CytR Repressor Research Articles

Transcriptional regulation of the cytR repressor gene of Escherichia coli: autoregulation and positive control by the cAMP/CAP complex

The Escherichia coli cytR-encoded repressor protein (CytR) controls the expression of several genes involved in nucleoside and deoxynucleoside uptake and metabolism. The cytR promoter was identified by determining the transcriptional initiation site of the cytR gene. A chromosomal cytR-lacZ+ operon fusion was isolated and used to study the regulation of cytR. We show that cytR expression is negatively controlled by the CytR protein and positively affected by the cAMP/CAP complex. Footprinting studies with purified CAP protein revealed two CAP binding sites upstream of the cytR promoter. A previously described mutation (cytR*) in the cloned cytR gene, which results in the phenotypic suppression of a CytR operator mutation in the tsx P2 promoter, was analysed. DNA sequence analysis of the cytR* mutation revealed a G-C to an A-T base pair transition at position -34 bp relative to the translational initiation site of cytR. This point mutation activates a cryptic promoter that is stronger than the wild-type cytR promoter and leads to overproduction of the CytR repressor.

Analysis of the tsx gene, which encodes a nucleoside-specific channel-forming protein (Tsx) in the outer membrane of Escherichia coli

The tsx gene of Escherichia coli encodes an outer membrane protein, Tsx, which constitutes the receptor for colicin K and bacteriophage T6, and functions as a substrate-specific channel for nucleosides and deoxynucleosides. The mini-Mu element pEG5005 was used to prepare a gene bank in vivo, and this bank was used to identify T6-sensitive strains carrying the cloned tsx gene. Subcloning of the tsx gene into the multicopy plasmid, pBR322, resulted in a strong overproduction of Tsx. The sequence of a 1477-bp DNA segment containing tsx and its flanking regions was determined. An open reading frame (ORF) was found which was followed by a pair of repetitive extragenic palindromic sequences. This ORF translated into a protein of 294 amino acids (aa), the first 22 aa of which showed the characteristic features of a bacterial signal sequence peptide. The putative mature form of Tsx is composed of 272 aa with a calculated M r of 31418. The aa sequence of Tsx shows an even distribution of charged residues (52 aa) and lacks extensive hydrophobic stretches. No significant homologies of Tsx to the channel-forming proteins OmpC, OmpF, PhoE and LamB from the E. coli outer membrane were detected. Using nuclease S1, we identified two transcription start points for the tsx mRNA which were separated by approx. 150 bp. Genetic data suggest that the synthesis of the larger mRNA species is directed by a weak promoter ( P1) that is controlled by the DeoR repressor, whereas the smaller mRNA species is directed by the main promoter P2, which is negatively controlled by the CytR repressor and positively affected by the cyclic AMP/catabolite activator protein complex.

CRP/cAMP- and CytR-regulated promoters in Escherichia coli K12: the cdd promoter.

Transcriptional regulation of the deoP2 promoter by the cyclic AMP/cyclic AMP receptor protein complex (cAMP/CRP) and the CytR repressor requires two high-affinity CRP targets located around -41 and -93 bp preceding the start site for transcription. Here we report the structure of cddP, another CRP/CytR-regulated promoter. In common with what was found in deo, the cdd promoter also contains multiple CRP targets. Thus, using the DNasel footprinting procedure, tandem CRP binding sites were identified around -41 and -93. These findings support a general model for CytR binding and CytR regulation, in which (i) CytR and the CRP/cAMP complex bind to similar or identical targets, (ii) two or more targets are necessary for proper binding of CytR to a promoter region, and (iii) CytR represses transcription by antagonizing cAMP/CRP activation.

Double negative and positive control of tsx expression in Escherichia coli.

The Escherichia coli tsx gene encodes an outer membrane protein that is involved in nucleoside uptake and serves as the receptor protein for colicin K and several bacteriophages. Regulation of its expression was studied by using tsx-lacZ protein and operon fusion strains carrying mutations in deoR, cytR, and crp. The cytR-encoded repressor had a stronger influence on tsx transcription than the DeoR repressor did, and the level of tsx expression in a deoR cytR double mutant was approximately the sum of those found in the single deoR and cytR strains. This double negative control of Tsx synthesis was superceded by a positive control mechanism mediated by the cyclic AMP-catabolite activator protein (cAMP-CAP) complex. Our results suggest that tsx expression is controlled at two separate and differently regulated promoters: the weaker promoter (P1) is repressible by DeoR, while the stronger promoter (P2) is subject to negative and positive control by the CytR repressor and the cAMP-CAP complex, respectively. A mutant was isolated that showed unaltered tsx regulation by DeoR and the cAMP-CAP complex but strongly reduced repression by CytR. This tsx operator mutant was used to obtain a suppressor mutation located on a plasmid carrying the cloned cytR gene that restored CytR control of tsx expression. The direction of tsx transcription was determined and found to be counterclockwise on the E. coli chromosome.

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.

Fusion of the lac genes to the promotor for the cytidine deaminase gene of Escherichia coli K-12.

Phage Mu has been inserted into the structural gene for cytidine deaminase (cdd). By the use of phage lambda (lac, Mu) the promoter for the cdd gene has been fused to lacZ. In these strains lacZ expression is regulated by the cytR repressor protein and is therefore induced by cytidine. The fusion strains were used for the isolation of cddo mutants. Plaque forming lambda phages carrying the different cdd-lacZ fusions were isolated. Studies of the cdd-Mu strains showed that the cdd gene is transcribed clockwise with respect to the Escherichia coli map.

Promoter-like mutants with increased expression of the Escherichia coli uridine phosphorylase structural gene

From an Escherichia coli K-12 strain lacking adenylate cyclase (cya) and cyclic AMP receptor protein (crp), two mutants were isolated that synthesize uridine phosphorylase constitutively. The mutations differ from one another and also from a wild type in the maximum rate of uridine phosphorylase synthesis. They have constitutive expression of the uridine phosphorylase gene (udp) in the presence of repressor protein coded by the cytR regulatory gene and decrease the sensitivity of the udp gene simultaneously with catabolite repression. Both mutations cause a high level of udp expression whether they are in a cya crp or in a cya+ crp+ background. Another mutation (udpP1) isolated previously alters the response of udp gene to the ctyR repressor and produces a higher constitutive level of uridine phosphorylase in a cytR+ than in a cytR background when bacteria are grown in glucose. The synthesis of uridine phosphorylase in this mutant is dependent on an intact cyclic AMP-cyclic AMP receptor protein complex. All mutations studied are cis-acting and extremely closely linked to the udp structural gene, and appear to affect the uridine phosphorylase promoter-operator region. The data obtained are in accordance with a suggestion that the cytR repressor protein normally asserts its function by preventing the positive action of cyclic AMP-cyclic AMP receptor protein complex.

Regulation of the deo operon in Escherichia coli

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.

Regulated in vitro synthesis of the enzymes of the deo operon of Escerichia coli. properties of the DNA directed system.

The four enzymes deoxyriboaldolase, thymidine phosporylase, deoxyribomutase, and purine nucleoside phosphorylase have been synthesized in substantial amounts in a DNA-dependent in vitro system programmed with DNA containing the deo operon. The synthesis is greatly stimulated by deoxyribose-5-phosphate and cyclic AMP indicating that the deoR repressor and the catabolite activating protein (CAP) are highly active under our cell-free conditions. In contrast it has not yet been possible to observe a reproducible effect of the cytR repressor in vitro. The sequential appearance of active enzymes has confirmed the direction of transcription as being dra-tpp-drm-pup and has indicated that the four genes are transcribed into a single tetracistronic message.