Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti

BMC Microbiology

Lorella Navazio³  Antonella Roveri²  Andrea Squartini¹  Ernesto Damiani⁴  Flavia Ercolin³  Mattia Zaccarin²  Roberto Moscatiello³ 

[1]Department of Agronomy, Food, Natural Resources, Animals and Environment, DAFNAE, University of Padova, Viale dell’Università 16, Padova, 35020, Legnaro, Italy

[2]Department of Molecular Medicine, University of Padova, Viale G. Colombo 3, Padova, 35131, Italy

[3]Department of Biology, University of Padova, Via U. Bassi 58/B, Padova, 35131, Italy

[4]Department of Biomedical Sciences, University of Padova, Viale G. Colombo 3, Padova, 35131, Italy

关键词: Rhizobium-legume symbiosis;    Nitrogen fixation;    Mesorhizobium loti;    Ferredoxin II;    Calcium homeostasis;    Calcium binding proteins;   

Others  :  1137577
DOI  :  10.1186/s12866-015-0352-5

 received in 2014-11-06, accepted in 2015-01-16,  发布年份 2015

【 摘 要 】

Background

Legumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca²⁺ concentration, which is a fundamental prerequisite for any Ca²⁺-based signalling system, is accomplished by complex mechanisms including Ca²⁺ binding proteins acting as Ca²⁺ buffers. In this work we investigated the occurrence of Ca²⁺ binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus.

Results

A soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca²⁺-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca²⁺-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca²⁺ binding ability of the M. loti protein was demonstrated by ⁴⁵Ca²⁺-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca²⁺ adducts.

Conclusions

The present data indicate that ferredoxin II is a major Ca²⁺ binding protein in M. loti that may participate in Ca²⁺ homeostasis and suggest an evolutionarily ancient origin for protein-based Ca²⁺ regulatory systems.

【 授权许可】

   
2015 Moscatiello et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Masson-Boivin C, Giraud E, Perret X, Batut J: Establishing nitrogen-fixing symbiosis with legumes: how many rhizobium recipes? Trends Microbiol 2009, 17:458-466.
• [2]Udvardi M, Poole PS: Transport and metabolism in legume-rhizobia symbioses. Annu Rev Plant Biol 2013, 64:781-805.
• [3]Kaneko T, Nakamura Y, Sato S, Asamizu E, Kato T, Sasamoto S, et al.: Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. DNA Res 2000, 7:331-338.
• [4]Sato S, Nakamura Y, Kaneko T, Asamizu E, Kato T, Nakao M, et al.: Genome structure of the legume, Lotus japonicus. DNA Res 2008, 15:227-239.
• [5]Saeki K, Kouchi H: The Lotus symbiont, Mesorhizobium loti: molecular genetic techniques and application. J Plant Res 2000, 113:457-465.
• [6]Oldroyd GE: Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol 2013, 11:252-263.
• [7]Moscatiello R, Alberghini S, Squartini A, Mariani P, Navazio L: Evidence for calcium-mediated perception of plant symbiotic signals in aequorin-expressing Mesorhizobium loti. BMC Microbiol 2009, 9:206. BioMed Central Full Text
• [8]Moscatiello R, Squartini A, Mariani P, Navazio L: Flavonoid-induced calcium signalling in Rhizobium leguminosarum bv. viciae. New Phytol 2010, 188:814-823.
• [9]Moscatiello R, Baldan B, Squartini A, Mariani P, Navazio L: Oligogalacturonides: novel signaling molecules in rhizobium-legume communications. Mol Plant Microbe Interact 2012, 25:1387-1395.
• [10]Arrigoni G, Tolin S, Moscatiello R, Masi A, Navazio L, Squartini A: Calcium-dependent regulation of genes for plant nodulation in Rhizobium leguminosarum detected by iTRAQ quantitative proteomic analysis. J Proteome Res 2013, 12:5323-5330.
• [11]Clapham DE: Calcium signaling. Cell 2007, 131:1047-1058.
• [12]Case RM, Eisner D, Gurney A, Jones O, Muallem S, Verkhatski A: Evolution of calcium homeostasis: from birth of the first cell to an omnipresent signalling system. Cell Calcium 2007, 42:345-350.
• [13]Berridge MJ, Lipp P, Bootman MD: The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 2000, 1:11-21.
• [14]Prins D, Michalak M: Organellar calcium buffers. Cold Spring Harb Perspect Biol 2011, 3:a004069.
• [15]Zhao Y, Yunming S, Zhao W, Huang X, Wang D, Brown N, et al.: CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation. Proc Natl Acad Sci U S A 2005, 102:5744-5748.
• [16]Shi Y, Zhao W, Zhang W, Ye Z, Zhao J: Regulation of intracellular free calcium concentration during heterocyst differentiation by HetR and NtcA in Anabaena sp. PCC 7120. Proc Natl Acad Sci U S A 2006, 103:11334-11339.
• [17]Hu Y, Zhang X, Shi Y, Zhou Y, Zhang W, Su X-D, et al.: Structures of Anabaena calcium-binding protein CcbP: insights into Ca2+ signaling during heterocyst differentiation. J Biol Chem 2011, 286:12381-12388.
• [18]Slupski JR, Ohnishi M, Carpenter MR, Reithmeier RA: Characterization of cardiac calsequestrin. Biochemistry 1987, 26:6539-6544.
• [19]Damiani E, Heilmann C, Salvatori S, Margreth A: Characterization of high-capacity low-affinity calcium binding protein of liver endoplasmic reticulum: calsequestrin-like and divergent properties. Biochem Biophys Res Commun 1989, 165:973-980.
• [20]Navazio L, Baldan B, Dainese P, James P, Damiani E, Margreth A, et al.: Evidence that spinach leaves express calreticulin but not calsequestrin. Plant Physiol 1995, 109:983-990.
• [21]Campbell KP, MacLennan DH, Jorgensen AO: Staining of the Ca2+-binding proteins, calsequestrin, calmodulin, troponin C, and S-100, with the carbocyanine dye “Stains-all”. J Biol Chem 1983, 258:11267-11273.
• [22]Green MR, Pastewka JV, Peacock AC: Differential staining of phosphoproteins on polyacrylamide gels with a cationic carbocyanine dye. Anal Biochem 1973, 56:43-51.
• [23]Green MR, Pastewka JV: Identification of sialic acid-rich glycoproteins on polyacrylamide gels. Anal Biochem 1975, 65:66-72.
• [24]Damiani E, Margreth A: Subcellular fractionation to junctional sarcoplasmic reticulum and biochemical characterization of 170 kDa Ca2+- and low-density-lipoprotein-binding protein in rabbit skeletal muscle. Biochem J 1991, 277:825-832.
• [25]Milner RE, Baksh S, Shemanko C, Carpenter MR, Smillie L, Vance JA, et al.: Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum. J Biol Chem 1991, 266:7155-7165.
• [26]Sigrist CJA, Cerutti L, Hulo N, Gattiker A, Falquet L, Pagni M, et al.: PROSITE: a documented database using patterns and profiles as motif descriptors. Brief Bioinform 2002, 3:265-274.
• [27]Rocha AG, Vothknecht UC: Identification of CP12 as a novel calcium-binding protein in chloroplasts. Plants 2013, 2:530-540.
• [28]Dodd AN, Kudla J, Sanders D: The language of calcium signaling. Annu Rev Plant Biol 2010, 61:593-620.
• [29]Cai X, Clapham DE: Ancestral Ca2+ signaling machinery in early animal and fungal evolution. Mol Biol Evol 2012, 29:91-100.
• [30]Michiels J, Xi C, Verhaert J, Vanderleyden J: The functions of Ca2+ in bacteria: a role for EF-hand proteins? Trends Microbiol 2002, 10:87-93.
• [31]Dominguez DC: Calcium signalling in bacteria. Mol Microbiol 2004, 54:291-297.
• [32]Dominguez DC, Guragain M, Patrauchan M. Calcium binding proteins and calcium signaling in prokaryotes. Cell Calcium, doi:10.1016/j.ceca.2014.12.006, in press.
• [33]Oldroyd GE, Murray JD, Poole PS, Downie JA: The rules of engagement in the legume-rhizobial symbiosis. Ann Rev Genet 2011, 45:119-144.
• [34]Granqvist E, Wysham D, Hazledine S, Kozlowski W, Sun J, Charpentier M, et al.: Buffering capacity explains signal variation in symbiotic calcium oscillations. Plant Physiol 2012, 160:2300-2310.
• [35]Tatsukami Y, Nambu M, Morisaka H, Kuroda K, Ueda M: Disclosure of the differences of Mesorhizobium loti under the free-living and symbiotic conditions by comparative proteome analysis without bacteroid isolation. BMC Microbiol 2013, 13:180. BioMed Central Full Text
• [36]Uchiumi T, Ohwada T, Itakura M, Mitsui H, Nukui N, Dawadi P, et al.: Expression islands clustered on the symbiosis island of the Mesorhizobium loti genome. J Bacteriol 2004, 186:2439-2448.
• [37]Karunakaran R, Ramachandran VK, Seaman JC, East AK, Mouhsine B, Mauchline TH, et al.: Transcriptomic analysis of Rhizobium leguminosarum biovar viciae in symbiosis with host plants Pisum sativum and Vicia cracca. J Bacteriol 2009, 191:4002-4014.
• [38]Young JP, Downer HL, Eardly BD: Phylogeny of the phototrophic rhizobium strain BTAi1 by polymerase chain reaction-based sequencing of a 16S rRNA gene segment. J Bacteriol 1991, 173:2271-2277.
• [39]Surek B, Kreimer G, Melkonian M, Latzko E: Spinach ferredoxin is a calcium-binding protein. Planta 1987, 171:565-568.
• [40]Dazzo FB: Leguminous root nodules. In Experimental Microbial Ecology. Edited by Burns R, Slater J. Oxford: Blackwell Scientific Publications; 1982:431-446.
• [41]Curioni A, Dal Belin Peruffo A, Furegon L: Electroendosmotic preparative electrophoresis: a one-step method for the purification of nearly all the proteins from complex mixtures. Basic Appl Myol 1993, 3:239-244.
• [42]Damiani E, Volpe P, Margreth A: Coexpression of two isoforms of calsequestrin in rabbit slow-twitch muscle. J Muscle Res Cell Motil 1990, 11:522-530.
• [43]Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685.
• [44]Maruyama K, Mikawa T, Ebashi S: Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J Biochem 1984, 95:511-519.
• [45]Wilkins MR, Lindskog I, Gasteiger E, Bairoch A, Sanchez JC, Hochstrasser DF, et al.: Detailed peptide characterisation using PEPTIDEMASS - a World-Wide-Web-accessible tool. Electrophoresis 1997, 18:403-408.