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The Formate Channel Foca Exports the Products of Mixed-Acid Fermentation

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The Formate Channel Foca Exports the Products of Mixed-Acid Fermentation The formate channel FocA exports the products of mixed-acid fermentation Wei Lü, Juan Du, Nikola J. Schwarzer, Elke Gerbig-Smentek, Oliver Einsle, and Susana L. A. Andrade1 Institute of Organic Chemistry and Biochemistry and BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany Edited by Christopher Miller, Howard Hughes Medical Institute, Brandeis University, Waltham, MA, and approved July 10, 2012 (received for review March 11, 2012) Formate is a major metabolite in the anaerobic fermentation of and is then oxidized to CO2 by a periplasmic formate de- glucose by many enterobacteria. It is translocated across cellular hydrogenase, FDH-N or FDH-O, resulting in the generation of membranes by the pentameric ion channel/transporter FocA that, proton motive force (11–13). In addition, the complex pathway together with the nitrite channel NirC, forms the formate/nitrite of mixed-acid fermentation produces acetate, ethanol, lactate, transporter (FNT) family of membrane transport proteins. Here we and succinate as further end products (Fig. 1A). FocA acts as have carried out an electrophysiological analysis of FocA from Sal- a passive exporter for formate anions generated in the cyto- monella typhimurium to characterize the channel properties and plasm, but a remarkable functional switch of transport mode was assess its specificity toward formate and other possible permeat- found for this protein when the pH of the growth medium ing ions. Single-channel currents for formate, hypophosphite and dropped below 6.8 (14). With ample protons available in the nitrite revealed two mechanistically distinct modes of gating that periplasm, the cell switches to active import of formate and reflect different types of structural rearrangements in the trans- again uses FocA for the task. Imported formic acid is then dis- port channel of each FocA protomer. Moreover, FocA did not con- proportionated into CO2 and H2 by the cytoplasmic formate: duct cations or divalent anions, but the chloride anion was hydrogen lyase complex (FHL), and hydrogen gas is either re- identified as further transported species, along with acetate, lac- leased or used as a low potential electron donor through mem- tate and pyruvate. Formate, acetate and lactate are major end brane-bound hydrogenases (15). Recently, high-resolution products of anaerobic mixed-acid fermentation, the pathway crystal structures were presented for FocA from E. coli (16) and BIOCHEMISTRY where FocA is predominantly required, so that this channel is ide- Vibrio cholerae (17) at high pH, and for S. typhimurium at low pH ally adapted to act as a multifunctional export protein to prevent (18). FocA forms stable pentamers with individual transport their intracellular accumulation. Because of the high degree of channels in each protomer (SI Appendix, Fig. S1) and shares conservation in the residues forming the transport channel among a remarkable structural homology to the (tetrameric) aquaporin FNT family members, the flexibility in conducting multiple mole- and glyceroporin channels (16, 19). The high-pH forms represent cules is most likely a general feature of these proteins. the passive channel conformation of FocA, whereas at pH values – less than 5, the passive transport of HCOO is terminated electrophysiology | planar lipid bilayer | formate channel through a pH-dependent gating mechanism that involves struc- tural rearrangements of the N-terminal helices in each protomer he formate/nitrite transporter family of integral membrane of the pentamer. This gating event was shown by direct elec- Ttransport proteins (FNT; transporter class 2.A.44; ref. 1) trophysiology of FocA reconstituted in planar lipid bilayer comprises the formate channel FocA (2), the nitrite channel membranes (18). We observed voltage-dependent currents, NirC (3), the formate uptake permease FdhC (4), and the commonly with a small difference in reversal potential from what hydrosulfide channel HSC (5). All members of the family facil- was expected for the applied gradient of formate, and we now itate the translocation of anions across a biological membrane show that this shift was then caused by the presence of chloride in their respective physiological context. NirC imports nitrite, anions in the buffer solutions. We conducted a detailed elec- – NO2 , into the cytoplasm. It is commonly found in an operon trophysiological analysis of anion transport by the formate with the cytoplasmic, assimilatory nitrite reductase NirBD (6) channel FocA, with the result that the protein exclusively + that produces ammonium, NH4 , for subsequent assimilation transports monovalent anions, albeit with unexpectedly low into biomolecules. Enteric bacteria such as Escherichia coli or specificity for the ion itself. Interestingly, the range of molecules Salmonella typhimurium also follow this route for the detox- transported by FocA largely matches the end products of mixed- ification of peroxynitrite, produced by the inducible NO synthase acid fermentation. in macrophages of their eukaryotic hosts as an aspect of the Results innate immune response (7, 8). A ΔnirC strain of Salmonella St was strongly inhibited for intracellular proliferation in spleen, Reversal Potential in Electrophysiology Studies of FocA Indicates liver, and lymph tissues, which indicates that NirC plays an im- Transport of Different Anionic Species. According to the Nernst portant role in enterobacterial pathogenesis and may thus be equation, a gradient of (20/200 mM) of the monovalent anion – a suitable target for specific, antimicrobial drugs (9). A recent formate should lead to a reversal potential of 58 mV in a volt- study on NirC reconstituted in proteoliposomes on solid-sup- age-clamp experiment. In our initial electrophysiological studies ported membranes further indicated that the protein can act + – as a secondary active H /NO2 antiporter (10). However, NirC would then actively export nitrite under physiological conditions, Author contributions: W.L., O.E., and S.L.A.A. designed research; W.L., J.D., N.J.S., and fi E.G.-S. performed research; W.L., J.D., N.J.S., and S.L.A.A. analyzed data; and W.L., O.E., a nding that is not obviously consistent with published data. The and S.L.A.A. wrote the paper. related formate channel FocA attains a very different physio- The authors declare no conflict of interest. logical role, because it is produced during anaerobic mixed-acid This article is a PNAS Direct Submission. fermentation (2). Here, pyruvate:formate lyase (PFL) converts 1 To whom correspondence should be addressed. E-mail: [email protected] pyruvate, the product of glycolysis, to acetyl-CoA and releases freiburg.de. – formate (HCOO ) that must be removed from the cytoplasm to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. avoid intracellular accumulation. The anion is exported via FocA 1073/pnas.1204201109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1204201109 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 concentration (Fig. 3A), showing a saturation behavior from which a maximum conductance G and an affinity k could be obtained by a hyperbolic fit (Fig. 3B). The recordings for formate yielded GFormate = 26.9 ± 0.5 pS, and kFormate = 11.7 ± 0.1 mM, a value close to the estimated maximal concentration of formate in the medium during mixed-acid fermentation (2, 13). No sig- nificant changes in the open probability were observed upon increase of ion concentration (Fig. 3A). N-Terminal Gating of FocA Is Subject to Precise pH Control. StFocA was shown to act as a passive channel for formate at pH 7.5. The anion currents ceased at pH 5.1, representing the unique func- tional switch of this protein from passive to active transport (18). However, whole-cells studies with variations of the pH of the Fig. 1. Mixed-acid fermentation and the FocA channel. (A) The metabolic pathway of anaerobic mixed-acid fermentation. Glucose is oxidized along growth medium located the functional switch at a pH value of the glycolytic pathway, and the product pyruvate is reduced in a minor 6.8 (14). To obtain a precise estimate, we conducted series of degree to lactate via lactate dehydrogenase (LDH). The bulk of pyruvate electrophysiology experiments at different pH values on the is cleaved into acetyl-CoA and formate by the key enzyme of the pathway, same bilayer membrane, by stepwise addition of acid to the cis PFL. Formate is then either exported via the channel FocA for periplasmic chamber. The permeability of FocA remained constant from reduction by formate dehydrogenases (FDH), or it is disproportionated to pH 7.4 to pH 5.8 but dropped steeply at pH 5.6 (Fig. 3C). This H2 and CO2 by the cytoplasmic formate:hydrogen lyase complex (FHL). Ap- specific gating thus occurred at a defined pH, and it can be proximately half of the generated acetyl-CoA is reduced in two steps by mechanistically rationalized by the rearrangement of the N-ter- aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) to the mini of the five protomers, as seen in the structure of StFocA at end product ethanol, whereas the remaining acetyl-CoA is converted to fi acetate via phosphotransacetylase (PTA) and acetate kinase (ACK). The pH 4 (18). The clear de nition of a switching point indicates that products formate, lactate, acetate, and ethanol are exported from the cell. the mechanism of pH sensing may involve a protonation event of (B) Detail of the transport channel of S. typhimurium FocA with the con- an amino acid side chain, and given the pH value of approxi- strictions and the amino acid residues involved in their formation. mately 5.7, a histidine may be a suitable candidate. The steepness of the current step itself might well be the result of strong cooperativity between individual subunits, in line with the ob- of StFocA, the observed reversal potential was slightly, but served direct interaction of the N-termini of monomers in the consistently, lower (18).
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