Transcription of the ContiguoussigB,dtxR, andgalEGenes inCorynebacterium diphtheriae: Evidence for Multiple Transcripts and Regulation by Environmental Factors

Abstract

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 factor. The second, located in the intergenic region between sigB and dtxR, is similar to promoters used by the primary vegetative 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.

Bacteriophage-based vectors for site-specific insertion of DNA in the chromosome of Corynebacteria

“…C7(β)ΔdtxR::pK-AIM suppressed production of siderophore in the presence of FeCl 3 by approximately 6-fold but it produced approximately 10 times more siderophore under high-iron conditions than strains that expressed dtxR. This result is equivalent to that observed previously with C7(β)ΔdtxR (Oram et al, 2006). In contrast when pK-AIMdtxR was integrated into attB2 in C7(β)ΔdtxR (to form C7(β)ΔdtxR::pK-AIMdtxR) the ability to repress siderophore production by approximately 50-fold in the presence of FeCl 3 was restored.…”

“…In fact, while we had previously demonstrated that an intact copy of dtxR was able to reverse the phenotype of the C7(β)ΔdtxR strain when introduced in trans on a replicating plasmid (Oram et al, 2006), this work is the first to establish that a single copy of dtxR integrated into attB2 is capable of restoring DtxR functions to levels seen in a wild-type control. In addition, the inclusion of a transcriptional reporter gene to an integrating vector would permit characterization of the activity of a promoter in single copy from the C. diphtheriae chromosome.…”

“…The combination of these functions with the oriT mobilization system for DNA transfer yielded a system with many potential applications. For example, the construction of gene deletions in the chromosome of C. diphtheriae has been reported by several groups (Oram et al, 2006;Schmitt and Drazek, 2001;Ton-That et al, 2004). This approach, however, is not likely to be successful if an essential gene is to be deleted.…”

“…Nevertheless, the ΔdtxR mutant has a growth defect compared with C7(β) in high iron medium and is unable to regulate sideophore production in response to iron availability (Oram et al, 2006). We thus determined if dtxR expressed from a novel chromosomal location (namely attB2) could complement these two DtxR-dependent phenotypes of C7(β)ΔdtxR.…”

“…Matings were performed as previously described (Oram et al, 2006) using E. coli S17-1 containing an RP4 mobilizable plasmid as the donor and strains of C. diphtheriae, C. glutamicum, or C. ulcerans as the recipients. Resistance to nalidixic acid was used to select for the Coryneform species.…”