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B. subtilis Elongation Factor Tu binds Calcium ions
Author(s) -
Dominguez Delfina C,
Lopes Rosana,
Palomino Javier
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.lb211
Subject(s) - ef hand , calmodulin , bacillus subtilis , calcium , polyclonal antibodies , biochemistry , calcium binding protein , chemistry , microbiology and biotechnology , biology , antibody , genetics , bacteria , organic chemistry
ABSTRACT An essential step toward the understanding of the role of calcium in prokaryotes is the identification of intracellular calcium‐binding proteins. In this report we present evidence that Bacillus subtilis elongation factor EF‐Tu is a calcium‐binding protein. A genomic analysis of EF‐hand related sequences in the genus Arabidopsis showed that elongation factors have EF‐hand domains. Considering the high degree of conservation across species and based on previous findings in our laboratory suggesting EF‐Tu may be a CaBP (unpublished results) we investigated further the B. subtilis EF‐Tu. We performed multiple sequence alignment and motif discovery of bacterial EF‐Tu and D. discoideum calmodulin sequences and the results suggested the presence of a possible calcium binding site in B. subtilis EF‐Tu. Secondary structure prediction indicated that the conserved acidic amino acids were found within a loop flanked by two helices. A recombinant B. subtilis EF‐Tu was partially purified. The protein was identified by Western blot assay using anti‐EF‐Tu antibodies and two‐dimensional electrophoresis followed by liquid chromatography and mass spectrometry. Calcium binding was examined, in vivo and in vitro , by 45 Ca‐autoradiography and immunodetection using monoclonal anti‐calmodulin and polyclonal anti‐EF‐hand antibodies the combined results indicated the B. subtilis EF‐Tu bindis calcium ions in vivo and in vitro . Addition of calcium chelators EGTA/BAPTA or CaCl 2 to cell cultures affected expression of B . subtilis EF‐Tu.