Specific transcriptional requirements for positive regulation of the anaerobically inducible pfl operon by ArcA and FNR

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Abstract
Expression of the pfl operon of Escherichia coli Is induced 12- to 15-fold by anaerobiosis and transcription is mediated by seven co-ordinately regulated promoters. The 5′ non-translated regulatory region of the operon is approximately 450 bp in length and contains two of the seven promoters, termed promoter 6 and promoter 7. Site-directed mutagenesis was used to aid the identification of DNA sequences important in directing transcription from the two promoters and to examine the effects such mutations had on the regulation of anaerobic pfl operon expression. Introduction of chromosomal mutations either in the FNR-binding site or -10 region of promoter 6 blocked transcription from this promoter, as determined by primer extension. Similarly, mutation of the -10 region or the putative FNR half-site located at -50 relative to the transcription start site of promoter 7 severely reduced transcription from that promoter. Prevention of transcription from promoter 6 by the -10 box mutation had no influence on promoter 7 transcription. Surprisingly, however, alteration of the FNR-binding site at promoter 6 did reduce transcription from promoter 7. Thus, a cis mutation located 280bp downstream on the DNA had a profound effect on promoter 7 transcription. This effect would be commensurate with this mutation disrupting an important interaction between proteins bound at promoter 7 with those bound at promoter 6. Primer extension demonstrated that the promoter 7 mutations had no apparent influence on promoter 6 transcription. By using pfl–lacZ gene fusions it could be shown that the FNR-binding site and -10 region mutations at promoter 6 abolished FNR-dependent anaerobic regulation of pfl operon expression. The equivalent mutations at promoter 7 caused a 25% reduction in anaerobic expression. The residual anaerobic expression in such constructs was FNR-, but no longer ArcA-dependent. A construct in which the -10 region of both promoters 6 and 7 was mutated showed no anaerobic induction of pfl operon expression. This indicates that transcription from both promoters is required for maximal anaerobic regulation by ArcA and FNR.