Identification, subcellular localization and functional interactions of PilMNOWQ and PilA4 involved in transformation competency and pilus biogenesis in the thermophilic bacterium Thermus thermophilus HB27 Judit Rumszauer, Cornelia Schwarzenlander and Beate Averhoff Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt ⁄ Main, Germany Keywords pilus biogenesis; Thermus thermophilus; transformation competency Correspondence B Averhoff, Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt ⁄ Main, Campus Riedberg, Max-von-Laue-Str 9, 60438 Frankfurt, Germany Fax: +49 69 79829306 Tel: +49 69 79829509 E-mail: Averhoff@em.uni-frankfurt.de (Received 10 April 2006, revised 17 May 2006, accepted 23 May 2006) doi:10.1111/j.1742-4658.2006.05335.x 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 [5] Despite the significance of natural transformation systems of thermophiles, information about transformation Abbreviations IPTG, isopropyl thio-b-D-galactoside; TFMS, trifluoromethanesulfonic acid; TM, Thermus medium FEBS Journal 273 (2006) 3261–3272 ª 2006 The Authors Journal compilation ª 2006 FEBS 3261 Analyses of novel Thermus competence proteins J Rumszauer et al 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 [4] 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 Gramnegative 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 [7] 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 3262 PilM, PilN, PilO, PilW, PilQ and PilA4 in the DNA translocator of T thermophilus HB27 Results Heterologous expression and purification of PilMNOWQ and PilA4 To perform biochemical analyses with the corresponding proteins, pilM, pilN, pilO, pilW, pilQ or pilA4 gene fragments were fused to malE and the fusion proteins were produced in Escherichia coli DH5a (Fig 1) The fusion proteins were purified on an amylose matrix The apparent molecular masses of the chimeric proteins were 82 kDa (MalE–PilM), 57 kDa (MalE–PilN), 60 kDa (MalE–PilO), 60 kDa (MalE–PilW), 72 kDa (MalE–PilQ), and 51 kDa (MalE–PilA4) These values correlate nicely with predicted molecular masses of the fusion proteins Antisera against the purified fusion proteins were generated in rabbits and tested by western blotting with purified fusion proteins Identification of PilM, PilN, PilO, PilW and PilQ in crude extracts The first goal of this study was to identify the individual proteins encoded by the pilMNOWQ competence gene cluster and pilA4 in T thermophilus HB27 Therefore, the polyclonal antisera raised against fragment fusions of PilM, PilN, PilO, PilW, PilQ, and PilA4, respectively, were applied to T thermophilus HB27 crude extracts separated by SDS ⁄ PAGE (Fig 2A–E) The antisera against PilM, PilN, PilO, and PilQ, Fig Organization of pilMNOWQ and generation of gene fragments fused to the gene for maltose-binding protein (malE) The arrows indicate the directions of transcription Numbers indicate base pairs of the complete genes FEBS Journal 273 (2006) 3261–3272 ª 2006 The Authors Journal compilation ª 2006 FEBS J Rumszauer et al Analyses of novel Thermus competence proteins A C could be due to the amount of PilA4 being below the detection limit, because of accumulation of PilA4 in the external medium as pili or attachment to the cell debris after cell disruption B D The competence proteins PilMOW are each required for natural transformation and piliation E Fig Detection of PilM, PilN, PilO, PilW and PilQ proteins in Thermus thermophilus HB27 Thermus thermophilus HB27 wild-type strain and mutant strains were grown to the exponential growth phase and subjected to crude extract preparation The crude extracts of wild-type (20 lg of protein) and mutant strains (20 lg of protein each) were analyzed by SDS ⁄ PAGE and western blotting by using PilM, PilN, PilO, PilW and PilQ antisera The results presented are the data from one experiment from a series of five independent experiments that gave identical results respectively, detected single protein species correlating with the predicted masses of 42, 23, 21 and 82 kDa PilW has a deduced molecular mass of 29.8 kDa, which is 10.2 kDa lower than the apparent molecular mass of 40 kDa (Fig 2D) Since the PilW antibodies are specific, and incorrect assignment of the start and stop sites of pilW can also be excluded, this difference is probably due to post-translational modifications resulting in a conformational change; alternatively, the separation in an SDS gel might be affected by the N-terminal hydrophobic region in PilW In contrast, PilA4 could not be detected in the crude extracts, although the antiserum was found to react specifically with the purified fusion proteins This A We previously reported that marker insertions in pilM, pilN, pilO, pilW, pilQ, and pilA4, respectively, resulted in a defect in natural transformation and absence of pilus structures These findings, together with the organization of these competence genes, suggested that PilA4 and PilQ are individually essential for transformation and piliation [7,8] In contrast to PilQ and PilA4, an individual role of PilM, PilN, PilO and PilW in natural transformation and piliation cannot be deduced from these data with confidence, since polar effects of marker insertions in pilM, pilN, pilO or pilW exerted on downstream-located genes could not be excluded, due to their head-to-tail organization [7,8] To analyze potential polar effects of marker insertions in pilM, pilN, pilO, and pilW, respectively, on downstream-located genes, we performed immunostaining with crude extracts of T thermophilus mutant strains Tt4 (pilM::kat), Tt5 (pilN::kat), Tt6 (pilO::kat), and Tt7 (pilW::kat) In crude extracts of mutants Tt4, Tt6, and Tt7, the proteins encoded by downstream-located genes, PilN, PilW, and PilQ, respectively, were detected (Fig 3A–C) Apparently, insertion of the kanamycin cassette in pilM, pilO or pilW has no polar effect on the downstream-located pilN, pilW or pilQ genes Taken together, these results provide clear evidence that pilM, pilO and pilW are individually essential for natural transformation and piliation of T thermophilus HB27 PilO was not detected in crude extracts of the pilN mutant (data not shown), whereas genes located downstream of pilO, such as pilW, were expressed This suggests that either biosynthesis or stability of the PilO protein is impaired in pilN mutants B C Fig PilN, PilW and PilQ production in Thermus thermophilus pilM, pilO or pilW mutant strains Thermus thermophilus HB27 wild-type and mutant strains were grown to the exponential growth phase and subjected to crude extract preparation The crude extracts (20 lg of protein) were analyzed by SDS ⁄ PAGE and western blotting by using PilN, PilW and PilQ antisera The results presented are the data from one experiment from a series of four independent experiments that gave identical results FEBS Journal 273 (2006) 3261–3272 ª 2006 The Authors Journal compilation ª 2006 FEBS 3263 Analyses of novel Thermus competence proteins J Rumszauer et al Subcellular localization of PilMNOWQ and PilA4 To determine the subcellular localization of the competence proteins PilM, PilN, PilO, PilW, PilQ, and PilA4, cells were lysed by sonification, total membranes were separated from the soluble fraction by ultracentrifugation, and inner and outer membranes were further separated by N-lauroylsarcosine extraction and subsequent ultracentrifugation The competence protein PilM is a rather hydrophilic protein, except for a short region of limited hydrophobicity close to the N-terminus To elucidate the subcellular localization of PilM, we performed western blot analyses of the cell fractions and found that PilM is exclusively localized in the inner membrane (Fig 4A) In addition, PilN and PilO are localized exclusively in the inner membrane These results, together with the rather hydrophilic character of PilN and PilO except for the N-terminal hydrophobic domain, suggest that PilN and PilO are inner membrane-anchored proteins, which may mediate recruitment and assembly of DNA translocator proteins at the inner membrane PilW, a Thermus competence protein with no similarities to known proteins, exhibits a hydrophobic region at the N-terminus To answer the question of whether this region is sufficient to mediate membrane anchoring, cell fractions were subjected to western blot analyses with PilW antiserum (Fig 4D) These studies revealed that PilW is distributed equally between the inner and outer membranes Major amounts of the secretin-like PilQ were detected in the outer membrane, whereas minor amounts of PilQ were also detected in the inner membrane (Fig 4E) The latter might result from transport of PilQ through the inner membrane to the outer membrane Although we could not detect PilA4 in cell-free extracts, it is clearly detectable in membrane fractions and found to be distributed equally between the inner and outer membranes (Fig 4F) The detection of PilA4 in the membranes could be due to an accumulation of high PilA4 levels in the membranes or attachment of the PilA4 to the membranes Interestingly, PilA4 had an apparent molecular mass of 23 kDa, which differs significantly from the deduced molecular mass of 14 kDa However, since no reaction of the antiserum was observed with membrane fractions of the pilA4 mutant, it is evident that the 23 kDa protein is PilA4, probably in a post-translationally modified form A B C D E F Fig Cellular localization of PilM, PilN, PilO, PilW, PilQ, and PilA4 Cells were harvested in the exponential growth phase, resuspended in lysis buffer and disrupted by sonification Soluble fractions and membrane fractions were separated by ultracentrifugation prior to separation of inner and outer membrane fractions by N-laurylsarcosine precipitation The resulting fractions were analyzed by SDS ⁄ PAGE and western blotting by using specific antisera against: (A) PilM; (B) PilN; (C) PilO; (D) PilW; (E) PilQ; and (F) PilA4 The data are the data from one experiment that was replicated three times with identical results S, soluble fraction; IM, inner membrane; OM, outer membrane 3264 FEBS Journal 273 (2006) 3261–3272 ª 2006 The Authors Journal compilation ª 2006 FEBS J Rumszauer et al Fig Analysis of PilA4 glycosylation Untreated proteins (– TFMS) and trifluoromethanesulfonic acid (TFMS)-treated proteins (+ TFMS) were separated by SDS ⁄ PAGE, transferred onto nitrocellulose membranes, and probed with MalE–PilA4 antibodies PilA4 undergoes glycosylation Structural subunits of type IV pili of Gram-negative bacteria are known to undergo different post-translational modifications such as glycosylation, and linkage to a-glycerophosphate or phosphorylcholine [13–17] Glycosidic bond cleavage by trifluoromethanesulfonic acid (TFMS) has been shown to be useful for the identification of polysaccharides linked to proteins, since the effect of TFMS on a glycoprotein is sufficiently specific that a change in molecular mass after treatment can be ascribed to removal of oligosaccharides Post-translational modifications other than glycosylation, such as by sulfate or phosphate, are stable to TFMS treatment To address the potential glycosylation of PilA4, we compared TFMS-treated and TFMS-untreated protein extracts of HB27 wild-type cells in western blot analyses These studies revealed that deglycosylation via TFMS treatment resulted in a shift of the apparent molecular mass of PilA4 to 18 and 14 kDa (Fig 5) This change in molecular mass after TFMS treatment suggests that PilA4 undergoes glycosylation The 14 kDa protein species corresponds to unmodified PilA4 protein, whereas the 18 kDa PilA4 might carry a further modification resistant to TFMS treatment PilQ and PilA4 copurify with pilus structures The similarities of PilM, PilN, PilO, PilQ and PilA4 to type IV pili proteins led to the question of whether these competence proteins are structural subunits of the T thermophilus pilus structures To address this question, we purified the pili structures by separating shear fractions of T thermophilus HB27 in a discontinuous sucrose gradient After centrifugation, the gradient was fractionated and inspected by electron microscopy Two fractions (corresponding to  50% sucrose) contained exclusively the pilus structures Analyses of novel Thermus competence proteins (Fig 6A) Inspection with respect to the presence of impurities and homogeneities of the pilus fractions revealed that small lipid vesicles were occasionally present Close inspection of representative areas revealed that 90% of the pilus structures were attached to a globular structure with a diameter of 20 nm at one end of the pilus structure (Fig 6B) To determine whether PilA4 is part of the pilus structures, immunogold labeling of the purified pili was performed with PilA4 antiserum raised against fragments of the native PilA4 protein Despite many different attempts, we never observed binding of gold-labeled antibodies to the pilus (data not shown) This finding suggests that either PilA4 is not part of the pilus, PilA4 is inaccessible in the native pilus, or the PilA4 antibody does not recognize the native protein To address this question, we analyzed the purified pilus fraction by SDS ⁄ PAGE and western blotting with PilA4 antibodies These studies revealed the presence of the 23 kDa PilA4 protein in the pilus fraction (Fig 6C) PilM, PilN and PilW were not detected in the pilus fraction (data not shown), indicating that these competence proteins are not structural subunits of the pili In contrast, PilQ was detected in the pilus fraction (Fig 6D), probably as a result of being torn out of the membrane together with the pilus during the shearing step Influence of PilM, PilN, PilO, PilW, PilQ and PilA4 on the subcellular localization of competence proteins In further studies, we addressed possible interactions between PilM, PilN, PilO, PilW, PilQ and PilA4 To this, we examined the influence of each protein on the subcellular localization of the other proteins We separated the inner and outer membrane fractions of pilM, pilN, pilO, pilW, pilQ and pilA4 mutants, respectively, from the soluble fractions (periplasm and cytoplasm) and performed western blot analyses to detect the competence proteins in the subcellular fractions Membrane fractions of the T thermophilus HB27 wild type were used as controls First, we compared the relative levels of PilM, PilN, and PilO in membrane fractions of mutants carrying insertions in pilM, pilN, pilO, pilW, or pilQ, but found no significant differences (Table 1) In contrast, mutation in pilQ led to the absence of PilW and PilA4 in the inner membrane In addition, pilW mutation resulted in the absence of PilQ and PilA4 in the outer membrane The abilities of PilW, PilQ and PilA4 to stably localize or accumulate in the outer membrane are strongly dependent one another, indicating interactions between PilW, PilQ and PilA4 in structure and assembly FEBS Journal 273 (2006) 3261–3272 ª 2006 The Authors Journal compilation ª 2006 FEBS 3265 Analyses of novel Thermus competence proteins J Rumszauer et al A B C D Fig Electron microscopy of Thermus thermophilus HB27 pili separated by sucrose density gradient and western blot analyses of purified pili fractions Pili were sheared off and separated as described in Experimental procedures Each fraction was analyzed by electron microscopy Major amounts of pili were detected in fractions containing  50% sucrose (A) Close inspection of the pili led to the detection of globular structures (indicated by arrows in A) at the base of the pili (B) SDS ⁄ PAGE was stained with Coomassie Western blot analyses of the pilus fraction revealed that pili were copurified with PilA4 (C) and PilQ (D) Table Subcellular localization of the PilM, PilN, PilO, PilW, PilQ and PilA4 competence factors OM, outer membrane; IM, inner membrane; ++, major amounts present in one of the membranes; +, present; –, absent Subcellular localization PilM PilN PilO PilW PilQ PilA4 Strains IM OM IM OM IM OM IM OM IM OM IM OM HB27 wild type Mutants Tt4 (pilM::kat) Tt5 (pilN::kat) Tt6 (pilO::kat) Tt7 (pilW::kat) Tt8 (pilQ::kat) Tt20 (pilA4::kat) + – + – + – + + + ++ + + – + + + + + – – – – – – + – + + + + – – – – – – + – – + + + – – – – – – + + + – + + + + + – – + + + + + – + ++ ++ ++ – – ++ + + + + + – + + + – – – Discussion 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, 3266 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 FEBS Journal 273 (2006) 3261–3272 ª 2006 The Authors Journal compilation ª 2006 FEBS J Rumszauer et al 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 a-glycerophosphate substituent attached to Ser93 by a phosphodiester linkage [17] 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 a-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 [21] and [22]) 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 [23], whereas MreB has been shown to perform dynamic motor-like movements extending along helical tracks [24] 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 Analyses of novel Thermus competence proteins 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 [28], ExeD of Aeromonas salmonicida [29], PilQ of P aeruginosa [25,30], and PilQ of N gonorrhoeae [31] 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 FEBS Journal 273 (2006) 3261–3272 ª 2006 The Authors Journal compilation ª 2006 FEBS 3267 Analyses of novel Thermus competence proteins J Rumszauer et al 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 compo- nents 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) [33], P aeruginosa (18.3 nm ± 1.2 nm) [34] or N meningitidis (15.5 nm) [33] In contrast, PilM, PilN and PilO are essential for transformation and piliation but 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) Fig 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 3268 FEBS Journal 273 (2006) 3261–3272 ª 2006 The Authors Journal compilation ª 2006 FEBS J Rumszauer et al Analyses of novel Thermus competence proteins 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 Experimental procedures Bacterial strains, growth conditions, and DNA manipulations Thermus thermophilus HB27 wild-type and mutant strains were grown at 68 °C under strong aeration in Thermus medium (TM) containing g of yeast extract, g of tryptone peptone and g of NaCl per liter, pH 7.5 [4] Escherichia coli strains were grown in LB medium (0.5% yeast extract, 1% tryptone peptone, 1% NaCl) at 37 °C Recombinant E coli strains were grown in the presence of ampicillin (100 lgỈmL)1) Thermus thermophilus HB27 mutants were grown in liquid media with 20 lgỈmL)1 kanamycin or on solid media with 40 lgỈmL)1 kanamycin DNA manipulations were perfomed with standard procedures [35] Generation of antibodies To avoid toxic effects of overproduced proteins on the E coli host cells, PilM, PilN, PilO, PilW, PilQ and PilA4 fragment fusions (Fig 1) were overproduced Therefore, T thermophilus HB27 pilA4, pilM, pilN, pilO, pilW and pilQ fragments were amplified from chromosomal DNA of T thermophilus HB27 (for primers see Table 2), cleaved with appropriate restriction enzymes, and cloned into the overexpression vector pMalc-2X (New England Biolabs GmbH, Frankfurt a M., Germany) The plasmid constructs were sequenced with custom-made primers Maltosebinding protein (MalE) fusion proteins were overproduced in E coli DH5a, by isopropyl thio-b-d-galactoside (IPTG) induction of the tac promoter and purified by immobilized amylose affinity chromatography performed as recommended by the manufacturer (New England Biolabs GmbH) Purified fusion proteins were used for immunization of rabbits Western blot analyses Thermus thermophilus HB27 cells were harvested in the exponential growth phase, resuspended in Laemmli sample buffer [36], and boiled for 10 to lyse the cells SDS ⁄ PAGE was performed in 15% (w ⁄ v) acrylamide separating gels [36] The proteins were electrotransferred onto nitrocellulose membranes [37] and stained with 0.2% PonceauS Red for detection of reference proteins, and membranes were blocked by incubation for h at room temperature in NaCl ⁄ Pi Tween-20 (140 mm NaCl, 10 mm KCl, 16 mm Na2HPO4, mm KH2PO4, 0.05% Tween-20) containing 0.5% skimmed milk powder Immunodetection of proteins in total cell lysates or in membrane fractions was performed with polyclonal PilA4 antiserum (dilution : 5000), PilM antiserum (dilution : 5000), PilN antiserum (dilution : 2500), PilO antiserum (dilution : 2000), PilW antiserum (dilution :10 000) or PilQ antiserum (dilution : 10 000) obtained from Davids Biotechnologie (Regensburg, Germany) ProteinA–horse radish Table PCR primer sequences Restriction sites for cloning are underlined Mismatches are indicated by bold type Gene designation pilM 5¢ ⁄ EcoRI 3¢ ⁄ BamHI pilN 5¢ ⁄ EcoRI 3¢ ⁄ XbaI pilO 5¢ ⁄ EcoRI 3¢ ⁄ SalI pilW 5¢ ⁄ EcoRI 3¢ ⁄ BamHI pilQ 5¢ ⁄ EcoRI 3¢ ⁄ HindIII pilA4 5¢ ⁄ EcoRI 3¢ ⁄ SalI Primer sequence (5¢- to 3¢) Size of fragments (bp) GGG CTT GGA ATT CGG GGC CTC GAG CTG GTC CGG ATC CGC CTT G 1246 TCG CCG AAT TCG GCC GCC GCA GGT AGT TCT AGA GGA GCC 426 AGG CAG GAA TTC GCC ACC GTC TCA GGC GTC GAC AGG CGT TCT 504 GAC CTG GGA TCC CGA GAC CTT G GTG GTG GAA TTC CCA CCC CCA 658 CAC CTT CCC CGA ATT CCT CGC C CAC CCT AAG CTT CAC CTA GGG CAC 832 GAC GCG GTG GAA TTC CAG GAG CAA CGC TAG CGT CGA CGC CAT AC 291 FEBS Journal 273 (2006) 3261–3272 ª 2006 The Authors Journal compilation ª 2006 FEBS 3269 Analyses of novel Thermus competence proteins J Rumszauer et al peroxidase (HRP) conjugate (Bio-Rad, Munchen, Geră many) as secondary antibody was used in combination with the BM Chemiluminescence Blotting Substrate Kit (Roche Diagnostics GmbH, Mannheim, Germany) to develop the chemiluminescence for visualization on Kodak X-AR film (Sigma-Aldrich, Saint-Quentin Falavier, France) Molecular weight markers, peqGOLD Protein-Marker II (10– 200 kDa), were obtained from Peqlab Biotechnologie GmbH, Erlangen, Germany Membrane isolation and subcellular fractionation Four hundred milliliter cultures were grown at 68 °C in TM, harvested in the mid-log-phase, washed with 20 mL of 10 mm Tris ⁄ HCl buffer (pH 8.0), and resuspended in mL of lysis buffer (10 mm Tris ⁄ HCl, mm EDTA, pH 7.8), containing 40 lgỈlL)1 DNase I and 40 lgỈlL)1 RNase A Cells were disrupted by sonification (3 · pulse), and mm MgCl2 was added immediately afterwards Intact cells and cell debris were removed by low-speed centrifugation (13 000 g, 15 min, °C; rotor type JA25.5, Beckman Coulter, Krefeld, Germany) The resulting crude cell extracts (supernatants) were subjected to western blot analyses Soluble and membrane proteins were separated by ultracentrifugation for h at 120 000 g at °C (rotor type Ti70, Beckman Coulter) Membrane pellets were washed and resuspended in mL of 10 mm Tris ⁄ HCl (pH 8.0) To separate inner and outer membrane fractions, the membranes were repeatedly pushed through a needle (0.45 · 25 mm) and subsequently incubated for 10 on ice in the presence of 2% N-lauroylsarcosine and 10 mm EDTA (pH 8.0) [38] After ultracentrifugation of the membranes for h at 120 000 g (4 °C; rotor type Ti70, Beckman Coulter), the outer membrane pellet was washed once with 10 mL of 10 mm Tris ⁄ HCl (pH 8.0) (120 000 g, h, °C; rotor type Ti70, Beckman Coulter), resuspended in H2O and stored at ) 20 °C To precipitate the inner membrane proteins, the supernatant was incubated for h at ) 20 °C with four volumes of cold acetone The inner membranes were precipitated by centrifugation for 30 at 16 000 g (0 °C; rotor type JA25.5, Beckman Coulter), resuspended in H2O, and stored at ) 20 °C Purity of the membrane fractions and use of the N-laurylsarcosine solubilization method in T thermophilus HB27 was verified by western blot analyses of membrane fractions with antibodies directed against the S-layer protein (outer membrane) (1AE1 antibodies) and with antibodies directed against the inner membrane-embedded cytochrome c1 (cytochrome bc1 complex) of the T thermophilus HB27 respiratory chain (inner membrane) Pili purification An L culture of T thermophilus HB27 was grown in TM medium without stirring The culture was harvested in the 3270 exponential growth phase (after h of incubation) and washed three times with 50 mm Tris ⁄ HCl (pH 7.5) (8000 g, min; rotor type JA10, Beckman Coulter) The cell suspension was pushed twice through a needle (0.45 · 25 mm) to shear off the pili After removal of the cells (20 000 g, · 10 min; rotor type JA25.5, Beckman Coulter) the pili fraction was pelleted via high-speed centrifugation (120 000 g, h; rotor type Ti70, Beckman Coulter) The pellet was resuspended in mL of H2O and subjected to sucrose density gradient centrifugation (30–70% sucrose gradient) for 24 h at 160 000 g (rotor type Ti70, Beckman Coulter) The gradient was fractionated into 1.2-mL samples, which were diluted with five volumes of 30 mm Tris ⁄ HCl, 0.9% (w ⁄ v) NaCl, pH 7.5, centrifuged (120 000 g, h; rotor type Ti70, Beckman Coulter) and dissolved in H2O, containing phenylmethanesulfonyl fluoride to inhibit proteinases Thermus pili were visualized by electron microscopy in samples containing 50% of sucrose Deglycosylation assay Proteins were deglycosylated by treatment with TFMS [39] Frozen cells (200 mg) of T thermophilus HB27 were freezedried overnight and resuspended in mL of 5% SDS The solution was refrozen before freeze-drying again for h The sample was slightly shaken in mL of anisole ⁄ TFMS (1 : 2) for h at °C The proteins were incubated (15 min, on ice) with mL of m sodium carbonate buffer (pH 9.2) and 22 mL of ethanol (96%) and precipitated by centrifugation (17 000 g, min; rotor type JA25.5, Beckman Coulter) The pellets were washed in H2O (47 000 g, 20 min; rotor type JA25.5, Beckman Coulter), resuspended in 200 lL of H2O and stored at )20 °C until the SDS ⁄ PAGE was performed Electron microscopy and immunogold labeling Negative staining and electron microscopy were performed as described [40] For immunogold labeling, the sheared pili were attached to Formvar-coated, glow-discharged, 0.01% poly-l-lysine-treated nickel grids After washing with NaCl ⁄ Pi buffer (2 mm KH2PO4, 16 mm Na2HPO4, 140 mm NaCl, 10 mm KCl, pH 7.2) and blocking with NaCl ⁄ Pi buffer containing 0.1% BSA, the grids were incubated for h with MalE–PilA4 antibodies The primary antibody was detected with the secondary antibody goat anti-rabbit and conjugated with gold (10 nm, Amersham Biosciences, Freiburg, Germany) Acknowledgements This work was supported by grants Av9 ⁄ 4-5 and Av9 ⁄ 5-1 from the Deutsche Forschungsgemeinschaft We are grateful to Gerhard Wanner (Ludwig-Maximi- FEBS Journal 273 (2006) 3261–3272 ª 2006 The Authors Journal compilation ª 2006 FEBS J Rumszauer et al lians-Universitat, Munchen), Winfried Haase and ă ă Werner Kuhlbrandt (Max-Planck-Institut fur Biophysă ă ik, Frankfurt) for the electron 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