyclic AMP↗

PubMed1981

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

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.

Bacteriophage lambda↗

PubMed1980

Construction and expression of hybrid plasmids containing Escherichia coli K-12 uxu genes.

The three genes of the Escherichia coli K-12 uxu region (uxu operon and regulatory gene) were isolated on a ColE1-uxu hybrid plasmid from the bank of Clarke and Carbon, and a restriction map of this region was established. In vitro recombination techniques were used to subclone the uxu restriction fragments into the plasmid vector pBR322 or pBR325. The various chimeric plasmids obtained were analyzed by restriction mapping and characterized genetically by introducing them in uxu mutant or wild-type strains. Differential rates of synthesis of the enzymes coded for by the uxu region were measured in the plasmid-containing strains; amplification of the products of the cloned genes was up to 40-fold the level found in haploid strains. The enzymes coded for by uxuA and uxuB were synthesized in vitro in a coupled transcription-translation system, confirming the results of the cloning experiments. The restriction analysis also suggests that the transcriptional direction of the uxu operon is from uxuA to uxuB and that the order of the loci in this region is: uxuR (regulatory gene), uxuB, uxuA, uxuAp (promoter), uxuAo (operator).

Cloning, Molecular↗