Growth phase-dependent regulation of nuoA-N expression in Escherichia coli K-12 by the Fis protein: upstream binding sites and bioenergetic significance

Abstract

The expression of the nuoA-N operon of Escherichia coli K-12, which encodes the proton-pumping NADH dehydrogenase I is modulated by growth phase-dependent regulation. Under respiratory growth conditions, expression was stimulated in early exponential, and to a lesser extent in late exponential and stationary growth phases. The stimulation in the early exponential growth phase was not observed in fis mutants, which are deficient for the growth phase-responsive regulator Fis. Neither the alternative σ factor RpoS nor the integration host factor (IHF) are involved in growth phase-dependent regulation of this operon. When incubated with nuo promoter DNA, isolated Fis protein formed three retarded complexes in gel mobility experiments. DNase I footprinting identified three distinct binding sites for Fis, 237 bp (fis1), 197 bp (fis2) and 139 bp (fis3) upstream of the start of the major transcript of nuoA-N, T1. The protein concentrations required for half-maximal binding to fis1, fis2 and fis3 were about 20 nM, 40 nM and 100 nM Fis, respectively. The IHF protein bound 82 bp upstream of the start of transcript T2 with a half-maximal concentration for binding of 50 nM. Due to the growth phase-dependent regulation by Fis, the synthesis of the coupling NADH dehydrogenase I is increased relative to that of the non-coupling NADH dehydrogenase II during early exponential growth. This ensures higher ATP yields under conditions where large amounts of ATP are required.