Suppression of FNR-dependent transcription activation at the Escherichia coli nir promoter by Fis, IHF and H-NS: modulation of transcription initiation by a complex nucleo-protein assembly

Expression from the Escherichia coli nir promoter is co-dependent on both the FNR protein (an anaerobically triggered transcription activator) and the NarL or NarP proteins (transcription activators triggered by nitrite and nitrate). Under anaerobic conditions, FNR binds to a site centred between positions -41 and -42, activating transcription of the nir operon. In previous work, we showed that this activation is suppressed by the binding of Fis protein, and at least one other protein, to sequence elements located upstream of the nir promoter. We proposed that the binding of NarL or NarP to a site centred between positions -69 and -70 counteracts this suppression, resulting in increased transcription in response to nitrite or nitrate. Here we have further investigated the different proteins that downregulate the nir promoter. We show that the nir promoter is repressed by three DNA binding proteins, Fis, IHF and H-NS. We demonstrate that, in addition to binding to its previously characterized upstream site located at position -142, Fis also binds to a second downstream site located at position +23. A second suppressing factor is IHF, that binds to a site located at position -88. Finally, the nucleoid associated protein, H-NS, preferentially binds to upstream sequences at the nir promoter and represses promoter activity. The association of Fis, IHF and H-NS suggests that nir promoter DNA is sequestrated into a highly ordered nucleo-protein structure that represses FNR-dependent transcription activation. NarL and NarP can relieve both IHF- and Fis-mediated repression, but are unable to counteract H-NS-mediated repression.