Genetic analysis of the stationary phase-induced mcb operon promoter in Escherichia coli.
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abstract
A combination of deletion analysis and random mutagenesis was used to identify regulatory elements in Pmcb, the stationary phase-induced promoter of the mcb operon. Our results indicate that Pmcb is controlled by at least three different factors, two previously identified and at least one unknown factor, which act at four different sites in the promoter. Sequences between -344 and -164 upstream of the transcriptional start site were required for wild-type levels of mcb transcription in stationary phase. More dramatic reductions in both exponential and stationary phase expression were observed when sequences from -164 to -54 were deleted. Point mutations located between -105 and -138 decreased both exponential and stationary phase expression. All but one of these mutations decreased OmpR-dependent activation of Pmcb transcription. EmrR, also known as MprA, acts directly or indirectly at sequences downstream of -54 to repress Pmcb. A minimal promoter containing sequences from -34 to +79 was still induced > or = 10-fold in stationary phase. Point mutations within this region identified sequences at -8, -11, -30, -31 and -32 as important for Pmcb activity. These bases are in the gearbox sequence, present in Pmcb and several other stationary phase-induced Escherichia coli promoters.
