Type IV pili (TFP) are flexible and versatile fibers on the surface of many gram-negative bacteria, primarily composed of multiple copies of protein subunits called major pilins. In addition to the cyanobacterial major pilin PilA1, Synechocystis sp. PCC 6803 encodes less abundant pilin-like proteins, known as minor pilins, which are crucial for pilus assembly and facilitate various functions, including natural competence, aggregation, adhesion, and motility.

Recent studies have highlighted the importance of different transcriptional units of minor pilins in distinct pilus functions. TU2300, which encodes PilA5 and PilA6, is essential for natural transformation but is dispensable for motility and flocculation. Similarly, we demonstrated that TU2301, which encodes PilA7 and PilA8, is crucial for natural transformation, but not for motility and flocculation. TU763, encoding PilA9-PilA12, is essential for motility and flocculation but dispensable for natural transformation. Investigations of the minor pilins PilX1, PilX2, and PilX3 indicate their importance for flocculation.

The absence of extracellular PilA1 in mutant strains lacking specific sets of minor pilins suggests that TFP assembly depends on the presence of either PilA5-8 or PilA9-12. We hypothesized that two different priming complexes are assembled, forming the tip of a pilus fiber with distinct functions. Here, we provide mass spectrometry (MS) data for sheared pili fractions and co-immunoprecipitation of specific minor pilins to elucidate the composition of these distinct pilus subtypes.

Furthermore, we visualized spatiotemporal dynamics of pilus subtypes by confocal microscopy employing a PilA1(T77C) cysteine mutant that can be stained with Alexa Fluor 488. The combination of this PilA1-variant with the deletion of minor pilin genes allowed us to detect phenotypic differences in cell surface piliation. Preliminary data showed long, less dynamic pilus fibers in pilA9-12 mutants, whereas pilA5-6 mutants exhibited short, dynamic fibers. The machine learning algorithm ILASTIK was used to automatically detect pili and analyze the pilus subtypes of knock-out mutants. We present a rapid and standardized method for analyzing the microscopic images of cells decorated with TFP.