PilB is the assembly ATPase for the bacterial type IV pilus (T4P), and as a consequence, it is essential for T4P-mediated bacterial motility. In some cases, PilB has been demonstrated to regulate the production of exopolysaccharide (EPS) during bacterial biofilm development independently of or in addition to its function in pilus assembly. While the ATPase activity of PilB resides at its C-terminal region, the N terminus of a subset of PilBs forms a novel cyclic-di-GMP (cdG)-binding domain. This multi-domain structure suggests that PilB binds cdG and adenine nucleotides through separate domains which may influence the functionality of PilB in both motility and biofilm development. Here, Chloracidobacterium thermophilum PilB is used to investigate ligand binding by its separate domains and by the full-length protein. Our results confirm the specificity of these individual domains for their respective ligands and demonstrate communications between these domains in the full-length protein. It is clear that when the N- and the C-terminal domains of PilB bind to cdG and ADP, respectively, they mutually influence each other in conformation and in their binding to ligands. We propose that the interactions between these domains in response to their ligands play critical roles in modulating or controlling the functions of PilB as a regulator of EPS production and as the T4P assembly ATPase.
The bacterial type IV pilus (T4P) is a versatile nanomachine that functions in pathogenesis, biofilm formation, motility, and horizontal gene transfer. T4P assembly is powered by the motor ATPase PilB which is proposed to hydrolyze ATP by a symmetrical rotary mechanism. This mechanism, which is deduced from the structure of PilB, is untested. Here, we report the first kinetic studies of the PilB ATPase, supporting co-ordination among the protomers of this hexameric enzyme. Analysis of the genome sequence of Chloracidobacterium thermophilum identified a pilB gene whose protein we then heterologously expressed. This PilB formed a hexamer in solution and exhibited highly robust ATPase activity. It displays complex steady-state kinetics with an incline followed by a decline over an ATP concentration range of physiological relevance. The incline is multiphasic and the decline signifies substrate inhibition. These observations suggest that variations in intracellular ATP concentrations may regulate T4P assembly and T4P-mediated functions in vivo in accordance with the physiological state of bacteria with unanticipated complexity. We also identified a mutant pilB gene in the genomic DNA of C. thermophilum from an enrichment culture. The mutant PilB variant, which is significantly less active, exhibited similar inhibition of its ATPase activity by high concentrations of ATP. Our findings here with the PilB ATPase from C. thermophilum provide the first line of biochemical evidence for the co-ordination among PilB protomers consistent with the symmetrical rotary model of catalysis based on structural studies.