Indicators for citrate, particularly those applicable to its in vivo detection and quantitation, have attracted much interest in both biochemical studies and industrial applications since citrate is a key metabolic intermediate playing important roles in living cells. We generated novel fluorescence indicators for citrate by fusing the circularly permuted fluorescent protein (cpFP) and the periplasmic domain of the bacterial histidine kinase CitA, which can bind to citrate with high specificity. The ratiometric fluorescent signal change was observed with one of these cpFP-based indicators, named CF98: upon addition of citrate, the excitation peak at 504 nm increased proportionally to the decrease in the peak at 413 nm, suitable for build-in quantitative estimation of the binding compound. We confirmed that CF98 can be used for detecting citrate in vitro at millimolar levels in the range of 0.1 to 50 mM with high selectivity; even in the presence of other organic acids such as isocitrate and malate, the fluorescence intensity of CF98 remains unaffected. We finally demonstrated the in vivo applicability of CF98 to estimation of the intracellular citrate concentration in Escherichia coli co-expressing the genes encoding CF98 and the citrate carrier CitT. The novel indicator CF98 can be a specific and simple detection tool for citrate in vitro and a non-invasive tool for real-time estimation of intracellular concentrations of the compound in vivo.
References
[1]
Krebs HA (1940) The citric acid cycle: A reply to the criticisms of F. L. Breusch and of J. Thomas. Biochem J 34: 460–463.
[2]
Kirimura K, Honda Y, Hattori T (2011) Citric acid. In: Moo-Young M, editor. Comprehensive Biotechnology. Second Edition: Elsevier. pp. 135–142.
[3]
Demain AL, Sanchez S (2011) Microbial synthesis of primary metabolites: current trends and future prospects. In: El-Mansi EMT, Bryce CFA, Dahhou B, Sanchez S, Demain AL, et al.., editors. Fermentation Microbiology and Biotechnology. Third Edition: CRC Press. pp. 77–100.
[4]
Geelen MJ, Schmitz MG (1993) The role of citrate in the regulation of hepatic fatty acid synthesis by insulin and glucagon. Horm Metab Res 25: 525–527.
[5]
Tosukhowong P, Borvonpadungkitti S, Prasongwatana V, Tungsanga K, Jutuporn S, et al. (2002) Urinary citrate excretion in patients with renal stone: roles of leucocyte ATP citrate lyase activity and potassium salts therapy. Clin Chim Acta 325: 71–78.
[6]
Fricke ST, Rodriguez O, Vanmeter J, Dettin LE, Casimiro M, et al. (2006) In vivo magnetic resonance volumetric and spectroscopic analysis of mouse prostate cancer models. Prostate 66: 708–717.
[7]
Newman RH, Fosbrink MD, Zhang J (2011) Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells. Chem Rev 111: 3614–3666.
[8]
Kirimura K, Ogawa S, Hattori T, Kino K (2006) Expression analysis of alternative oxidase gene (aox1) with enhanced green fluorescent protein as marker in citric acid-producing Aspergillus niger. J Biosci Bioeng 102: 210–214.
[9]
Hattori T, Honda Y, Kino K, Kirimura K (2008) Expression of alternative oxidase gene (aox1) at the stage of single-cell conidium in citric acid-producing Aspergillus niger. J Biosci Bioeng 105: 55–57.
[10]
Honda Y, Kobayashi K, Kirimura K (2011) Increases in gene-targeting frequencies due to disruption of kueA as a ku80 homolog in citric acid-producing Aspergillus niger. Biosci Biotechnol Biochem 75: 1594–1596.
[11]
Bermejo C, Ewald JC, Lanquar V, Jones AM, Frommer WB (2011) In VIVO biochemistry: quantifying ion and metabolite levels in individual cells or cultures of yeast. Biochem J 438: 1–10.
[12]
Evellin S, Mongillo M, Terrin A, Lissandron V, Zaccolo M (2004) Measuring dynamic changes in cAMP using fluorescence resonance energy transfer. Methods Mol Biol 284: 259–270.
[13]
Imamura H, Nhat KP, Togawa H, Saito K, Iino R, et al. (2009) Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators. Proc Natl Acad Sci USA 106: 15651–15656.
[14]
Dulla C, Tani H, Okumoto S, Frommer WB, Reimer RJ, et al. (2008) Imaging of glutamate in brain slices using FRET sensors. J Neurosci Methods 168: 306–319.
[15]
Fehr M, Frommer WB, Lalonde S (2002) Visualization of maltose uptake in living yeast cells by fluorescent nanosensors. Proc Natl Acad Sci USA 99: 9846–9851.
[16]
Fehr M, Lalonde S, Ehrhardt DW, Frommer WB (2004) Live imaging of glucose homeostasis in nuclei of COS-7 cells. J Fluoresc 14: 603–609.
[17]
Ewald JC, Reich S, Baumann S, Frommer WB, Zamboni N (2011) Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations. PLoS One 6: e28245.
[18]
Baird GS, Zacharias DA, Tsien RY (1999) Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci USA 96: 11241–11246.
[19]
Nagai T, Sawano A, Park ES, Miyawaki A (2001) Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci USA 98: 3197–3202.
[20]
Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, et al. (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3: 281–286.
[21]
Markvicheva KN, Bilan DS, Mishina NM, Gorokhovatsky AY, Vinokurov LM, et al. (2011) A genetically encoded sensor for H2O2 with expanded dynamic range. Bioorg Med Chem 19: 1079–1084.
[22]
Berg J, Hung YP, Yellen G (2009) A genetically encoded fluorescent reporter of ATP:ADP ratio. Nat Methods 6: 161–166.
[23]
Bott M, Meyer M, Dimroth P (1995) Regulation of anaerobic citrate metabolism in Klebsiella pneumoniae. Mol Microbiol 18: 533–546.
[24]
Bott M (1997) Anaerobic citrate metabolism and its regulation in enterobacteria. Arch Microbiol 167: 78–88.
[25]
Kaspar S, Perozzo R, Reinelt S, Meyer M, Pfister K, et al. (1999) The periplasmic domain of the histidine autokinase CitA functions as a highly specific citrate receptor. Mol Microbiol 33: 858–872.
[26]
Gerharz T, Reinelt S, Kaspar S, Scapozza L, Bott M (2003) Identification of basic amino acid residues important for citrate binding by the periplasmic receptor domain of the sensor kinase CitA. Biochem 42: 5917–5924.
[27]
Reinelt S, Hofmann E, Gerharz T, Bott M, Madden DR (2003) The structure of the periplasmic ligand-binding domain of the sensor kinase CitA reveals the first extracellular PAS domain. J Biol Chem 278: 39189–39196.
[28]
Sevvana M, Vijayan V, Zweckstetter M, Reinelt S, Madden DR, et al. (2008) A ligand-induced switch in the periplasmic domain of sensor histidine kinase CitA. J Mol Biol 377: 512–523.
[29]
McNicholas S, Potterton E, Wilson KS, Noble ME (2011) Presenting your structures: the CCP4mg molecular-graphics software. Acta Crystallogr D Biol Crystallogr 67: 386–394.
[30]
Pos KM, Dimroth P, Bott M (1998) The Escherichia coli citrate carrier CitT: a member of a novel eubacterial transporter family related to the 2-oxoglutarate/malate translocator from spinach chloroplasts. J Bacteriol 180: 4160–4165.
[31]
Thomas JA, Buchsbaum RN, Zimniak A, Racker E (1979) Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochem 18: 2210–2218.
[32]
Albe KR, Butler MH, Wright BE (1990) Cellular concentrations of enzymes and their substrates. J Theor Biol 143: 163–195.
[33]
Bennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ, et al. (2009) Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol 5: 593–599.
[34]
Lagunas R, Gancedo C (1983) Role of phosphate in the regulation of the Pasteur effect in Saccharomyces cerevisiae. Eur J Biochem 137: 479–483.
[35]
Gnoni GV, Priore P, Geelen MJ, Siculella L (2009) The mitochondrial citrate carrier: metabolic role and regulation of its activity and expression. IUBMB Life 61: 987–994.
[36]
Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, et al. (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6: 325–330.
[37]
Maruyama J, Nakajima H, Kitamoto K (2001) Visualization of nuclei in Aspergillus oryzae with EGFP and analysis of the number of nuclei in each conidium by FACS. Biosci Biotech Biochem 65: 1504–1510.
[38]
Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, et al. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotech 20: 87–90.
