In the Alphaproteobacterium Hyphomicrobium denitrificans SoxR Serves a Sulfane Sulfur-Responsive Repressor of Sulfur Oxidation

Abstract

In organisms that use reduced sulfur compounds as alternative or additional electron donors to organic compounds, transcriptional regulation of genes for enzymes involved in sulfur oxidation is needed to adjust metabolic flux to environmental conditions. However, little is known about the sensing and response to inorganic sulfur compounds such as thiosulfate in sulfur-oxidizing bacteria. In the Alphaproteobacterium Hyphomicrobium denitrificans, one strategy is the use of the ArsR–SmtB-type transcriptional regulator SoxR. We show that this homodimeric repressor senses sulfane sulfur and that it is crucial for the expression not only of sox genes encoding the components of a truncated periplasmic thiosulfate-oxidizing enzyme system but also of several other sets of genes for enzymes of sulfur oxidation. DNA binding and transcriptional regulatory activity of SoxR are controlled by polysulfide-dependent cysteine modification. The repressor uses the formation of a sulfur bridge between two conserved cysteines as a trigger to bind and release DNA and can also form a vicinal disulfide bond to orchestrate a response to oxidizing conditions. The importance of the sulfur bridge forming cysteines was confirmed by site-directed mutagenesis, mass spectrometry, and gel shift assays. In vivo, SoxR interacts directly or indirectly with a second closely related repressor, sHdrR.

A cascade of sulfur transferases delivers sulfur to the sulfur‐oxidizing heterodisulfide reductase‐like complex

“…The polypeptides sHdrA and sHdrC2 as well as LbpA2 (aq_402) were identified, but were also present in the control sample (Table S4 ). Of the remaining proteins, 85 were associated with protein biosynthesis and degradation, and 24 were related to known or proposed functions of TusA other than sulfur oxidation, such as tRNA modification, molybdopterin cofactor biosynthesis and regulation (Dahl et al, 2013 ; Ikeuchi et al, 2006 ; Li, Törkel, et al, 2023 ). To gain further insight into the interaction partners of AqTusA, the re‐purified samples were applied to SDS–PAGE (Figure 8a ).…”

“…Hm. denitrificans possesses two candidate transporters (SoxT1A and SoxT1B, Figure 1 ) encoded in close proximity to the genes encoding Sox proteins that are involved in the initial steps of thiosulfate oxidation and the genes for the cytoplasmic sHdr system (Li, Koch, et al, 2023 ; Li, Törkel, et al, 2023 ). However, evidence supporting the proposed sulfur transport has yet to be presented.…”

“…The proteins were then transferred to salt‐free 50 mM Tris–HCl buffer (pH 7.5) or to 20 mM Tris–HCl buffer pH 7.4 for AqTusA and stored at −70°C. Size exclusion chromatography on HiLoad 16/60 Superdex™ 75 (Cytiva, Freiburg, Germany) was performed as described in Li, Törkel, et al ( 2023 ).…”

“…sibirica , 1.5 nmol of the proteins were incubated with 5 mM polysulfide, thiosulfate, tetrathionate, or GSSG in 20 μL 50 mM Tris–HCl, pH 7.5. The polysulfide stock solution needed for these experiments was prepared, diluted, and used as previously described (Ikeda et al, 1972 ; Li, Törkel, et al, 2023 ). For sulfur transfer experiments, 1.5 nmol of the putative sulfur‐donating proteins were incubated with 0.5 mM polysulfide for 30 min at room temperature in 20 μL 50 mM Tris–HCl, pH 7.5.…”

“…His-tagged and Strep-tagged proteins were purified with Ni-NTA Agarose (Jena Bioscience, Jena, Germany) (except for AqTuA that was purified with a HiScreen Ni FF column (Cytiva, Freiburg, Germany)) and Strep-Tactin Superflow (IBA Lifesciences, Göttingen, Germany), respectively, according to the manufacturer's instructions. The proteins were then transferred to salt-free 50 mM Tris-HCl buffer (pH 7.5) or to 20 mM Tris-HCl buffer pH 7.4 for AqTusA and stored at À70 C. Size exclusion chromatography on HiLoad 16/60 Superdex™ 75 (Cytiva, Freiburg, Germany) was performed as described in Li, Törkel, et al (2023).…”