High rate of early virological failure with the once-daily tenofovir/lamivudine/nevirapine combination in naive HIV-1-infected patients.

BACKGROUND: The combination of one non-nucleoside reverse transcriptase inhibitor (NNRTI) with two nucleoside reverse transcriptase inhibitors is a validated first-line antiretroviral (ARV) therapy. The once-daily combination of lamivudine, tenofovirDF and nevirapine has not been evaluated in a clinical trial. METHODS: Randomized, open-label, multicentre, non-inferiority trial comparing lamivudine, tenofovirDF and nevirapine once daily (Group 2) with zidovudine/lamivudine and nevirapine twice daily (Group 1), in naive HIV-1-infected patients with a CD4 count <350/mm(3). We planned to enroll 250 patients. RESULTS: As of May 2006, 71 patients had been enrolled (35 in Group 1 and 36 in Group 2) and an unplanned interim analysis was done. The groups were comparable at baseline: median CD4 count was 195 and 191/mm(3) and median plasma viral load was 4.9 log(10) and 5.01 log(10), respectively, in Groups 1 and 2. Eight early non-responses (22.2%) were observed, all in Group 2, while two later viral rebounds occurred. Resistance genotypes for the nine Group 2 failing patients showed the mutations M184V/I (n = 3), K65R (n = 6), one or more NNRTI resistance mutations in all cases. At baseline, the nine Group 2 patients who failed had higher median plasma viral load (5.4 log(10)) and lower median CD4 count (110/mm(3)) than the other Group 2 patients (4.7 log(10), P = 0.002 and 223/mm(3), P = 0.004). Nevirapine trough concentrations were not different between the two groups, nor between patients with full viral suppression or those who failed in Group 2. Due to slow recruitment, and those results, the steering committee decided to stop the trial at 12 months. CONCLUSIONS: In ARV-naive HIV-1-infected patients, the once-daily lamivudine, tenofovirDF and nevirapine regimen resulted in a high rate of early virological failures. The reasons for the failures remain unclear.

Three-dimensional structure of the diphtheria toxin repressor in complex with divalent cation co-repressors.

BACKGROUND: When Corynebacterium diphtheriae encounters an environment with a low concentration of iron ions, it initiates the synthesis of several virulence factors, including diphtheria toxin. The diphtheria toxin repressor (DtxR) plays a key role in this iron-dependent, global regulatory system and is the prototype for a new family of iron-dependent repressor proteins in Gram-positive bacteria. This study aimed to increase understanding of the general regulatory principles of cation binding to DtxR. RESULTS: The crystal structure of dimeric DtxR holo-repressor in complex with different transition metals shows that each subunit comprises an amino-terminal DNA-binding domain, an interface domain (which contains two metal-binding sites) and a third, very flexible carboxy-terminal domain. Each DNA-binding domain contains a helix-turn-helix motif and has a topology which is very similar to catabolite gene activator protein (CAP). Molecular modeling suggests that bound DNA adopts a bent conformation with helices alpha 3 of DtxR interacting with the major grooves. The two metal-binding sites lie approximately 10 A apart. Binding site 2 is positioned at a potential hinge region between the DNA-binding and interface domains. Residues 98-108 appear to be crucial for the functioning of the repressor; these provide four of the ligands of the two metal-binding sites and three residues at the other side of the helix which are at the heart of the dimer interface. CONCLUSIONS: The crystal structure of the DtxR holorepressor suggests that the divalent cation co-repressor controls motions of the DNA-binding domain. In this way the metal co-repressor governs the distance between operator recognition elements in the two subunits and, consequently, DNA recognition.

Sequence of a conditionally essential region of bacteriophage T3, including the primary origin of DNA replication.

The 3526 base-pair nucleotide sequence from near the end of bacteriophage T3 gene 1 to within the coding sequence of gene 2.5 is given. It includes the complete coding sequences for nine known or presumptive proteins, most of which are only conditionally essential for phage growth. The sequence includes five promoters for the phage RNA polymerase, the terminator for early (host enzyme-catalyzed) transcription, and two recognition sites for RNAase III. The primary origin of T3 DNA replication that is utilized by the phage in vivo has been localized to a 142 base-pair region. It has several features in common with the phage T7 origin of DNA replication, and exhibits considerable homology to recognition sites for the mRNA processing enzyme RNAase III. It is proposed that the primary origin of T3 DNA replication may have evolved directly from an RNAase III recognition site. The deletions present in a number of T3 mutant strains and the location of the nucleotide changes in several T3 strains that are defective in their ability to grow on F+-containing strains or on optA mutant hosts have been determined. We discuss how T3 may have become genetically isolated from its relatives in the T7-T3 group and simultaneously acquired novel biological and biochemical properties.

Zidovudine resensitization and dual HIV-1 resistance to zidovudine and lamivudine in the delta lamivudine roll-over study.

OBJECTIVE: To study zidovudine resensitization and dual resistance to zidovudine/lamivudine in HIV-1 isolates from nucleoside reverse transcriptase (RT) inhibitor-experienced patients during selective pressure exerted by zidovudine/lamivudine combination therapy. DESIGN AND METHODS: HIV-1 isolates from 29 patients receiving zidovudine/lamivudine combination therapy in the Delta roll-over study were analysed at entry and during a 1 year follow-up period for phenotypic susceptibility to zidovudine and lamivudine in the ANRS PBMC assay. The RT gene from codon 20 to 230 and at codon 333 was analysed by nucleotide sequencing of the corresponding isolates. RESULTS: HIV-1 isolates from 23 of the 29 patients were phenotypically resistant to zidovudine at baseline; 61% of these patients showed significant zidovudine resensitization during follow-up. The zidovudine IC50 value correlated positively with log10 plasma HIV-1 RNA (P = 0.02) and negatively with the CD4 cell count (P = 0.004). Zidovudine resensitization (related to acquisition of the M184V mutation) was transient, with evolution towards dual resistance to zidovudine and lamivudine in 20 of the 29 patients. The phenotype of certain dually resistant isolates coincided with the emergence of multiple mutations in the 5' part of the RT gene. CONCLUSIONS: M184V-mediated zidovudine resensitization of HIV-1 is transient in most patients who are given zidovudine/lamivudine combination therapy when zidovudine resistance has already emerged. The subsequent evolution towards dual phenotypic resistance to zidovudine/lamivudine corresponds to complex genotypic profiles.

Permissivity of primary cultures of human Kupffer cells for HIV-1.

Kupffer cells (liver macrophages) represent the largest reservoir of fixed macrophages in the body. Accordingly, we have undertaken a study to evaluate their susceptibility to human immunodeficiency virus type 1 (HIV-1). Five-day-old primary cultures of Kupffer cells (KC) were infected with HIV-1, and as the infection progressed, syncytia appeared. Within the cells, viral proteins were detected by immunofluorescence using monoclonal antibodies directed against gp120 and p24. Electron microscopic examinations revealed the presence of typical Lentivirinae particles. The particles released from KC in the extracellular medium showed reverse transcriptase activity and p24 antigen; they could infect lymphocytic cells and were neutralized by a HIV+ patient's serum or an anti-gp120 monoclonal antibody. Our results thus demonstrate that the interaction of HIV-1 with KC in vitro leads to a productive infection. They suggest that the KC may be involved in the pathogenesis of HIV-1 infection and may (i) participate in the transmission of the infection to the peripheral blood cells, (ii) play a role in the depletion of uninfected CD4+ cells.

HIV-1 sensitivity to zidovudine: a consensus culture technique validated by genotypic analysis of the reverse transcriptase.

In order to select and standardize a reliable assay for the analysis of sensitivity of HIV isolates to AZT, we have compared two culture methods. The first assay (Cell-Associated Isolate Sensitivity Assay: CAISA) quantified AZT-resistant HIV isolates by end-point dilution cultures of peripheral blood mononuclear cells (PBMCs) in the presence of various concentrations of AZT. In the second assay (Cell-Free Isolate Sensitivity Assay: CFISA), following a conventional isolation of HIV, dilutions of infected cell-free supernatants were cultivated with fresh normal donor PBMCs in the presence of increasing concentrations of AZT. Samples from 64 untreated and AZT-treated patients were studied by CAISA (41), CFISA (43) or both assays (20). The CFISA, which allows the determination of titration parameters with respect to various kinetics patterns of viral replication was selected, and some of the CFISA phenotypically characterized isolates were further studied by nucleotide sequence analysis of the reverse transcriptase gene. CFISA showed that isolates from untreated patients were susceptible to AZT while the frequency of resistance increased with the duration of therapy. Genotypic analysis of CFISA-resistant isolates exhibited mutations at crucial positions, particularly at residue 215. We consider CFISA as a consensus culture technique for longitudinal studies of isolates from patients receiving AZT or other analogs of nucleosides.

Utilization of host iron sources by Corynebacterium diphtheriae: identification of a gene whose product is homologous to eukaryotic heme oxygenases and is required for acquisition of iron from heme and hemoglobin.

Corynebacterium diphtheriae was examined for the ability to utilize various host compounds as iron sources. C. diphtheriae C7(-) acquired iron from heme, hemoglobin, and transferrin. A siderophore uptake mutant of strain C7 was unable to utilize transferrin but was unaffected in acquisition of iron from heme and hemoglobin, which suggests that C. diphtheriae possesses a novel mechanism for utilizing heme and hemoglobin as iron sources. Mutants of C. diphtheriae and Corynebacterium ulcerans that are defective in acquiring iron from heme and hemoglobin were isolated following chemical mutagenesis and streptonigrin enrichment. A recombinant clone, pCD293, obtained from a C7(-) genomic plasmid library complemented several of the C. ulcerans mutants and three of the C. diphtheriae mutants. The nucleotide sequence of the gene (hmuO) required for complementation was determined and shown to encode a protein with a predicted mass of 24,123 Da. Sequence analysis revealed that HmuO has 33% identity and 70% similarity with the human heme oxygenase enzyme HO-1. Heme oxygenases, which have been well characterized in eukaryotes but have not been identified in prokaryotes, are involved in the oxidation of heme and subsequent release of iron from the heme moiety. It is proposed that the HmuO protein is essential for the utilization of heme as an iron source by C. diphtheriae and that the heme oxygenase activity of HmuO is involved in the release of iron from heme. This is the first report of a bacterial gene whose product has homology to heme oxygenases.

Transcription of the Corynebacterium diphtheriae hmuO gene is regulated by iron and heme.

The hmuO gene is required for the utilization of heme and hemoglobin as iron sources by Corynebacterium diphtheriae. The product of hmuO has homology to eukaryotic heme oxygenases which are involved in the degradation of heme and the release of iron. To investigate the mechanism of hmuO regulation, a promoterless lacZ gene present on the promoter-probe vector pCM502 was placed under transcriptional control of the hmuO promoter. In C. diphtheriae C7, optimal expression from the hmuO promoter was obtained only in the presence of heme or hemoglobin under low-iron conditions. Expression of hmuO in high-iron medium containing heme was repressed five- to sixfold from that seen under low-iron conditions in the presence of heme. Transcription from the hmuO promoter in the absence of heme or hemoglobin was fully repressed in high-iron medium and was expressed at very low levels in iron-depleted conditions. Expression studies with tile hmuO-lacZ fusion construct in C7hm723, a dtxR mutant of C7, and in a hmuO mutant of C. diphtheriae HC1 provided further evidence that transcription of the hmuO promoter is repressed by DtxR and iron and activated by heme. In Escherichia coli, the hmuO promoter was expressed at very low levels under all conditions examined. Gel mobility shift assays and DNase I footprinting experiments indicated that DtxR binds in a metal-dependent manner to a sequence that overlaps the putative hmuO promoter. Total cellular RNA isolated from C. diphtheriae was used to identify the transcriptional start site for the hmuO gene. Northern blot analysis suggested that the hmuO mRNA was monocistronic and that transcription was heme inducible.

Identification of a two-component signal transduction system from Corynebacterium diphtheriae that activates gene expression in response to the presence of heme and hemoglobin.

Corynebacterium diphtheriae, the causative agent of diphtheria, utilizes various host compounds to acquire iron. The C. diphtheriae hmuO gene encodes a heme oxygenase that is involved in the utilization of heme and hemoglobin as iron sources. Transcription of the hmuO gene in C. diphtheriae is controlled under a dual regulatory mechanism in which the diphtheria toxin repressor protein (DtxR) and iron repress expression while either heme or hemoglobin is needed to activate transcription. In this study, two clones isolated from a C. diphtheriae chromosomal library were shown to activate transcription from the hmuO promoter in Escherichia coli. Sequence analysis revealed that these activator clones each carried distinct genes whose products had significant homology to response regulators of two-component signal transduction systems. Located upstream from each of these response regulator homologs are partial open reading frames that are predicted to encode the C-terminal portions of sensor kinases. The full-length sensor kinase gene for each of these systems was cloned from the C. diphtheriae chromosome, and constructs each carrying one complete sensor kinase gene and its cognate response regulator were constructed. One of these constructs, pTSB20, which carried the response regulator (chrA) and its cognate sensor kinase (chrS), was shown to strongly activate transcription from the hmuO promoter in a heme-dependent manner in E. coli. A mutation in chrA (chrAD50N), which changed a conserved aspartic acid residue at position 50, the presumed site of phosphorylation by ChrS, to an asparagine, abolished heme-dependent activation. These findings suggest that the sensor kinase ChrS is involved in the detection of heme and the transduction of this signal, via a phosphotransfer mechanism, to the response regulator ChrA, which then activates transcription of the hmuO promoter. This is the first report of a bacterial two-component signal transduction system that controls gene expression through a heme-responsive mechanism.

Construction and consequences of directed mutations affecting the hemin receptor in pathogenic Corynebacterium species.

Genes encoding an ATP-binding cassette transporter system involved in hemin iron utilization from Corynebacterium ulcerans were cloned and characterized. The genes are homologous to a hemin transport system previously identified in Corynebacterium diphtheriae. Disruption of the hmuT gene, which encodes the putative hemin receptor, resulted in greatly reduced ability of C. ulcerans to use hemin or hemoglobin as an iron source. Inactivation of hmuT in C. diphtheriae by site-specific recombination had no effect on hemin utilization, which suggests that C. diphtheriae has an additional system for transporting hemin.

Cloning, sequence, and footprint analysis of two promoter/operators from Corynebacterium diphtheriae that are regulated by the diphtheria toxin repressor (DtxR) and iron.

DtxR is an iron-dependent sequence-specific DNA-binding protein that binds to the tox operator, an inverted-repeat nucleotide sequence located upstream from the diphtheria toxin gene. In this study, two additional iron-regulated promoter/operator sequences (IRP1 and IRP2) that are controlled by DtxR were cloned from the chromosome of Corynebacterium diphtheriae and characterized. Operon fusions to lacZ were used to analyze expression from IRP1 and IRP2 in Escherichia coli. Transcription from both promoters was strongly repressed in high-iron medium in the presence of the cloned dtxR gene; however, transcription in the absence of dtxR was 50- to 100-fold greater, regardless of the iron concentration. Purified DtxR altered the electrophoretic mobility of DNA fragments carrying IRP1 or IRP2, and the nucleotide sequences of the two promoter/operator regions indicated that they are both homologous with the tox operator. DtxR protected an approximately 30-bp region on both IRP1 and IRP2 from DNase I digestion. A 19-bp consensus DtxR-binding site was derived from a comparison of the various DtxR-regulated operator/promoter sequences. Footprinting experiments using hydroxyl radicals and dimethyl sulfate demonstrated that DtxR interacted with these operators in a symmetrical manner, probably as a dimer or multimer. The deduced amino acid sequence of an open reading frame (ORF1) located downstream from IRP1 was homologous with a family of periplasmic proteins involved in iron transport in gram-negative bacteria and with the ferrichrome receptor, FhuD, from Bacillus subtilis. These findings suggest that ORF1 encodes a membrane-associated lipoprotein that may serve as the receptor for a ferric-siderophore complex in C. diphtheriae.

Characterization of a defective diphtheria toxin repressor (dtxR) allele and analysis of dtxR transcription in wild-type and mutant strains of Corynebacterium diphtheriae.

The production of diphtheria toxin and siderophore by the Corynebacterium diphtheriae regulatory mutant C7(beta)hm723 is resistant to the inhibitory effects of iron, and the mutant strain is defective for function of the regulatory gene dtxR. A 2.8-kb HindIII fragment carrying the C7(beta)hm723 dtxR allele was cloned and characterized in Escherichia coli. The restriction endonuclease maps of the 2.8-kb HindIII fragment from C7(beta)hm723 and the corresponding fragment from wild-type C. diphtheriae C7 were identical. RNA dot blot analysis with total RNA isolated from wild-type C. diphtheriae C7 and C7(beta)hm723 indicated that the dtxR gene was transcribed at very low but equivalent levels in both strains and was not regulated by iron. beta-Galactosidase synthesis from a tox-lacZ translational fusion construct in E. coli in high-iron medium was not repressed by the C7(beta)hm723dtxR allele, but was strongly repressed by the wild-type dtxR gene. The 28- to 29-kDa polypeptide expressed from the mutant dtxR allele in E. coli had the same electrophoretic mobility as the wild-type dtxR gene product in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The nucleotide sequence of the coding region and the 5' upstream region of the C7(beta)hm723 dtxR allele was determined and compared with the wild-type nucleotide sequence. The dtxR allele from C7(beta)hm723 contained a single-base change located 140 nucleotides from the 5' start of the gene, which resulted in replacement of arginine in the wild-type sequence by histidine in the mutant protein. These data demonstrate that C7(beta)hm723 expresses a mutant DtxR repressor protein that is severely defective in repressor activity.

Analysis of diphtheria toxin repressor-operator interactions and characterization of a mutant repressor with decreased binding activity for divalent metals.

The diphtheria toxin repressor (DtxR) is an Fe(2+)-activated protein with sequence-specific DNA-binding activity for the diphtheria toxin (tox) operator. Under high-iron conditions in Corynebacterium diphtheriae, DtxR represses toxin and siderophore biosynthesis as well as iron uptake. DtxR and a mutant repressor with His-47 substituted for Arg-47, designated DtxR-R47H, were purified and compared. Six different divalent cations (Cd2+, Co2+, Fe2+, Mn2+, Ni2+, and Zn2+) activated the sequence-specific DNA-binding activity of DtxR and enabled it to protect the tox operator from DNase I digestion, but Cu2+ failed to activate DtxR. Hydroxyl radical footprinting experiments indicated that DtxR binds symmetrically about the dyad axis of the tox operator. Methylation protection experiments demonstrated that DtxR binding alters the susceptibility to methylation of three G residues within the AT-rich tox operator. These findings suggest that two or more monomers of DtxR are involved in binding to the tox operator, with symmetrical DNA-protein interactions occurring at each end of the palindromic operator. In this regard, DtxR resembles several other well-characterized prokaryotic repressor proteins but differs dramatically from the Fe(2+)-activated ferric uptake repressor protein (Fur) of Escherichia coli. The concentration of Co2+ required to activate DtxR-R47H was at least 10-fold greater than that needed to activate DtxR, but the sequence-specific DNA binding of activated DtxR-R47H was indistinguishable from that of wild-type DtxR. The markedly deficient repressor activity of DtxR-R47H is consistent with a significant decrease in its binding activity for divalent cations.

Genetics and regulation of enterobactin genes in Shigella flexneri.

Although Shigella flexneri possesses the genes for two siderophore systems, enterobactin and aerobactin, the enterobactin system is only rarely utilized. To investigate the regulation of enterobactin expression in S. flexneri, all of the genes specifically required for synthesis and transport of enterobactin were cloned from both an expressing (Ent+) and a nonexpressing (Ent-) strain. Notable differences between the cloned genes included endonuclease restriction site changes and the presence of an IS1 element in the Ent- DNA. Southern hybridization revealed that this IS1 element, present at the 3' end of the entF gene, is conserved at this location in different strains and serotypes of Ent- S. flexneri. The Ent- cloned genes were tested for their ability to complement the defect in 11 different Escherichia coli enterobactin mutants. The Ent- genes fully complemented nine mutants but failed to complement the entF mutant AN117 and only partially complemented the entE mutant AN93. Whole-cell RNA isolated from E. coli and the Shigella strains was hybridized to 32P-labeled DNA containing the entB gene or a fragment carrying a portion of the entF gene. E. coli and the Ent+ Shigella strains exhibited derepression of transcription of these genes in low-iron media. Transcription in the Ent- strain remained repressed regardless of iron concentration. Expression of the entB and entF genes was also examined in an Ent- Shigella fur mutant. Expression of entF was only partially derepressed and entB remained fully repressed at all iron concentrations, suggesting that factors other than Fur are responsible for the repression of these enterobactin genes in the Ent- Shigella strains.

Iron-dependent regulation of diphtheria toxin and siderophore expression by the cloned Corynebacterium diphtheriae repressor gene dtxR in C. diphtheriae C7 strains.

A regulatory gene (dtxR) responsible for iron-dependent repression of the toxin (tox) and siderophore genes in Corynebacterium diphtheriae was cloned and characterized. A DNA fragment carrying dtxR repressed expression of a tox-lacZ gene fusion in Escherichia coli DH5 alpha in a high-iron environment but not under low-iron conditions. A protein with mobility corresponding to approximately 28 to 29 kDa was identified as the product of the dtxR gene by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A shuttle vector designated pCM2.6 was constructed which carries the origin of replication from C. diphtheriae plasmid pNG2 and confers resistance to chloramphenicol in E. coli and C. diphtheriae. DNA fragments carrying dtxR were cloned into pCM2.6, and the hybrid shuttle plasmids were transformed by electroporation into wild-type C. diphtheriae C7(beta) and the regulatory mutant C7(beta)hm723, which produces toxin and siderophore constitutively under high-iron conditions. Expression of the cloned dtxR determinant did not affect the phenotype of C. diphtheriae C7(beta). In C. diphtheriae C7(beta)hm723, expression of cloned dtxR restored full repression of siderophore production and partial repression of diphtheria toxin production during growth in a high-iron environment.

Genetic analysis of the enterobactin gene cluster in Shigella flexneri.

The genes for transport and synthesis of the phenolate siderophore enterobactin are present on the chromosomes of both Ent+ and Ent- clinical isolates of Shigella flexneri. To determine why Ent- S. flexneri isolates fail to express a functional enterobactin system, the structure and expression of enterobactin genes were examined. Several alterations may be responsible for the inability of S. flexneri to express enterobactin. (i) The mRNA levels produced from the entC and fepB genes were not derepressed in low-iron media. (ii) DNA sequence analysis of the entC-fepB intergenic region revealed an 83-bp noncontiguous deletion in the putative fepB leader sequence. The deleted sequences are in a region which would be capable of forming extensive stem-and-loop structures. (iii) An amber codon in the 5' portion of the entC gene was also detected. (iv) An IS1 element, previously mapped to the Ent- S. flexneri enterobactin gene cluster, was found to lie within a potential transcriptional termination sequence in the entF-fepE intergenic region. (v) A mutation responsible for the inactivation of the entF gene was mapped to the entF coding region by using entF hybrid gene fusions. (vi) A comparison of outer membrane profiles from an E. coli strain harboring the cloned fepA gene from either an Ent+ or Ent- Shigella isolate revealed that the Ent- FepA protein is present in the outer membrane but at greatly reduced levels than that of the Ent+ FepA protein. This observation, along with additional studies, suggests that the Ent- FepA may be defective in translation and/or translocation.

Expression and characterization of a heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Iron acquisition requires oxidative cleavage of the heme macrocycle.

A full-length heme oxygenase gene from the pathogenic bacterium Corynebacterium diphtheriae has been subcloned and expressed in Escherichia coli. The enzyme is expressed at high levels as a soluble catalytically active protein that results in the accumulation of biliverdin within the E. coli cells. The purified heme oxygenase forms a 1:1 complex with heme (Kd = 2.5 +/- 1 microM) and has hemeprotein spectra similar to those previously reported for the purified eukaryotic heme oxygenases. In the presence of an E. coli NADPH-dependent reductase isolated during the purification of Hmu O, the heme-Hmu O complex is catalytically turned over to yield biliverdin IXalpha and carbon monoxide. A number of redox partners were investigated for their ability to reconstitute Hmu O activity in vitro. Of these the most efficient appeared to be the recombinant NADH-dependent putidaredoxin/putidaredoxin reductase from Pseudomonas putida. As with the E. coli NADPH-dependent reductase the final products of the reaction were biliverdin IXalpha and carbon monoxide. This is the first bacterial heme oxygenase to be described to date. The close relationship between iron acquisition and pathogenesis suggests that the release of iron from heme by heme oxygenase may play a crucial role in the pathogenicity of C. diphtheriae.

Characterization of mutations that inactivate the diphtheria toxin repressor gene (dtxR).

The diphtheria toxin repressor (DtxR) is an iron-dependent regulator of diphtheria toxin production and iron uptake in Corynebacterium diphtheriae. It is activated in vitro by divalent metal ions including Fe2+, Cd2+, Co2+, Mn2+, Ni2+, and Zn2+. We characterized 20 different mutations in dtxR induced by bisulfite mutagenesis, 18 of which caused single-amino-acid substitutions in DtxR and two of which were chain-terminating mutations. Six of the amino acid replacements were clustered between residues 39 and 52 in a predicted helix-turn-helix motif that exhibits homology with several other repressors and is identified as the putative DNA-binding domain of DtxR. Three substitutions occurred within a predicted alpha-helical region with the sequence His-98-X3-Cys-102-X3-His-106 that resembles metal-binding motifs in several other proteins and is identified as the putative metal-binding site of DtxR. Several purified variants of DtxR with decreased repressor activity failed to bind in gel retardation assays to DNA fragments that contained the tox operator. A quantitative assay for binding of DtxR to 63Ni2+ was also developed. Scatchard analysis revealed that DtxR has a single class of high-affinity 63Ni(2+)-binding sites with a Kd of 2.11 x 10(-6) M and a maximum binding capacity of approximately 1.2 atoms of Ni2+ per DtxR monomer. The P39L, T40I, T44I, and R47H variants of DtxR exhibited normal to slightly decreased 63Ni(2+)-binding activity, but H106Y, which has an amino acid substitution in the presumed metal-binding domain, exhibited markedly decreased 63Ni(2+)-binding activity.

Purification and characterization of the diphtheria toxin repressor.

The diphtheria toxin repressor gene (dtxR) encodes a protein (DtxR) that regulates transcription of the diphtheria toxin gene (tox) by an iron-dependent mechanism. Cloned dtxR was expressed in Escherichia coli from the phage T7 gene 10 promoter, and DtxR was purified. Specific binding of DtxR to the tox+ operator was dependent on reduction of DtxR and the presence of ferrous ions. DtxR protected a sequence of approximately 30 nucleotide pairs, partially overlapping the tox promoter and containing a region of dyad symmetry, from digestion by DNase I. DtxR exhibited very little binding to the mutant tox-201 operator region and failed to bind to the promoter/operator region of the ferric uptake regulation (fur) gene of E. coli.