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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.
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 . Despite the significance of natural transformation systems of thermophiles, information about transformation systems of thermophiles and extreme thermophiles is very scarce.
To get insights into the transformation systems of thermophilic bacteria, we chose T. thermophilus HB27, which exhibits the highest transformation frequencies among the Thermus strains, as a model strain . On the basis of the complete genome sequence of T. thermophilus HB27, we have identified by directed gene disruption seven distinct competence gene loci [6–8]. Sequence analyses revealed that several of the deduced proteins are similar to proteins of the type IV pili and type II secretion machineries. PilA1, PilA2, PilA3 and PilA4 are similar to the precursors of the structural subunits of type IV pili, the prepilins, PilD exhibits similarities to the prepilin-processing prepilin peptidases, and PilQ is similar to members of the secretin family, which is a large family whose members form multimeric pores in the outer membranes of Gram-negative bacteria [9–12]. These similarities, together with the finding that transformation-defective pilA4, pilD and pilQ mutants, respectively, are devoid of pilus structures, suggest a functional link between pili and natural transformation in T. thermophilus HB27, although the functions of the type IV pili-related competence proteins in the process of DNA uptake are still unknown.
The pilMNOWQ competence genes are located in a competence locus comprising five tandemly arranged analogously orientated genes, pilM, pilN, pilO, pilW, and pilQ. Mutant studies with T. thermophilus HB27 mutants, carrying marker insertions in pilM, pilN, pilO, pilW, and pilQ, respectively, revealed that the pilMNOWQ cluster is essential for natural transformation and piliation. Owing to the head-to-tail organization of the genes, potential polar effects of marker insertions on downstream-located genes of the pil cluster could not be excluded, and therefore the question of whether the products of pilM, pilN, pilO and pilW each play a role in natural transformation and piliation is still open.
Here, we present the identification of the competence proteins PilM, PilN, PilO, PilW, PilQ and PilA4 in T. thermophilus HB27; the last of these was found to undergo glycosylation. We show that the individual proteins of the pilMNOWQ competence cluster are each essential for natural transformation of T. thermophilus HB27. Furthermore, we present the first information on the subcellular localization of the PilMNOWQ and PilA4 competence proteins and on the effect of mutations in distinct competence proteins on the subcellular localization of other proteins. Taken together, the data presented here provide the first insights into the function of the competence proteins PilM, PilN, PilO, PilW, PilQ and PilA4 in the DNA translocator of T. thermophilus HB27.
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We recently reported on the identification and characterization of seven distinct competence gene loci in the genome of T. thermophilus HB27 comprising a total of 16 potential genes of the DNA translocator [6–8]. However, so far, none of the competence proteins has been detected or analyzed in T. thermophilus HB27, and nothing is known with respect to their function in DNA translocation.
Therefore, in the first part of this study we produced fragments of the PilM, PilN, PilO, PilW, PilQ and PilA4 competence proteins and raised antisera against these proteins to visualize the proteins in the T. thermophilus wild-type strain. This is the first report on the detection of competence proteins in T. thermophilus HB27.
An interesting finding was that PilA4 protein undergoes glycosylation. This is a trait of many pili proteins and has also been detected in pilin-like proteins of DNA transformation systems [18–20]. The detection of an 18 kDa PilA4 after TFMS treatment suggests that PilA4 may undergo a further modification. This has been shown for the meningococcal pilin; it contains an α-glycerophosphate substituent attached to Ser93 by a phosphodiester linkage . It has been suggested that glycerol residues might serve as a substrate for fatty acylation and, thereby, be involved in membrane anchoring of the pilin. Since PilA4 is similar to the meningococcal pilin and contains several central serine residues, it is tempting to speculate that PilA4 might also contain an α-glycerophosphate substituent. Taken together, the studies clearly show that the 23 kDa PilA4 protein undergoes glycosylation and that the glycosylated PilA4 protein is active in the DNA translocator.
Where are the PilM, PilN, PilO, PilW, PilQ and PilA4 competence proteins located in the cell and what could be their function? Several of the selected proteins contain only a few or no hydrophobic segments, and therefore their subcellular localization was not obvious. Here, we show that PilM is exclusively located in the inner membrane. PilM contains a conserved C-terminal ATPase domain of actin-like ATPases, such as FtsA and MreB, which are involved in cell division and cell morphogenesis (for reviews, see  and ). FtsA, the only septum protein without a membrane anchor, is required in bacteria for the assembly and stabilization of Z-rings comprising tubulin-like FtsZ filaments , whereas MreB has been shown to perform dynamic motor-like movements extending along helical tracks . Owing to the similarities of PilM with members of the actin family, together with the inner membrane localization of PilM, it is tempting to speculate that PilM might represent a dynamic motor protein involved in the assembly of the DNA translocator complex in the inner membrane. The Thermus competence proteins PilN and PilO show very weak similarities to PilN and PilO proteins of unknown function in type IV pili of Gram-negative bacteria. Like PilO and PilN of Pseudomonas aeruginosa and Neisseria gonorrhoeae[25–27], the T. thermophilus PilO and PilN proteins each have a hydrophobic N-terminal domain which may act as an inner or outer membrane anchor. This is in accordance with their localization in the inner membrane. PilN and PilO may mediate recruitment and assembly of DNA translocator proteins at the inner membrane.
We found that the nonconserved PilW is distributed equally between the inner and outer membranes. PilW is likely to form integral parts of a transmembrane DNA translocator structure and it may interact via its hydrophobic N-terminus with other proteins in the membranes such as PilQ and PilA4. In addition, its extended hydrophilic C-terminus may interact with other DNA translocator proteins in the periplasm. Consistent with this suggestion is our finding that a pilW mutation results in the absence of PilA4 and PilQ from the outer membrane. Taken together, our results indicate that PilW may interact with PilQ and PilA4 in the outer membrane and that this interaction is required for biogenesis of the DNA translocator and/or is involved in the stabilization of PilQ and PilA4 proteins in the outer membrane. Moreover, the absence of any PilW-like proteins in the transformation machineries of mesophilic bacteria, together with the effect of a pilW mutation on the biogenesis and/or stability of PilA4 and PilQ in the outer membrane, indicate that PilW is a special feature of the transformation machinery in T. thermophilus that is probably essential for the adaptation of the DNA translocator to high temperature.
The secretin-like PilQ was detected in sufficient amounts in the inner and outer membranes. The presence of PilQ in inner membranes is interesting, because secretin-like proteins of type IV pili and type II protein translocation machineries are known to form ring-like structures in outer membranes. The presence of PilQ in T. thermophilus inner and outer membranes suggests that the secretin-like PilQ protein is accumulated and may be assembled into ring-like structures at the inner membrane prior to transport through the periplasm to the outer membrane. The secretin-like PilQ protein of T. thermophilus has a conserved C-terminal part, very similar to the C-termini of other members of the secretion family, such as PilQ of Myxococcus xanthus, ExeD of Aeromonas salmonicida, PilQ of P. aeruginosa[25,30], and PilQ of N. gonorrhoeae. This C-terminal stretch has been shown to be required for multimer formation of the corresponding PulD of Klebsiella and PilQ of N. gonorrhoeae[31,32]. Taken together, the conserved C-terminus of PilQ and its outer membrane localization are in agreement with our suggestion that Thermus secretin-like PilQ monomers may form a multimeric ring-like structure acting in the translocation of DNA through the outer membrane or functioning as a scaffold for the DNA translocator spanning the outer membrane. However, it has to be noted that the N-terminus of T. thermophilus PilQ does not exhibit any similarities to conserved N-terminal domains of secretins that are proposed to mediate interaction with other proteins not related to type II secretion or type IV pili biogenesis pathways. Owing to the nonconserved N-terminal domain of PilQ, and the colocalization of PilQ with PilW in inner and outer membranes and the results from the pilW and pilQ mutant studies, it is tempting to speculate that the nonconserved PilW protein is implicated in the assembly and stability of PilQ multimers at the inner membrane and transport of these subassemblies to the outer membrane.
The presence of the pilin-like PilA4 protein in the inner and outer membranes suggests that PilA4 may represent a structural subunit of a DNA translocator anchored in the inner membrane and extending through the periplasm and the outer membrane. The finding that a PilQ mutant no longer has PilA4 in the outer membrane is in support of a PilQ-comprising scaffold in the outer membrane guiding the PilA4-consisting translocator through the outer membrane.
The copurification of PilA4 and PilQ with the pilus structures indicates that both are structural components of the pilus. Moreover, it is tempting to speculate that PilQ might form the globular structure at the pilus base, since it corresponds in diameter with the PilQ complex of N. gonorrhoeae (15.5–16.5 nm) , P. aeruginosa (18.3 nm ± 1.2 nm)  or N. meningitidis (15.5 nm) . In contrast, PilM, PilN and PilO are essential for transformation and piliation but do not copurify with the pili, indicating that they may contribute to the biogenesis of the pilus, the stability of pilus structures, and/or inner membrane association of the pilus. PilW, which we found to be nearly equally distributed between inner and outer membranes but not in the purified pilus fraction, may be involved in inner and outer membrane associations of pilus proteins and/or stability of the pilus structure.
On the basis of our current knowledge, we propose a model for the DNA translocation process in T. thermophilus HB27 (Fig. 7).
Figure 7. Model for DNA uptake in Thermus thermophilus HB27. DNA is bound to a so far unknown DNA-binding protein close to the potential ring-like structure of secretin-like PilQ proteins in the outermost layer, which comprises S-layer and lipids and does not represent a classic outer membrane. The DNA is transported through the ring-like structure, the periplasmic space and peptidoglycan by a DNA translocator comprising pilin-like (PilA4) proteins. PilW is an inner and outer membrane protein that may be essential for assembly, stabilization and piloting of the PilQ/PilA4-comprising DNA translocator complex, spanning the outer membrane and periplasmic space, whereas PilM, PilN and PilO are inner membrane proteins that probably form part of the assembly platform and are involved in the assembly of the DNA translocator complex in the inner membrane. The potential traffic NTPase PilF is essential for transformation and may be implicated in retraction of the PilA4-comprising DNA translocator transporting the DNA through the periplasmic space. Binding of the DNA to the DNA-binding protein ComEA on the surface of the inner membrane may be a prerequisite for DNA translocation across the inner membrane, which could be performed through a ComEC-comprising channel. dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.
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Current studies are underway to answer the question of whether the pilus structures themselves are implicated in DNA translocation. Future work will purify different subassemblies of the DNA transporter in T. thermophilus, and develop assays for its functional units.