1887

Abstract

During mixed-acid fermentation, initially translocates formate out of the cell, but re-imports it at lower pH. This is performed by FocA, the archetype of the formate-nitrite transporter (FNT) family of pentameric anion channels. Each protomer of FocA has a hydrophobic pore through which formate/formic acid is bidirectionally translocated. It is not understood how the direction of formate/formic acid passage through FocA is controlled by pH. A conserved histidine residue (H209) is located within the translocation pore, suggesting that protonation/deprotonation might be linked to the direction of formate translocation. Using a formate-responsive -based reporter system we monitored changes in formate levels when H209 in FocA was exchanged for either of the non-protonatable amino acids asparagine or glutamine, which occur naturally in some FNTs. These FocA variants (with N or Q) functioned as highly efficient formate efflux channels and the bacteria could neither accumulate formate nor produce hydrogen gas. Therefore, the data in this study suggest that this central histidine residue within the FocA pore is required for pH-dependent formate uptake into cells. We also address why H209 is evolutionarily conserved and provide a physiological rationale for the natural occurrence of N/Q variants of FNT channels.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001132
2022-01-27
2022-05-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/168/1/mic001132.html?itemId=/content/journal/micro/10.1099/mic.0.001132&mimeType=html&fmt=ahah

References

  1. Sawers RG. Formate and its role in hydrogen production in Escherichia coli . Biochem Soc Trans 2005; 33:42–46 [View Article] [PubMed]
    [Google Scholar]
  2. Sargent F. The Model [NiFe]-Hydrogenases of Escherichia coli . Adv Microb Physiol 2016; 68:433–507 [View Article] [PubMed]
    [Google Scholar]
  3. Pinske C, Sawers RG. Anaerobic formate and hydrogen metabolism. EcoSal Plus 2016; 7: [View Article] [PubMed]
    [Google Scholar]
  4. Knappe J, Sawers G. A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli . FEMS Microbiol Rev 1990; 6:383–398 [View Article] [PubMed]
    [Google Scholar]
  5. Rossmann R, Sawers G, Böck A. Mechanism of regulation of the formate-hydrogenlyase pathway by oxygen, nitrate, and pH: definition of the formate regulon. Mol Microbiol 1991; 5:2807–2814 [View Article] [PubMed]
    [Google Scholar]
  6. Suppmann B, Sawers G. Isolation and characterization of hypophosphite--resistant mutants of Escherichia coli: identification of the FocA protein, encoded by the pfl operon, as a putative formate transporter. Mol Microbiol 1994; 11:965–982 [View Article] [PubMed]
    [Google Scholar]
  7. Mukherjee M, Vajpai M, Sankararamakrishnan R. Anion-selective Formate/nitrite transporters: taxonomic distribution, phylogenetic analysis and subfamily-specific conservation pattern in prokaryotes. BMC Genomics 2017; 18:560 [View Article] [PubMed]
    [Google Scholar]
  8. Wang Y, Huang Y, Wang J, Cheng C, Huang W et al. Structure of the formate transporter FocA reveals a pentameric aquaporin-like channel. Nature 2009; 462:467–472 [View Article] [PubMed]
    [Google Scholar]
  9. Waight AB, Love J, Wang D-N. Structure and mechanism of a pentameric formate channel. Nat Struct Mol Biol 2010; 17:31–37 [View Article] [PubMed]
    [Google Scholar]
  10. W, Du J, Wacker T, Gerbig-Smentek E, Andrade SLA et al. pH-dependent gating in a FocA formate channel. Science 2011; 332:352–354 [View Article] [PubMed]
    [Google Scholar]
  11. W, Schwarzer NJ, Du J, Gerbig-Smentek E, Andrade SLA et al. Structural and functional characterization of the nitrite channel NirC from Salmonella typhimurium . Proc Natl Acad Sci U S A 2012; 109:18395–18400 [View Article] [PubMed]
    [Google Scholar]
  12. Czyzewski BK, Wang D-N. Identification and characterization of a bacterial hydrosulphide ion channel. Nature 2012; 483:494–497 [View Article] [PubMed]
    [Google Scholar]
  13. Wu B, Rambow J, Bock S, Holm-Bertelsen J, Wiechert M et al. Identity of a Plasmodium lactate/H(+) symporter structurally unrelated to human transporters. Nat Commun 2015; 6:6284 [View Article] [PubMed]
    [Google Scholar]
  14. Golldack A, Henke B, Bergmann B, Wiechert M, Erler H et al. Substrate-analogous inhibitors exert antimalarial action by targeting the Plasmodium lactate transporter PfFNT at nanomolar scale. PLoS Pathog 2017; 13:e1006172 [View Article] [PubMed]
    [Google Scholar]
  15. Zeng JM, Hapuarachchi SV, Shafik SH, Martin RE, Kirk K et al. Identifying the major lactate transporter of Toxoplasma gondii tachyzoites. Sci Rep 2021; 11:6787 [View Article] [PubMed]
    [Google Scholar]
  16. Lyu M, Su C-. C, Kazura JW, Yu EW. Structural basis of transport and inhibition of the Plasmodium falciparum transporter PfFNT. EMBO Rep 2021; 22:e51628 [View Article] [PubMed]
    [Google Scholar]
  17. Waight AB, Czyzewski BK, Wang D-. N. Ion selectivity and gating mechanisms of FNT channels. Curr Opin Struct Biol 2013; 23:499–506 [View Article] [PubMed]
    [Google Scholar]
  18. W, Du J, Schwarzer NJ, Wacker T, Andrade SLA et al. The formate/nitrite transporter family of anion channels. Biol Chem 2013; 394:715–727 [View Article] [PubMed]
    [Google Scholar]
  19. Helmstetter F, Arnold P, Höger B, Petersen LM, Beitz E. Formate-nitrite transporters carrying nonprotonatable amide amino acids instead of a central histidine maintain pH-dependent transport. J Biol Chem 2019; 294:623–631 [View Article] [PubMed]
    [Google Scholar]
  20. Kammel M, Hunger D, Sawers RG. The soluble cytoplasmic N-terminal domain of the FocA channel gates bidirectional formate translocation. Mol Microbiol 2021; 115:758–773 [View Article] [PubMed]
    [Google Scholar]
  21. Hunger D, Doberenz C, Sawers RG. Identification of key residues in the formate channel FocA that control import and export of formate. Biol Chem 2014; 395:813–825 [View Article] [PubMed]
    [Google Scholar]
  22. Wiechert M, Beitz E. Mechanism of formate-nitrite transporters by dielectric shift of substrate acidity. EMBO J 2017; 36:949–958 [View Article] [PubMed]
    [Google Scholar]
  23. Beyer L, Doberenz C, Falke D, Hunger D, Suppmann B et al. Coordination of FocA and pyruvate formate-lyase synthesis in Escherichia coli demonstrates preferential translocation of formate over other mixed-acid fermentation products. J Bacteriol 2013; 195:1428–1435 [View Article] [PubMed]
    [Google Scholar]
  24. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: a Laboratory Manual, 2nd edn. NY (Cold Spring Harbor Laboratory, NY: Cold Spring Harbor; 1989
    [Google Scholar]
  25. Begg YA, Whyte JN, Haddock BA. The identification of mutants of Escherichia coli deficient in formate dehydrogenase and nitrate reductase activities using dye indicator plates. FEMS Microbiol Lett 1977; 2:47–50 [View Article]
    [Google Scholar]
  26. Falke D, Schulz K, Doberenz C, Beyer L, Lilie H et al. Unexpected oligomeric structure of the FocA formate channel of Escherichia coli: a paradigm for the formate-nitrite transporter family of integral membrane proteins. FEMS Microbiol Lett 2010; 303:69–75 [View Article] [PubMed]
    [Google Scholar]
  27. Hamilton CM, Aldea M, Washburn BK, Babitzke P, Kushner SR. New method for generating deletions and gene replacements in Escherichia coli . J Bacteriol 1989; 171:4617–4622 [View Article] [PubMed]
    [Google Scholar]
  28. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685 [View Article] [PubMed]
    [Google Scholar]
  29. Schägger H, von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 1991; 199:223–231 [View Article] [PubMed]
    [Google Scholar]
  30. Doberenz C, Zorn M, Falke D, Nannemann D, Hunger D et al. Pyruvate formate-lyase interacts directly with the formate channel FocA to regulate formate translocation. J Mol Biol 2014; 426:2827–2839 [View Article] [PubMed]
    [Google Scholar]
  31. Miller J. Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1972
    [Google Scholar]
  32. Pinske C. The ferredoxin-like proteins HydN and YsaA enhance redox dye-linked activity of the formate dehydrogenase H component of the formate hydrogenlyase complex. Front Microbiol 2018; 9:1238 [View Article] [PubMed]
    [Google Scholar]
  33. Pravda L, Sehnal D, Toušek D, Navrátilová V, Bazgier V et al. MOLEonline: a web-based tool for analyzing channels, tunnels and pores (2018 update). Nucleic Acids Res 2018; 46:W368–W373 [View Article] [PubMed]
    [Google Scholar]
  34. Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome Res 2004; 14:1188–1190 [View Article] [PubMed]
    [Google Scholar]
  35. Hopper S, Böck A. Effector-mediated stimulation of ATPase activity by the sigma 54-dependent transcriptional activator FHLA from Escherichia coli. J Bacteriol 1995; 177:2798–2803 [View Article] [PubMed]
    [Google Scholar]
  36. Plaga W, Frank R, Knappe J. Catalytic-site mapping of pyruvate formate lyase. Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate). Eur J Biochem 1988; 178:445–450 [View Article] [PubMed]
    [Google Scholar]
  37. Atkovska K, Hub JS. Energetics and mechanism of anion permeation across formate-nitrite transporters. Sci Rep 2017; 7:12027 [View Article] [PubMed]
    [Google Scholar]
  38. Theobald DL, Miller C. Membrane transport proteins: surprises in structural sameness. Nat Struct Mol Biol 2010; 17:2–3 [View Article] [PubMed]
    [Google Scholar]
  39. Armstrong CM. Packaging life: the origin of ion-selective channels. Biophys J 2015; 109:173–177 [View Article] [PubMed]
    [Google Scholar]
  40. Bot CT, Prodan C. Quantifying the membrane potential during E. coli growth stages. Biophys Chem 2010; 146:133–137 [View Article] [PubMed]
    [Google Scholar]
  41. Thauer RK, Kirchniawy FH, Jungermann KA. Properties and function of the pyruvate-formate-lyase reaction in clostridiae. Eur J Biochem 1972; 27:282–290 [View Article] [PubMed]
    [Google Scholar]
  42. Merlin C, Masters M, McAteer S, Coulson A. Why is carbonic anhydrase essential to Escherichia coli?. J Bacteriol 2003; 185:6415–6424 [View Article] [PubMed]
    [Google Scholar]
  43. Lv X, Liu H, Ke M, Gong H. Exploring the pH-dependent substrate transport mechanism of FocA using molecular dynamics simulation. Biophys J 2013; 105:2714–2723 [View Article] [PubMed]
    [Google Scholar]
  44. ten Brink B, Otto R, Hansen UP, Konings WN. Energy recycling by lactate efflux in growing and nongrowing cells of Streptococcus cremoris . J Bacteriol 1985; 162:383–390 [View Article] [PubMed]
    [Google Scholar]
  45. Stephenson M, Stickland LH. Hydrogenlyases: Bacterial enzymes liberating molecular hydrogen. Biochem J 1932; 26:712–724 [View Article] [PubMed]
    [Google Scholar]
  46. Smeulders MJ, Peeters SH, van Alen T, de Bruijckere D, Nuijten GHL et al. Nutrient limitation causes differential expression of transport- and metabolism genes in the compartmentalized anammox bacterium Kuenenia stuttgartiensis . Front Microbiol 2020; 11:1959 [View Article] [PubMed]
    [Google Scholar]
  47. Fu D, Libson A, Miercke LJ, Weitzman C, Nollert P et al. Structure of a glycerol-conducting channel and the basis for its selectivity. Science 2000; 290:481–486 [View Article] [PubMed]
    [Google Scholar]
  48. Sui H, Han BG, Lee JK, Walian P, Jap BK. Structural basis of water-specific transport through the AQP1 water channel. Nature 2001; 414:872–878 [View Article] [PubMed]
    [Google Scholar]
  49. Agre P, King LS, Yasui M, Guggino WB, Ottersen OP et al. Aquaporin water channels--from atomic structure to clinical medicine. J Physiol 2002; 542:3–16 [View Article] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.001132
Loading
/content/journal/micro/10.1099/mic.0.001132
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error