A mutational study of the ArcA-P binding sequences in the aldA promoter of Escherichia coli

The aldA gene (encoding aldehyde dehydrogenase) of Escherichia coli is anaerobically repressed by ArcA-P, the phosphorylated response regulator of the ArcB/A two-component signal transduction system. The region of aldA contains two 10-bp sequences (5'-TGTTAATTAA-3') that perfectly match the proposed ArcA-P binding consensus (5'-[A/T]GTTAAT-TA[A/T]-3'). One consensus sequence is on the coding strand (-13 to -4 from the transcriptional start point), whereas the other is on the template strand (position -2 to -11). In this study we used the aldA promoter to test the validity of the proposed consensus sequence. DNase I protection experiments confirmed the TO-bp sequence to be a strong ArcA-P binding site. Alteration of the wild-type sequence from 5'-T (G) under bar TTAATTAA (C) under bar-3' to 5'-T (C) under bar TTAATTAA (G) under bar-3' or 5'-T (A) under bar TTAATTAA (T) under bar-3' by site-directed mutagenesis markedly decreased the in vitro affinity of the promoter region for ArcA-P, and abolished the anaerobic repression of mutant att lambda::Phi (aldA'-lacZ) transcriptional reporter constructs. Both the in vitro and in vivo results therefore support the proposed consensus sequence.