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Iron regulation of toxA and regA transcript accumulation was examined in Pseudomonas aeruginosa PA103 containing the regA gene on a multicopy plasmid. The patterns of transcript accumulation for toxA and regA were found to be positively correlated. Dot blot and Northern (RNA) blot analysis of total RNA isolated throughout the bacterial growth cycle indicated that multiple copies of the regA gene uncoupled iron repression of the first phase of transcript accumulation for both regA and toxA genes. However, regulation by iron of the second phase of transcript accumulation for each gene was unaffected by several regA gene copies. Total toxin production was increased in cells with multiple copies of regA grown in either low- or high-iron medium. Primer extension analysis of regA mRNA extracted from cells grown in high- and low-iron medium and examined at different points in the cell growth cycle supported the hypothesis that iron regulation of regA transcription occurs at the level of transcriptional initiation. Two start sites were shown for regA transcription at -164 and -75 base pairs from the ATG start codon. The differential regulation of regA transcript accumulation when regA is present in single or multiple copy and the mapping of independent start sites for regA mRNA support the evidence that regA transcription is directed by independently regulated promoter regions.

Differential regulation by iron of regA and toxA transcript accumulation in Pseudomonas aeruginosa