DeoR Repressor Research Articles

Crystallization of the Effector-Binding Domain of Repressor DeoR from Bacillus subtilis

The DeoR repressor of Bacillus subtilis negatively regulates the expression of the enzymes needed for the metabolism of deoxyribonucleosides and deoxyribose. To gain structural information on the regulation of DeoR function by a small molecular ligand, we prepared crystals of the C-terminal domain of DeoR in its free form and in complex with the effector molecule deoxyribose-5-phosphate. To develop the optimal procedure for crystallization, we have employed the thermofluor assay as a tool for the optimization of protein crystallizability. The monocrystals of three crystal forms were used to collect complete sets of diffraction data at a synchrotron radiation source and will be used to determine the DeoR crystal structure.

The association of DNA damage response and nucleotide level modulation with the antibacterial mechanism of the anti-folate drug Trimethoprim

BackgroundTrimethoprim is a widely prescribed antibiotic for a variety of bacterial infections. It belongs to a class of anti-metabolites - antifolates - which includes drugs used against malarial parasites and in cancer therapy. However, spread of bacterial resistance to the drug has severely hampered its clinical use and has necessitated further investigations into its mechanism of action and treatment regimen. Trimethoprim selectively starves bacterial cells for tetrahydrofolate, a vital cofactor necessary for the synthesis of several metabolites. The outcome (bacteriostatic or bactericidal) of such starvation, however, depends on the availability of folate-dependent metabolites in the growth medium. To characterize this dependency, we investigated in detail the regulatory and structural components of Escherichia coli cellular response to trimethoprim in controlled growth and supplementation conditions.ResultsWe surveyed transcriptional responses to trimethoprim treatment during bacteriostatic and bactericidal conditions and analyzed associated gene sets/pathways. Concurrent starvation of all folate dependent metabolites caused growth arrest, and this was accompanied by induction of general stress and stringent responses. Three gene sets were significantly associated with the bactericidal effect of TMP in different media including LB: genes of the SOS regulon, genes of the pyrimidine nucleotide biosynthetic pathway and members of the multiple antibiotic resistance (mar) regulon controlled by the MarR repressor. However, the SOS response was identified as the only universal transcriptional signature associated with the loss of viability by direct thymine starvation or by folate stress. We also used genome-wide gene knock-out screen to uncover means of sensitization of bacteria to the drug. We observed that among a number of candidate genes and pathways, the effect of knock-outs in the deoxyribose nucleotide salvage pathway, encoded by the deoCABD operon and under the control of the DeoR repressor, was most informative.ConclusionTranscriptional induction of DNA damage response is an essential feature of the bactericidal effect of trimethoprim. Either the observation of the transcriptional response or DNA damage itself, or both, is made possible by thymine starvation when other folate-dependent metabolites are not limited. The effect of DNA damage by the drug takes place prior to its bactericidal effect, at the beginning of the lag stage of the treatment. Mutations in the deoxyribose nucleotide salvage pathway can affect duration of the lag as well as the rate of killing. This information can be used to postulate certain mechanistic differences between direct thymine starvation in thymidylate synthase deficient mutants and thymine starvation by anti-folate inhibitors.

Purification and characterization of the DeoR repressor of Bacillus subtilis.

Transcription of the Bacillus subtilis dra-nupC-pdp operon is repressed by the DeoR repressor protein. The DeoR repressor with an N-terminal His tag was overproduced with a plasmid under control of a phage T5 promoter in Escherichia coli and was purified to near homogeneity by one affinity chromatography step. Gel filtration experimental results showed that native DeoR has a mass of 280 kDa and appears to exist as an octamer. Binding of DeoR to the operator DNA of the dra-nupC-pdp operon was characterized by using an electrophoretic gel mobility shift assay. An apparent dissociation constant of 22 nM was determined for binding of DeoR to operator DNA, and the binding curve indicated that the binding of DeoR to the operator DNA was cooperative. In the presence of low-molecular-weight effector deoxyribose-5-phosphate, the dissociation constant was higher than 1,280 nM. The dissociation constant remained unchanged in the presence of deoxyribose-1-phosphate. DNase I footprinting exhibited a protected region that extends over more than 43 bp, covering a palindrome together with a direct repeat to one half of the palindrome and the nucleotides between them.

Identification and characterization of a DeoR-specific operator sequence essential for induction of dra-nupC-pdp operon expression in Bacillus subtilis.

The deoR gene located just upstream the dra-nupC-pdp operon of Bacillus subtilis encodes the DeoR repressor protein that negatively regulates the expression of the operon at the level of transcription. The control region upstream of the operon was mapped by the use of transcriptional lacZ fusions. It was shown that all of the cis-acting elements, which were necessary for full DeoR regulation of the operon, were included in a 141-bp sequence just upstream of dra. The increased copy number of this control region resulted in titration of the DeoR molecules of the cell. By using mutagenic PCR and site-directed mutagenesis techniques, a palindromic sequence located from position -60 to position -43 relative to the transcription start point was identified as a part of the operator site for the binding of DeoR. Furthermore, it was shown that a direct repeat of five nucleotides, which was identical to the 3' half of the palindrome and was located between the -10 and -35 regions of the dra promoter, might function as a half binding site involved in cooperative binding of DeoR to the regulatory region. Binding of DeoR protein to the operator DNA was confirmed by a gel electrophoresis mobility shift assay. Moreover, deoxyribose-5-phosphate was shown to be a likely candidate for the true inducer of the dra-nupC-pdp expression.

Dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein.

The genes encoding deoxyriboaldolase (dra), nucleoside uptake protein (nupC), and pyrimidine nucleoside sequences were determined. Sequence analysis showed that the genes were localized immediately downstream of the hut operon. Insertional gene disruption studies indicated that the three genes constitute an operon with the gene order dra-nupC-pdp. A promoter mapping immediately upstream of the dra gene was identified, and downstream of the pdp gene the nucleotide sequence indicated the existence of a factor-independent transcription terminator structure. In wild-type cells growing in succinate minimal medium, the pyrimidine nucleoside phosphorylase and deoxyriboaldolase levels were five- to eightfold higher in the presence of thymidine and fourfold higher in the presence of deoxyadenosine. By the use of lacZ fusions, the regulation was found to be at the level of transcription. The operon expression was subject to glucose repression. Upstream of the dra gene an open reading frame of 313 amino acids was identified. Inactivation of this gene led to an approximately 10-fold increase in the levels of deoxyriboaldolase and pyrimidine nucleoside phosphorylase, and no further induction was seen upon the addition of deoxyribonucleosides. The upstream gene most likely encodes the regulator for the dra-nupC-pdp operon and was designated deoR (stands for deoxyribonucleoside regulator).

DeoR repression at-a-distance only weakly responds to changes in interoperator separation and DNA topology

The interoperator distance between a synthetic operator Os and the deoP2O2-galK fusion was varied between 46 and 176 bp. The repression of the deoP2 directed galK expression as a function of the interoperator distance (center-to-center) was measured in vivo in a single-copy system. The results show that the DeoR repressor efficiently can repress transcription at all the interoperator distances tested. The degree of repression depends very little on the spacing between the operators, however, a weak periodic dependency of 8-11 bp may exist.

DeoP1 promoter and operator mutants in Escherichia coli: isolation and characterization

Plasmid DNA containing deoP1, one of the two major promoters of the deo operon, has been mutagenized using hydroxylamine, and promoter down-mutations and operator mutations were selected. The isolated mutants are all located within a 16 bp palindromic sequence containing the -10 region of deoP1. The results show that RNA polymerase and DeoR repressor compete for the same DNA target. The deoP1 promotor activity is dependent on a TG motif one base pair upstream of the -10 consensus sequence. The sequence of the deo operator site was further verified by use of a synthetic linker.

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.

Single and double loop formation when deoR repressor binds to its natural operator sites

Distal effects on the in vivo repression of the deo operon are thought to be mediated by the deoR repressor with DNA loop formation. Such loops are easily observed by electron microscopy when the oligomeric deoR repressor is added to a DNA fragment carrying the three genetically defined operators at their chromosomal distances. Upon binding of deoR to any two operators, single loops are formed, 280, 600, and 880 bp in size. With the deo operon, double loops are also formed, which are the combination of the 280 bp and 600 bp loops and the result of simultaneous binding of the protein to its three sites. The formation of both single and double loops is consistent with the long-range effects observed in vivo and with the cooperative involvement of all three operator sites in the repression.

Purification and characterization of the deoR repressor of Escherichia coli.

The deoR gene, which encodes the deor repressor protein in Escherichia coli, was fused to the strong Ptrc promoter in plasmid pKK233-2. The Ptrc promoter is kept repressed by lacI repressor to prevent cell killing. Induction of the Ptrc--deoR fusion plasmid resulted in the accumulation of 4% of the soluble protein as deoR protein. The deoR repressor protein was purified to 80% purity using conventional techniques; it has a mass of 28.5 kd and appears to exist as an octamer in solution. The deoR repressor is shown by DNase I footprinting to bind to the 16 bp palindromic sequence in the Pribnow box region of the deoP1 promoter. Also, the deoR repressor binds cooperatively in vitro to a DNA template with two deoR binding sites separated by 224 bp in keeping with the conclusion from genetic experiments that more than one operator is required for efficient repression of the deo operon.

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.

DNA-protein recognition: demonstration of three genetically separated operator elements that are required for repression of the Escherichia coli deoCABD promoters by the DeoR repressor.

The sequences required for full repression of the Escherichia coli deoP1 and P2 promoters by the deoR repressor (DeoR) have been analyzed in vivo. Using recombinant techniques, we have constructed a set of deo-lacZ fusions which contain different parts of the sequences involved in the regulation of deo expression on low copy number fusion vectors. Since these vectors are present in only one copy per chromosome at temperatures below 37 degrees C, this vector system allows very accurate studies of gene control signals. Our results show that three DeoR operator sites exist in the deoP1-P2 regulatory region. Two of these loci overlap the initiation sites for deoP1 (O1) and deoP2 (O2) transcription located 599 bp apart, whereas the third site (OE) is present approximately 270 bp upstream of P1. DeoR repression of both P1 and P2 transcription is weak on promoter fragments which only contain one operator site (O1 or O2). Enhanced repression by deoR is observed on promoter fragments containing two operator sites. However, all three sites are needed for full repression. These findings are discussed with respect to upstream and downstream control regions of eukaryotic genes.

Two operator sites separated by 599 base pairs are required for deoR repression of the deo operon of Escherichia coli.

DeoP1 and deoP2 promoter fragments from the deo operon of Escherichia coli have been transcriptionally fused to the galactokinase gene. From single-copy expression of these fusions it is shown that the deoR binding site of both deoP1 and deoP2 are necessary to achieve full repression of the deo operon by the deoR repressor. Repression of the promoters can be achieved either by supplying extra deoR repressor in trans or by introduction of an extra deoR binding site at a position between 224 and 997 bp upstream of the promoter. Furthermore, the deoP2 promoter is shown to be regulated in a cumulative way by both the deoR and the cytR repressors, while deoP1 is only regulated by the deoR repressor. DeoP2 is a strong promoter being 20 times stronger than araPBAD and four times stronger than deoP1.

The structure of tandem regulatory regions in the deo operon of Escherichia coli K12

The transcription initiation sites of the deo operon of Escherichia coli K12 have been determined in vitro and in vivo. Initiation of transcription occurs at two, tandemly arranged, homologous regions which are located 600 base pairs apart from each other. Evidence for a novel regulation mechanism of deo gene expression involving the deoR repressor is presented.

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.