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Review article |
1 Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, USA
2 Unité de Génétique Moléculaire, CNRS URA 2172, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France
Correspondence
Milton H. Saier Jr
msaier{at}ucsd.edu
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ABSTRACT |
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 TOP ABSTRACT OverviewComputer methodsT2S, T4P and Fla...T2S systemsPilins (T2SG, H, I,...T2SE/T4PC/FlaI ATPase phylogenyT2SF/T4PC/FlaJ transmembrane...Multispanning TM protein...ConclusionsREFERENCES |
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Present address: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA.
Present address: Department of Life Science, Jeonju University, Chonju, Korea.
Present address: Institute of Molecular Biology, National Chung Hsing University, Taichung, 402, Taiwan, Republic of China.
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Overview |
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TOPABSTRACTOverviewComputer methodsT2S, T4P and Fla...T2S systemsPilins (T2SG, H, I,...T2SE/T4PC/FlaI ATPase phylogenyT2SF/T4PC/FlaJ transmembrane...Multispanning TM protein...ConclusionsREFERENCES |
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), represents the major pathway for exoprotein transport from the periplasm across the outer membrane in a wide variety of Gram-negative bacteria (Pugsley, 1993a). The type II secreton is composed of a core of around 12 proteins, some of which are not present in all type II secretons and others of which appear to be dispensable for secreton function (Filloux et al., 1998; Pugsley, 1993a; Pugsley et al., 1997; Sandkvist, 2001). In this review, secreton components will be referred to according to their specific, four-letter gene designation or by the designation T2S and the last letter of their gene designation. For example, the products of the Klebsiella pneumoniae (Klebsiella oxytoca) pulD gene (d'Enfert et al., 1989), the Aeromonas hydrophila exeD gene (Ast et al., 2002) and the Xanthomonas campestris xpsD gene (Hu et al., 1995) are all referred to as members of the T2SD family.
Surprisingly, the T2SD family of proteins (members of the secretin superfamily; TC #1.B.22) (Martinez et al., 1998; Nguyen et al., 2000; Thanassi, 2002; Yen et al., 2002) are the only integral outer-membrane secreton components. Therefore, they are the only ones capable of forming channels in bacterial outer membranes to permit exoprotein efflux (Bitter et al., 1998; Hardie et al., 1996a; Nouwen et al., 1999, 2000). The well-established multimeric state and low-resolution structures of secretins (Bitter et al., 1998; Brok et al., 1999; Collins et al., 2001, 2003) are consistent with this idea. A role in pilus biogenesis has been proposed (Sauvonnet et al., 2000).
The other secreton components include the following. (1) A peripheral outer-membrane lipoprotein (the T2SS protein or pilotin) (Hardie et al., 1996a, b) that has so far been found only in a small number of secreton systems. (2) A peripheral plasma membrane protein (the T2SE protein), a putative ATP-binding protein that, in one case, is reported to be monomeric and to have both ATPase and autokinase activities (Sandkvist et al., 1995). T2SE proteins have characteristic signature sequences, including a highly conserved region that is flanked by aspartate residues as well as an essential zinc-finger-like motif (Possot & Pugsley, 1994, 1997). They are part of a superfamily of ATPases that includes a subfamily of multimeric proteins (often referred to as the VirB11 subfamily) involved in type IV secretion/bacterial conjugation (Cao & Saier, 2001; Krause et al., 2000; Yeo et al., 2000). (3) Predicted integral plasma membrane proteins (T2SA, B, C, F, G, H, I, J, K, L, M, N and O). T2SG through K (the pseudopilins) have N-terminal domains that are similar to those of type IV pilins (Nunn, 1999; Pugsley, 1993a). According to modelling based on the structure of a type IV pilin (Parge et al., 1995), they may mediate subunit interactions that lead to filament formation. T2SO is the prepilin peptidase that cleaves and then N-methylates pseudopilins/pilins at a conserved site N-terminal to the hydrophobic region (Bleves et al., 1998; Nunn & Lory, 1992, 1993; Pugsley, 1993b; Pugsley et al., 2001). T2SL is required for the T2SE protein to associate with the plasma membrane and is stabilized by T2SM (Michel et al., 1998; Possot et al., 2000; Py et al., 1999, 2001; Sandkvist et al., 1995, 1999, 2000).
The precise functions of the plasma membrane protein constituents of the secreton other than T2SO remain largely a matter of conjecture although, in view of the established similarity with the T4P systems, many of them are probably involved in the assembly of a pilus-like structure (see below). T2SC, T2SL and T2SM have relatively large periplasmic domains, leading to the notion that they might form part of a trans-periplasmic complex that controls the opening of the secretin channel and/or recognizes and directs the substrate exoproteins to this secretin (Possot et al., 2000). Nevertheless, all three of these proteins are required for pilus formation by the T2S. Other proteins, such as the T2SE ATPase and/or a proton-channel-forming constituent (possibly T2SF), could be involved in energizing secreton/pseudopilus assembly or exoprotein transport through the outer membrane (Bleves et al., 1999; Letellier et al., 1997; Possot et al., 1997, 2000). This latter suggestion is based, in part, on a superficial analogy between protein secretion and the import of bulky ligands (e.g. siderophores and cyanocobalamin) across the outer membrane of Escherichia coli. The latter process is driven by the proton-motive force (pmf) via an integral plasma membrane protein complex, the TonB/ExbBD complex (Postle & Kadner, 2003). However, it is also possible that ATP hydrolysis plays a direct role in the secretory process, especially in secretons that have two ATPases, like those in Aeromonas species (Schoenhofen et al., 1998).
The long-recognized similarity between the T2S and T4P systems (Hobbs & Mattick, 1993; Pugsley, 1993a) was strengthened by the recent observation that increased expression of the major pseudopilin (T2SG) caused bacteria expressing secreton genes to assemble a pilus composed of this protein (Sauvonnet et al., 2000). The similarities between the T2S and T4F systems extend beyond the pilins/pseudopilins and prepilin peptidase to include T2SD (secretin) (Bitter et al., 1998; Collins et al., 2001; Schmidt et al., 2001) as well as T2SE and T2SF (Nunn et al., 1990). In addition, a pilotin whose sequence is unrelated to that of identified T2S proteins is required for secretin assembly and stability in T4P systems (Drake et al., 1997). However, some secreton components that are needed for pilus assembly by the T2S (e.g. T2SC; Sauvonnet et al., 2000) appear to be absent from the T4P system. Additionally, certain T4P systems have unique components that are required for pilus assembly (see later). These observations probably reflect the ancient separation during divergent evolution of the T2S and T4P systems.
A uniform system of nomenclature for T4P system components remains to be established. In the following sections, we will refer extensively to three relatively well-characterized T4P systems. These are from Pseudomonas aeruginosa (Pil), Neisseria (Pil) and the E. coli EAF plasmid (Bfp) (see footnote 3 in Table 1 for nomenclature of major T4P components in these bacteria). Many T4P systems, including these three, have two or even three ATPases that are related to T2SE. In these bacteria, T4P systems cause ‘twitching’ motility by cycles of pilus extrusion (assembly) and retraction (disassembly) (Merz et al., 2000; Skerker & Berg, 2001). PilT/BfpF have been proposed to be the force-generating proteins (Merz et al., 2000). The pilus might span the outer membrane by passing though the centre of the secretin channel (Wolfgang et al., 2000).
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) are less well characterized than either the T2S or T4P systems. The most prominent component of the Fla system is the flagellin, which shares similarities near its N-terminal end with type IV pilins and pseudopilins and is processed by an enzyme with similar substrate recognition properties to prepilin peptidase (Bardy & Jarrell, 2002). ATPases and TM proteins homologous to those found in T2S and T4P systems of bacteria can also be identified. The numbers of protein constituents in the archaeal flagellar organelles are comparable to those in the bacterial T2S and T4P systems.
In this paper, we identify recognizable homologues in the current databases of the protein constituents of a generic T2S system that includes all secreton components irrespective of the bacterium in which they were identified, the related T4P systems of P. aeruginosa and other Gram-negative bacteria, and the related archaeal flagellar systems of Methanococcus voltae and other archaea. The sequences of the most conserved of these proteins are analysed for structural and phylogenetic attributes, and the conclusions resulting from these analyses are presented. Tables of proteins as well as the corresponding multiple alignments and some supplementary phylogenetic trees can be found on our website (www-biology.ucsd.edu/
msaier/supmat).
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Computer methods |
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TOPABSTRACTOverviewComputer methodsT2S, T4P and Fla...T2S systemsPilins (T2SG, H, I,...T2SE/T4PC/FlaI ATPase phylogenyT2SF/T4PC/FlaJ transmembrane...Multispanning TM protein...ConclusionsREFERENCES |
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) with iterations to convergence was used to screen the databases for homologues of the three systems (T2S, T4P and Fla) that represent the focus of this study. The query sequences were the Pul (K. oxytoca), Pil (P. aeruginosa) and Fla systems (M. voltae) (see our website www-biology.ucsd.edu/msaier/supmat). The homologues found and reported in this review represent those proteins in the databases as of February 2002. (2) The Clustal X program (Thompson et al., 1997) and (3) the TREE program (Feng & Doolittle, 1990) were used for multiple alignment of homologous sequences and derivation of phylogenetic trees with the aid of the BLOSUM30 scoring matrix and the TREEVIEW drawing program (Page, 1996; see Young et al., 1999 for evaluation of these and other relevant programs). (4) The TMPred program (Hofmann & Stoffel, 1993) and (5) the TopPred2 program (von Heijne, 1992) were used for prediction of the integral membrane topologies of individual proteins. (6) The DAS program was used for prediction of secondary structure. (7) The WHAT program (Zhai & Saier, 2001b), with a sliding window of from 7 to 21 residues, was used to simultaneously predict hydropathy, amphipathicity (angle of 100° for 
-helix; angle of 180° for 
-strand), topology and secondary structure of individual proteins. (8) The AveHAS program (Zhai & Saier, 2001a) was used for plotting mean hydropathy, similarity and amphipathicity as a function of alignment position in the multiple alignments. These programs are available on our ‘software’ and ‘biotools' websites (http://www-biology.ucsd.edu/msaier/transport/ and http://www-biology.ucsd.edu/yzhai/biotools.html, respectively). (9) The GAP program was used to establish homology (Devereux et al., 1984; Saier, 1994).
In this paper, we use the WHAT (Zhai & Saier, 2001b) and AveHAS (Zhai & Saier, 2001a) programs in combination to predict transmembrane segments (TMSs). These programs combine several established programs to make structural predictions about transmembrane proteins. For example, the WHAT program examines individual proteins, using JNET (Cuff et al., 1998) and MEMSAT (Jones et al., 1994) for secondary structure and transmembrane topology prediction, respectively. Both of these programs are among the best available for these purposes. The AveHAS program first generates a multiple alignment for a collection of homologous sequences (Thompson et al., 1997) and then averages (1) hydropathy, (2) amphipathicity and (3) similarity plots to provide structural information that is much more reliable than possible when evaluating a single protein sequence (Zhai & Saier, 2001a). Transmembrane -strands can thus be accurately predicted because they exhibit (1) predicted -structure using JNET, (2) increased hydrophobicity, relative to other portions of the polypeptide chain, and (3) increased amphipathicity when the angle is set at 180° as is appropriate for -strands (Le et al., 1999; Zhai & Saier, 2002). This method predicts transmembrane -strands with about 80 % accuracy.
Supplementary material which can be found on our website (www-biology.ucsd.edu/msaier/supmat) includes: (1) tables of all homologues of the different protein types included in this study, (2) the multiple alignments for these homologues, (3) the phylogenetic trees for these same families of proteins, (4) a 16S rRNA phylogenetic tree for all bacteria from which proteins included in this study were derived and (5) a tabulation of known protein constituents of all T2S systems for which homologues of all or most constituents of the secreton have been identified.
Complementation of the pulF deletion in the complete pul gene cluster was carried out using pBR322 derivatives by homologous genes under lacp control in a compatible plasmid, as described by Possot et al. (2000). gspF was amplified using specific primers that incorporated restriction endonuclease cleavage sites for cloning, as previously described (Possot et al., 2000).
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T2S, T4P and Fla system constituents |
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TOPABSTRACTOverviewComputer methodsT2S, T4P and Fla...T2S systemsPilins (T2SG, H, I,...T2SE/T4PC/FlaI ATPase phylogenyT2SF/T4PC/FlaJ transmembrane...Multispanning TM protein...ConclusionsREFERENCES |
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presents a summary of the protein constituents of representative T2S, T4P and Fla systems. Four of the proteins in the T2S and T4P systems are demonstrably homologous. These are (1) the prepilin peptidase/N-methyltransferase, (2) the ATPase, (3) the secretin and (4) the multispanning transmembrane (TM) protein. Three of these, the ATPase, the TM protein and the prepilin peptidase, have been identified in Fla systems. The first two are clearly homologous to their T2S/T4P counterparts (Patenge et al., 2001; Thomas & Jarrell, 2001). As indicated in Tables 2 (T2S) and 3 (T4P), these are the constituents of both the T2S and T4P systems with the largest numbers of recognizable homologues. Archaeal FlaK shows only weak similarity to established bacterial prepilin peptidases (Bardy & Jarrell, 2002). Otherwise, homology between constituents could not be demonstrated.
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T2S systems |
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TOPABSTRACTOverviewComputer methodsT2S, T4P and Fla...T2S systemsPilins (T2SG, H, I,...T2SE/T4PC/FlaI ATPase phylogenyT2SF/T4PC/FlaJ transmembrane...Multispanning TM protein...ConclusionsREFERENCES |
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) that represent some of the best-defined secreton constituents from functional standpoints (Fig. 1a–d). Fig. 1(a) presents the outer-membrane secretins (T2SD); Fig. 1(b) presents the cytoplasmic ATPases (T2SE); Fig. 1(c) presents the putative multispanning TM plasma membrane proteins (T2SF); and Fig. 1(d) presents the largest of the prepilin subunits (T2SK). Examination of these four trees reveals that, within experimental error, they all have the same configuration. Thus, proceeding from the top of these trees in the clockwise direction: (1) The same 
-proteobacterial proteins always cluster together at the top of the trees. (2) Next, two P. aeruginosa proteins cluster together. (3) These are followed by the Caulobacter homologue, Ccr. (4) The Aquifex homologues, Aae, are next (displaced in Fig. 1d). (5) These are followed by the closely related Xylella and Xanthomonas proteins. (6) The chlamydial clusters (absent in the tree shown in Fig. 1d; see Table 3) are together in Fig. 1(a–c) in the expected positions. (7) The loose cluster including the P. aeruginosa Hxc system constituents together with the tightly clustering Bce and Bps proteins are next observed. (8) Finally, the Lpn proteins can be found just to the left of the -proteobacterial clusters. The similar relative configurations and branch lengths exhibited in these four trees suggest that the constituents of these secreton systems have evolved in parallel from a single common ancestral system without shuffling of constituents between systems throughout most of evolutionary history.
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), are absent from several species of bacteria that produce T2SD-type secretins. A complete phylogenetic tree of PulS homologues cannot be constructed because of the limited number of known examples, possibly due to their poor conservation which renders them non-identifiable by sequence comparisons (see www-biology.ucsd.edu/msaier/supmat). One of the difficulties in determining whether PulS-like pilotins exist in other T2S systems is that the C-terminal regions of the secretins, to which they probably bind, are among the least conserved regions of these proteins, suggesting that the pilotins may also be poorly conserved. Other proteins, notably T2SA and T2SB (Ast et al., 2002; Condemine & Shevchik, 2000), have been proposed to perform PulS-like functions. Interestingly, E. coli K-12 has genes for T2SA and T2SB proteins within its secreton gene cluster (Francetic et al., 2000) as well as an unlinked gene, yacC, with limited similarity to pulS.
Examination of the phylogenetic trees for the other constituents of these systems (see Table 3 and www-biology.ucsd.edu/msaier/supmat) revealed that they exhibit essentially the same configurations and relative branch lengths within experimental error. Thus we conclude that secreton systems have probably evolved by whole gene cluster duplication and by speciation without appreciable exchange of constituents between systems.
The T2SC family of proteins deserves special mention for two reasons. First, the Xanthomonas campestris gene designated xpsN (Lee et al., 2000, 2001) was clearly misnamed, since it is similar to genes for T2SC proteins (and, therefore, should be called xpsC) and is unrelated to genes for T2SN proteins. This allows one to rationalize recent data showing that XpsC(N) is essential and interacts with proteins D, M and/or L (Lee et al., 2000, 2001). PulN is not essential while T2SC proteins are essential and interact with T2SD, L and/or M proteins (Bleves et al., 1999; Possot et al., 2000). Second, the T2SC family of proteins can be divided into several distinct clusters depending on whether they possess (1) a coiled-coil segment, (2) a PDZ-type structure (Gerard-Vincent et al., 2002; Pallen & Ponting, 1997) or (3) neither, close to the C-terminal ends of the proteins (Fig. 2). It is interesting to note that at least one member of the last class, HxcC from P. aeruginosa, is apparently functional (Ball et al., 2002), indicating that neither the coiled-coil domain nor the PDZ domain is essential for secretion. Furthermore, the PDZ domain, predicted to exist in PulC (Pallen & Ponting, 1997), is also predicted to be a coiled-coil by the algorithms we used (Fig. 2).
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Pilins (T2SG, H, I, J and K) and the pilin processing enzyme (T2SO) |
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TOPABSTRACTOverviewComputer methodsT2S, T4P and Fla...T2S systemsPilins (T2SG, H, I,...T2SE/T4PC/FlaI ATPase phylogenyT2SF/T4PC/FlaJ transmembrane...Multispanning TM protein...ConclusionsREFERENCES |
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, homologues of the five prepilin constituents of T2S systems (T2SG, H, I, J and K) are found in similar numbers (18–25). Phylogenetic trees for these five families of homologues resembled those shown in Fig. 1(a–c) although only data for 20 T2SK homologues are shown in Fig. 1(d). (The other trees can be viewed on our website at www-biology.ucsd.edu/msaier/supmat). The tree for T2SO homologues, prepilin peptidase/N-methyltransferase that cleave and methylate prepseudopilins, revealed that they also cluster as shown in Fig. 1(a–d), within experimental error (see www-biology.ucsd.edu/msaier/supmat). Thus sequence-related pseudopilins from different bacteria always fell into the same clusters, irrespective of the class of pseudopilin (G, H, I, J or K) analysed. In addition to the authenticated Gram-negative bacterial T2SO proteins, members of the prepilin peptidase family are derived from both high- and low-G+C Gram-positive bacteria as well as very diverse Gram-negative bacteria (e.g. Chlorobium, Deinococcus, Synechocystis and Thermatoga) and might have different functions with related or unrelated substrate specificities. Moreover, a single organism may have multiple paralogues. For example, seven have been identified in E. coli, all very divergent in sequence, branching from points near the centre of the phylogenetic tree. They must have resulted from early gene duplication events or possibly were acquired by lateral transfer. On the other hand, only one prepilin peptidase gene is present in P. aeruginosa [PilD/XcpO(A)] and many other bacteria with fully sequenced genomes.
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T2SE/T4PC/FlaI ATPase phylogeny |
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TOPABSTRACTOverviewComputer methodsT2S, T4P and Fla...T2S systemsPilins (T2SG, H, I,...T2SE/T4PC/FlaI ATPase phylogenyT2SF/T4PC/FlaJ transmembrane...Multispanning TM protein...ConclusionsREFERENCES |
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msaier/supmat, is presented in Fig. 3. This figure represents an expansion of two previously published trees exhibiting similar overall topology (Cao & Saier, 2001; Planet et al., 2001). The tree includes 12 well-defined clusters. Cluster 1 has five subclusters designated A–E. All of these proteins likely function as secreton ATPases. Clusters 2, 3, 4, 5 and 6 include sequence divergent ATPases, all possibly concerned with bacterial pilus assembly/biogenesis/function. Clusters 7 and 8 A–C are all archaeal proteins; cluster 8B includes the recognized archaeal flagellar ATPases; clusters 9A–E and 10 are type IV protein secretion/conjugation ATPases (see Cao & Saier, 2001 and the Overview). Cluster 11 is a mixture of Gram-negative and high-G+C Gram-positive bacterial homologues including the TadA protein involved in tight adherence to surfaces in Actinobacillus actinomycetemcomitans (Kachlany et al., 2000). Cluster 12 consists of competence-related proteins from low-G+C Gram-positive bacteria. Thus the primary clusters correspond to specific functional types, and most clusters are restricted to a particular type of organism (i.e. Gram-negative bacteria, Gram-positive bacteria or archaea). In many cases, the functions of the proteins are not known. Eukaryotic homologues were not found.
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summarizes the size differences (averaged, ± standard deviations) observed for the clusters depicted in Fig. 3. The proposed functions of these clustered proteins, usually based on functional assignments for just a few homologues, and their classification according to the organisms in which they are found, are also presented in Table 4. Of greatest interest is the fact that most of the 12 clusters (subdivided in the cases of clusters 1, 8 and 9) consist of proteins with a characteristic and very restricted size range. The one exception is cluster 6, which consists of two sequence-divergent E. coli proteins that in actuality branch from points near the centre of the tree. It is therefore clear that both protein size and function consistently correlate with phylogeny, and often with organismal type as well.
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were examined to determine if the phylogenies of the proteins within any of the clusters follow those of the 16S rRNAs. Most of these clusters include two or more paralogues from a single organism, and, in general, protein phylogeny does not follow that of the 16S rRNAs (see the online version of this review at http://mic.sgmjournals.org for the bacterial 16S rRNA tree). For example, in the secreton ATPases of cluster 1A, two paralogues are each observed for E. coli and P. aeruginosa. However, in clusters 7, 8A and 8B, each cluster displays only one protein from each archaeon with a fully sequenced genome, and these follow the phylogenies of the organisms fairly closely (see Fig. 4 for the archaeal 16S rRNA tree). Thus, with the possible exception of the archaeal homologues, no cluster can be considered to consist exclusively of orthologues. We predict that horizontal transfer as well as late gene duplication events were responsible for the configurations of the portions of the tree that include bacterial homologues.
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T2SF/T4PC/FlaJ transmembrane (TM) protein phylogeny |
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TOPABSTRACTOverviewComputer methodsT2S, T4P and Fla...T2S systemsPilins (T2SG, H, I,...T2SE/T4PC/FlaI ATPase phylogenyT2SF/T4PC/FlaJ transmembrane...Multispanning TM protein...ConclusionsREFERENCES |
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. The proteins fall into seven clusters which are labelled according to the probable corresponding cluster designations presented in Fig. 3 for the ATPases. Cluster 1 includes almost all currently recognized secreton constituents, and most of these proteins are derived from proteobacteria of the -, - and -subclasses (cluster 1A). Paralogues in this cluster are found only for E. coli and P. aeruginosa as was observed for the ATPases (Fig. 3). Such observations suggest that each ATPase functions with a TM protein. The chlamydial proteins (cluster 1B) and the Xylella fastidiosa and Xanthomonas campestris proteins (cluster 1C) compose two additional but divergent groups, as observed for the ATPases shown in Fig. 3. Additional proteins included in cluster 1 are sequence divergent proteins from Vibrio cholerae and P. aeruginosa, both -proteobacteria (cluster E), and several proteins from other phylogenetically divergent bacteria (Aquifex, Thermatoga, Deinococcus and Synechocystis) (clusters D1 and D2). Corresponding proteins are found in cluster 1 of Fig. 3.
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-subclass. Progressing around the tree in the clockwise direction, cluster 12 consists of competence proteins from low-G+C Gram-positive bacteria. Corresponding ATPases are found in cluster 12 of Fig. 3. Cluster 4 consists of a distinct group of -proteobacterial homologues, probably all concerned with pilus function or biogenesis. Clusters 11A and 11B represent two other primarily proteobacterial clusters with distant homologues from the high-G+C Gram-positive bacterium Streptomyces coelicolor. Included within these two clusters are the TadB and TadC homologues from the disease-causing Gram-negative bacterium Actinobacillus actinomycetemcomitans. Both of these two proteins are concerned with tight adherence to surfaces (Kachlany et al., 2000). The corresponding ATPase, TadA, is found in cluster 11 in Fig. 3. It is interesting to note that the two integral membrane proteins TadB and TadC apparently function with a single ATPase, TadA. This observation might suggest that these integral membrane constituents function as homo- or hetero-oligomeric (possibly dimeric) structures. A dimeric structure would be in agreement with the fact that the archaeal homologues are internally duplicated proteins, twice as large as the bacterial homologues with approximately twice as many TMSs (see next section).
Cluster 8 includes proteins derived exclusively from archaea. The functions of cluster 8A and cluster 8D proteins are unknown, and they may or may not have counterparts in Fig. 3. Cluster 8A in Fig. 3 includes only one protein per organism, except for Archaeoglobus fulgidus where three paralogues are found. However, in cluster 8A of Fig. 5, two sets of homologues are found for most represented organisms. Most of the proteins in cluster 8B are probably constituents of archaeal flagellar systems. These proteins are represented only once per organism, have counterparts in Fig. 3, and exhibit phylogenetic relationships that reflect those of the 16S rRNAs (compare Figs 3, 4 and 5). These proteins are therefore likely to be orthologues with a single ATPase per TM protein. It should be noted that many clusters of ATPases found in Fig. 3 are not represented in Fig. 5. These ATPases probably function in a process and by a mechanism that is independent of a multispanning TM protein homologue. Alternatively, they may act with multispanning TM proteins that are too divergent in sequence for us to recognize.
The data summarized in Table 5 reveal that, as for the ATPases, each major cluster of multispanning TM proteins exhibits its own characteristic size range. However, there is no direct or inverse size relationship between the ATPases tabulated in Table 4 and the membrane proteins tabulated in the corresponding clusters in Table 5.
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Multispanning TM protein topologies and the occurrence of internal repeats |
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TOPABSTRACTOverviewComputer methodsT2S, T4P and Fla...T2S systemsPilins (T2SG, H, I,...T2SE/T4PC/FlaI ATPase phylogenyT2SF/T4PC/FlaJ transmembrane...Multispanning TM protein...ConclusionsREFERENCES |
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), indicates three TM segments, as predicted by most hydrophobicity algorithms. However, it should be noted that the second predicted hydrophobic segment of these proteins is rather long and that some algorithms predict that it is extended to form two TM segments (TMSs), making four in all. One such protein is GspF from E. coli K-12 (GspFEco), which was predicted to have four TMSs by all algorithms tested. The distribution of positively charged amino acids at each end of the hydrophobic segments beyond TMS1 did not allow us to predict their orientation in the membrane according to the ‘positive-inside rule’ (von Heijne & Gavel, 1988). Furthermore, the gspF gene exhibited very low complementation of a deletion of the pulF gene in the pul gene cluster (10–25 % restoration of pullulanase secretion in four independent experiments; data not shown) compared with pulFKox and outFEch (>80 % restoration under identical conditions; Possot et al., 2000). Whether or not GspFEco differs from all other T2SF proteins remains to be determined. However, if GspFEco topology is partially reversed compared to that of other T2SF proteins, a well-conserved segment predicted to be cytoplasmic in OutF (Thomas et al., 1997) would be located in the periplasm in GspFEco, which seems unlikely.
The topology of T2SF homologues in the T4P system is equally unclear. The experimentally determined protein topology of one member of this group, the E. coli protein BfpE, a member of the PilR cluster of pilus-related proteins in Fig. 5, gives four TMSs, with TMSs 2 and 3 being nearly contiguous (Blank & Donnenberg, 2001). Indeed, most algorithms predict the same four TMSs in this protein (not shown). However, as with GspFEco, the topology of BfpE cannot be predicted from the positive-inside rule. Furthermore, most algorithms predict three TMSs for the closely related V. cholerae protein, TcpE, and three to five TMSs for other proteins in the PilR cluster. Topological predictions for the PilC/G cluster also indicate three and five TMSs, with three TMSs most frequently predicted for the archetypal protein of this cluster, P. aeruginosa PilC (Fig. 6). Once again, a large domain that is predicted to be periplasmic by the 3 TMS model is predicted to be cytoplasmic by the 4 TMS model, as discussed above for T2SF proteins. Although not as highly conserved as the corresponding segment of the T2SF proteins, this region of almost all proteins under consideration (including the T2SF proteins) contains several highly or absolutely conserved residues. The possible exceptions are all in the PilR cluster, including TcpE and BfpE, in which only some of these highly conserved residues are present. Furthermore, these regions of TcpE and BfpE are almost totally unrelated, which is in marked contrast, for example, to proteins in the T2SF cluster. Therefore, in contrast to GspFEco noted above, it is quite conceivable that BfpE does have a topology different from the predicted 3 TM proteins such as PilC and TcpE.
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The GAP program (Devereux et al., 1984) predicted that regions within the first halves of the TM proteins are homologous to regions in the second halves. One such alignment for P. aeruginosa PilC is shown in Fig. 7(a). This 91 residue binary comparison shows 33 % identity and 55 % similarity with an e value of 3x10-8. Comparison scores of 23–25 SD for these portions of the two halves of pilc and of 10 SD for corresponding portions of the two halves of PulF of K. pneumoniae were obtained. These values are sufficient to establish that the two halves of these proteins arose from a common origin, probably by an internal gene duplication event. Interestingly, part of this duplicated region includes a diagnostic motif for members of the T2SF-PilC/G-PilR protein clusters, while other residues are well conserved in all clusters (Fig. 7).
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. Nine peaks of hydropathy (black bars) are evident. Peaks 1–4 appear similar in arrangement to peaks 5–8. Indeed, comparison scores using the GAP program suggested that these proteins also arose by an internal duplication event since, if TMS9 is omitted, the first halves appear to be homologous to the second halves. Thus, in comparing the most conserved portions of the first halves of these proteins with those of the second halves, comparison scores of up to 8 SD were obtained. A value of 8 SD corresponds to a probability of 10-15 that this degree of similarity arose by chance. This value is strongly suggestive of homology. Alignments of a bacterial TM protein (C372 of Agrobacterium tumefaciens) with the two halves of an archaeal TM protein (FlaJ of M. voltae) are shown in Fig. 7(b, c). These alignments were generated using the PSI-BLAST program with two iterations (Altschul et al., 1997). The conserved residues in each of the archaeal halves thus represent a matrix of conserved motifs between each of the archaeal halves and the bacterial protein.
Based on these results, we suggest that the smaller bacterial proteins arose by one internal gene duplication event, and that a second internal gene duplication event gave rise to the larger archaeal homologues. Judging from the relative degrees of sequence similarity, however, the bacterial duplication event(s) probably occurred after the archaeal duplication event(s). This fact suggests that these duplication events have occurred more than once during the evolution of this family of TM proteins. A similar situation has been observed for other transmembrane protein families (Tseng et al., 1999). It should be noted that the topologies of these proteins can not be deduced with certainty from the hydropathy plots alone.
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Conclusions |
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TOPABSTRACTOverviewComputer methodsT2S, T4P and Fla...T2S systemsPilins (T2SG, H, I,...T2SE/T4PC/FlaI ATPase phylogenyT2SF/T4PC/FlaJ transmembrane...Multispanning TM protein...ConclusionsREFERENCES |
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). This conclusion suggests the importance of strict protein–protein interactions for the maintenance of proper function. Similar conclusions have been reached for other protein secretion systems as well as other types of multicomponent transporters (Cao & Saier, 2001; Kuan et al., 1995; Nguyen et al., 2000). The implications of these findings have recently been discussed (Saier, 2003a). (3) The protein constituents of the various systems analysed have undergone sequence divergence at different rates, where the ATPases and multispanning TM proteins are among the largest and were the most slowly diverging. These facts allowed the most reliable phylogenetic analyses with these constituents and also facilitated homologue identification. (4) Between T2S and T4P systems, four proteins [the ATPases (T2SE homologues), the multispanning TM proteins (T2SF homologues), the secretins (T2SD homologues) and the prepilin peptidases (T2SO homologues)] proved to be demonstrably homologous. However, only three of these constituents proved to be demonstrably homologous between these bacterial systems and the archaeal Fla systems. Secretins are apparently lacking in the archaeal Fla systems as expected since archaea lack an outer membrane. (5) When the two most conserved families, those of the ATPases and the multispanning TM proteins, were analysed phylogenetically, it became clear that there is not a one-to-one correspondence between these two constituents. Some phylogenetic clusters of the ATPases appear to function with TM proteins while others either do not, or they utilize a TM protein that is too divergent in sequence to be recognized using the search tools employed. Nevertheless, we could identify clusters of ATPase that consistently function with TM proteins in both bacteria and archaea. (6) Size comparisons of proteins within both the ATPase clusters and the multispanning TM protein clusters (Tables 4 and 5) revealed that each phylogenetic cluster exhibits a characteristic size range. However, the sizes of the ATPase and TM protein homologues do not correlate with each other either directly or inversely. We propose that the members of each cluster serve a unified function. This last suggestion is substantiated by limited experimental data (see Tables 4 and 5). (7) Information about the evolution of the multispanning TM proteins was forthcoming. Thus the bacterial homologues clearly arose by an internal gene duplication event, and the same is probably true of the archaeal homologues. However, these duplication events were not the same, suggesting that gene duplication occurred at least twice during the evolution of this protein family. The same conclusion has been reached for some integral membrane transport protein families but not for others (Kuan & Saier, 1993; Pao et al., 1998; Saier, 1999a, b, 2000, 2001a, b, 2003b). This report presents sequence comparisons that allow us to establish relationships between several of the protein constituents of the T2S, T4P and Fla systems of bacteria and archaea. Further analyses will be required to establish the functional significance of many of the provocative observations made here.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTOverviewComputer methodsT2S, T4P and Fla...T2S systemsPilins (T2SG, H, I,...T2SE/T4PC/FlaI ATPase phylogenyT2SF/T4PC/FlaJ transmembrane...Multispanning TM protein...ConclusionsREFERENCES |
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D. J. VanDyke, J. Wu, S. Y. M. Ng, M. Kanbe, B. Chaban, S.-I. Aizawa, and K. F. Jarrell Identification of a Putative Acetyltransferase Gene, MMP0350, Which Affects Proper Assembly of both Flagella and Pili in the Archaeon Methanococcus maripaludis J. Bacteriol., August 1, 2008; 190(15): 5300 - 5307. [Abstract] [Full Text] [PDF]
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X. Zogaj, S. Chakraborty, J. Liu, D. G. Thanassi, and K. E. Klose Characterization of the Francisella tularensis subsp. novicida type IV pilus Microbiology, July 1, 2008; 154(7): 2139 - 2150. [Abstract] [Full Text] [PDF]
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S. Chakraborty, M. Monfett, T. M. Maier, J. L. Benach, D. W. Frank, and D. G. Thanassi Type IV Pili in Francisella tularensis: Roles of pilF and pilT in Fiber Assembly, Host Cell Adherence, and Virulence Infect. Immun., July 1, 2008; 76(7): 2852 - 2861. [Abstract] [Full Text] [PDF]
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K. E. Kram, G. A. Hovel-Miner, M. Tomich, and D. H. Figurski Transcriptional Regulation of the tad Locus in Aggregatibacter actinomycetemcomitans: a Termination Cascade J. Bacteriol., June 1, 2008; 190(11): 3859 - 3868. [Abstract] [Full Text] [PDF]
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X. Han, R. M. Kennan, J. K. Davies, L. A. Reddacliff, O. P. Dhungyel, R. J. Whittington, L. Turnbull, C. B. Whitchurch, and J. I. Rood Twitching Motility Is Essential for Virulence in Dichelobacter nodosus J. Bacteriol., May 1, 2008; 190(9): 3323 - 3335. [Abstract] [Full Text] [PDF]
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V. Jakovljevic, S. Leonardy, M. Hoppert, and L. Sogaard-Andersen PilB and PilT Are ATPases Acting Antagonistically in Type IV Pilus Function in Myxococcus xanthus J. Bacteriol., April 1, 2008; 190(7): 2411 - 2421. [Abstract] [Full Text] [PDF]
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J. M. Boyd, A. Dacanay, L. C. Knickle, A. Touhami, L. L. Brown, M. H. Jericho, S. C. Johnson, and M. Reith Contribution of Type IV Pili to the Virulence of Aeromonas salmonicida subsp. salmonicida in Atlantic Salmon (Salmo salar L.) Infect. Immun., April 1, 2008; 76(4): 1445 - 1455. [Abstract] [Full Text] [PDF]
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E. Shimoda, T. Muto, T. Horiuchi, N. Furuya, and T. Komano Novel Class of Mutations of pilS Mutants, Encoding Plasmid R64 Type IV Prepilin: Interface of PilS-PilV Interactions J. Bacteriol., February 15, 2008; 190(4): 1202 - 1208. [Abstract] [Full Text] [PDF]
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S. A. Clock, P. J. Planet, B. A. Perez, and D. H. Figurski Outer Membrane Components of the Tad (Tight Adherence) Secreton of Aggregatibacter actinomycetemcomitans J. Bacteriol., February 1, 2008; 190(3): 980 - 990. [Abstract] [Full Text] [PDF]
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 K. S. Auernik, Y. Maezato, P. H. Blum, and R. M. Kelly The Genome Sequence of the Metal-Mobilizing, Extremely Thermoacidophilic Archaeon Metallosphaera sedula Provides Insights into Bioleaching-Associated Metabolism Appl. Envir. Microbiol., February 1, 2008; 74(3): 682 - 692. [Abstract] [Full Text] [PDF]
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J. Zupan, C. A. Hackworth, J. Aguilar, D. Ward, and P. Zambryski VirB1* Promotes T-Pilus Formation in the vir-Type IV Secretion System of Agrobacterium tumefaciens J. Bacteriol., September 15, 2007; 189(18): 6551 - 6563. [Abstract] [Full Text] [PDF]
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P. S. Duggan, P. Gottardello, and D. G. Adams Molecular Analysis of Genes in Nostoc punctiforme Involved in Pilus Biogenesis and Plant Infection J. Bacteriol., June 15, 2007; 189(12): 4547 - 4551. [Abstract] [Full Text] [PDF]
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Z. Szabo, M. Sani, M. Groeneveld, B. Zolghadr, J. Schelert, S.-V. Albers, P. Blum, E. J. Boekema, and A. J. M. Driessen Flagellar Motility and Structure in the Hyperthermoacidophilic Archaeon Sulfolobus solfataricus J. Bacteriol., June 1, 2007; 189(11): 4305 - 4309. [Abstract] [Full Text] [PDF]
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M. Podar, C. B. Abulencia, M. Walcher, D. Hutchison, K. Zengler, J. A. Garcia, T. Holland, D. Cotton, L. Hauser, and M. Keller Targeted Access to the Genomes of Low-Abundance Organisms in Complex Microbial Communities Appl. Envir. Microbiol., May 15, 2007; 73(10): 3205 - 3214. [Abstract] [Full Text] [PDF]
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J. Arts, A. de Groot, G. Ball, E. Durand, M. E. Khattabi, A. Filloux, J. Tommassen, and M. Koster Interaction domains in the Pseudomonas aeruginosa type II secretory apparatus component XcpS (GspF) Microbiology, May 1, 2007; 153(5): 1582 - 1592. [Abstract] [Full Text] [PDF]
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A. Lykidis, K. Mavromatis, N. Ivanova, I. Anderson, M. Land, G. DiBartolo, M. Martinez, A. Lapidus, S. Lucas, A. Copeland, et al. Genome Sequence and Analysis of the Soil Cellulolytic Actinomycete Thermobifida fusca YX J. Bacteriol., March 15, 2007; 189(6): 2477 - 2486. [Abstract] [Full Text] [PDF]
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J. Arts, R. van Boxtel, A. Filloux, J. Tommassen, and M. Koster Export of the Pseudopilin XcpT of the Pseudomonas aeruginosa Type II Secretion System via the Signal Recognition Particle-Sec Pathway J. Bacteriol., March 1, 2007; 189(5): 2069 - 2076. [Abstract] [Full Text] [PDF]
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Z. Szabo, A. O. Stahl, S.-V. Albers, J. C. Kissinger, A. J. M. Driessen, and M. Pohlschroder Identification of Diverse Archaeal Proteins with Class III Signal Peptides Cleaved by Distinct Archaeal Prepilin Peptidases J. Bacteriol., February 1, 2007; 189(3): 772 - 778. [Abstract] [Full Text] [PDF]
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S. A. Frye, R. Assalkhou, R. F. Collins, R. C. Ford, C. Petersson, J. P. Derrick, and T. Tonjum Topology of the outer-membrane secretin PilQ from Neisseria meningitidis Microbiology, December 1, 2006; 152(12): 3751 - 3764. [Abstract] [Full Text] [PDF]
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F. H. Login and V. E. Shevchik The Single Transmembrane Segment Drives Self-assembly of OutC and the Formation of a Functional Type II Secretion System in Erwinia chrysanthemi J. Biol. Chem., November 3, 2006; 281(44): 33152 - 33162. [Abstract] [Full Text] [PDF]
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M. Tomich, D. H. Fine, and D. H. Figurski The TadV Protein of Actinobacillus actinomycetemcomitans Is a Novel Aspartic Acid Prepilin Peptidase Required for Maturation of the Flp1 Pilin and TadE and TadF Pseudopilins. J. Bacteriol., October 1, 2006; 188(19): 6899 - 6914. [Abstract] [Full Text] [PDF]
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C. Saarimaa, M. Peltola, M. Raulio, T. R. Neu, M. S. Salkinoja-Salonen, and P. Neubauer Characterization of Adhesion Threads of Deinococcus geothermalis as Type IV Pili. J. Bacteriol., October 1, 2006; 188(19): 7016 - 7021. [Abstract] [Full Text] [PDF]
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S. DebRoy, V. Aragon, S. Kurtz, and N. P. Cianciotto Legionella pneumophila Mip, a Surface-Exposed Peptidylproline cis-trans-Isomerase, Promotes the Presence of Phospholipase C-Like Activity in Culture Supernatants Infect. Immun., September 1, 2006; 74(9): 5152 - 5160. [Abstract] [Full Text] [PDF]
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J. Andrzejewska, S. K. Lee, P. Olbermann, N. Lotzing, E. Katzowitsch, B. Linz, M. Achtman, C. I. Kado, S. Suerbaum, and C. Josenhans Characterization of the Pilin Ortholog of the Helicobacter pylori Type IV cag Pathogenicity Apparatus, a Surface-Associated Protein Expressed during Infection. J. Bacteriol., August 1, 2006; 188(16): 5865 - 5877. [Abstract] [Full Text] [PDF]
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A. Daniel, A. Singh, L. J. Crowther, P. J. Fernandes, W. Schreiber, and M. S. Donnenberg Interaction and localization studies of enteropathogenic Escherichia coli type IV bundle-forming pilus outer membrane components. Microbiology, August 1, 2006; 152(Pt 8): 2405 - 2420. [Abstract] [Full Text] [PDF]
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S. de Bentzmann, M. Aurouze, G. Ball, and A. Filloux FppA, a Novel Pseudomonas aeruginosa Prepilin Peptidase Involved in Assembly of Type IVb Pili J. Bacteriol., July 1, 2006; 188(13): 4851 - 4860. [Abstract] [Full Text] [PDF]
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P. R. Chowdhury and J. A. Heinemann The General Secretory Pathway of Burkholderia gladioli pv. agaricicola BG164R Is Necessary for Cavity Disease in White Button Mushrooms. Appl. Envir. Microbiol., May 1, 2006; 72(5): 3558 - 3565. [Abstract] [Full Text] [PDF]
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N. Buddelmeijer, O. Francetic, and A. P. Pugsley Green Fluorescent Chimeras Indicate Nonpolar Localization of Pullulanase Secreton Components PulL and PulM. J. Bacteriol., April 1, 2006; 188(8): 2928 - 2935. [Abstract] [Full Text] [PDF]
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 P. Youderian and P. L. Hartzell Transposon Insertions of magellan-4 That Impair Social Gliding Motility in Myxococcus xanthus Genetics, March 1, 2006; 172(3): 1397 - 1410. [Abstract] [Full Text] [PDF]
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M. Kostakioti, C. L. Newman, D. G. Thanassi, and C. Stathopoulos Mechanisms of Protein Export across the Bacterial Outer Membrane J. Bacteriol., July 1, 2005; 187(13): 4306 - 4314. [Full Text] [PDF]
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L. J. Crowther, A. Yamagata, L. Craig, J. A. Tainer, and M. S. Donnenberg The ATPase Activity of BfpD Is Greatly Enhanced by Zinc and Allosteric Interactions with Other Bfp Proteins J. Biol. Chem., July 1, 2005; 280(26): 24839 - 24848. [Abstract] [Full Text] [PDF]
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O. Rossier and N. P. Cianciotto The Legionella pneumophila tatB Gene Facilitates Secretion of Phospholipase C, Growth under Iron-Limiting Conditions, and Intracellular Infection Infect. Immun., April 1, 2005; 73(4): 2020 - 2032. [Abstract] [Full Text] [PDF]
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S.-V. Albers and A. J. M. Driessen Analysis of ATPases of putative secretion operons in the thermoacidophilic archaeon Sulfolobus solfataricus Microbiology, March 1, 2005; 151(3): 763 - 773. [Abstract] [Full Text] [PDF]
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