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Detecting Posttranslational Modifications of Bacterial SSB Proteins

Post-translational modifications of single-stranded DNA-binding proteins (SSBs) have been reported from eukaryotic to prokaryotic systems. While eukaryotic SSBs are regulated by phosphorylation on serine and threonine residues, bacterial SSB proteins are also phosphorylated on a tyrosine residue. This was initially observed during systematic search for global phosphotyrosine-containing proteins in Streptomyces, complex lifecycle bacteria, involving mycelial growth and spore formation. Tyrosine phosphorylation was further confirmed on SSB proteins from spore forming bacterium Bacillus subtilis and in simpler prokaryote, Escherichia coli. However, thorough study of this modification and its cognate kinase was performed only on SSB proteins from Bacillus subtilis. It was shown that phosphorylation of B. subtilis SSB significantly increased binding to single-stranded DNA in vitro. Mass spectrometry analysis of SSB from B. subtilis identified Tyr82 as the phosphorylation site. Analyses of the resolved and predicted crystal structures of SSB proteins from B. subtilis, E. coli and S. coelicolor revealed that identified phosphorylation site occupies similar positions. Our results indicate that tyrosine phosphorylation of bacterial SSBs is a conserved modification in taxonomically distant bacteria

detection of phosphorylated SSBs ; purification of His tagged SSB proteins. western blotting, identification of phosphorylation sites by mass spectrometry

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