Members of the extremely thermophilic genus Thermus belong to one of the oldest branches of bacterial evolution and, together with the genus Deinococcus, form a distinctive group within the Bacteria deserving the taxonomic status of a phylum [1,2]. Thermus representatives, such as Thermus thermophilus strain HB27, Thermus thermophilus HB8, Thermus flavus AT62, Thermus caldophilus, and Thermus aquaticus YT1, exhibit the extraordinary trait of high transformation competence [3,4]. The high transformation frequencies, together with the high thermotolerance, suggest a significant impact of the Thermus transformation system on DNA transfer in extreme environments and therefore on the evolution of life. This is supported by recent data from comparative genomics and phylogenetic analyses in the thermophilic bacterium T. thermophilus HB27. This strain seems to have acquired numerous genes from (hyper)thermophilic bacteria and archaea, suggesting that horizontal gene transfer was probably decisive in its thermophilic adaptation [5]. Despite the significance of natural transformation systems of thermophiles, information about transformation The natural transformation system of the thermophilic bacterium Thermus thermophilus HB27 comprises at least 16 distinct competence proteins encoded by seven distinct loci. In this article, we present for the first time biochemical analyses of the Thermus thermophilus competence proteins PilMNOWQ and PilA4, and demonstrate that the pilMNOWQ genes are each essential for natural transformation. We identified three different forms of PilA4, one with an apparent molecular mass of 14 kDa, which correlates with that of the deduced protein, an 18-kDa form and a 23-kDa form; the last was found to be glycosylated. We demonstrate that PilM, PilN and PilO are located in the inner membrane, whereas PilW, PilQ and PilA4 are located in the inner and outer membranes. These data show that PilMNOWQ and PilA4 are components of a DNA translocator structure that spans the inner and outer membranes. We further show that PilA4 and PilQ both copurify with pilus structures. Possible functions of PilQ and PilA4 in DNA translocation and in pilus biogenesis are discussed. Comparative mutant studies revealed that mutations in either pilW or pilQ significantly affect the location of the other protein in the outer membrane. Furthermore, no PilA4 was present in the outer membranes of these mutants. From these findings, we conclude that the abilities of PilW, PilQ and PilA4 to stably localize or accumulate in the outer membrane fraction are strongly dependent on one another, which is in accord with an outer membrane DNA translocator complex comprising PilW, PilQ, and PilA4.Abbreviations IPTG, isopropyl thio-b-D-galactoside; TFMS, trifluoromethanesulfonic acid; TM, Thermus medium.

Thermus thermophilus HB27 is well known for its extraordinary trait of high frequencies of natural transformation, which is considered a major mechanism of horizontal gene transfer. We show that the DNA translocator of T. thermophilus binds and transports DNA from members of all three domains. These results, together with the data obtained from genome comparisons, suggest that the DNA translocator of T. thermophilus has a major impact in adaptation of Thermus to thermal stress conditions and interdomain DNA transfer in extreme hot environments. DNA transport in T. thermophilus is mediated by a macromolecular transport machinery that consists of at least 16 subunits and spans the cytoplasmic membrane and the entire cell periphery. Here, we have addressed the role of single subunits in DNA binding and transport. PilQ is involved in DNA binding, ComEA, PilF and PilA4 are involved in transport of DNA through the outer membrane and PilM, PilN, PilO, PilA1-3, PilC and ComEC are essential for the transport of DNA through the thick cell wall layers and/or through the inner membrane. These data are discussed in the light of the subcellular localization of the proteins. A topological model for DNA transport across the cell wall is presented.

Horizontal gene transfer has been a major force for genome plasticity over evolutionary history, and is largely responsible for fitness‐enhancing traits, including antibiotic resistance and virulence factors. In particular, for adaptation of prokaryotes to extreme environments, lateral gene transfer seems to have played a crucial role. Recently, by performing a genome‐wide mutagenesis approach with Thermus thermophilus HB27, we identified the first genes in a thermophilic bacterium for the uptake of free DNA, a process called natural transformation. Here, we present the first data on the biochemistry and bioenergetics of the DNA transport process in this thermophile. We report that linear and circular plasmid DNA are equally well taken up with a high maximal velocity of 1.5 µg DNA·(mg protein)−1·min−1, demonstrating an extremely efficient binding and uptake rate of 40 kb·s−1·cell−1. Uncouplers and ATPase inhibitors immediately inhibited DNA uptake, providing clear evidence that DNA translocation in HB27 is an energy‐dependent process. DNA uptake studies with genomic DNA of Bacteria, Archaea and Eukarya revealed that Thermus thermophilus HB27 takes up DNA from members of all three domains of life. We propose that the extraordinary broad substrate specificity of the highly efficient Thermus thermophilus HB27 DNA uptake system may contribute significantly to thermoadaptation of Thermus thermophilus HB27 and to interdomain DNA transfer in hot environments.