Structural and functional characterization of the nitrite channel NirC from Salmonella typhimurium

Abstract

) is a central intermediate in the nitrogen metabolism of microorganisms and plants, and is used as a cytotoxin by macrophages as part of the innate immune response. The bacterial membrane protein NirC acts as a specific channel to facilitate the transport of nitrite anions across lipid bilayers for cytoplasmic detoxification. Despite NirC's importance in nitrogen metabolism and in the pathogenicity of enteric bacteria, available biochemical data are scarce. Here we present a functional and structural characterization of NirC from Salmonella typhimurium by lipid bilayer electrophysiology and X-ray crystallography. NirC is a pentameric member of the formate/nitrite transporter family of membrane proteins that operates as a channel with high conductance. Single-channel measurements reveal fast and slow gating events but, in contrast to the related FocA formate channel, no pH-dependent gating. A 2.4Å crystal structure of NirC at pH 5 shows similarity to FocA and aquaporins, but lacks the structural asymmetry observed in the formate channel at similarly low pH. Resolved water molecules in the protomers suggest a transport mechanism that also permits a facultative NO 2 − /H + symport.ion channels | protein crystallography | planar lipid bilayer

Beyond Antimicrobial Resistance: Evidence for a Distinct Role of the AcrD Efflux Pump in Salmonella Biology

“…Salmonella has two nitrate reductases, one located in the cytoplasm and the other in the periplasm (NapACF) (47). NirBD is an NADH-dependent nitrite reductase located in the cytoplasm, while NirC is a nitrite/proton antiporter in the bacterial membrane, that allows transport of nitrite into the cytoplasm for detoxification (48, 49), and contributes to Salmonella virulence in both mice and macrophages (50). However, there was no difference in the ability of the acrD mutant to grow anaerobically compared to wild-type Salmonella , nor was there a significant difference in the ability of the mutant to grow in the presence of sodium nitrate or survive in acidified nitrite.…”

Ion selectivity and gating mechanisms of FNT channels

“…As a result of structural conservation, the essential aspects of the pore in the center of the FNT protomer are very similar among the five FocA, HSC and NirC proteins[9,17–20]. In all structures, the proposed translocation pathway is defined by a narrow central pore region that opens into a funnel on both the cytoplasmic and periplasmic sides (Figure 2e).…”

“…Over the past few years, crystal structures of five FNT proteins have been reported from the three subfamilies identified thus far (Figures 1–3): FocA from E. coli , Vibrio cholera and S. typhimurium [17–19], HSC from C.difficile [9] and NirC from S. typhimurium [20]. All of these structures are highly similar, showing a homopentamer, and the fold of each protomer resembles that of the tetrameric aquaporin and glyceroporin channels [21–24], likely indicating a common evolutionary history (Figure 1c–f).…”

Pyruvate Formate-Lyase Interacts Directly with the Formate Channel FocA to Regulate Formate Translocation

“…FNT proteins are widely distributed among the archaea, bacteria and certain fungi and the family includes several thousand members that can be divided into minimally three sub-classes, with diverse cellular functions [5–7], suggesting that this family is evolutionarily ancient. As well as a conserved pentameric structure, the FNT proteins are characterized by their translocation of small monovalent anionic species, such as formate, nitrite or hydrosulfide, across bacterial cytoplasmic membranes [2,3,5,8,9]. As many of the microorganisms that synthesize FNT channels are anaerobes, these proteins likely provide microbial cells with the capability of energy-efficient and controlled uptake (or export) of anions [10].…”