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DNA translocators of natural transformation systems are complex systems critical for the uptake of free DNA and provide a powerful mechanism for adaptation to changing environmental conditions. In natural transformation machineries, outer membrane secretins are suggested to form a multimeric pore for the uptake of external DNA. Recently, we reported on a novel structure of the DNA translocator secretin complex, PilQ, in Thermus thermophilus HB27 comprising a stable cone and cup structure and six ring structures with a large central channel. Here, we report on structural and functional analyses of a set of N-terminal PilQ deletion derivatives in T. thermophilus HB27. We identified 136 N-terminal residues exhibiting an unusual ααβαββα fold as a ring-building domain. Deletion of this domain had a dramatic effect on twitching motility, adhesion, and piliation but did not abolish natural transformation. These findings provide clear evidence that the pilus structures of T. thermophilus are not essential for natural transformation. The truncated complex was not affected in inner and outer membrane association, indicating that the 136 N-terminal residues are not essential for membrane targeting. Analyses of complex formation of the truncated PilQ monomers revealed that the region downstream of residue 136 is required for multimerization, and the region downstream of residue 207 is essential for monomer stability. Possible implications of our findings for the mechanism of DNA uptake are discussed.
Secretins form multimeric channels across the outer membrane of Gram-negative bacteria that mediate the import or export of substrates and/or extrusion of type IV pili. The secretin complex of Thermus thermophilus is an oligomer of the 757-residue PilQ protein, essential for DNA uptake and pilus extrusion. Here, we present the cryo-EM structure of this bifunctional complex at a resolution of ~7 Å using a new reconstruction protocol. Thirteen protomers form a large periplasmic domain of six stacked rings and a secretin domain in the outer membrane. A homology model of the PilQ protein was fitted into the cryo-EM map. A crown-like structure outside the outer membrane capping the secretin was found not to be part of PilQ. Mutations in the secretin domain disrupted the crown and abolished DNA uptake, suggesting a central role of the crown in natural transformation.