Insights into assembly of the type IVa pilus machine in Myxococcus xanthus

Type IVa pili (T4aP) are widely distributed and highly versatile bacterial cell surface structures that function in motility, adhesion, biofilm formation and virulence. Key to their function is their ability to undergo extension/adhesion/retraction cycles powered by the cell envelope-spanning T4aP machine. Assembly of the T4aP machine in Myxococcus xanthus follows an outside-in parthway, starting with the incorporation of the PilQ secretin in the OM, which then recruits the periplasmic, inner membrane and cytoplasmic components. Additionally, a complex composed of four minor pilins and PilY1 primes T4aP extension and is also present at the pilus tip mediating adhesion. Here we focuse on the assembly of the bipolarly localized T4aP machine in the rod-shaped bacterium M. xanthus. The genome of M. xanthus encodes for three sets of minor pilins and PilY1. Here, we find that one of these minor pilin and PilY1 gene clusters includes a noncanonical cytochrome c, which we named TfcP. While TfcP has an unusually low redox potential that makes a function in respiration unlikely, it is conditionally essential for T4aP dependent motility by promoting the accumulation of PilY1.1 in the presence of low calcium concentrations. We suggest that TfcP extends the range of calcium concentration at which PilY1.1 can function, making its function more robust against environmental fluctuations. Next, we focused on the assembly of new T4aP machines in the new poles of cells after cell division. We demonstrate that PilQ starts to be recruited to the nascent poles during cytokinesis, but mostly is recruited to the new poles after completion of cytokinesis. This recruitment depends on the peptidoglycan-binding AMIN domains of PilQ and we propose that this mechanism is general for AMIN domain containing secretins. Additionally, PilQ transiently recruits the pilotin Tgl to the nascent and new poles, which then induces the multimerization of PilQ in the outer membrane. We suggest that the transient interaction between PilQ and Tgl is mediated by the unfolded β-lip of PilQ, the domain that is integrated into the outer membrane. In addition, we uncover that the diguanylate cyclase DmxA is important for the symmetric assembly of the T4aP machine at the new cell poles after cytokinesis. In the absence of DmxA, cells exhibit a misregulated cell polarity and a very heterogeneous polar localization of the T4aP machine, which leads to an increased reversal frequency. DmxA is recruited to the division site by components of the divisiome shortly before the completion of cytokinesis and rapidly elevates the cellular c-di-GMP level. We suggest that this burst of c-di-GMP regulates the symmetric incorporation of polar landmarks at the new cell poles in both daughter cells. Lastly, we establish a detailed protocol for the application of miniTurboID-based proximity labeling in M. xanthus. We apply this protocol to the polarity regulator MglA, and utilize proximity labeling to compare the conditional interactome of MglA.