Plant pathogenic bacteria utilize biofilm growth-associated repressor (BigR), a novel winged-helix redox switch, to control hydrogen sulfide detoxification under hypoxia

Abstract

Winged-helix transcriptional factors play important roles in the control of gene expression in many organisms. In the plant pathogens Xylella fastidiosa and Agrobacterium tumefaciens, the winged-helix protein BigR, a member of the ArsR/SmtB family of metal sensors, regulates transcription of the bigR operon involved in bacterial biofilm growth. Previous studies showed that BigR represses transcription of its own operon through the occupation of the RNA polymerase-binding site; however, the signals that modulate its activity and the biological function of its operon are still poorly understood. Here we show that although BigR is a homodimer similar to metal sensors, it functions as a novel redox switch that derepresses transcription upon oxidation. Crystal structures of reduced and oxidized BigR reveal that formation of a disulfide bridge involving two critical cysteines induces conformational changes in the dimer that remarkably alter the topography of the winged-helix DNA-binding interface, precluding DNA binding. This structural mechanism of DNA association-dissociation is novel among winged-helix factors. Moreover, we demonstrate that the bigR operon is required for hydrogen sulfide detoxification through the action of a sulfur dioxygenase (Blh) and sulfite exporter. As hydrogen sulfide strongly inhibits cytochrome c oxidase, it must be eliminated to allow aerobic growth under low oxygen tension, an environmental condition found in bacterial biofilms, xylem vessels, and root tissues. Accordingly, we show that the bigR operon is critical to sustain bacterial growth under hypoxia. These results suggest that BigR integrates the transcriptional regulation of a sulfur oxidation pathway to an oxidative signal through a thiol-based redox switch.

FIGURE 1.

Structure and conformational changes of oxidized and reduced BigR. A, structural comparison of the oxidized (blue) and reduced (red) monomers. The intrachain disulfide bond is represented as sticks, and the corresponding cysteine residues are labeled. Secondary structure elements are indicated. B, 2F_o − F_c electron density map contoured at 1.2σ showing the disulfide between Cys-42 and Cys-108 in the oxidized monomer A. C, superposition of the BigR dimers. Light and dark colors are used to distinguish the homodimer subunits. The figure was produced by superposing the C-α atoms of residues 21–32 (helix 1) from both monomers. The distances between the Gln-67 C-α atoms of helix 4 from opposite subunits are indicated.

FIGURE 2.

Oxidized BigR ceases from binding to DNA and releases transcription. A, gel-shift assay showing that oxidized (S–S) BigR does not bind to the target DNA as reduced (S–H) BigR; however, binding is restored upon tris(2-carboxyethyl)phosphine (TCEP) treatment. Shifted bands are indicated by arrows, and FP is the free probe. B, gel-shift assay showing that both the C42S (M1) and the C108S (M2) mutants bind to the target DNA as the wild type (Wt) protein. Shifted bands are indicated by arrows, and FP is the free probe. C, GFP fluorescence as a measurement of the transcriptional activity of the bigR operon reporter plasmid alone (Rep) or in the presence of the wild type BigR, M1, or M2 proteins. D, GFP reporter gene assay of E. coli cell extracts expressing the wild type or mutated BigR proteins, in the presence (S–S) or absence (S–H) of GSSG. Error bars indicate S.E.

FIGURE 3.

The structural basis for the redox-regulated DNA binding in BigR. A, basic residues potentially involved in DNA interaction in oxidized (blue) and reduced BigR (red) are represented in ball-and-sticks format. Dark and light colors are used to distinguish monomers A and B, respectively. Residues marked with an asterisk are replaced by alanines in the refined structure, and their side chains were modeled to produce this image. B, electrostatic surface of reduced (top) and oxidized (bottom) BigR showing differences in the basic DNA-binding region as well as in the negatively charged surface of the opposite face of the dimers. The bonds for potential contour map visualization are ± 2 kT/e. C, comparison of the secondary structure elements in oxidized (blue) and reduced (red) BigR showing the N terminus of helix 1 in between helices 2 and 5 in the reduced structure. Dark and light colors correspond to monomers A and B, respectively. D, stereo view of the Cys-42 and Cys-108 neighborhood depicting a network of sulfur-containing residues.

FIGURE 4.

The bigR operon is required for hydrogen sulfide detoxification. A, growth of A. tumefaciens wild type and bigR⁻ and blh⁻ insertion mutants in BiGGY agar medium, showing that the blh⁻ cells accumulate higher levels of hydrogen sulfide relative to wild type (wt) and bigR⁻ mutant. B and C, growth of the wild type, bigR⁻, and blh⁻ cells in thiosulfate (0–25 mm) or ammonium sulfide (0–1 mm) gradient plates, respectively. D, effect of pH on the toxicity of 1 mm ammonium sulfite relative to control (no ammonium sulfide). The growth of the blh⁻ cells is affected by the acidic pH only. Bacterial cells were plated as indicated in A. E, sulfite levels in the culture supernatants of wild type, bigR⁻, and blh⁻ cells estimated by the sulfite test strip, according to the scale. Culture medium without bacterial growth (control) is shown for comparison.

FIGURE 5.

The bigR operon is critical for growth under oxygen-limiting conditions. A, growth of A. tumefaciens wild type and bigR⁻ and blh⁻ mutants under nitrogen-purged atmospheres (N₂). Bacterial cells were grown for the time periods indicated either under atmospheric oxygen or after a purge of nitrogen. B, after incubation under nitrogen-purged air (0.75 or 1.5 liters of N₂) for the time periods indicated, the flasks were opened, and the cells were further grown for 2–4 days to show that oxygen restored their growth. Atmos. O₂, under atmospheric oxygen.

FIGURE 6.

Conformational changes of BigR and related metal sensors. A, superposition of the BigR and SmtB dimers showing the conformational changes in the quaternary structures of reduced (red) and oxidized (blue) BigR relative to the structures of SmtB in its apo (purple) and zinc-bound forms (pink). Oxidized BigR adopts a more compact conformation than the apo and zinc SmtBs, as judged by the distances between the C-α atoms of Gln-67 (34.8 Å) and equivalent His-78 in the apo (40.9 Å) and zinc SmtB (37.4 Å). B, superposition of BigR and CzrA dimers showing that oxidized and reduced BigR have quaternary conformations similar to zinc CzrA (orange) and CzrA bound to DNA (yellow), respectively, indicating that the more compact (closed) conformation associated with high DNA binding affinity is conserved between BigR and CzrA. The figures were produced by superposing the C-α atoms of helix 1 of both monomers.