Question

Describe the process of gene regulation using the diphtheria toxin gene. More correct, relevant detail is better.

Answer 1

Transcription of the bacteriophage-borne diphtheria toxin gene tox is negatively regulated, in response to intracellular Fe²⁺ concentration, by the chromosomally encoded diphtheria toxin repressor (DtxR). Due to a scarcity of tools, genetic analysis of Corynebacterium diphtheriae has primarily relied on analysis of chemically induced and spontaneously occurring mutants and on the results of experiments with C. diphtheriae genes cloned in Escherichia coli or analyzed in vitro. We modified a Tn5-based mutagenesis technique for use with C. 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 of dtxR and the other within an essential region of the dtxR coding sequence, indicating for the first time that dtxR is a dispensable gene in C. diphtheriae. Both mutant strains secrete diphtheria toxin when grown in medium containing sufficient iron to repress secretion of diphtheria toxin by wild-type C. 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 within dtxR does 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 within dtxR abolished DtxR activity. This is the first evidence that DtxR plays a role in protecting the cell from oxidative stress.

The Corynebacterium diphtheriae diphtheria toxin (DT) repressor (DtxR) is the prototype for a group of bacterial metal-dependent regulator proteins . Examples of this DtxR-defined regulator protein group are found in a variety of bacterial species, including the AT-rich gram-positive staphylococcal species in which it is called SirR , acid-fast mycobacterial and rhodococcal species , and the commercially relevant Brevibacterium and Streptomyces species . Many members of this metal-dependent regulator family bind and are activated by Fe²⁺ in vivo, but TroR from Treponema pallidum and MntR from Bacillus subtilis are activated by Mn2+ .

In addition to their function in iron-dependent gene regulation, a role has been suggested for DtxR-like metal-dependent regulators in protecting the bacterial cell from oxidative stress. Mycobacterium smegmatis cells containing the wild-type DtxR homologue IdeR were better able to survive in the presence of hydrogen peroxide than those containing a null mutation in ideR . Further characterization demonstrated that IdeR-deficient cells produce fewer RNA messages for katG, encoding catalase and peroxidase, and sodA, encoding manganese superoxide dismutase, suggesting a positive role for IdeR in regulating these genes . Similarly, in gram-negative bacteria, the ferric uptake regulatory protein Fur, which is the prototype for a family of metal-dependent regulators that is distinct from DtxR, regulates genes in response to both iron and oxidative stress.

The DNA binding, metal specificity, and physical structure of DtxR have been studied extensively in vitro and in Escherichia coli, leading to several important observations. DtxR binds a 19-bp operator sequence that overlaps the promoters of the genes that it regulates . In both E. coli and C. diphtheriae, gene regulation by DtxR is observed in response to changing iron concentrations, but in vitro, DtxR can be activated by other divalent transition metals, including cadmium, cobalt, manganese, nickel, and zinc . Crystallographic studies have shown that DtxR has three domains . Amino acids 1 to 73 form the DNA binding domain 1, which contains a classical helix-turn-helix motif, amino acids 74 to 140 form domain 2, which is required for dimerization and metal binding, and amino acids 140 to 226 form domain 3, which has the same topology as the SH3 domains found in signal transduction proteins.

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