Identification, subcellular localization and functional interactions of PilMNOWQ and PilA4 involved in transformation competency and pilus biogenesis in the thermophilic bacterium Thermus thermophilus HB27

Abstract

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.

Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis

“…We then examined whether these inner membrane complexes were able to bind to the major type IV pilin from T. thermophilus, TtPilA4 (14). To generate a form of TtPilA4 that would be soluble and tractable to binding studies, we engineered an expression construct that had 36 residues removed from the N terminus of the mature protein (designated TtPilA4 ).…”

“…The consequence would be that, when a major pilin binds to this assembly, pilins from adjacent PilMNO complexes are brought into close spatial proximity, where they could associate and thus be immediately available for incorporation into a pilus fiber. We should note that there is no equivalent of the PilP lipoprotein in T. thermophilus, although there is a separate protein, PilW, which has no orthologs in proteobacteria but may carry out a similar function (14).…”

“…Here, we report a structural investigation of the PilMNO complex from T. thermophilus (denoted as TtPilMNO; we will use the prefix "Tt" to identify the pilus biogenesis proteins from T. thermophilus). The T4P biogenesis machinery has been well characterized in T. thermophilus, providing an excellent basis for structural investigations (14)(15)(16). We show that the complex can be assembled in an incremental manner, starting with association of TtPilM with TtPilN, then TtPilO, and, finally, the major pilin (TtPilA4).…”

“…TTHA1221 in the T. thermophilus HB8 genome sequence was identified as having the highest sequence similarity to PilA4 from T. thermophilus HB27, which is the major pilin protein in that strain (14). TTHA1221 was therefore designated as TtPilA4, to ensure consistency.…”

“…Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 4BHR (TtPilA ) and 4BHQ (TtPilN Experiments conducted in Thermus thermophilus have shown that pilM, pilN, or pilO mutants are all nonpiliated and exhibit phenotypes characteristic of the absence of T4P (14,15). Given their location in the inner membrane, it is reasonable to propose that they form a platform for T4P formation, but the details whereby they might contribute to this process are unclear.…”