Analysis of the upstream sequences of the Rhodobacter sphaeroides 2.4.1 hemA gene: in vivo evidence for the presence of two promoters that are both regulated by fnrL.

Abstract

The presumed DNA target for the Rhodobacter sphaeroides 2.4.1 FnrL regulatory protein is the FNR consensus sequence, TTGAT-N(4)-ATCAA, based on (1) similarities between the helix-turn-helix motifs of FnrL and the Escherichia coli homologue, Fnr, and (2) the established FnrL dependence for anaerobic induction of six gene clusters all having upstream FNR consensus-like sequences. We are interested in understanding the regulation of one among these; namely, the hemA gene, which codes for one of two isozymes of 5-aminolevulinate (ALA) synthase in this organism. Here, we present in vivo evidence that the hemA gene is transcribed from two promoters. Both are under oxygen control, and disabling the fnrL gene abolishes induction of each promoter in response to lowering oxygen tension. Based on the 5' position of the FNR consensus sequence relative to the downstream promoter, we had hypothesized that activation of that promoter is mediated by binding of FnrL to the FNR consensus sequence. Consistent with this hypothesis, we found here that transcription from the downstream promoter is no longer inducible when the FNR consensus sequence is deleted. With respect to the upstream promoter, based on the fact that the +1 site of transcription from that promoter is within the FNR consensus sequence, we propose an indirect role for FnrL. This possibility is discussed, together with other unresolved aspects of hemA expression.