Development of method for preliminary identification of cyclic dinucleotides in bacterial cultures

Abstract

Cyclic dinucleotides are perspective second messengers in Bacteria. Known compounds of the class play various roles in many processes in bacteria from cell cycle to pathogenesis. In vivo compounds are synthesized with cyclic dinucleotide cyclases and degrade with specific phosphodiesterases. Many of discovered proteins of these classes do not interact with known cyclic dinucleotide compounds, which suggest another compounds of the class may be also in the bacterial cell. In this work a method was developed , based on liquid chromatography coupled mass spectrometry with several columns, which is capable to distinguish possible compound of interest in the bacterial extract based on the chemically synthesized compounds of the class. Method is shown to have LOD lower, than in any method proposed before for single cyclic dinucleotide compound. As a proof of principle, several bacteria reported to produce cyclic diguanylate or cyclic diadenylate were tested. In addition, it was demonstrated that levels of some of cyclic dinucleotides are dependent on growing phase of bacteria.