Unusual N-terminal ααßαßßα fold of PilQ from Thermus thermophilus mediates ring formation and is essential for piliation.

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 ααßαßßα 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.

Structure and function of PilQ, a secretin of the DNA transporter from the thermophilic bacterium Thermus thermophilus HB27.

Secretins are a family of large bacterial outer membrane protein complexes mediating the transport of complex structures, such as type IV pili, DNA and filamentous phage, or various proteins, such as extracellular enzymes and pathogenicity determinants. PilQ of the thermophilic bacterium Thermus thermophilus HB27 is a member of the secretin family required for natural transformation. Here we report the isolation, structural, and functional analyses of a unique PilQ from T. thermophilus. Native PAGE, gel filtration chromatography, and electrophoretic mobility shift analyses indicated that PilQ forms a macromolecular homopolymeric complex that binds dsDNA. Electron microscopy showed that the PilQ complex is 15 nm wide and 34 nm long and consists of an extraordinary stable "cone" and "cup" structure and five ring structures with a large central channel. Moreover, the electron microscopic images together with secondary structure analyses combined with structural data of type II protein secretion system and type III protein secretion system secretins suggest that the individual rings are formed by conserved domains of alternating α-helices and ß-sheets. The unprecedented length of the PilQ complex correlated well with the distance between the inner and outer membrane of T. thermophilus. Indeed, PilQ was found immunologically in both membranes, indicating that the PilQ complex spans the entire cell periphery of T. thermophilus. This is consistent with the hypothesis that PilQ accommodates a PilA4 comprising pseudopilus mediating DNA transport across the outer membrane and periplasmic space in a single-step process.

Synthesis of glycine betaine from choline in the moderate halophile Halobacillus halophilus: co-regulation of two divergent, polycistronic operons.

The moderately halophilic bacterium Halobacillus halophilus can synthesize glycine betaine from choline. Oxidation of choline is induced by salinity and repressed by exogenous glycine betaine. The genes encoding the choline dehydrogenase (gbsB) and the glycine betaine aldehyde dehydrogenase (gbsA) were identified and shown to constitute an operon. Divergent to this operon is another operon containing gbsR and gbsU that encode proteins with similarities to a transcriptional regulator and a glycine betaine-binding protein respectively. Synthesis of the four Gbs proteins was strictly dependent on the choline concentration of the medium. Salinity was essential for the production of GbsB and increased the production of GbsA, GbsR and GbsU. Glycine betaine repressed the production of all four Gbs proteins with half maximal inhibition at 0.1 mM glycine betaine.