Characterization of the signaling domain of the NO-responsive regulator NorR from Ralstonia eutropha H16 by site-directed mutagenesis

Abstract

In Ralstonia eutropha H16, the nitric oxide (NO)-responsive transcriptional activator NorR controls the expression of a dicistronic operon that encodes a membrane-bound NO reductase, NorB, and a protein of unknown function, NorA. The N-terminal domain (NTD) of NorR is responsible for perception of the signal molecule, nitric oxide. Thirteen out of 29 conserved residues of the NTD were exchanged by site-directed mutagenesis. Replacement of R63, R72, D93, D96, C112, D130, or F137 strongly decreased NorR-dependent promoter activation, while the exchange of Y95 or H110 led to an increase in promoter activity compared to that of the wild type. A purified truncated NorR comprising only the NTD (NorR-NTD) contained one iron atom per molecule and was able to bind NO in the as-isolated state. Based on the iron content of NorR-NTD proteins with single amino acid replacements, residues R72, D93, D96, C112, and D130 are likely candidates for iron ligands. Residues R63, Y95, and H110 appear not to be involved in NO binding but may take part in subsequent steps of the signal transduction mechanism of NorR.

FIG. 1.

Conserved residues in NorR-NTD and amino acid exchanges. Boxed residues are strictly conserved in NorR orthologs; encircled residues are conserved in the majority of orthologs. Sequence positions of marked residues are indicated. Examples of alternative residues are given below the sequence along with the corresponding organism: Bxv, Burkholderia xenovorans LB400; Ppr, Photobacterium profundum SS9; Avn, Azotobacter vinelandii AvOP; Prm, Polaromonas sp. strain JS666. A conserved tyrosine that is replaced by phenylalanine in a distinct subset of NorR orthologs (see text for details) is marked by a diamond-shaped outline (middle sequence row). Arrows denote exchanges constructed in this study.

FIG. 2.

Transcriptional activation of the norA promoter by NorR and mutated derivatives of NorR. Promoter activation by NorR was induced by the addition of 2 mM SNP (+) and compared to uninduced cells (−). Activities (expressed in Miller units) were determined at 4 h (white bars) and 20 h (gray bars) after induction. The wild type (WT) and NorR mutants are indicated at the bottom. Standard deviations (error bars) were estimated from three to six independent experiments. The panel at the right is presented at a different scale to fit mutants with high activities.

FIG. 3.

Growth of mutant strains by denitrification as shown in a microtiter plate assay. Each curve represents the mean of four to six individual cultures. Turbidity was measured at 436 nm. Δ, wild type; •, NorR-negative mutant; +, NorR-F85L; *, NorR-D96N; ⧫, NorR-Y95F; ×, NorR-H110Q; -, NorR-C112S. The inflection point of the curves around 12 h reflects a lag phase of cell growth during adaptation from aerobic growth to denitrification, which was due to residual oxygen in the culture wells.

FIG. 4.

Optical spectra of NorR-NTD (200 μM). The main panel shows NorR-NTD in the as-isolated state. The inset shows difference spectra of NorR-NTD incubated with NO in vitro and in vivo, respectively: dashed line, NorR-NTD incubated with NO (200 μM) minus as-isolated NorR-NTD; solid line, NorR-NTD purified from cultures amended with NO minus NorR-NTD purified from cultures without the addition of NO. Abs, absorption.