Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands
Correspondence
Arnold J. M. Driessen
a.j.m.driessen{at}biol.rug.nl
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ABSTRACT |
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INTRODUCTION |
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Around 4·5 % of the genome of the thermoacidophilic archaeon S. solfataricus encodes proteins predicted to be secreted to the external medium (Albers & Driessen, 2002). These proteins contain putative secretory signal peptides, double arginine-containing signal peptides that bear the signature for the twin arginine export pathway, lipoprotein signal peptides, and type IV pilin-like signals. This latter group is exceptionally large in S. solfataricus and consists of 16 proteins (Albers et al., 2003
). In addition to the flagellin subunit, this group includes precursors of both characterized and predicted substrate-binding proteins that function as extracellular components of ABC transporters (Albers et al., 2003
; Albers & Driessen, 2002
). GlcS, the glucose-binding protein, AraS, the arabinose-binding protein, and TreS, the trehalose-binding protein, have been isolated from S. solfataricus membranes and N-terminal sequenced. This has confirmed that they are processed in the same manner as the flagellin (Albers et al., 1999
; Elferink et al., 2001
). Moreover, by means of an in vitro assay it has been shown that PibD, the type IV pilin peptidase of S. solfataricus, processes both the flagellin and the glucose-binding protein precursors (Albers et al., 2003
).
Proteins containing type IV pilin signal peptides can be assembled into multimeric structures, such as the archaeal flagellum or type IV pili. In both cases, the positive short N-terminal part of the signal peptide is cleaved from the precursor, and this is believed to prevent transmembrane translocation of the protein before processing. The mature proteins are subsequently assembled by dedicated secretion systems into the flagellum or pilus structure (Bardy et al., 2003; Mattick, 2002
). The structures of both multimeric complexes have been solved (Cohen-Krausz & Trachtenberg, 2002
; Craig et al., 2003
; Samatey et al., 2001
). This shows that the N-terminal hydrophobic domain of both proteins is used as a scaffold for the assembly of the subunits. Considering the role of the signal peptide in achieving the oligomeric state of these proteins, it is an intriguing question why a certain group of substrate-binding proteins, which are part of an ABC transporter, are equipped with a type-IV-pilin signal peptide. We hypothesize that these proteins assemble into a multimeric complex using the hydrophobic domain at the N-terminus as a scaffold, so that the sugar-binding domain is facing outwards. We tentatively call such a complex the bindosome. Preliminary data indeed suggest that the binding proteins are contained in a large complex, but little is known about its exact size and structure (S. V. Albers, unpublished results). By analogy with the bacterial systems, we expect that the subunits of such a putative bindosome are assembled by a dedicated secretion system with homology to the bacterial type II, type IV, or type IV pilin secretion systems. One common component of the Gram-negative bacterial systems is a cytoplasmic ATPase. These are essential for the formation of the multimeric structure and are believed to energize the assembly process (Cascales & Christie, 2003
; Sandkvist, 2001
). This extensively studied class of proteins is collectively named after the VirB11 protein of the Ti-transfer system of Agrobacterium tumefaciens (Christie et al., 1989
; Sagulenko et al., 2001
). VirB11 proteins are related in sequence to the GspE class of ATPases, the second class of secretion ATPases, which power the type II secretion system (Possot & Pugsley, 1994
). A third class is constituted by PilT, the ATPase of the type IV pilin system, which mediates the retraction of adhesive type IV pili (Merz et al., 2000
). All these ATPases are hydrophilic proteins and contain the typical conserved boxes as the Walker A and B motifs. Most of the characterized Virb11-like secretion ATPases have been shown to hydrolyse ATP and found to exist as hexamers in nucleotide-bound form (Krause et al., 2000
; Yeo et al., 2000
). Structures of different nucleotide-bound states of HP0525, the secretion ATPase of the Cag system of Helicobacter pylori, show dynamic changes upon nucleotide binding (Savvides et al., 2003
; Yeo et al., 2000
). The PilT homologues of Aquifex aeolicus and Legionella pneumophila have been shown to hydrolyse ATP and form hexameric structures, such as the VirB11 proteins (Herdendorf et al., 2002
; Sexton et al., 2004
). It is, however, unclear whether type II secretion ATPases also form multimers or not, because EpsE, the ATPase of the cholera toxin secretion system of Vibrio cholerae, crystallizes as a monomer in both liganded and unliganded form (Robien et al., 2003
).
To identify the secretion system(s) involved in the assembly of type IV pilin-like proteins in S. solfataricus, we screened the genome and identified five putative candidate genes encoding secretion ATPases. We determined which genes are co-transcribed with the secretion ATPases to form a functional secretion system. The putative secretion ATPases were expressed in Escherichia coli, purified and shown to exhibit ATPase activity. The possible function of these systems is discussed.
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METHODS |
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RNA isolation and analysis.
Total RNA was isolated from S. solfataricus cells by using TRIZOL reagent (Invitrogen). Cell pellets were resuspended in TEN buffer (20 mM Tris/HCl, pH 8·0, 100 mM NaCl, 1 mM EDTA). Lysis was achieved by incubation for 5 min at room temperature in equal amounts of TEN buffer supplemented with 1·6 % sodium N-lauroylsarcosine and 0·12 % Triton X-100 (v/v). Subsequent steps were performed according to the protocol of the manufacturer. Isolated total RNA was treated for 1 h with RNase-free DNase at 37 °C. Isolated total RNA (1 µg) was added to one RT-PCR Ready-to-Go bead (Amersham). Sequences of primers used to identify specific transcripts are listed in Supplementary Table S1 with the online version of this paper at http://mic.sgmjournals.org. The reaction was performed according to the protocol of the manufacturer. Amplified RNA was analysed on 2 % agarose gels.
Cloning and plasmid construction.
Chromosomal DNA was isolated as described previously (Albers et al., 1999). Vectors and plasmids used in this study are summarized in Table 1
. Oligonucleotide primers for the genes sso0120, sso0572, sso2316 (flaI), sso2387 and sso2680 were designed based on the genome sequence of S. solfataricus P2 (http://www-archbac.u-psud.fr/projects/sulfolobus/) and listed in Supplementary Table S1. The genes were amplified with primers containing the appropriate restriction sites to ligate the PCR products into the expression vector pBADHisA (Stratagene). The C-terminal 6xHis epitope tags of the reverse primers were introduced in-frame to the gene products. All gene products were sequenced.
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Purification of recombinant proteins.
Cells were rapidly thawed and 1 mM PMSF and DNase were added. Subsequently cells were broken in a cell disruptor at 30 000 p.s.i. (207 000 kPa). After a low spin for 15 min at 8000 g to remove unbroken cell material and inclusion bodies, a high spin for 1 h at 100 000 g was performed to pellet the membranes. In the case of FlaI, the supernatant was adjusted to 100 mM NaCl and then subjected to heat incubation for 20 min at 70 °C. The sample was cooled on ice and precipitated proteins were spun down. The supernatant was applied to a nickel-loaded chelating column (Pharmacia). An imidazole gradient from 5 to 250 mM was applied, and the protein was found to be eluted at around 230 mM imidazole. Fractions containing FlaI were pooled and dialysed overnight at 4 °C against 20 mM Tris/HCl, pH 7·5, 100 mM NaCl and 10 % (v/v) glycerol. The crude membranes containing the other four recombinant proteins were subjected to a sucrose gradient to isolate inner-membrane vesicles. The inner-membrane vesicles were solubilized at a protein concentration of 5·5 mg ml1 using 1 % (w/v) N-dodecyl-D--maltopyranoside (DDM) for 30 min at room temperature. After spinning down the aggregates and non-solubilized proteins, the supernatant was applied to a nickel-loaded chelating column. After extensive washing, the proteins were eluted with an imidazole gradient from 5 to 250 mM imidazole. Fractions containing the recombinant proteins were pooled and dialysed overnight at 4 °C against 20 mM Tris/HCl, pH 7·5, 100 mM NaCl, 10 % (v/v) glycerol, 0·05 % (w/v) DDM. SSO0572 was dialysed against the same buffer containing 20 mM Tris/HCl, pH 9, to prevent precipitation.
Fractionation of Sulfolobus cells.
S. solfataricus cells grown on 400 ml minimal medium containing only the desired carbon source were collected by centrifugation, and resuspended in 3·5 ml of 10 mM Tris/HCl, pH 7·5, 1 mM EDTA. PMSF (1 mM) was added and cells were lysed by sonication (8 times, 15 s on at 8 µm amplitude, 45 s off) using a Soniprep 150 Ultrasonic Disintegrator (MSE Scientific Instruments) equipped with a microprobe. The samples were centrifuged for 30 min at 100 000 g at 4 °C, and the supernatant and pellet were taken to be the cytoplasmic and membrane fractions, respectively. Membranes were resuspended in 300 µl of 10 mM Tris/HCl, pH 7·5.
ATPase activity measurements.
Purified proteins (3 µg) were pre-heated at 60 °C for 2 min in 100 µl assay buffer (25 mM MES, pH 6·5, 150 mM NaCl and 1 mM MgCl2). Reactions were initiated by adding 1 mM ATP, GTP, CTP or UTP and stopped after 15 min by freezing in liquid nitrogen. For measurements in the presence of other divalent cations, MgCl2 was replaced by either MnCl2 or CaCl2. The amount of released inorganic phosphate was determined using a colorimetric method (Lanzetta et al., 1979). The data were corrected for non-enzymic ATP hydrolysis. For the autophosphorylation assay, the proteins were incubated in assay buffer containing Mn2+ with 50 µM ATP [300 mCi (4·81 GBq) of [
-32P]ATP ml1] for 45 min at 50 °C. The proteins were run on a 12 % SDS-PAGE and analysed by phosphor imaging.
Other methods and materials.
DDM was from Anatrace. All other chemicals were purchased from Sigma. Polyclonal antisera were raised in chickens against purified secretion ATPases by Agrisera. Protein concentration determinations were carried out using the DC Kit (Bio-Rad).
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RESULTS |
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DISCUSSION |
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The bacterial type II and IV secretion systems usually contain between 12 and 15 subunits (Cascales & Christie, 2003; Christie, 2001
; Sandkvist, 2001
). The putative systems identified in S. solfataricus seem to contain a much lower number of components, although we do not know whether or not other genes outside of the operons contribute to the secretion systems as well. In all operons, the secretion ATPases were accompanied by integral membrane proteins with homology to TadB, one of the membrane proteins of the tight adherence system (Kachlany et al., 2000
). Moreover, three operons contain proteins with a type IV pilin signal peptide. FlaB, the subunit of the archaeal flagellum, is likely to be the substrate of the FlaHIJ system. SSO2681 and SSO0117/0118 might be substrates for their cognate secretion systems or might have a similar function as so-called pseudopilins. The latter are thought to form a translocation piston to push proteins across protein pores in the outer membrane of Gram-negative bacteria (Mattick et al., 1996
; Sandkvist, 2001
; Shevchik et al., 1997
). In the archaeal system, such a piston needs to bridge the distance between the cytoplasmic membrane and the S-layer. It has been described that the S-layer of Sulfolobus species contains pores of 35 nm (Koenig, 1988
; Prüschenk et al., 1987
). These could be the exit points for secreted proteins, thereby eliminating the need for specific outer-membrane pore proteins, which in Gram-negative bacteria are formed by the secretin family of proteins (Thanassi, 2002
).
The ATPase activities measured for the five recombinant secretion ATPases are comparable to the ones characterized in bacteria. Aquifex aeolicus PilT, plasmid R388 TrwD, Actinobacillus actinomycetemcomitans TadA and Legionella pneumophila DotB display a similar low activity of 115 nmol mg1 min1 (Bhattacharjee et al., 2001; Herdendorf et al., 2002
; Rivas et al., 1997
; Sexton et al., 2004
). The low activity may be due to the lack of an interaction with other components of the secretion systems, possibly the integral membrane components and/or the substrate proteins. The addition of phospholipids has been shown to increase the activity of HP0252, TadA or RP4 TrbB two- to tenfold (Bhattacharjee et al., 2001
; Krause et al., 2000
). The ATPase activity is dependent on the presence of divalent cations, with an apparent preference for Mn2+. SSO0572 was slightly more active with Ca2+ than Mn2+. A preference for Mn2+ has also been demonstrated for the autophosphorylation or protein phosphorylation activity of SSO2387 by Lower & Kennelly (2003)
. However, in contrast to this initial report, we observe that Ca2+ is almost as stimulatory as Mn2+. The current assay is not performed in 5 M urea which might explain the low activity with Ca2+ observed by Lower & Kennelly. SSO2387 has been proposed to function as a protein kinase, but its homology to secretion ATPase was not previously noted, despite the fact that it contains all conserved motifs. Nevertheless, SSO2387 was the only ATPase that exhibited an autophosphorylation activity when incubated with ATP. VirB11 was shown to have ATPase and autophosphorylation activity (Christie et al., 1989
), and with EpsE (Sandkvist et al., 1995
) autophosphorylation was also demonstrated. The function of the autophosphorylation activity during protein secretion, however, remains obscure. We tried to assess the oligomeric behaviour of these ATPases, since in bacteria they form a hexameric complex that interacts with the membrane components of the secretion systems (Herdendorf et al., 2002
; Krause et al., 2000
; Savvides et al., 2003
). However, the proteins precipitated upon concentration, which precluded further oligomerization experiments.
For many of the described systems of the main terminal branch in Gram-negative bacteria, the ATPases play an essential role in the biological function, that is, protein secretion, pilus assembly or pilus retraction. For the archaeal flagella secretion system, FlaI was shown to be essential for the assembly of the flagellar structure in Halobacterium salinarum and M. voltae (Patenge et al., 2001; Thomas et al., 2002
). The production of FlaB, the structural subunit of the flagellum, was not affected and the protein was normally processed by the peptidase in the FlaI knockout strain (Thomas et al., 2002
). The same was noted in M. voltae for the FlaJ knockout strain (Thomas et al., 2002
). FlaJ is a membrane-spanning protein, which might form the minimal secretion pore with FlaI. Apart from SSO572, all secretion ATPases are either preceded or followed by a protein that shows some similarity to FlaJ. Because of this operon organization, we speculate that the ATPase and the membrane protein constitute the minimal cores of the secretion systems.
Except for the FlaHIJ system, the substrates of the other systems in S. solfataricus are unknown. Since the operons containing SSO0120 and SSO2680 are expressed under all conditions under which the sugar-binding proteins are expressed, it appears that these two are the best candidates to form an assembly machinery of the bindosome. When we searched the genome of S. tokodaii, a close relative of S. solfataricus, for the presence of ABC transporters, we found a variety of homologues of the systems which contain substrate-binding proteins belonging to the di/oligopeptide cluster in S. solfataricus. We could not identify any homologues to the transport systems of S. solfataricus from which the substrate-binding proteins are synthesized as precursors containing a type IV pilin-like signal peptide. Strikingly, a comparison between the two species of the presence of the various secretion operons showed that S. tokodaii contains all the operons except the SSO2680 operon. Therefore, we consider this as the most likely candidate for the assembly of the bindosome. This operon also contains a putative substrate gene, SSO2681, that is expressed under all growth conditions. This protein is synthesized as a precursor with a type IV pilin-like signal sequence, but has no further homology to any known proteins.
Further functional elucidation will depend on gene deletions in S. solfataricus to identify the substrates and subunits of the different secretion systems. In this respect, a method for gene inactivation in S. solfataricus has very recently been reported (Worthington et al., 2003). This may now enable a more direct test of the involvement of SSO2680 and the other ATPases in the secretion of the binding proteins.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 October 2004;
revised 2 November 2004;
accepted 8 November 2004.
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