In vivo detection of molybdate-binding proteins using a competition assay with ModE in Escherichia coli

• Published in: FEMS microbiology letters Vol. 218; no. 1; pp. 187 - 193
• Main Authors: Kuper, Jochen, Meyer zu Berstenhorst, Sonja, Vödisch, Bernd, Mendel, Ralf R., Schwarz, Günter, Boxer, David H.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 21.01.2003; Blackwell; Oxford University Press
• Subjects: Analytical, structural and metabolic biochemistry; Arabidopsis Proteins - genetics; Assaying; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Binding; Binding and carrier proteins; Binding, Competitive; Biological and medical sciences; Biosynthesis; Calnexin - genetics; Cloning, Molecular; Cnx1; Competition; Cytoplasm; E coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Gene regulation; In vivo methods and tests; Metabolism. Enzymes; Microbiological Techniques; Microbiology; Miller assay; ModE; ModE protein; Molybdate; Molybdate binding; Molybdenum; Molybdenum - metabolism; Molybdopterin; Plants (botany); Proteins; Trace elements; Transcription; Transcription Factors - genetics; Transcription Factors - metabolism; Miller assay; Cnx1; Molybdate binding; Molybdenum; ModE; Eucaryote; Escherichia coli; Method; Molybdates; Screening; Binding protein; Bacteria; Enterobacteriaceae
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.2003.tb11517.x

Abstract Molybdenum is an important trace element as it forms the essential part of the active site in all molybdenum-containing enzymes. We have designed an assay for the in vivo detection of molybdate binding to proteins in Escherichia coli. The assay is based on (i) the molybdate-dependent transcriptional regulation of the moa operon by the ModE protein, and (ii) the competition for molybdate between ModE and other molybdate-binding proteins in the cytoplasm of E. coli. We were able to verify in vivo molybdate binding to three different bacterial proteins that are known to bind molybdate. This sensitive in vivo system allows the testing of different proteins for molybdate binding under in vivo conditions and will facilitate the identification of other cellular factors needed for molybdate binding. As a first example, we examined the eukaryotic protein Cnx1 that is involved in the last step of molybdenum cofactor biosynthesis in plants, and show that it is able to compete with ModE for molybdate in a molybdopterin-dependent fashion.