Nitrate and Nitrite Regulation of the Fnr-dependent aeg-46.5Promoter of Escherichia coliK-12 is Mediated by Competition Between Homologous Response Regulators (NarL and NarP) for a Common DNA-binding Site

Abstract

The NarL and NarP proteins are homologous response regulators that function to regulate anaerobic respiratory gene expression in response to nitrate and nitrite in Escherichia coli. Expression of the aeg-46.5operon (anaerobically expressed gene at 46.5 minutes on the genetic map) is induced during anaerobic growth by the global transcriptional regulatory protein Fnr. aeg-46.5operon expression is further induced by the NarP protein in response to nitrate or nitrite and this induction is antagonized by NarL. We used in vivoand in vitrotechniques to investigate how these three transcriptional regulatory proteins control the activity of single promoter. Deletion and mutational analysis of the aeg-46.5operon control region identified two distinct cis-acting elements. A sequence with similarity to the Fnr-binding site consensus, centered at position 64.5, was essential for Fnr-dependent anaerobic induction of aeg-46.5operon expression. In all other naturally occuring Fnr-dependent promoters the primary Fnr-binding site is centered between 40 and 50. The second cis-acting element, a region of perfect symmetry centered at -44.5, shares sequence similarity with the NarL-binding site consensus. This region was required for nitrate and nitrite induction of aeg-46.5operon expression. We purified the NarP and NarL proteins as maltose-binding protein (MBP) fusion proteins and investigated their interaction with the aeg-46.5operon control region. Incubation with the phospho-donor, acetyl phosphate, allowed both MBP-NarP and MBP-NarL to protect the 44.5 region of the aeg-46.5operon control region from DNase I cleavage. Single and double nucleotide substitutions in the 44.5 region reduced or abolished nitrate and nitrite induction of aeg-46.5operon expression in vivoand prevented the binding of MBP-NarP and MBP-NarL to the control region in vitro. Presumably the NarP and NarL proteins compete for the 44.5 binding site to regulate aeg-46.5operon expression in response to nitrate and nitrite. Apparently, only the NarP protein is competent to activate transcription of the aeg-46.5operon when bound to the 44.5 region. f2 f2 Abbreviations used: MBP, maltose-binding protein; IPTG, isopropyl-β-d-thiogalactopyranoside; PEG, polyethylene glycol; c.f.u., colony-forming units; CIP, calf intestinal phosphatase.