Coordinate regulation of the Escherichia coli formate dehydrogenase fdnGHI and fdhF genes in response to nitrate, nitrite, and formate: roles for NarL and NarP

Abstract

Escherichia coli possesses three distinct formate dehydrogenase enzymes encoded by the fdnGHI, fdhF, and fdoGHI operons. To examine how two of the formate dehyrogenase operons (fdnGHI and fdhF) are expressed anaerobically in the presence of low, intermediate, and high levels of nitrate, nitrite, and formate, chemostat culture techniques were employed with fdnG-lacZ and fdhF-lacZ reporter fusions. Complementary patterns of gene expression were seen. Optimal fdhF-lacZ expression occurred only at low to intermediate levels of nitrate, while high nitrate levels caused up to 10-fold inhibition of gene expression. In contrast, fdnG-lacZ expression was induced 25-fold in the presence of intermediate to high nitrate concentrations. Consistent with prior reports, NarL was able to induce fdnG-lacZ expression. However, NarP could not induce expression; rather, it functioned as an antagonist of fdnG-lacZ expression under low-nitrate conditions (i.e., it was a negative regulator). Nitrite, a reported signal for the Nar sensory system, was unable to stimulate or suppress expression of either formate dehydrogenase operon via NarL and NarP. The different gene expression profiles of the alternative formate dehydrogenase operons suggest that the two enzymes have complementary physiological roles under environmental conditions when nitrate and formate levels are changing. Revised regulatory schemes for NarL- and NarP-dependent nitrate control are presented for each operon.

FIG. 1.

Effect of nitrate on fdnG-lacZ and fdhF-lacZ expression during steady-state anaerobic cell growth. At each level of nitrate, the chemostat was sampled, and β-galactosidase activity was determined as described in Materials and Methods. After a shift to new conditions, a steady state was generally achieved within five residence times. Expression of the fdhF-lacZ fusion (○) and expression of the fdnG-lacZ fusion (•) were determined relative to the maximum level achieved for each fusion. The maximal fdnG-lacZ expression was 5,000 U, and the maximal fdhF-lacZ expression was 1,450 U.

FIG. 2.

Effects of nitrite and formate on fdnG-lacZ and fdhF-lacZ expression under anaerobic conditions. The chemostat was sampled after each steady state was achieved, and β-galactosidase activity was determined as described in Materials and Methods. The cell growth conditions were identical to those used in the experiment whose results are shown Fig. 1, except that nitrate was omitted and different levels of nitrite (A), formate (B), or nitrate and formate (C) were present in the nutrient supply.

FIG. 3.

Effects of nitrate, nitrite, and formate on fdhF-lacZ expression in narL and narP strains. The cell growth conditions, harvesting methods, and enzyme assays were identical to those used in the experiment whose results are shown Fig. 1, except that different concentrations of nitrate (A), nitrate and formate (B), or nitrite (C) were added. •, expression in the wild-type strain; ○, expression in narP strain; ▵, expression in narL strain.

FIG. 4.

Effects of nitrate and nitrite on fdnG-lacZ expression in narL and narP strains. The cell growth conditions, harvesting methods, and enzyme assays were identical to those used in the experiment whose results are shown Fig. 1, except that different concentrations of nitrate (A) or nitrite (B) were added. •, expression in the wild-type strain; ○, expression in narP strain; ▵, expression in narL strain.

FIG. 5.

DNase I footprint analysis of the fdhF regulatory region with NarL and NarL phosphate. Footprinting procedures and NarL phosphorylation were performed as described in Materials and Methods. The amount of protein used in each lane is indicated above the lane. The open box indicates the DNase I-protected regions. The arrows indicate the positions of potential NarL heptamer-like recognition sequences based on the NarL heptamer sequence of Darwin et al. (10).

FIG. 6.

Steady-state levels of formate remaining in the growth vessel after different levels of formate were added. The chemostat conditions were identical to those described in the legend to Fig. 2. The vessel was sampled after each steady state was achieved, and the formate concentration was determined as described in Materials and Methods. The level of formate remaining (○) is indicated in panel A, while the solid line indicates the amount of added formate plus the amount of initial formate produced (3.25 mM) when no formate was added. The dotted line indicates the amount of formate expected if no formate was produced or consumed by the cells. (B) Optical density at 600 nm [OD (600 nm)] of the cells in the vessel.

FIG. 7.

Model for regulation of fdnG operon expression. Under anaerobic conditions in the absence of nitrate the FNR protein induces fdnG operon expression. The presence of a low nitrate level weakly induces fdnG expression due to the low level of phosphorylated NarL protein. However, phosphorylated NarP protein bound at the same heptamer sites at positions −109 and −100 competes with NarL to antagonize its ability to stimulate transcription. NarP phosphate is unable to perform activation. At increased levels of nitrate, the NarL phosphate protein binds to the sites at positions −109 and −100 and replaces the NarP protein; NarL phosphate then maximally induces fdnG expression.

FIG. 8.

Proposed model for anaerobic regulation of fdhF gene expression by nitrate. FHLA is the formate-dependent transcriptional activator of fdhF gene expression. The fdhF promoter is sigma-54 protein dependent. Gene expression is low in the absence of added formate. However, in the presence of a high formate concentration, FHLA binds to the UAS region to activate fdhF gene expression. On the other hand, nitrate suppresses fdhF gene expression. The binding of the NarL phosphate protein at the fdhF promoter somehow interferes with binding of FHLA and/or sigma-54 RNA polymerase. Under these conditions, fdhF expression is repressed.