Identification of trimethylation at C-terminal lysine of pilin in the cyanobacterium Synechocystis PCC 6803

Various post-translational modifications (PTMs) of pilin in Synechocystis sp. PCC 6803 have been proposed. In this study, we investigated previously unidentified PTMs of pilin by mass spectrometry (MS). MALDI-TOF MS and TOF/TOF MS showed that the molecular mass of the C-terminal lysine of pilin was increased by 42 Da, which could represent acetylation (ΔM = 42.0470) or trimethylation (ΔM = 42.0106). To discriminate between these isobaric modifications, the molecular mass of the C-terminal tryptic peptide was measured using 15T Fourier transform ion cyclotron resonance (FT-ICR) MS. The high magnetic field FT-ICR provided sub-ppm mass accuracy, revealing that the C-terminal lysine was modified by trimethylation. We could also detect the existence of mono- and di-methylation of the C-terminal lysine. Cells expressing a pilin point mutant with glutamine replacing the C-terminal lysine showed dramatically reduced motility and short pili. These findings suggest that trimethylation of pilin at the C-terminal lysine may be essential for the biogenesis of functional pili.

Research highlights
► Pilin in Synechocystis sp. PCC 6803 was analyzed by mass spectrometry.
► The C-terminal lysine of pilin is post-translationally modified by trimethylation.
► The pilA1K168Q mutant cells showed reduced gliding motility.
► Thick pili on the pilA1K168Q cell surface were few in number and very short.
► The C-terminal trimethylation of pilin may be required for biogenesis of thick pili.