Transcription analysis and nucleotide sequence of tox promoter/operator mutants of corynebacteriophage β

Abstract

The production of diphtheria toxin (DT) by Corynebacterium diphtheriae C7(β) is transcriptionally regulated by the iron-dependent diphtheria toxin repressor, DtxR. Transcription of the tox gene was studied in wild-type C. diphtheriae C7(β) and in lysogens carrying mutants of β that determine insensitivity to inhibition of DT production by iron. Under low iron conditions in all strains, tox-specific mRNA appeared and DT production began during late-log phase, and they increased to maximal levels at stationary phase. Under high iron conditions, tox-specific mRNA and DT production were strongly repressed in C7(β) but only partially repressed in C7( β tox-202 ) and C7( β tox-201 ). Under high and low iron conditions, DT production and tox-specific mRNA levels were greater in C7( β tox-201 ) and C7( β tox-202 ) than in wild-type C7(β). Addition of iron or rifampicin to low iron cultures of C. diphtheriae C7(β) repressed tox-mRNA production promptly and with a similar time course. In contrast, repression of tox-mRNA synthesis in C. diphtheriae C7( β tox-201 ) occurred promptly after addition of rifampicin but more slowly after addition of iron. Nucleotide sequence analysis revealed single G to A mutations at positions −47 and −48, within the preferred ‘−10’ sequence of the tox promoter, in β tox-201 and β tox -202, respectively. The single nucleotide substitutions in the tox-201 and tox-202 regulatory alleles, therefore, have pleiotropic effects, causing increased activity of the promoter and partial resistance of the operator to iron-dependent repression.