Skip to main content
SpringerLink
Log in

Characterization of Bacillus subtilis YfkE (ChaA): a calcium-specific Ca2+/H+ antiporter of the CaCA family

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

YfkE, a protein from Bacillus subtilis, exhibits homology to the Ca2+:Cation Antiporter (CaCA) Family. In a fluorescence-based assay of everted membrane vesicles prepared from Na+(Ca2+)/H+ antiporter-defective mutant Escherichia coli KNabc, YfkE exhibited robust Ca2+/H+ antiport activity, with a K m for Ca2+ estimated at 12.5 μM at pH 8.5 and 113 μM at pH 7.5. Neither Na+ nor K+ served as a substrate. Mg2+ also did not serve as a substrate, but inhibited the Ca2+/H+ antiporter activity. The Ca2+ transport capability of YfkE was also observed directly by transport assays in everted membrane vesicles using radiolabeled 45Ca2+. Transcriptional analysis from the putative yfkED operon using β-garactosidase activity as a reporter revealed that both of the yfkE and yfkD genes are regulated by forespore-specific sigma factor, SigG, and the general stress response regulator, SigB. These results suggest that YfkE may be needed for Ca2+ signaling in the sporulation or germination process in B. subtilis. ChaA is proposed as the designation for YfkE of B. subtilis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

DPA:

Dipicolinic acid

CaCA:

Ca2+:Cation Antiporter

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

References

  • Ambudkar SV, Zlotnick GU, Rosen BP (1984) Calcium efflux from Escherichia coli: evidence for two systems. J Biol Chem 259:6142–6146

    PubMed  CAS  Google Scholar 

  • Cheng J, Guffanti AA, Wang W, Krulwich TA, Bechhofer DH (1996) Chromosomal tetA(L) gene of Bacillus subtilis: regulation of expression and physiology of a tetA(L) deletion strain. J Bacteriol 178:2853–2860

    PubMed  CAS  Google Scholar 

  • Chung YJ, Krueger C, Metzgar D, Saier MH Jr (2001) Size comparisons among integral membrane transport protein homologues in Bacteria, Archaea, and Eucarya. J Bacteriol 183:1012–1021

    Article  PubMed  CAS  Google Scholar 

  • Daniel RA, Errington J (1993) Cloning, DNA sequence, functional analysis and transcriptional regulation of the genes encoding dipicolinic acid synthetase required for sporulation in Bacillus subtilis. J Mol Biol 232:468–483

    Article  PubMed  CAS  Google Scholar 

  • Daniel RA, Haiech J, Denizot F, Errington J (1997) Isolation and characterization of the lacA gene encoding β-galactosidase in Bacillus subtilis and a regulator gene, lacR. J Bacteriol 179:5636–5638

    PubMed  CAS  Google Scholar 

  • Dominguez DC (2004) Calcium signaling in bacteria. Mol Microbiol 54:291–297

    Article  PubMed  CAS  Google Scholar 

  • Fujisawa M, Kusumoto A, Wada Y, Tsuchiya T, Ito M (2005) NhaK, a novel monovalent cation/H+ antiporter of Bacillus subtilis. Arch Microbiol 183:411–420

    Article  PubMed  CAS  Google Scholar 

  • Futsaether CM, Johnsson A (1994) Using fra-2 to measure intracellular free calcium in Propionibacterium acnes. Can J Microbiol 40:439–445

    Article  PubMed  CAS  Google Scholar 

  • Gangola P, Rosen BP (1987) Maintenance of intracellular calcium in Escherichia coli. J Biol Chem 262:12570–12574

    PubMed  CAS  Google Scholar 

  • Goldberg EB, Arbel T, Chen J, Karpel R, Mackie GA, Schuldiner S, Padan E (1987) Characterization of a Na+/H+ antiporter gene of Escherichia coli. Proc Nat Acad Sci USA 84:2615–2619

    Article  PubMed  CAS  Google Scholar 

  • Guffanti AA, Cheng J, Krulwich TA (1998) Electrogenic antiport activities of the Gram-positive Tet proteins include a Na+(K+)/K+ mode that mediates net K+ uptake. J Biol Chem 273:26447–26454

    Article  PubMed  CAS  Google Scholar 

  • Helmann JD, Wu MF, Kobel PA, Gamo FJ, Wilson M, Morshedi MM, Navre M, Paddon C (2001) Global transcriptional response of Bacillus subtilis to heat shock. J Bacteriol 183:7318–7328

    Article  PubMed  CAS  Google Scholar 

  • Herbaud ML, Guiseppi A, Denizot F, Haiech J, Kilhoffer MC (1998) Calcium signalling in Bacillus subtilis. Biochim Biophys Acta 1448:212–226

    Article  PubMed  CAS  Google Scholar 

  • Igura N, Kamimura Y, Islam MS, Shimoda M, Hayakawa I (2003) Effects of minerals on resistance of Bacillus subtilis spores to heat and hydrostatic pressure. Appl Environ Microbiol 69:6307–6310

    Article  PubMed  CAS  Google Scholar 

  • Ito M, Guffanti AA, Krulwich TA (2001) Mrp-dependent Na+/H+ antiporters of Bacillus exhibit characteristics that are unanticipated for completely secondary active transporters. FEBS Lett 496:117–120

    Article  PubMed  CAS  Google Scholar 

  • Ivey DM, Guffanti AA, Zemsky J, Pinner E, Karpel R, Padan E, Schuldiner S, Krulwich TA (1993) Cloning and characterization of a putative Ca2+/H+ antiporter gene from Escherichia coli upon functional complementation of Na+/H+ antiporter-deficient strains by the overexpressed gene. J Biol Chem 268:11296–11303

    PubMed  CAS  Google Scholar 

  • Jones HE, Holland IB, Baker HL, Campbell AK (1999) Slow changes in cytosolic free Ca2+ in Escherichia coli highlight two putative influx mechanisms in response to changes in extracellular calcium. Cell Calcium 25:265–274

    Article  PubMed  CAS  Google Scholar 

  • Knight MR, Campbell AK, Smith SM, Trewavas AJ (1991) Recombinant aequorin as a probe for cytosolic free Ca2+ in Escherichia coli. FEBS Lett 282:405–408

    Article  PubMed  CAS  Google Scholar 

  • Kraev A, Quednau BD, Leach S, Li XF, Dong H, Winkfein R, Perizzolo M, Cai X, Yang R, Philipson KD, Lytton J (2001) Molecular cloning of a third member of the potassium-dependent sodium-calcium exchanger gene family, NCKX3. J Biol Chem 276:23161–23172

    Article  PubMed  CAS  Google Scholar 

  • Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, Bertero MG, Bessières P, Bolotin A, Borchert S, Borriss R, Boursier L, Brans A, Braun M, Brignell SC, Bron S, Brouillet S, Bruschi CV, Caldwell B, Capuano V, Carter NM, Choi SK, Codani JJ, Connerton IF, Cummings NJ, Daniel RA, Denizot F, Devine KM, Düsterhöft A, Ehrlich SD, Emmerson PT, Entian KD, Errington J, Fabret C, Ferrari E, Foulger D, Fritz C, Fujita M, Fujita Y, Fuma S, Galizzi A, Galleron N, Ghim SY, Glaser P, Goffeau A, Golightly EJ, Grandi G, Guiseppi G, Guy BJ, Haga K, Haiech J, Harwood CR, Hénaut A, Hilbert H, Holsappel S, Hosono S, Hullo MF, Itaya M, Jones L, Joris B, Karamata D, Kasahara Y, Klaerr-Blanchard M, Klein C, Kobayashi Y, Koetter P, Koningstein G, Krogh S, Kumano M, Kurita K, Lapidus A, Lardinois S, Lauber J, Lazarevic V, Lee SM, Levine A, Liu H, Masuda S, Mauël C, Médigue C, Medina N, Mellado RP, Mizuno M, Moestl D, Nakai S, Noback M, Noone D, O’Reilly M, Ogawa K, Ogiwara A, Oudega B, Park SH, Parro V, Pohl TM, Portetelle D, Porwollik S, Prescott AM, Presecan E, Pujic P, Purnelle B, Rapoport G, Rey M, Reynolds S, Rieger M, Rivolta C, Rocha E, Roche B, Rose M, Sadaie Y, Sato T, Scanlan E, Schleich S, Schroeter R, Scoffone F, Sekiguchi J, Sekowska A, Seror SJ, Serror P, Shin BS, Soldo B, Sorokin A, Tacconi E, Takagi T, Takahashi H, Takemaru K, Takeuchi M, Tamakoshi A, Tanaka T, Terpstra P, Tognoni A, Tosato V, Uchiyama S, Vandenbol M, Vannier F, Vassarotti A, Viari A, Wambutt R, Wedler E, Wedler H, Weitzenegger T, Winters P, Wipat A, Yamamoto H, Yamane K, Yasumoto K, Yata K, Yoshida K, Yoshikawa HF, Zumstein E, Yoshikawa H, Danchin A (1997) The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390:249–256

    Google Scholar 

  • Kuwana R, Kasahara Y, Fujibayashi M, Takamatsu H, Ogasawara N, Watabe K (2002) Proteomics characterization of novel spore proteins of Bacillus subtilis. Microbiology 148:3971–3982

    PubMed  CAS  Google Scholar 

  • Leighton TJ, Doi RH (1971) The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis. J Biol Chem 246:3189–3195

    PubMed  CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  • Matsushita T, Ueda T, Kusaka I (1986) Purification and characterization of Ca2+/H+ antiporter from Bacillus subtilis. Eur J Biochem 156:95–100

    Article  PubMed  CAS  Google Scholar 

  • Michiels J, Xi C, Verhaert J, Vanderleyden J (2002) The functions of Ca2+ in bacteria: a role for EF-hand proteins? Trends Microbiol 10:87–93

    Article  PubMed  CAS  Google Scholar 

  • Moir A (2006) How do spores germinate? J Appl Microbiol 101:526–530

    Article  PubMed  CAS  Google Scholar 

  • Nakamura LK, Roberts MS, Cohan FM (1999) Relationship of Bacillus subtilis clades associated with strains 168 and W23: a proposal for Bacillus subtilis subsp. Subtilis subsp. nov. and Bacillus subtilis subsp. Spizizenii subsp. nov. Int. J Syst Bacteriol 49:1211–1215

    Article  CAS  Google Scholar 

  • Nicholson WL, Setlow P (1990) Sporulation, germination and outgrowth. In: Harwood CR, Cutting SM (eds) Molecular biological methods for Bacillus. Wiley, Chichester, pp 391–450

    Google Scholar 

  • Nozaki K, Inaba K, Kuroda T, Tsuda M, Tsuchiya T (1996) Cloning and sequencing of the gene for Na+/H+ antiporter of Vibrio parahaemolyticus. Biochem Biophys Res Commun 24:774–779

    Article  Google Scholar 

  • O’hara MB, Hageman JH (1990) Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells. J Bacteriol 172:4161–4170

    PubMed  Google Scholar 

  • Price CW, Fawcett P, Cérémonie H, Su N, Murphy CK, Youngman P (2001) Genome-wide analysis of the general stress response in Bacillus subtilis. Mol Microbiol 41:757–774

    Article  PubMed  CAS  Google Scholar 

  • Radchenko MV, Tanaka K, Waditee R, Oshimi S, Matsuzaki Y, Fukuhara M, Kobayashi H, Takabe T, Nakamura T (2006) Potassium/proton antiport system of Escherichia coli. J Biol Chem 281:19822

    Article  PubMed  CAS  Google Scholar 

  • Raeymaekers L, Wuytack E, Willems I, Michiels CW, Wuytack F (2002) Expression of a P-type Ca2+-transport ATPase in Bacillus subtilis during sporulation. Cell Calcium 32:93–103

    Article  PubMed  CAS  Google Scholar 

  • Rosen BP (1986) Ion extrusion systems in Escherichia coli. Methods Enzymol 125:328–336

    Article  PubMed  CAS  Google Scholar 

  • Saier MH Jr, Eng BH, Fard S, Garg J, Haggerty DA, Hutchinson WJ, Jack DL, Lai EC, Liu HJ, Nusinew DP, Omar AM, Pao SS, Paulsen IT, Quan JA, Sliwinski M, Tseng TT, Wachi S, Young GB (1999) Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim Biophys Acta 1422:1–56

    PubMed  CAS  Google Scholar 

  • Saier MH Jr, Goldman SR, Maile RR, Moreno MS, Weyler W, Yang N, Paulsen IT (2002) Transport capabilities encoded within the Bacillus subtilis genome. J Mol Microbiol Biotechnol 4:37–67

    PubMed  CAS  Google Scholar 

  • Setlow P (2006) Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol 101:514–525

    Article  PubMed  CAS  Google Scholar 

  • Seto-Young DL, Ellar DJ (1981) Studies on calcium transport during growth and sporulation. Microbios 30:191–208

    PubMed  CAS  Google Scholar 

  • Silver S, Toth K, Scribner H (1975) Facilitated transport of calcium by cells and subcellular membranes of Bacillus subtilis and Escherichia coli. J Bacteriol 122:880–885

    PubMed  CAS  Google Scholar 

  • Smith RJ (1995) Calcium and bacteria. Adv Microb Physiol 37:83–133

    Article  PubMed  CAS  Google Scholar 

  • Steil L, Serrano M, Henriques AO, Völker U (2005) Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis. Microbiology 151:399–420

    Article  PubMed  CAS  Google Scholar 

  • Tjalsma H, Bolhuis A, Jongbloed JD, Bron S, van Dijl JM (2000) Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev 64:515–547

    Article  PubMed  CAS  Google Scholar 

  • Torrecilla I, Leganés F, Bonilla I, Fernández-Piñas F (2000) Use of recombinant aequorin to study calcium homeostasis and monitor calcium transients in response to heat and cold shock in cyanobacteria. Plant Physiol 123:161–176

    Article  PubMed  CAS  Google Scholar 

  • Tsuchiya T, Rosen BP (1975) Characterization of an active transport system for calcium in inverted membrane vesicles of Escherichia coli. J Biol Chem 250:7687–7692

    PubMed  CAS  Google Scholar 

  • Vagner V, Dervyn E, Ehrlich SD (1998) A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144:3097–3104

    Article  PubMed  CAS  Google Scholar 

  • Vrij W, Bulthuis R, Postma E, Konings WN (1985) Calcium transport in membrane vesicles of Bacillus subtilis. J Bacteriol 164:1294–1300

    PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by Grant-in-Aid for Scientific Research on Priority Areas (C) “Genome Biology” from the Ministry of Education, Culture, Sports, Science and Technology of Japan to M.I. and GM52837 from the National Institutes of Health to Terry A. Krulwich, in whose laboratory at the Mount Sinai School of Medicine the initial fluorescence experiments and the 45Ca2+ uptake assays were conducted. The helpful suggestions of Terry A. Krulwich and Arthur A Guffanti about these experiments and the article are appreciated.

Author information

Authors and Affiliations

  1. Bio-Nano Electronics Research Center, Toyo University, 2100, Kujirai, Kawagoe, Saitama, 350-8585, Japan

    Makoto Fujisawa & Masahiro Ito

  2. Faculty of Food Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Oura-gun, Gunma, 374-0193, Japan

    Makoto Fujisawa

  3. Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Oura-gun, Gunma, 374-0193, Japan

    Yuko Wada, Takahiro Tsuchiya & Masahiro Ito

Authors
  1. Makoto Fujisawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuko Wada
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takahiro Tsuchiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Masahiro Ito
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Masahiro Ito.

Additional information

Communicated by Arnold Driessen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fujisawa, M., Wada, Y., Tsuchiya, T. et al. Characterization of Bacillus subtilis YfkE (ChaA): a calcium-specific Ca2+/H+ antiporter of the CaCA family. Arch Microbiol 191, 649–657 (2009). https://doi.org/10.1007/s00203-009-0494-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-009-0494-7

Keywords

Search

Navigation