Diana Marra Oram

Corynebacterium diphtheriae , a Gram-positive, aerobic bacterium, is the causative agent of diphtheria and cutaneous infections. While mechanisms required for heme iron acquisition are well known in C. diphtheriae , systems involved in the acquisition of other metals such as zinc and manganese remain poorly characterized. In this study, we identified a genetic region that encodes an ABC-type transporter ( iutBCD ) and that is flanked by two genes ( iutA and iutE ) encoding putative substrate binding proteins of the cluster 9 family, a related group of transporters associated primarily with the import of Mn and Zn. We showed that IutA and IutE are both membrane proteins with comparable Mn and Zn binding abilities. We demonstrated that the iutABCD genes are cotranscribed and repressed in response to iron by the iron-responsive repressor DtxR. Transcription of iutE was positively regulated in response to iron availability in a DtxR-dependent manner and was repressed in response to Zn by the Zn-dependent repressor Zur. Electrophoretic mobility shift assays showed that DtxR does not bind to the iutE upstream region, which indicates that DtxR regulation of iutE is indirect and that other regulatory factors controlled by DtxR are likely responsible for the iron-responsive regulation. Analysis of the iutE promoter region identified a 50-bp sequence at the 3′ end of the iutD gene that is required for the DtxR-dependent and iron-responsive activation of the iutE gene. These findings indicate that transcription of iutE is controlled by a complex mechanism that involves multiple regulatory factors whose activity is impacted by both Zn and Fe. IMPORTANCE Vaccination against diphtheria prevents toxin-related symptoms but does not inhibit bacterial colonization of the human host by the bacterium. Thus, Corynebacterium diphtheriae remains an important human pathogen that poses a significant health risk to unvaccinated individuals. The ability to acquire iron, zinc, and manganese is critical to the pathogenesis of many disease-causing organisms. Here, we describe a gene cluster in C. diphtheriae that encodes a metal importer that is homologous to broadly distributed metal transport systems, some with important roles in virulence in other bacterial pathogens. Two metal binding components of the gene cluster encode surface exposed proteins, and studies of such proteins may guide the development of second-generation vaccines for C. diphtheriae .

Regulation of metal ion homeostasis is essential to bacterial cell survival, and in most species it is controlled by metal-dependent transcriptional regulators. In this study, we describe a Corynebacterium diphtheriae ferric uptake regulator-family protein, Zur, that controls expression of genes involved in zinc uptake. By measuring promoter activities and mRNA levels, we demonstrate that Zur represses transcription of three genes ( zrg , cmrA , and troA ) in zinc-replete conditions. All three of these genes have similarity to genes involved in zinc uptake. Transcription of zrg and cmrA was also shown to be regulated in response to iron and manganese, respectively, by mechanisms that are independent of Zur. We demonstrate that the activity of the zur promoter is slightly decreased under low zinc conditions in a process that is dependent on Zur itself. This regulation of zur transcription is distinctive and has not yet been described for any other zur . An adjacent gene, predicted to encode a metal-dependent transcriptional regulator in the ArsR/SmtB family, is transcribed from a separate promoter whose activity is unaffected by Zur. A C. diphtheriae zur mutant was more sensitive to peroxide stress, which suggests that zur has a role in protecting the bacterium from oxidative damage. Our studies provide the first evidence of a zinc specific transcriptional regulator in C. diphtheriae and give new insights into the intricate regulatory network responsible for regulating metal ion concentrations in this toxigenic human pathogen.

This report describes the development and use of TnKnXSp, a selectable broad-host-range reporter transposon with a promoterless aphA gene. Insertion

The iron-dependent transcriptional regulator DtxR from Corynebacterium diphtheriae is the prototype for a family of metal-dependent regulators found in diverse bacterial species. The structure of DtxR and its action as a repressor have been extensively characterized, but little is known about expression of dtxR. In the current study, we investigated transcription of dtxR as well as the sigB and galE genes located immediately upstream and downstream from dtxR, respectively. We identified two promoters that determine transcription of dtxR. The first, located upstream of sigB, appears to be controlled by an extracytoplasmic function sigma factor. The second, located in the intergenic region between sigB and dtxR, is similar to promoters used by the primary vegetative sigma factors in other actinomycete species. Using quantitative real-time assays, we demonstrated that the number of transcripts initiated upstream from sigB is affected by several environmental factors. In contrast, the presence of sodium dodecyl sulfate was the only factor tested that conclusively affects the number of transcripts initiated in the sigB-dtxR intergenic region. Additionally, we provided evidence for the existence of transcripts that contain sigB, dtxR, and galE. Our studies provide the first quantitative transcriptional analysis of a gene encoding a DtxR family regulator and give new insights into transcriptional regulation in C. diphtheriae.

Iron dependent regulatory proteins of the diphtheria toxin repressor family regulate transcription in a variety of bacterial species. These regulator

Metal-dependent transcriptional regulators of the diphtheria toxin repressor (DtxR) family have been identified in a wide variety of bacterial genera, where they control gene expression in response to one of two metal ions, Fe²⁺or Mn²⁺. DtxR ofCorynebacterium diphtheriaeis the best characterized of these important metal-dependent regulators. The genusCorynebacteriumincludes many phenotypically diverse species, and the prevalence of DtxR-like regulators within the genus is unknown. We assayed chromosomal DNA from 42 different corynebacterial isolates, representing 33 different species, for the presence of a highly conserved region of thedtxRgene that encodes the DNA-binding helix-turn-helix motif and metal-binding site 1 within domains 1 and 2 of DtxR. The chromosome of all of the isolates contained this conserved region ofdtxR, and DNA sequencing revealed a high level of nucleotide sequence conservation within this region in all of the corynebacterial species (ranging from 62 to 100% identity and averaging 70% identity with thedtxRprototype). The level of identity was even greater for the predicted protein sequences encoded by thedtxR-like genes, ranging from 81 to 100% identity and averaging 91% identity with DtxR. Using a DtxR-specific antiserum we confirmed the presence of a DtxR-like protein in extracts of most of the corynebacterial isolates and determined the precise amount of DtxR per cell inC. diphtheriae. The high level of identity at both DNA and protein levels suggests that all of the isolates tested encode a functional DtxR-like Fe²⁺-activated regulatory protein that can bind homologs of the DtxR operator and regulate gene expression in response to iron.

Transcription of the bacteriophage-borne diphtheria toxin genetoxis negatively regulated, in response to intracellular Fe²⁺concentration, by the chromosomally encoded diphtheria toxin repressor (DtxR). Due to a scarcity of tools, genetic analysis ofCorynebacterium diphtheriaehas primarily relied on analysis of chemically induced and spontaneously occurring mutants and on the results of experiments withC. diphtheriaegenes cloned inEscherichia colior analyzed in vitro. We modified a Tn5-based mutagenesis technique for use withC. diphtheriae, and we used it to construct the first transposon insertion libraries in the chromosome of this gram-positive pathogen. We isolated two insertions that affected expression of DtxR, one 121 bp upstream ofdtxRand the other within an essential region of thedtxRcoding sequence, indicating for the first time thatdtxRis a dispensable gene inC. diphtheriae. Both mutant strains secrete diphtheria toxin when grown in medium containing sufficient iron to repress secretion of diphtheria toxin by wild-typeC. diphtheriae. The upstream insertion mutant still produces DtxR in decreased amounts and regulates siderophore secretion in response to iron in a manner similar to its wild-type parent. The mutant containing the transposon insertion withindtxRdoes not produce DtxR and overproduces siderophore in the presence of iron. Differences in the ability of the two mutant strains to survive oxidative stress also indicated that the upstream insertion retained slight DtxR activity, whereas the insertion withindtxRabolished DtxR activity. This is the first evidence that DtxR plays a role in protecting the cell from oxidative stress.