Abstract

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.

Highlights

  • Secretins are key components of complex DNA and protein transport machineries

  • N-terminal Ring of PilQ Is Not Essential for Inner Membrane (IM) Association of PilQ Complex—Recently, we have shown that the wild type PilQ complex was mainly associated with the outer membrane but was detectable in IM fractions [16]

  • Secretins comprise a family of large, homomultimeric, and dynamic protein channels that are localized in the outer membranes of Gram-negative bacteria and are essential for transport of macromolecular structures, such as proteins or DNA, across the outer membranes via diverse multiprotein systems (28 –34)

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Summary

Background

Secretins are key components of complex DNA and protein transport machineries. Results: An unusual secretin ␣␣␤␣␤␤␣ fold was identified as a ring-building motif essential for piliation but not for transformation. 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. We identified 136 N-terminal residues exhibiting an unusual ␣␣␤␣␤␤␣ 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. To get insights into the structure and function of natural transformation systems, we chose the transformable thermophilic Thermus thermophilus HB27 as a model bacterium for molecular analyses of the DNA translocator. These studies led to a tentative model of the DNA translocation process.

The abbreviations used are
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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