Lysine methyltransferase Sll0171 modulates motility through PilA1 methylation in <i>Synechocystis</i> sp. PCC 6803

Synechocystis sp. PCC 6803 (Synechocystis), a unicellular and motile cyanobacterium, exhibits directed motility toward or away from light stimuli through type IV pili (T4P)-mediated locomotion. However, the regulatory mechanisms governing T4P-dependent motility in Synechocystis remain incompletely understood. In this study, we demonstrated that Sll0171 can catalyze lysine methylation both in vivo and in vitro. Notably, the loss of sll0171 resulted in enhanced motility, accompanied by impaired photosynthetic capacity. We identified 65 endogenous methylation sites targeted by Sll0171 in Synechocystis via label-free quantitative proteomic analysis. Subsequent functional characterization revealed that Sll0171 specifically methylates lysine 168 (K168) of PilA1, the primary structural subunit of T4P. To elucidate the role of PilA1 methylation, we employed site-directed mutagenesis to generate mutants mimicking constitutively methylated (pilA1K168Q) or nonmethylated (pilA1K168R) states. Compared to wild-type Synechocystis, the pilA1K168R mutant displayed markedly more T4P and enhanced motility, whereas the pilA1K168Q mutant exhibited near-complete inhibition of T4P and motility. Collectively, these findings identify Sll0171 as a previously uncharacterized lysine methyltransferase in Synechocystis and reveal a methylation-dependent regulatory mechanism governing T4P and motility in cyanobacteria. This work advances our understanding of post-translational modifications in modulating microbial motility and environmental adaptation.