Regulation of the deo operon in Escherichia coli: the double negative control of the deo operon by the cytR and deoR repressors in a DNA directed in vitro system.

Summary The synthesis of the four enzymes of the deo operon in Escherichia coli is known from in vivo experiments to be subject to a double negative control, exerted by the products of the cytR and deoR genes.A DNA-directed in vitro protein synthesizing system makes the deo enzymes (exemplified by thymidine phosphorylase) in agreement with in vivo results. Enzyme synthesis is stimulated by cyclic AMP and repressed by the cytR and deoR gene products. Repression by the cytR repressor is reversed by cytidine or adenosine in the presence of cyclic AMP, while repression by the deoR repressor is reversed by deoxyribose-5-phosphate.Assays for the presence of the cytR and deoR repressors were established by use of S-30 extracts prepared from the regulatory mutants.Dissociation constants for repressor-operator binding as well as for repressor-inducer interactions have been estimated from the results.Abbreviations and Symbols deoA (previously designated tpp) Genes coding for: thymidine, phosphorylase - deoB (previously designated drm) deoxyribomutase - deoC (previously designated dra) deoxyriboaldolase - deoD (previously designated pup) purine nucleoside phosphorylase - udp uridine phosphorylase - cytR regulatory gene for cdd, udp, deoC, deoA, deoB, and deoD - deoR (previously designated nucR) regulatory gene for deoC, deoA, deoB, and deoD Enzymes (EC 2.4.2.1) Purine nucleoside phosphorylase or purine nucleoside: orthophosphate(deoxy)ribosyltansferase - (EC 2.4.2.4) thymidine phosphorylase or thymidine: orthophosphate deoxyribosyltransferase - (EC 2.4.2.3) uridine phosphorylase or uridine: orthophosphate ribosyltransferase - (EC 4.1.2.4) deoxyriboaldolase or 2-deoxy-D-ribose-5-phosphate: acetaldehydelyase - (EC 2.7.5.6) phosphodeoxyribomutase The deo operon is defined as the gene cluster consisting of deoC deoA deoB deoD. The deo enzymes are the four enzymes encoded by the four genes of the deo operon. cAMP: cyclic adenosine 3,5-monophosphate. CRP: cyclic AMP receptor protein. dRib-5P: deoxyribose-5-phosphate. THUR: 3,4,5,6-tetrahydrouridine; EDTA: ethylene-diamine-tetra-acetate.     

Identification of the Escherichia coli deoR and cytR gene products.

The protein products encoded by the Escherichia coli deoR and cytR structural genes have been identified based on results obtained from E. coli maxicells harboring (i) recombinant plasmids carrying wild-type deoR and cytR genes, (ii) deletion derivatives of the deoR+ and cytR+ plasmids, (iii) plasmids containing site-specific mutations in the deoR and cytR structural genes, and (iv) plasmids which have transposon Tn1000 inserted into the deoR and cytR structural genes. Analysis of the protein profiles obtained from all the maxicell experiments demonstrated that the deoR gene encodes a protein with a subunit molecular weight of 30,500 and that the product of the cytR gene is a protein with a subunit molecular weight of 37,000.     

Tandem CRP binding sites in the deo operon of Escherichia coli K-12

The locations of DNA binding by the cyclic AMP receptor protein (CRP) in the deo operon of Escherichia coli have been determined by the DNase I footprinting procedure. Two high affinity sites were found around positions -35 and -90, preceding the second deo promoter. In vitro data on induction of gene fusions that join different parts of the deoP -2 regulatory region to the lac genes suggest that: (1) both CRP binding sites are needed for high expression from the deoP -2 region; and (2) negative regulation by the cytR repressor is accomplished by preventing the cAMP-CRP complex from binding to the second target.     

Bacillus subtilis citM, the structural gene for dihydrolipoamide transsuccinylase: cloning and expression in Escherichia coli

The 2-oxoglutarate dehydrogenase multienzyme complex is composed of three different subenzymes: 2-oxoglutarate dehydrogenase (E1o), dihydrolipoamide transsuccinylase (E2o), and dihydrolipoamide dehydrogenase (E3). Bacillus subtilis E1o and E2o are encoded by the citK and citM genes, respectively. A 3.4-kb BamHI DNA fragment containing citK and citM markers was isolated from a library of B. subtilis DNA in Escherichia coli. Functional E2o was expressed from the cloned DNA both in B. subtilis and E. coli. E2o had an apparent Mr of 60,000 when expressed in E. coli. The B. subtilis E2o component complemented an E. coli E2o-defective mutant in vivo and in vitro. It is concluded that functional B. subtilis E2o can be produced in E. coli and can interact with E. coli and E1o and E3 to form an active chimeric enzyme complex.     

Transposition of the deo operon structural genes in Escherichia coli K-12 to plasmid RP4 using bacteriophage mu]

Transposition of the structural genes of the deo operon of Escherichia coli K-12 into plasmid RP4 by means of temperate bacteriophage Mu was carried out. Some variants of composite RP4-deo-Mu plasmids were obtained and the expression of the deo genes integrated into the RP4 plasmid genome was studied. It was shown that the expression of these genes remains under the control of the chromosomal regulatory genes (deoR and cytR); although the activity of thymidine phosphorilase in the strain E. coli which contains hybrid plasmid is 4-6 fold greater than that in strains of E. coli with chromosomal localization of the deo operon.     

A control element within a structural gene: the gal operon of Escherichia coli

The gal operon of Escherichia coli is transcribed from two overlapping promoters, PG1 and PG2. Cyclic AMP and its receptor protein (CRP) modulate the two promoters in opposite directions by binding to a single cat locus. Both the promoters are negatively regulated by a single repressor, the product of the galR gene. An operator site, defined by several mutations, has previously been located upstream from the cat locus. We have isolated and characterized a new set of cis-dominant constitutive mutations of the gal operon and determined their locations by DNA sequencing. From these studies, we propose the existence of a second functional gal operator element at an extraordinary site--within galE, the first structural gene. Both the operators, OE (exterior) and OI (interior), are involved in the repression of PG1 and PG2. This would be the first example of the presence of a functional operator element within a structural protein-coding region.     

CS3纤毛抗原表达调控机理的研究

CS3亚基结构基因的核苷酸序列分析表明,在其翻译起始位点的上游存在着rbs位点及原核启动子的—10区和—35区DNA序列。凝胶阻滞和启动报告基因表达的实验确证了CS3亚基结构基因具有自身的启动子(Ps),怛它的作用受其上游区域的抑制。核苷酸序列分析发现,在Ps—35区上游550bp和840bp处各存在一个富A-T簇。结合原核启动子的一般作用规律推知,CS3的表达可能受DNA结合蛋白型的正向调节因子的作用,互补实验结果表明cfaD基因不仅可消除上游区对Ps的抑制,而且可大幅度地提高Ps的启动能力。在分析表达调控的基础上获得CS3重组高效表达。同时提出了其表达调控模型。     

Investigation of the regulation of the Escherichia coli btuB gene using operon fusions

Operon fusions were isolated between Mu dX (lac CmR ApR) and btuB, the gene encoding the multivalent vitamin B12 outer membrane receptor. Using these fusions, vitamin B12-mediated repression of btuB in Escherichia coli was demonstrated. Mutations in metH, metE and ompR as well as exogenous methionine, membrane pertubants, high osmolar conditions and temperature had no major effect on the expression of the btuB gene.     

Molecular cloning of polyhydroxyalkanoate synthesis operon from Aeromonas hydrophila and its expression in Escherichia coli

The polyhydroxyalkanoate synthesis operon was cloned from Aeromonas hydrophila CGMCC 0911. Heterogeneous expression of the cloned polyhydroxyalkanoate synthesis operon in Escherichia coli resulted in accumulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) consisting of 13.9 mol % 3-hydroxyhexanoate up to 29.2 wt % of cell dry weight when grown in lauric acid. The cell dry weight of recombinant E. coli harboring the polyhydroxyalkanoate synthesis operon was improved to 1.7 g L (-1), which was much higher than that of 0.3 g L (-1) of the wild type E. coli. Coexpression of acyl-CoA dehydrogenase gene (yafH) from E. coli and Vitreoscilla hemoglobin gene (vgb) from Vitreoscilla together with the whole A. hydrophila CGMCC 0911 polyhydroxyalkanoate synthesis operon facilitated cell growth and polyhydroxyalkanoate accumulation in E. coli. When yafH was coexpressed together with the polyhydroxyalkanoate synthesis operon, the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) content was increased from 29.2 to 52.1 wt %, and the cell dry weight was also increased slightly from 1.70 to 1.86 g L (-1). Coexpression of vgb gene could further enhance the cell dry weight to 2.0 g L(-1) and the polyhydroxyalkanoate content to 60.7 wt %.     

大肠杆菌棉子糖操纵子α—半乳糖苷酶表达的调节控制

The alpha-galactosidase, coded for by the first structural gene rafA in the plasmid determined raf operon was an inducible enzyme. In contrast to lac or mel operon, raf operon has more strict structural specificity for inducers. The enzyme can be induced by melibiose and raffinose, or weakly by D-galactose, but not by structurally related sugars such as lactose, PNPG etc.. The alpha-galactosidase forming capacity as function of growth curve reached a single peak at the end of the logarithmic phase of the growth. The structure and regulation of raf operon is similar to those of lac operon. The repressormor-mediated negative control plays a major role in the regulation of raf operon, and cAMP-CAP mediated positive control is also involved in the regulation. When 0.4% glucose was added into the medium with other carbon sources, the expression of the enzyme was repressed by 2-3 fold. Transient catabolite repression has been observed neither in inducible nor constitutive alpha-galactosidase expression. Based on alpha-galactosidase assay, in mutant strains CA8306(cya) and CA8445 (cya, crp) the expression level of raf operon was only 9% and 2.5% of that in wild type strain respectively. The glucose effect or the repression in cya mutant can be abolished by 1-5 mmol cAMP. The constitutive alpha-galactosidase expression in cya and cry double mutant (CA8445) remains repressible by glucose, but irreversible by cAMP, suggesting cAMP-CAP complex is not the exclusive mediator of the catablite repression.(ABSTRACT TRUNCATED AT 250 WORDS)