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Volume 169, Issue 1

Iron homeostasis in relies on three differentially expressed efflux systems Open Access

Abstract

In iron homeostasis is maintained by the ferric uptake regulator (Fur) and manganese homeostasis relies on the manganese transport regulator (MntR). Both Fur and MntR function as bi-functional metalloregulators that repress import and activate metal ion efflux systems. The ferrous iron efflux ATPase, PfeT, is derepressed by hydrogen peroxide (HO) as sensed by PerR and induced by iron as sensed by Fur. Mutants lacking PfeT are sensitive to iron intoxication. Here, we show that mutants are also iron-sensitive, largely due to decreased expression of the MntR-activated MneP and MneS cation diffusion facilitator (CDF) proteins previously defined for their role in Mn export. The ability of MneP and MneS to export iron is apparent even when their expression is not induced by Mn. Our results demonstrate that PfeT, MneP and MneS each contribute to iron homeostasis, and a triple mutant lacking all three is more iron-sensitive than any single mutant. We further show that sensitivity to HO does not correlate with iron sensitivity. For example, an mutant is HO-sensitive due to elevated Mn(II) that increases PerR-mediated repression of peroxide resistance genes, and this repression is antagonized by elevated Fe in an mutant. Thus, HO-sensitivity reflects the relative levels of Mn and Fe as sensed by the PerR regulatory protein. These results underscore the complex interplay between manganese, iron and oxidative stress in .

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Bacillus subtilis , cation diffusion facilitator , iron , manganese , metal homeostasis , oxidative stress and regulation
Funding
This study was supported by the:
  • NIH (Award R35GM122461)
    • Principle Award Recipient: JohnD Helmann
© 2023 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2023-01-17
2024-05-17
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References

  1. Helmann JD. Specificity of metal sensing: iron and manganese homeostasis in Bacillus subtilis. J Biol Chem 2014; 289:28112–28120 [View Article]
     [Google Scholar]
  2. Chandrangsu P, Rensing C, Helmann JD. Metal homeostasis and resistance in bacteria. Nat Rev Microbiol 2017; 15:338–350 [View Article] [PubMed]
     [Google Scholar]
  3. Sarvan S, Butcher J, Stintzi A, Couture JF. Variation on a theme: investigating the structural repertoires used by ferric uptake regulators to control gene expression. Biometals 2018; 31:681–704 [View Article] [PubMed]
     [Google Scholar]
  4. Sevilla E, Bes MT, Peleato ML, Fillat MF. Fur-like proteins: Beyond the ferric uptake regulator (Fur) paralog. Arch Biochem Biophys 2021; 701:108770 [View Article]
     [Google Scholar]
  5. Bsat N, Herbig A, Casillas-Martinez L, Setlow P, Helmann JD. Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors. Mol Microbiol 1998; 29:189–198 [View Article]
     [Google Scholar]
  6. Ollinger J, Song KB, Antelmann H, Hecker M, Helmann JD. Role of the Fur regulon in iron transport in Bacillus subtilis. J Bacteriol 2006; 188:3664–3673 [View Article]
     [Google Scholar]
  7. Pi H, Helmann JD. Sequential induction of Fur-regulated genes in response to iron limitation in Bacillus subtilis. Proc Natl Acad Sci U S A 2017; 114:12785–12790 [View Article] [PubMed]
     [Google Scholar]
  8. Gaballa A, Antelmann H, Aguilar C, Khakh SK, Song K-B et al. The Bacillus subtilis iron-sparing response is mediated by a Fur-regulated small RNA and three small, basic proteins. Proc Natl Acad Sci U S A 2008; 105:11927–11932 [View Article]
     [Google Scholar]
  9. Smaldone GT, Antelmann H, Gaballa A, Helmann JD. The FsrA sRNA and FbpB protein mediate the iron-dependent induction of the Bacillus subtilis lutABC iron-sulfur-containing oxidases. J Bacteriol 2012; 194:2586–2593 [View Article]
     [Google Scholar]
  10. Pinochet-Barros A, Helmann JD. Bacillus subtilis Fur Is a transcriptional activator for the PerR-repressed pfeT Gene, encoding an iron efflux pump. J Bacteriol 2020; 202:e00697-19 [View Article]
     [Google Scholar]
  11. Guan G, Pinochet-Barros A, Gaballa A, Patel SJ, Argüello JM et al. PfeT, a P1B4 -type ATPase, effluxes ferrous iron and protects Bacillus subtilis against iron intoxication. Mol Microbiol 2015; 98:787–803 [View Article]
     [Google Scholar]
  12. Pi H, Helmann JD. Ferrous iron efflux systems in bacteria. Metallomics 2017; 9:840–851 [View Article] [PubMed]
     [Google Scholar]
  13. Brown JB, Lee MA, Smith AT. Ins and Outs: Recent advancements in membrane protein-mediated prokaryotic ferrous iron transport. Biochemistry 2021; 60:3277–3291 [View Article]
     [Google Scholar]
  14. Oh YK, Freese E. Manganese requirement of phosphoglycerate phosphomutase and its consequences for growth and sporulation of Bacillus subtilis. J Bacteriol 1976; 127:739–746 [View Article]
     [Google Scholar]
  15. Que Q, Helmann JD. Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Mol Microbiol 2000; 35:1454–1468 [View Article]
     [Google Scholar]
  16. Glasfeld A, Guedon E, Helmann JD, Brennan RG. Structure of the manganese-bound manganese transport regulator of Bacillus subtilis. Nat Struct Biol 2003; 10:652–657 [View Article]
     [Google Scholar]
  17. McGuire AM, Cuthbert BJ, Ma Z, Grauer-Gray KD, Brunjes Brophy M et al. Roles of the A and C sites in the manganese-specific activation of MntR. Biochemistry 2013; 52:701–713 [View Article]
     [Google Scholar]
  18. Guedon E, Moore CM, Que Q, Wang T, Ye RW et al. The global transcriptional response of Bacillus subtilis to manganese involves the MntR, Fur, TnrA and sigmaB regulons. Mol Microbiol 2003; 49:1477–1491 [View Article]
     [Google Scholar]
  19. Huang X, Shin JH, Pinochet-Barros A, Su TT, Helmann JD. Bacillus subtilis MntR coordinates the transcriptional regulation of manganese uptake and efflux systems. Mol Microbiol 2017; 103:253–268 [View Article]
     [Google Scholar]
  20. Chen L, Keramati L, Helmann JD. Coordinate regulation of Bacillus subtilis peroxide stress genes by hydrogen peroxide and metal ions. Proc Natl Acad Sci U S A 1995; 92:8190–8194 [View Article]
     [Google Scholar]
  21. Herbig AF, Helmann JD. Roles of metal ions and hydrogen peroxide in modulating the interaction of the Bacillus subtilis PerR peroxide regulon repressor with operator DNA. Mol Microbiol 2001; 41:849–859 [View Article]
     [Google Scholar]
  22. Lee JW, Helmann JD. The PerR transcription factor senses H₂O₂ by metal-catalysed histidine oxidation. Nature 2006; 440:363–367 [View Article]
     [Google Scholar]
  23. Jacquamet L, Traoré DAK, Ferrer J-L, Proux O, Testemale D et al. Structural characterization of the active form of PerR: insights into the metal-induced activation of PerR and Fur proteins for DNA binding. Mol Microbiol 2009; 73:20–31 [View Article] [PubMed]
     [Google Scholar]
  24. Traoré DAK, El Ghazouani A, Jacquamet L, Borel F, Ferrer J-L et al. Structural and functional characterization of 2-oxo-histidine in oxidized PerR protein. Nat Chem Biol 2009; 5:53–59 [View Article] [PubMed]
     [Google Scholar]
  25. Ahn BE, Baker TA. Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR. Proc Natl Acad Sci U S A 2016; 113:E23–31 [View Article] [PubMed]
     [Google Scholar]
  26. Pinochet-Barros A, Helmann JD. Redox sensing by Fe²⁺ in bacterial fur family metalloregulators. Antioxid Redox Signal 2018; 29:1858–1871
     [Google Scholar]
  27. Ma Z, Lee JW, Helmann JD. Identification of altered function alleles that affect Bacillus subtilis PerR metal ion selectivity. Nucleic Acids Res 2011; 39:5036–5044 [View Article]
     [Google Scholar]
  28. Ji C-J, Kim J-H, Won Y-B, Lee Y-E, Choi T-W et al. Staphylococcus aureus PerR Is a hypersensitive hydrogen peroxide sensor using iron-mediated histidine oxidation. J Biol Chem 2015; 290:20374–20386 [View Article]
     [Google Scholar]
  29. Helmann JD, Wu MFW, Gaballa A, Kobel PA, Morshedi MM et al. The global transcriptional response of Bacillus subtilis to peroxide stress is coordinated by three transcription factors. J Bacteriol 2003; 185:243–253 [View Article]
     [Google Scholar]
  30. Faulkner MJ, Helmann JD. Peroxide stress elicits adaptive changes in bacterial metal ion homeostasis. Antioxid Redox Signal 2011; 15:175–189 [View Article] [PubMed]
     [Google Scholar]
  31. Faulkner MJ, Ma Z, Fuangthong M, Helmann JD. Derepression of the Bacillus subtilis PerR peroxide stress response leads to iron deficiency. J Bacteriol 2012; 194:1226–1235 [View Article]
     [Google Scholar]
  32. Chen L, Helmann JD. Bacillus subtilis MrgA is a Dps(PexB) homologue: evidence for metalloregulation of an oxidative-stress gene. Mol Microbiol 1995; 18:295–300 [View Article]
     [Google Scholar]
  33. Pi H, Patel SJ, Argüello JM, Helmann JD. The Listeria monocytogenes Fur-regulated virulence protein FrvA is an Fe(II) efflux P1B4 -type ATPase. Mol Microbiol 2016; 100:1066–1079 [View Article] [PubMed]
     [Google Scholar]
  34. VanderWal AR, Makthal N, Pinochet-Barros A, Helmann JD, Olsen RJ et al. Iron efflux by PmtA is critical for oxidative stress resistance and contributes significantly to group a Streptococcus virulence. Infect Immun 2017; 85:e00091-17 [View Article]
     [Google Scholar]
  35. Patel SJ, Lewis BE, Long JE, Nambi S, Sassetti CM et al. Fine-tuning of substrate affinity leads to alternative roles of Mycobacterium tuberculosis Fe2+ -ATPases. J Biol Chem 2016; 291:11529–11539 [View Article]
     [Google Scholar]
  36. Turner AG, Djoko KY, Ong C-LY, Barnett TC, Walker MJ et al. Group A Streptococcus co-ordinates manganese import and iron efflux in response to hydrogen peroxide stress. Biochem J 2019; 476:595–611 [View Article] [PubMed]
     [Google Scholar]
  37. Turner AG, Ong C-LY, Djoko KY, West NP, Davies MR et al. The PerR-Regulated P1B-4-Type ATPase (PmtA) acts as a ferrous iron efflux pump in Streptococcus pyogenes. Infect Immun 2017; 85:e00140-17 [View Article]
     [Google Scholar]
  38. Slack FJ, Mueller JP, Sonenshein AL. Mutations that relieve nutritional repression of the Bacillus subtilis dipeptide permease operon. J Bacteriol 1993; 175:4605–4614 [View Article]
     [Google Scholar]
  39. Quisel JD, Burkholder WF, Grossman AD. In vivo effects of sporulation kinases on mutant Spo0A proteins in Bacillus subtilis. J Bacteriol 2001; 183:6573–6578 [View Article]
     [Google Scholar]
  40. Davies BW, Walker GC. Disruption of sitA compromises Sinorhizobium meliloti for manganese uptake required for protection against oxidative stress. J Bacteriol 2007; 189:2101–2109 [View Article]
     [Google Scholar]
  41. Fleming MD, Trenor CC, Su MA, Foernzler D, Beier DR et al. Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet 1997; 16:383–386 [View Article]
     [Google Scholar]
  42. Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 1997; 388:482–488 [View Article] [PubMed]
     [Google Scholar]
  43. Runyen-Janecky L, Dazenski E, Hawkins S, Warner L. Role and regulation of the Shigella flexneri sit and MntH systems. Infect Immun 2006; 74:4666–4672 [View Article] [PubMed]
     [Google Scholar]
  44. Sabri M, Léveillé S, Dozois CM. A SitABCD homologue from an avian pathogenic Escherichia coli strain mediates transport of iron and manganese and resistance to hydrogen peroxide. Microbiology 2006; 152:745–758 [View Article]
     [Google Scholar]
  45. Yeowell HN, White JR. Iron requirement in the bactericidal mechanism of streptonigrin. Antimicrob Agents Chemother 1982; 22:961–968 [View Article] [PubMed]
     [Google Scholar]
  46. Imlay JA. The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium. Nat Rev Microbiol 2013; 11:443–454 [View Article] [PubMed]
     [Google Scholar]
  47. Frawley ER, Fang FC. The ins and outs of bacterial iron metabolism. Mol Microbiol 2014; 93:609–616 [View Article] [PubMed]
     [Google Scholar]
  48. Khademian M, Imlay JA. How microbes evolved to tolerate oxygen. Trends Microbiol 2021; 29:428–440 [View Article]
     [Google Scholar]
  49. Randazzo P, Anba-Mondoloni J, Aubert-Frambourg A, Guillot A, Pechoux C et al. Bacillus subtilis regulators MntR and Zur participate in redox cycling, antibiotic sensitivity, and cell wall plasticity. J Bacteriol 2020; 202:e00547-19 [View Article]
     [Google Scholar]
  50. Jordan MR, Wang J, Capdevila DA, Giedroc DP. Multi-metal nutrient restriction and crosstalk in metallostasis systems in microbial pathogens. Curr Opin Microbiol 2020; 55:17–25 [View Article] [PubMed]
     [Google Scholar]
  51. Osman D, Martini MA, Foster AW, Chen J, Scott AJP et al. Bacterial sensors define intracellular free energies for correct enzyme metalation. Nat Chem Biol 2019; 15:241–249 [View Article] [PubMed]
     [Google Scholar]
  52. Hohle TH, O’Brian MR. Magnesium-dependent processes are targets of bacterial manganese toxicity. Mol Microbiol 2014; 93:736–747 [View Article] [PubMed]
     [Google Scholar]
  53. Kolaj-Robin O, Russell D, Hayes KA, Pembroke JT, Soulimane T. Cation Diffusion Facilitator family: Structure and function. FEBS Lett 2015; 589:1283–1295 [View Article] [PubMed]
     [Google Scholar]
  54. Montanini B, Blaudez D, Jeandroz S, Sanders D, Chalot M. Phylogenetic and functional analysis of the Cation Diffusion Facilitator (CDF) family: improved signature and prediction of substrate specificity. BMC Genomics 2007; 8:107 [View Article] [PubMed]
     [Google Scholar]
  55. Lu M, Chai J, Fu D. Structural basis for autoregulation of the zinc transporter YiiP. Nat Struct Mol Biol 2009; 16:1063–1067 [View Article] [PubMed]
     [Google Scholar]
  56. Lu M, Fu D. Structure of the zinc transporter YiiP. Science 2007; 317:1746–1748 [View Article] [PubMed]
     [Google Scholar]
  57. Martin JE, Giedroc DP. Functional determinants of metal ion transport and selectivity in paralogous cation diffusion facilitator transporters CzcD and MntE in Streptococcus pneumoniae. J Bacteriol 2016; 198:1066–1076 [View Article]
     [Google Scholar]
  58. Cubillas C, Vinuesa P, Tabche ML, Dávalos A, Vázquez A et al. The cation diffusion facilitator protein EmfA of Rhizobium etli belongs to a novel subfamily of Mn(2+)/Fe(2+) transporters conserved in α-proteobacteria. Metallomics 2014; 6:1808–1815 [View Article]
     [Google Scholar]
  59. Lam LN, Wong JJ, Chong KKL, Kline KA. Enterococcus faecalis manganese exporter MntE alleviates manganese toxicity and is required for mouse gastrointestinal colonization. Infect Immun 2020; 88:e00058-20 [View Article]
     [Google Scholar]
  60. Ouyang A, Gasner KM, Neville SL, McDevitt CA, Frawley ER. MntP and YiiP contribute to manganese efflux in Salmonella enterica serovar typhimurium under conditions of manganese overload and nitrosative stress. Microbiol Spectr 2022; 10:e0131621 [View Article]
     [Google Scholar]
  61. Anjem A, Imlay JA. Mononuclear iron enzymes are primary targets of hydrogen peroxide stress. J Biol Chem 2012; 287:15544–15556 [View Article] [PubMed]
     [Google Scholar]
  62. Sobota JM, Gu M, Imlay JA. Intracellular hydrogen peroxide and superoxide poison 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, the first committed enzyme in the aromatic biosynthetic pathway of Escherichia coli. J Bacteriol 2014; 196:1980–1991 [View Article]
     [Google Scholar]
  63. Juttukonda LJ, Skaar EP. Manganese homeostasis and utilization in pathogenic bacteria. Mol Microbiol 2015; 97:216–228 [View Article] [PubMed]
     [Google Scholar]
  64. Turner AG, Ong C-L, Gillen CM, Davies MR, West NP et al. Manganese homeostasis in group A Streptococcus is critical for resistance to oxidative stress and virulence. mBio 2015; 6:e00278-15 [View Article]
     [Google Scholar]
  65. Grunenwald CM, Choby JE, Juttukonda LJ, Beavers WN, Weiss A et al. Manganese detoxification by MntE is critical for resistance to oxidative stress and virulence of Staphylococcus aureus. mBio 2019; 10:e02915-18 [View Article]
     [Google Scholar]
  66. Rosch JW, Gao G, Ridout G, Wang YD, Tuomanen EI. Role of the manganese efflux system mntE for signalling and pathogenesis in Streptococcus pneumoniae. Mol Microbiol 2009; 72:12–25 [View Article]
     [Google Scholar]
  67. Begg SL. The role of metal ions in the virulence and viability of bacterial pathogens. Biochem Soc Trans 2019; 47:77–87 [View Article] [PubMed]
     [Google Scholar]
  68. Becker KW, Skaar EP. Metal limitation and toxicity at the interface between host and pathogen. FEMS Microbiol Rev 2014; 38:1235–1249 [View Article] [PubMed]
     [Google Scholar]
  69. Lopez CA, Skaar EP. The impact of dietary transition metals on host-bacterial interactions. Cell Host Microbe 2018; 23:737–748 [View Article]
     [Google Scholar]
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Iron homeostasis in Bacillus subtilis relies on three differentially expressed efflux systems
Microbiology 169, 001289 (2023); https://doi.org/10.1099/mic.0.001289
/content/journal/micro/10.1099/mic.0.001289
/content/journal/micro/10.1099/mic.0.001289
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