The molybdate-responsive Escherichia coli ModE transcriptional regulator coordinates periplasmic nitrate reductase (napFDAGHBC) operon expression with nitrate and molybdate availability

Abstract

Expression of the Escherichia coli napFDAGHBC operon (also known as aeg46.5), which encodes the periplasmic molybdoenzyme for nitrate reduction, is increased in response to anaerobiosis and further stimulated by the addition of nitrate or to a lesser extent by nitrite to the cell culture medium. These changes are mediated by the transcription factors Fnr and NarP, respectively. Utilizing a napF-lacZ operon fusion, we demonstrate that napF gene expression is impaired in strain defective for the molybdate-responsive ModE transcription factor. This control abrogates nitrate- or nitrite-dependent induction during anaerobiosis. Gel shift and DNase I footprinting analyses establish that ModE binds to the napF promoter with an apparent K(d) of about 35 nM at a position centered at -133.5 relative to the start of napF transcription. Although the ModE binding site sequence is similar to other E. coli ModE binding sites, the location is atypical, because it is not centered near the start of transcription. Introduction of point mutations in the ModE recognition site severely reduced or abolished ModE binding in vitro and conferred a modE phenotype (i.e., loss of molybdate-responsive gene expression) in vivo. In contrast, deletion of the upstream ModE region site rendered napF expression independent of modE. These findings indicate the involvement of an additional transcription factor to help coordinate nitrate- and molybdate-dependent napF expression by the Fnr, NarP, NarL, and ModE proteins. The upstream ModE regulatory site functions to override nitrate control of napF gene expression when the essential enzyme component, molybdate, is limiting in the cell environment.

FIG. 1.

Deletion analysis and mapping of the ModE binding site at the E. coli napF promoter. Shown in panel A are the various DNA fragments, with relevant restriction sites, used in the construction of the napF-lacZ operon fusions detailed in the text. Restriction site locations relative to the start site of transcription are indicated in parentheses. Shown below is a schematic representation of the napF promoter region. The transcription start site is indicated (5), and coordinates relative to this start site are given in base pairs. The locations of Fnr, NarP (7), and ModE binding sites are indicated with brackets. (B) The DNA fragments used to map the ModE binding site are shown. The ability (+) or inability (−) of ModE to bind a particular fragment in a gel shift assay with 128 nM ModE is indicated.

FIG. 2.

Interaction of ModE with napF promoter DNA. Increasing amounts of purified ModE protein were incubated with a labeled napF promoter fragment from λHW2. (A) Wild-type napF promoter DNA and ModE without molybdate added. (B) Wild-type napF promoter DNA and ModE with 100 μM molybdate added. (C) Mutated napF promoter DNA from λPM54. (D) Mutated napF promoter DNA from λPM55.

FIG. 2.

Interaction of ModE with napF promoter DNA. Increasing amounts of purified ModE protein were incubated with a labeled napF promoter fragment from λHW2. (A) Wild-type napF promoter DNA and ModE without molybdate added. (B) Wild-type napF promoter DNA and ModE with 100 μM molybdate added. (C) Mutated napF promoter DNA from λPM54. (D) Mutated napF promoter DNA from λPM55.

FIG. 3.

DNase I footprint analysis of ModE interaction at napF. The pattern of protection when ModE is bound at napF in the presence of 100 μM molybdate is shown. The vertical bracket indicates the region of protection. Coordinates relative to the start site of transcription are given in base pairs.

FIG. 4.

Alignment of the ModE binding site at the napF, dmsA, modA, and moaA promoters with the proposed ModE consensus sequence. Nucleotides protected from DNaseI digestion are bracketed, and nucleotide matches to the ModE consensus sequence are shown in uppercase (2, 20, 23).