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Type Four Pili (T4P) are extracellular appendages mediating several bacterial functions such as motility, biofilm formation and infection. The ability to adhere to substrates is essential for all these functions. In Myxococcus xanthus, during twitching motility, the binding of polar T4P to exopolysaccharides (EPS), induces pilus retraction and the forward cell movement. EPS are produced, secreted and weakly associated to the M. xanthus cell surface or deposited on the substrate. In this study, a genetic screen allowed us to identify two factors involved in EPS-independent T4P-dependent twitching motility: the PilY1.1 protein and the HsfBA phosphorelay. Transcriptomic analyses show that HsfBA differentially regulates the expression of PilY1 proteins and that the down-regulation of pilY1.1 together with the accumulation of its homologue pilY1.3, allows twitching motility in the absence of EPS. The genetic and bioinformatic dissection of the PilY1.1 domains shows that PilY1.1 might be a bi-functional protein with a role in priming T4P extension mediated by its conserved N-terminal domain and roles in EPS-dependent motility mediated by an N-terminal DUF4114 domain activated upon binding to Ca2+. We speculate that the differential transcriptional regulation of PilY1 homologs by HsfBA in response to unknown signals, might allow accessorizing T4P tips with different modules allowing twitching motility in the presence of alternative substrates and environmental conditions. Author summary: In the motile bacterium Myxococcus xanthus, T4P mediate twitching motility by binding to the sugar moiety of the extracellular matrix deposited on the neighboring cells or on the substrate. The binding of T4P to these sugars also termed exopolysaccharides (EPS) stimulates the pilus fiber retraction pulling the cell forwards. In this study, we performed a series of genetic analyses leading to the discovery that M. xanthus cells can move in the absence of EPS if two conditions are fulfilled: the pilY1.1 gene is down-regulated and the PilY1.3 protein is accumulated on pili. RNAseq, qRT-PCR and gel retardation assays show that the differential accumulation of PilY1 proteins is under the control of the HsfBA phosphorelay, which up-regulates the expression of pilY1.1 and down-regulates that of a homologue, pilY1.3. We also found that PilY1.1 has a domain at the N terminus probably requiring Ca2+ to be active in EPS-dependent motility, and a conserved domain at the C terminus essential for T4P assembly. Conversely, PilY1.3 contains a Von Willebrand factor A (VWA) domain and is potentially involved in the binding to proteins. We speculate that thanks to the HsfBA regulation, T4P can be equipped with different PilY1 homologues to allow twitching motility in the presence of different substrates. [ABSTRACT FROM AUTHOR]