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A Versatile Class of Cell Surface Directional Motors Gives Rise to Gliding Motility and Sporulation in Myxococcus xanthus
research-article
Author(s):
Morgane Wartel
1 ,
Adrien Ducret
1
,
2 ,
Shashi Thutupalli
3 ,
Fabian Czerwinski
3 ,
Anne-Valérie Le Gall
1 ,
Emilia M. F. Mauriello
1 ,
Ptissam Bergam
4 ,
Yves V. Brun
2 ,
Joshua Shaevitz
3 ,
Tâm Mignot
1
,
*
Publication date (Electronic):
10 December 2013
Journal:
PLoS Biology
Publisher:
Public Library of Science
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Abstract
Abstract
Eukaryotic cells utilize an arsenal of processive transport systems to deliver macromolecules to specific subcellular sites. In prokaryotes, such transport mechanisms have only been shown to mediate gliding motility, a form of microbial surface translocation. Here, we show that the motility function of the Myxococcus xanthus Agl-Glt machinery results from the recent specialization of a versatile class of bacterial transporters. Specifically, we demonstrate that the Agl motility motor is modular and dissociates from the rest of the gliding machinery (the Glt complex) to bind the newly expressed Nfs complex, a close Glt paralogue, during sporulation. Following this association, the Agl system transports Nfs proteins directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by Agl-Nfs ensures its distribution around the spore. Thus, the Agl-Glt/Nfs machineries may constitute a novel class of directional bacterial surface transporters that can be diversified to specific tasks depending on the cognate cargo and machinery-specific accessories.
Author Summary
Many living cells use processive cytoskeletal motors to transport proteins and subcellular organelles to specific subcellular sites. In bacteria, this type of transport has yet to be identified and it is generally thought that random protein collisions underlie most biochemical processes. In recent years, our view of the bacterial cell was changed by the discovery of subcellular compartmentalization and a cytoskeleton, suggesting that processive motors might also operate in prokaryotes. We previously characterized a mechanism of intracellular transport that drives cell motility across solid surfaces in the gram-negative bacterium Myxococcus xanthus. Since the transport apparatus was also found in bacterial species that do not move on surfaces, we postulated that intracellular transport underlies other cellular processes in bacteria. Indeed, we show here that the Myxococcus motility motor can be adapted to transport sporulation-specific proteins around the nascent spore surface. Because the transported proteins are linked to the main spore coat, this motion assists the assembly of a protective spore coat. In conclusion, the Myxococcus motility/sporulation transport machinery defines an emerging class of versatile transport systems, suggesting that processive transport has been overlooked and may well orchestrate many processes in bacteria.
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Most cited references27
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The Non-Flagellar Type III Secretion System Evolved from the Bacterial Flagellum and Diversified into Host-Cell Adapted Systems
Sophie Abby, Eduardo P. C. Rocha (2012)
Introduction Microbial protein secretion facilitates environmental exploitation and manipulation [1], [2]. The most frequent bacterial motility machinery, the flagellum, uses a type 3 secretion system (T3SS) to secrete its extracellular components. The non-flagellar type 3 protein secretion system (NF-T3SS), often named injectisome, is homologous to the flagellum and also acts as a T3SS. This system is used to secrete its extracellular components and to deliver effectors into host cells. The NF-T3SS is an important virulence factor for animal pathogens within Salmonella, Escherichia, Chlamydia and Yersinia and plant pathogens within Xanthomonas, Ralstonia or Burkholderia (for reviews see [3]–[8]). Some genomes encode several NF-T3SSs. In Burkholderia pseudomallei discrete NF-T3SSs are involved in animal and plant pathogenesis [9]–[11], and in Salmonella enterica two NF-T3SSs are used in different phases of infection [12]. NF-T3SSs are exposed at the cell surface and are therefore targeted by the immune response. As a result, NF-T3SSs are being studied as targets for vaccines, e.g. for protection against Shigella [13], and for new antibacterial drugs [14]. They have also been used to deliver vaccine antigens to the cytosol of eukaryotic cells [15], [16]. NF-T3SSs have been most thoroughly studied for their role in antagonistic associations between pathogens and their hosts, but they also play an important role in mutualistic associations between bacteria and insects [17], plants [18], or fungi [19]. Phylogenetic evidence indicates that NF-T3SSs originated before most multicellular eukaryotes, possibly to favor interactions with early unicellular eukaryotes [20]. Each model NF-T3SS has its unique nomenclature in the literature. Hence, we use a unifying nomenclature throughout this publication [3], naming the NF-T3SS core components with the prefix sct for secretion and cellular translocation, followed by the suffixes used in the Yersinia Ysc system (see Table S1 for correspondence with other systems). When no unique name has been proposed we use the name of the Yersinia system by default, unless specifically specified. To avoid ambiguity, we follow Desvaux et al. [21] and use “translocation” for transport through a lipid bilayer and “secretion” for transport from the interior to the exterior of the cell. We use the term “delivery” to refer to the active transport from the interior of one cell to the cytosol of a second cell. The NF-T3SS is a complex protein structure that spans the cytoplasmic and outer membranes of bacteria and the cell envelope of the eukaryotic host to deliver effectors directly into its cytosol (Figure 1A). The basal body of the NF-T3SS provides a structural basis for the secretion machinery involved in protein delivery. The outer membrane component of the basal body is formed by a homo-polymeric ring of secretins (SctC) [22], [23], whose inactivation leads to the accumulation of effectors in the periplasm [24]. This protein is part of a large family of pore-forming secretins, also found in Type IV pili (T4P), Type II Secretion Systems (T2SS), Flp pili encoded by the tight adherence (Tad) system and the extrusion machinery of filamentous phages [25]. At the inner-membrane the basal body inner ring is composed of SctJ and SctD [22], [26]. The needle of the NF-T3SS is connected to the basal body at the inner membrane and extends outward from the cell [27]. In Salmonella, Shigella and Yersinia the needle is made of a major subunit (SctF) [26], [28], [29]. Phytopathogens do not have a needle but a flexible pilus-like structure encoded by HrpA [30], [31]. HrpA shares several traits with SctF and its pilus presumably represents an adaptation of the NF-T3SS to the thick plant cell wall [32]. The needle is capped by a tip (LcrV) involved in regulating secretion and in positioning the translocation pore in the host cell membrane [33], [34]. When the system is in contact with a target cell and delivery is activated, the translocon (YopB and YopD) acts as a pore-former in the eukaryotic membrane [35]. A few highly conserved proteins underneath the basal body are essential for the function of the NF-T3SS. One is a member of the F-/V- ATPase family (SctN), with homologs in flagella and F0F1 proton-translocating ATPases [36]–[39]. This ATPase functions in the recognition and unfolding of secreted proteins and possibly participates in energizing the process of secretion [40]–[43]. In S. enterica Typhimurium, a protein similar to the flagellum C-ring “sorting” platform (SctQ) was suggested to orchestrate the order of protein secretion in interaction with SctK and SctL [44]. The remaining highly conserved proteins (SctRSTUV) form the secretion apparatus. Their functions are poorly understood but they are thought to include substrate selection and molecular switching between modes of secretion [45], [46]. In Salmonella, Shigella, and Yersinia, another protein of importance, SctP, controls the needle-length during its assembly [26], [47], [48]. In Yersinia, SctP and SctU are involved in substrate-switching [45], [49], possibly by regulating the export of the inner rod protein (SctI) [49]. 10.1371/journal.pgen.1002983.g001 Figure 1 Identification of T3SS core genes in complete genomes. (A) Representation of the proposed organization of a NF-T3SS system, inspired from the Salmonella system [139]. For consistency with the main text, nomenclature follows that of Yersinia for some genes (see Table S1 for equivalence). Genes in red have homologs in the flagellum (main text, Table S1). “Euka” stands for eukaryotic, “mb” stands for membrane, “IM” for inner membrane, and “OM” for outer membrane. (B) Distribution of the number of different protein profiles of the 9 core genes of NF-T3SS matched per replicon (i-evalue 97% in gene-by-gene analyses, and >99% for combined protein profiles (see Materials and Methods), and that all mis-classified NF-T3SSs were in Myxo and Chlamy systems (see below). The accuracy of discrimination between the two types of T3SS for single proteins shows that these profiles are potentially useful for unassembled genomic data, including metagenomic data. We have therefore implemented a web server that allows detection of NF-T3SS and flagellar genes with our profiles (http://mobyle.pasteur.fr/cgi-bin/portal.py#forms::T3SSscan-FLAGscan). The NF-T3SSs described here can be queried and visualized from http://secreton.web.pasteur.fr. 10.1371/journal.pgen.1002983.g002 Figure 2 Discrimination between NF-T3SSs and flagella based on Hmmer profile scores. Each T3SS core protein was scored using the protein profiles for flagella (Y axis) and for NF-T3SSs (X axis). One sees a clear separation around the main diagonal. The color indicates the system (blue for flagellar proteins, red for NF-T3SS proteins). A circle indicates a correct prediction, and a triangle a wrong prediction. The analysis is described in Materials and Methods, and the learning dataset is shown in Figure S1. The origin(s) of T3SS We now turn to the evolutionary origins of the T3SS, a topic that has been extensively debated [75], [82], [93], [94], in order to decide between three scenarios: the early split of the two systems, the flagellum-first or the NF-T3SS-first hypotheses (Figure 3A). Our analysis showed that of the T3SS proteins with clear homologs between flagella and NF-T3SSs, only the ATPase also has homologs in other cell machineries with significant sequence similarity to allow rooting the T3SS tree. A set of homologs of the T3SS ATPase and F- and V- ATPase catalytic subunits [39] (Protocol S1) were selected and aligned, resulting in sequences with an average length of 459 amino-acids. We selected 296 informative positions from the multiple alignment with BMGE [95] (Dataset S1), and chose the tree with the highest maximum likelihood from 200 phylogenies built with RAxML [96]. This tree supports the T3SS monophyly with high support (100% bootstrap), and places the root of the T3SS tree within flagellar sequences (Figure 3B, Protocol S1). NF-T3SS sequences emerge in one clade within the flagellar T3SSs. We also counted the proportion of the 1000 bootstrap trees fitting each of the three evolutionary hypotheses (Figure 3A, Protocol S1). 84% of the trees support the flagellum-first hypothesis, arguing strongly against an early split between flagella and NF-T3SSs or that flagella were derived from an NF-T3SS. We confirmed this result by an analysis on a larger dataset including all curated systems. It is often difficult to obtain clear bootstrap supports for inner branches in very large trees spanning sequences with limited similarity and/or few sites. However, even this very large dataset clearly supported the flagellum-first scenario (>72% of the bootstrap trees, Text S1, Figure S2). 10.1371/journal.pgen.1002983.g003 Figure 3 Rooting the history of T3SS. (A) Representation of the three possible scenarios for the relative emergence of the flagellum and of the NF-T3SS. The numbers represent the percentage of each scenario in the bootstrap analysis of the ATPase family (out of 997 bootstrap trees where outgroup sequences were monophyletic, Figure S2, Protocol S1, Text S1). (B) The maximum likelihood tree of the ATPase family shows that the NF-T3SS derives from the flagellum. Support values are shown only for relationships important to discriminate between the three scenarios in the panel (A). They correspond to the occurrence of the splits in 1000 bootstrap replicates. The multiple alignment used to build this tree had 296 informative sites and is supplied in Dataset S1. The phylogeny of NF-T3SS In order to investigate early steps in the evolution of the NF-T3SS we reconstructed the phylogenies of the eight core proteins present in both NF-T3SSs and flagella for both single genes and their concatenated sequences. Initially we included the flagellar proteins to root the NF-T3SS tree. Then we restricted the analyses to the NF-T3SS proteins to obtain longer and more conserved multiple alignments allowing more accurate phylogenetic inference. The individual phylogenies of the eight core proteins support a common descent of all NF-T3SSs from a single ancestor and identify the same NF-T3SS groupings (Figure S3, Table S4). These groupings extend and clarify a previous classification of NF-T3SSs [62] (Figure 4, Figure S4, Text S2). The use of the program Prunier [97] showed that any topological differences among the eight individual gene trees and the concatenate trees were supported by less than 90% of the bootstraps (Text S3). Thus, gene-wise and concatenated “rooted” and “unrooted” phylogenetic analyses all support a similar history for the eight core NF-T3SS genes showing that they evolved together, apart from their flagellar homologs. 10.1371/journal.pgen.1002983.g004 Figure 4 Concatenate phylogeny of the core NF-T3SS proteins with flagellar homologs. The eight individual gene trees are displayed in Figure S3. The large monophyletic clades highlighted by different colors correspond to the NF-T3SS families (names in bold, see Text S2). Colored circles indicate the bootstrap support of these families and of the relationships between them. The “Ecology/Host” panel indicates the predominant bacterium/eukaryote associations for each family (see main text and Figure S4). The external branch indicates the position of the flagellum sequences and was inferred with the “rooted” dataset (“rooted” tree is in Figure S5). “Chlamy” stands for Chlamydiales, “Desulfo” for Desulfovibrionales, “Myxo” for Myxococcales, and “Rhizo” for Rhizobiales. The scale of the tree is given in substitutions per site. The first NF-T3SS was probably not a contact-dependent secretion system The NF-T3SS tree places the root between one group of systems found in Delta-proteobacteria of the Myxococcales order (the “Myxo” group), and all remaining systems (≥98% bootstrap support, Figure 4 and Figures S4, S5). The early diverging Myxo group includes both a long and a short variant of the NF-T3SS locus. These variants lack core NF-T3SS genes, resulting in their prior annotations within the genome of Myxococcus xanthus as relics of NF-T3SS undergoing degradation [98]. If this interpretation were correct, then these genes should evolve quickly and our assignment of these variants to a basal phylogenetic position might be artifactual. However, we identified six additional genomes from the same clade (Cystobacterineae) having homologous systems (Figure 5) (see [99] for the taxonomy of Myxococcales). All fully sequenced genomes of Cystobacterineae have the “short” locus and a monophyletic group of three of these genomes also possesses the “long” locus. These loci have G+C contents within the 25–75% range of the G+C genomic content (SeqinR [100] analysis with a 1 kb sliding window), and a conserved gene order (Figure 5). The core genes that are common to these variants correspond to the secretion apparatus, the ATPase, the smaller inner ring protein and the inner rod protein (short locus). The long locus also contains the large inner ring protein. These core proteins interact with each other and also correspond to the most conserved NF-T3SS core proteins. The long and short loci each correspond to distinct monophyletic groupings (Figure S4) that have probably been inherited vertically because the topology and branching structures of these phylogenies resemble those of the 16S rDNA tree (Figure 5). The conservation of these systems in sequence, gene composition and genetic organization is striking because the species harboring them diverged a long time ago. For example, the 16S rDNA subunits of Anaeromyxobacter and Myxococcus show lower sequence similarity than that between Escherichia coli and Vibrio cholerae. The strong conservation of these loci in sequence and organization over such long time scales suggests that they are functional. 10.1371/journal.pgen.1002983.g005 Figure 5 Genomic organization of the Myxo NF-T3SS. The gene contents of the “short” and “long” Myxo NF-T3SSs are displayed on the 16S rRNA phylogeny of the group. The 16S rRNA maximum likelihood tree was built with RAxML [96] (General time-reversible model + 4-categories-discretized Gamma distribution for rate variations among sites) and chosen from the best of 200 starting trees. The numbers in the tree nodes refer to bootstrap values out of 100 replicates. The identified NF-T3SS genes are represented by boxes containing the last letter of the “sct” gene name, and colored according to the color code of Figure 1A. Dashed transparent boxes correspond to genes showing hits for Hmmer sequence profiles with i-evalue above the 10−3 threshold. Empty boxes correspond to genes not annotated in our analysis, and boxes with three dots correspond to several (4 to 16) consecutive non-annotated genes. Proteins encoded in the loci are colored on the NF-T3SS structure drawings, whereas parts of the structure whose corresponding gene could not be found remain unfilled. We argue that the Myxo NF-T3SSs probably derived from ancestors that were neither flagella nor protein delivery systems. Myxo systems lack proteins that are indispensable for flagellar function, such as FlgBCDEK, FliEG, FlgH and MotAB. They also lack NF-T3SS genes such as the secretin, the major needle subunit (SctF), the tip protein (LcrV) and the translocon (YopB/YopD). Homologs of the tip and translocon proteins may have been missed in our sequence similarity searches because of their rapid evolution. However, the flagellar proteins, the secretin and SctF are highly conserved and should have been found by our sequence similarity searches had they been present. Our profiles show significant sequence similarity between SctF, the major needle subunit of the NF-T3SS, and FliC/FlgL (the flagellin and a hook-associated protein), whose homology was previously suggested based on structural data [71], [101]. Hence, the earliest NF-T3SS probably contained an ancestral SctF that was lost in Myxo systems. The lack of an outer membrane channel and of SctF suggests that Myxo systems are not able to deliver effectors to eukaryotic cells and possibly not even to secrete proteins to the extracellular space. Multiple origins of secretins We next analyzed the diversification of NF-T3SS within the main branch of its phylogeny, i.e. among loci including a secretin. This branch includes all NF-T3SSs shown experimentally to deliver effectors into the eukaryotic cytosol. The first split along this branch separates the Chlamy from the other systems with 100% bootstrap support (Figure 4, Figure S3, Table S4). Subsequent diversification was very rapid within the other taxa as shown by a succession of short branches with weak bootstrap support (Figure 4 and Figures S3, S4, S5). To investigate the early NF-T3SS diversification we made a phylogenetic analysis of the NF-T3SS secretin together with secretins from T2SS, T4P, Tad system and filamentous phages [102]. Surprisingly, the phylogeny shows that secretins have been independently recruited to the NF-T3SS on at least three occasions (Figure 6A). The Rhizo secretin RhcC2 branches together with secretins from the Tad loci (e.g. RcpA and CpaC) [103]. The secretin domain of Chlamy NF-T3SSs (excluding a large unique N-ter region, see Figure 6B and [81], [92]) clusters with the gene IV secretin of filamentous phages within T2SS secretins. The secretins from the remaining NF-T3SSs cluster together in a third group (Figure 6A) hereafter referred as “NF-T3SS-like” secretins. The most parsimonious explanation for these results is that the last common ancestor of extant NF-T3SSs lacked a secretin. 10.1371/journal.pgen.1002983.g006 Figure 6 Evolutionary history, domain architecture, and genetic organization of the NF-T3SS secretins. (A) Phylogeny of the secretin family. The numbers represent bootstrap support values for branches separating the different types of secretins found in NF-T3SSs (highlighted by colored bars). Gray boxes indicate the main secretin groups. The two genes encoding the Rhizo secretin are indicated in orange and yellow boxes (see panel B). NF-T3SS types are named as in Figure 4. (B) Domain architecture of the secretins related to NF-T3SS secretins. PFAM domains were identified using InterProScan [140], except for the Chlamy-specific N-terminal domain of the NF-T3SS secretin CdsC (burgundy box), and the dashed box “N-domain”, which were identified by sequence similarity with the other secretins using Blast. Signal peptides were detected with InterProScan and PsortB [141]. Dotted lines correspond to the boundaries of the alignments of Blast hits. See Text S4 for more analyses of Rhizo secretins. (C) Analysis of the genetic organization of the Rhizo loci mapped on their phylogeny. The phylogeny was inferred from the concatenation of the eight NF-T3SS core genes (Figure 4, Figure S4). Conventions are the same as in Figure 5. Numbers in white boxes correspond to the number of genes separating the block of genes with conserved order (light green box) from the RhcC2 secretin (outside the box), or to the number of consecutive genes not annotated in this study (inside the box). The secretin of Rhizo NF-T3SSs is encoded by two genes, rhcC1 and rhcC2 (Figure 6B) [104], and we show that they have distinct origins. The gene rhcC2 encodes a protein whose architecture is similar to the secretin of the Tad locus of Caulobacter crescentus CpaC (Figure 6B, Text S4), in agreement with its phylogenetic position within Tad loci (Figure 6A). It includes the “secretin” domain, absent from RhcC1, and an N-terminal “BON” domain [105]. The gene rhcC1 is found at a conserved position in Rhizo loci, among other NF-T3SS core genes (Figure 6C). The protein RhcC1 is homologous to the N-terminal part of the NF-T3SS-like secretins (Blast search with e-value 0.05). We also did not find significant sequence homologs of YopB/YopD in the NF-T3SS of Myxo or plant-associated bacteria. Since the translocon is required for protein delivery but not for secretion [35], it might have been acquired after the secretin. Balloons indicate gene losses and accretions. Only genes mentioned in the main text are shown. Abbreviated names of taxa are as in Figure 4. Our results contradict the proposal that NF-T3SS evolved in Chlamydiales before being transferred to Proteobacteria [62], [93]. Transfer of the multiple loci within the Chlamy system would require multiple events, whereas the unique loci of other NF-T3SSs can be transferred in one single event. All Chlamy NF-T3SSs include a T2SS-like secretin whereas other NF-T3SSs share a secretin from a distinct origin (Figure 6). Finally, key elements between these systems have no homologs, e.g. the proposed tip protein for Chlamy (CT584) cannot be aligned to tip proteins from other systems [120]. Hence, we propose that the last common ancestor of the NF-T3SSs derived from a flagellum, lacked a secretin and included a periplasmic or extracellular structure based on SctF. Interestingly, Spirochetes have periplasmic flagella, and in some cases lack the outer membrane protein FlgH [121]. Inactivation of the secretin in S. enterica SPI1 NF-T3SS prevents the formation of extracellular appendages but does not prevent the secretion of the main needle filament subunit (SctF) to the periplasm, where it remains associated with the inner ring proteins [53]. Hence, it is conceivable that a needle-like structure might have assembled at the periplasm in the ancestral NF-T3SS even in the absence of an outer membrane channel. We argue that this system was subsequently transferred across taxa prior to independent acquisitions of a secretin. The atypical Myxo system The early evolution of the NF-T3SS was accompanied by the accretion of new genes that are present in even the most basal clades of the NF-T3SS (Figure 7). These include the structural genes sctD and sctI and probably also system-specific chaperones and regulators, whose evolutionary patterns are obscured by their fast evolutionary rates. The Myxo NF-T3SS was the first group to split from the others (Figure 7). Myxo systems are only present in Cystobacterineae and should have significant adaptive value because they are present in all available genomes of this taxon, show strict vertical inheritance and are highly conserved in sequence and genetic organization. The absence of the secretin and translocon as well as other proteins essential for protein delivery by other NF-T3SSs suggests that the Myxo NF-T3SS is unable to deliver effectors into the cytosol of eukaryotes. This is consistent with Cystobacterineae ecology, which are free-living bacteria that prey on other bacteria [122]. These systems could thus be involved in protein translocation and/or make some sort of periplasmic or extracellular structure. Functional analysis of these systems would be needed to understand their function. Multiple recruitments of secretins to NF-T3SSs Acquisition of the secretin was the next major step in NF-T3SS evolution for which clear evidence is available. Our analysis shows that the secretin was recruited three times: in Chlamy (from T2SS), in the other systems (possibly from T4P) and then again in Rhizo (from Tad systems). This is consistent with the remarkable versatility of secretins as outer-membrane channels [123]. The interaction between the inner membrane rings and the secretin takes place between the C-terminal region of SctD and the N-terminus domains of the secretin [124]–[127]. Interestingly, the only part of the early secretin RhcC1 that remains in Rhizo after its partial replacement is the N-terminal domain that interacts with SctD (Figure 6, Text S4). The Tad and the RhcC2 secretins lack this N-terminal domain and have a membrane-interaction, “BON” domain [105], that is present in SctD but absent in other secretins. This domain architecture might interfere with a direct interaction between RhcC2 and SctD, which led to the conservation of RhcC1 as a linker between the two. In that case, interactions of the secretin during the assembly of the NF-T3SS might be critical for successful acquisitions of secretins from other systems. Flagellum assembly resembles the inside-out model of NF-T3SS assembly [128], [129]. The ancestral NF-T3SS probably also assembled inside-out because it lacked a secretin. The inside-out assembly of the secretion apparatus and especially of the newly acquired SctD protein at the inner membrane might have led to interactions with secretins from other systems that stabilized an outer membrane channel in the NF-T3SS complex. Indeed, extant NF-T3SS secretins are inserted in membranes independently of the assembly of the inner ring. The inner ring assembles, and then stabilizes the secretin multimer [53]. These interactions presumably evolved towards a stable genetic linkage of the secretin with the NF-T3SS. Outside-in assembly modes, e.g. as proposed for Yersinia NF-T3SS, might have evolved after the acquisition of the secretin. Studies on the assembly of Myxo systems might therefore elucidate the mechanisms of assembly of the ancestral NF-T3SS and also of the evolution of the assembly process of the other NF-T3SSs. Rapid adaptation of NF-T3SSs to diverse eukaryotic cell envelopes Acquisition of the secretin and of the translocon proteins allowed bacteria to deliver effectors to eukaryotic cells. This was followed by very rapid diversification of NF-T3SSs into groups with different characteristics and tropisms (Figure 7). Such rapid radiation hinders robust phylogenetic inference after the split of the Chlamy systems at the base of the remaining NF-T3SSs (Figure 4), even though we were able to infer older splits with strong confidence. Needle/pilus, tip and translocon proteins, all of which promote intimate interactions with the host, evolved quickly and were probably key determinants of NF-T3SSs radiation. The original NF-T3SS included a needle (SctF) resembling the flagellum homolog. The replacement of the needle by a pilus was a key late adaptation in NF-T3SSs that specifically interact with the cell walls of plants and fungi. Sequence evolution of the NF-T3SS pilus of some plant-associated bacteria is driven by strong positive/diversifying selection in response to host defenses [130], [131]. It might be expected that vertebrate immune systems would have had similar effects on the evolution of animal-associated NF-T3SSs. Hence, diversifying selection on plant-associated NF-T3SSs is probably not the cause of the differences between pili and needles. Instead, the pilus probably reflects adaptation to the thick cell walls of plants and fungi. More genomic, functional and structural data on NF-T3SSs involved in plant interactions will be necessary to understand whether all these plant-associated NF-T3SSs arose independently or derived from the same ancestral NF-T3SS. This work shows how a key protein secretion system arose from the recruitment of a structure that evolved for another purpose. The T3SS was initially adapted to the transport of flagellar components through the membrane and was probably exapted on multiple occasions to transport other proteins. One of those exaptations resulted in the ancestral NF-T3SS. This system adapted to its new function by a series of gene losses and acquisitions. Strikingly, it acquired secretins multiple times. Thereafter, the system was able to deliver effectors directly to eukaryotic cells, which dramatically increased its rapid diversification due to the adaptive value of NF-T3SS for mutualistic and antagonistic interactions with eukaryotes. This evolutionary reconstruction represents a remarkable example of how successive losses and recruitments of components from a series of existing molecular machines can lead to the evolution of a new complex system. Materials and Methods Genome data Genomes were extracted from GenBank Refseq. To extend the taxon sampling, we added to the dataset some draft genomes of interest. We analyzed 1385 genomes with 2575 replicons (1483 chromosomes and 1092 plasmids). Data availability The NF-T3SS clusters identified in this study can be queried using different criteria (including taxonomy and NF-T3SS family name), and visualized along with the results of our Hmmer profiles searches at http://secreton.web.pasteur.fr. The profiles of NF-T3SS and flagellum proteins can be queried at http://mobyle.pasteur.fr/cgi-bin/portal.py#forms::T3SSscan-FLAGscan. The list of NF-T3SSs and flagella included in phylogenetic analyses can be found in Dataset S2. Construction of sequence profiles for NF-T3SSs and flagella We selected one sequenced model organism from each described NF-T3SS family (genomes marked in red on Figure S4, list in Table S5 [6]). We extracted NF-T3SS protein sequences according to their genome sequence annotations and the literature. We performed similarity searches between these sequences with a Blast “all against all” search and applied a clustering algorithm with stringent parameters on the transformed e-value (-log(e-value), MCL [132] inflation parameter I = 1.5) to sequences showing hits with an e-value lower than 10−3. We obtained nine families that were found in all model systems, which corresponded to the nine previously described NF-T3SS core proteins: SctC, SctJ, SctN, SctQ, SctR, SctS, SctT, SctU, SctV. We aligned these nine protein families with Muscle [133], manually edited the alignments with Seaview [134], and built sequence profiles with Hmmer [86]. A similar approach was conducted for flagella from phylogenetically distinct model organisms (MCL clustering, I = 1.8) (List in Table S3). Out of 14 protein families widely conserved in flagella, eight were homologous to NF-T3SS core proteins (Table S1, protein clustering of protein families obtained from NF-T3SS and flagellar model systems, MCL parameter I = 2.5), and were extracted to build Hmmer sequence profiles. We also selected three widely conserved flagellar families with no NF-T3SS homolog (confirmed by the clustering above and Hmmer profile searches): FliE, FlgB, FlgC (rod proteins), and built sequence profiles to identify other occurrences of these proteins. Additional profiles were also built for FlgDEKL, FliG, MotA and MotB (MCL parameter I = 1.5) that are essential flagellar-specific genes [71]. NF-T3SSs/flagella identification To discriminate between homologous genes in flagella and NF-T3SSs, we performed a Hmmer search with the profiles of the eight proteins shared between them (“core proteins”), the secretin (the NF-T3SS-specific core proteins), and the three selected flagellum-specific proteins. Hmmer hits with Evalue and best-1-domain Evalue (or i-evalue) lower than 10−3 were selected. Two hits were said to be contiguous when separated by less than 35 genes (average size of a flagellum cluster). We searched for clusters of contiguous hits to separate NF-T3SSs and flagellar systems. Clusters of genes showing positive hits for at least seven of the eight core genes, including a secretin, and lacking all three flagellum-specific genes were classed as NF-T3SSs. Sets of clusters of NF-T3SS core genes (core genes+sctC) not containing flagellum-specific genes were inferred as scattered NF-T3SSs. Flagellum clusters contained no secretin and had hits for at least 10 flagellar genes (core genes+flagellum-specific genes). Scattered flagella had hits for at least 10 flagellar genes, and had at least one cluster containing flagellum-specific genes with one core gene. All detected NF-T3SSs, plus other clusters close to the definition above (i.e. having fewer of the core genes clustered) were manually curated and checked according to the available literature. Thus, the Myxo systems were retrieved even if they lacked a secretin and flagellum-specific genes. We did not include in our analysis the system of Lawsonia intracellularis because its flagellar genes and NF-T3SS genes were intermingled in different positions of its genome, rendering the reconstruction of the two systems more hazardous (Table S2). Discriminant analysis with hits for both NF-T3SS/flagellar genes We attributed two types of Hmmer scores to genes with homologs in both systems: one score corresponding to the NF-T3SS profile and the other to the flagellar profile. We made a learning dataset including a third of the T3SS genes and their predicted role (NF-T3SS or flagellum). This subset was randomly drawn from the set of all T3SS. Using linear discriminant analysis [135], we predicted the type of system for the remaining two thirds of the dataset. We performed such an analysis with the combined dataset (all eight genes common to the flagellum and NF-T3SS together) as described above, and also in a gene-by-gene analysis. Accuracy is defined as the number of true predictions over the total number of predictions. Individual phylogenetic analyses We extracted from the genomes the genes encoding proteins homologous to T3SS core genes that were detected as part of a NF-T3SS or flagellum system. In a given system, when multiple Hmmer hits were available for a single gene, we kept the one displaying the lower Evalue and the maximal length. Many flagellar systems had multiple hits for the same genes scattered in the genome. We manually curated a subset of these flagella (357 out of 699 detected, the list of strains is in Dataset S2). We aligned sequences with Muscle (default parameters, [133]) and selected informative sites with BMGE (BLOSUM30 similarity matrix, gap rate cut-off = 0.20, sliding window size = 3, entropy score cut-off = 0.5 [95]). We built phylogenetic trees with RAxML ([96], Le and Gascuel [136] matrix + 4-categories-discretized Gamma distribution for rate variation among sites + empirical frequencies of amino-acids): we selected the best maximum likelihood tree among 200 different starting tree inferences, and computed 1000 bootstrap trees (i.e. trees based on bootstrap alignments, consisting of randomized sites drawn with replacement from the original alignment, and of the same size of the original alignment). In the case of the ATPase SctN, we built an extra dataset that we extended with previously described outgroup sequences [39] (see Text S1) and built a tree as described above. We also ran an extra phylogenetic analysis in a similar way on a subset of these sequences (see Protocol S1, Text S1). We built a tree as indicated above with a secretin dataset that included i) sequences identified in a previously described dataset [102] that were retrieved using their accession numbers; ii) SctC of detected NF-T3SSs; iii) all the secretins we found in Myxo and Chlamy genomes. Sequences displaying branch lengths longer than 1 substitution per site were excluded from phylogenetic analyses, and the phylogenetic reconstruction was run again with the cleansed dataset. This led to the exclusion of several flagellar systems and of five potential NF-T3SSs. Some of these systems are probably undergoing degradation (Table S2). Concatenated phylogenetic analyses The protein alignments of the genes were concatenated and phylogenetic trees were built with RAxML [96] (Le and Gascuel matrix [136] + 4-categories-discretized Gamma distribution for rate variation among sites + empirical frequencies of amino-acids) with 100 rapid bootstraps [96] for the rooted tree. We made a more thorough phylogenetic search for the unrooted dataset: we performed 100 bootstrap replicates, and mapped them on the best (i.e. with the highest likelihood) among 100 phylogenies obtained from distinct start trees. We attributed families to predicted NF-T3SSs according to previously defined families ([62]; list of genomes in [6]). We searched a posteriori for putative significant inconsistencies between phylogenies of individual unrooted trees and the concatenate tree using the program Prunier [97] (bootstrap threshold = 80% and 90% in both gene trees and in the reference tree, see Text S3). Building families of neighboring genes We extended the cluster of NF-T3SS core genes by 10 genes upstream and downstream in the replicon sequence. All these protein sequences were extracted, and a similarity search (Blast “all against all”) was performed between them. Pairs of sequences having hits with e-value lower than 10−3 were clustered based on Blast alignments using the Silix program ([137], parameters used: minimal percentage identity = 20, minimal percentage of sequence overlap = 50, and minimal accepted length for sequences = 50). The most abundant protein families, considering both replicons and NF-T3SS families, were extracted and Hmmer profiles were built from them to extend the search in NF-T3SS neighboring genes. Programs used for graphics We used the Scriptree program [138] to draw annotated trees (Figure 4; Figures S3, S4, S5) and Figtree (http://tree.bio.ed.ac.uk/software/figtree) to draw trees (Figure 3B, Figure 5, Figure 6). Graphics on Figure 1B, Figure 2, and Figure S1 were drawn with R (http://www.r-project.org). All figures were modified with Inkscape (http://www.inkscape.org). Supporting Information Dataset S1 The filtered alignment of the ATPase family used for phylogenetic analyses and the tree in Figure 3B (Fasta format). Sequence names correspond to codes described in Dataset S2 for flagella and NF-T3SS, and to Genbank accession numbers for F-/V- ATPases. (TXT) Click here for additional data file. Dataset S2 List of systems analyzed along with taxonomic information, and equivalence for system codes found in Figure S5 and Dataset S1. (XLS) Click here for additional data file. Figure S1 Learning dataset for discrimination between NF-T3SS and flagellar proteins based on Hmmer profile scores. For the subset of T3SS proteins used as a training dataset for the linear discriminant analysis shown in Figure 2 we show their protein profile scores using the NF-T3SS-specific profiles (X axis) and the flagellum-specific profiles (Y axis). Color codes indicate the different gene families. Filled circles indicate flagellar system proteins whereas open squares indicate NF-T3SS proteins. (PDF) Click here for additional data file. Figure S2 Representation of each scenario for T3SS evolution in the phylogenetic analysis of the F-/V- ATPase family. The number of bootstrap trees in agreement with each scenario is indicated in cells. These numbers are shown for both the dataset used for the tree on Figure 3B (out of 997 bootstrap trees) and for a wider dataset including all curated systems (out of 974 trees), after the mark “&” (Text S1). (PDF) Click here for additional data file. Figure S3 Individual phylogenies of the NF-T3SS core proteins with flagellar homologs. These trees were obtained with NF-T3SS proteins, and did not include flagellar proteins. (A) The eight collapsed individual trees. Branches with low support value (bootstrap<80%) were collapsed. Supports for some critical relationships in the rooted trees can be found in Table S4, black arrows indicate position of the flagellum sequences in the “rooted” versions of these trees (not shown). (B) These trees are the same as the trees presented in panel A, except that branches with bootstrap values below 80% were not collapsed. (PDF) Click here for additional data file. Figure S4 Maximum likelihood phylogeny of the NF-T3SS using the concatenation of the eight core genes with homologs in the flagellum. The eight individual trees are displayed in Figure S3. For more clarity, strain names were replaced by the corresponding species names when the systems were monophyletic. Names in red correspond to the model NF-T3SSs used to build the protein profiles of the NF-T3SS core genes. Bootstrap supports are all shown for relationships between families, but within families bootstrap supports are indicated only when below 80%. The “Gene content” panel indicates the presence (black for clear homologs, or brown if found in a distant or unrelated clade-specific family with weak or no sequence similarity) and absence (grey) of other conserved NF-T3SS genes (see Materials and Methods). When not found in the protein dataset with our Hmmer search or with our clustering method, traces of genes were sought by performing tBlastn searches on complete genomes. These hits could correspond either to pseudogenes, fast evolving sequences, or sequencing errors. If traces were retrieved, the gene appears with a salmon box. The “Ecology” panel indicates bacterium/eukaryote associations. Large monophyletic clades correspond to the NF-T3SS families (colored boxes). “Chlamy” stands for Chlamydiales, “Desulfo” for Desulfovibrionales, “Myxo” for Myxococcales, “Rhizo” for Rhizobiales, and “Bups” for Burkholderia pseudomallei group. The black arrow indicates the position of the flagellum sequences in the “rooted” version of this tree (Figure S5), and the scale of the tree is given in substitutions per site. (PDF) Click here for additional data file. Figure S5 Concatenate phylogeny of flagellar and non-flagellar T3SSs based on SctNJQRSTUV and their respective flagellar homologs (see Tables S1 or S4 for gene names). Branch supports correspond to the occurence of the splits in 100 rapid bootstrap trees. Color boxes surround the different NF-T3SS families. Yellow for Myxo, brown for Chlamy, red for Hrp2, orange for Ysc, green for Rhizo, cyan for Hrp1, violet for SPI2, and dark purple for SPI1. Two levels of taxonomy are indicated on the right. See Dataset S2 for equivalence between system codes and replicon names. (PDF) Click here for additional data file. Protocol S1 Analysis of the F-/V- ATPase family. (DOC) Click here for additional data file. Table S1 Nomenclature of NF-T3SS and flagellar components in various systems. (DOC) Click here for additional data file. Table S2 List and justification of excluded NF-T3SS systems. (DOC) Click here for additional data file. Table S3 List of model genomes used to build flagellar protein families and profiles. (DOC) Click here for additional data file. Table S4 Support values (percentage of rapid bootstraps) for critical relationships in rooted phylogenies of T3SS genes. (DOC) Click here for additional data file. Table S5 List of model genomes used to build NF-T3SS protein families and profiles (in red on Figure S4). (DOC) Click here for additional data file. Text S1 Analysis of the F-/V- ATPase trees. (DOC) Click here for additional data file. Text S2 The phylogeny of NF-T3SS. (DOC) Click here for additional data file. Text S3 Checking phylogenetic signal consistency with Prunier. (DOC) Click here for additional data file. Text S4 Tracing the origins of Rhizo secretins. (DOC) Click here for additional data file.
- Record: found
- Abstract: found
- Article: not found
Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein.
Changjiang Dong, L L LaMontagne, James Naismith … (2006)
Many types of bacteria produce extracellular polysaccharides (EPSs). Some are secreted polymers and show only limited association with the cell surface, whereas others are firmly attached to the cell surface and form a discrete structural layer, the capsule, which envelopes the cell and allows the bacteria to evade or counteract the host immune system. EPSs have critical roles in bacterial colonization of surfaces, such as epithelia and medical implants; in addition some EPSs have important industrial and biomedical applications in their own right. Here we describe the 2.26 A resolution structure of the 340 kDa octamer of Wza, an integral outer membrane lipoprotein, which is essential for group 1 capsule export in Escherichia coli. The transmembrane region is a novel alpha-helical barrel. The bulk of the Wza structure is located in the periplasm and comprises three novel domains forming a large central cavity. Wza is open to the extracellular environment but closed to the periplasm. We propose a route and mechanism for translocation of the capsular polysaccharide. This work may provide insight into the export of other large polar molecules such as DNA and proteins.
- Record: found
- Abstract: found
- Article: not found
Evolution of increased complexity in a molecular machine.
Many cellular processes are carried out by molecular 'machines'-assemblies of multiple differentiated proteins that physically interact to execute biological functions. Despite much speculation, strong evidence of the mechanisms by which these assemblies evolved is lacking. Here we use ancestral gene resurrection and manipulative genetic experiments to determine how the complexity of an essential molecular machine--the hexameric transmembrane ring of the eukaryotic V-ATPase proton pump--increased hundreds of millions of years ago. We show that the ring of Fungi, which is composed of three paralogous proteins, evolved from a more ancient two-paralogue complex because of a gene duplication that was followed by loss in each daughter copy of specific interfaces by which it interacts with other ring proteins. These losses were complementary, so both copies became obligate components with restricted spatial roles in the complex. Reintroducing a single historical mutation from each paralogue lineage into the resurrected ancestral proteins is sufficient to recapitulate their asymmetric degeneration and trigger the requirement for the more elaborate three-component ring. Our experiments show that increased complexity in an essential molecular machine evolved because of simple, high-probability evolutionary processes, without the apparent evolution of novel functions. They point to a plausible mechanism for the evolution of complexity in other multi-paralogue protein complexes.
Author and article information
Contributors
Michael T. Laub: Role: Academic Editor
Journal
Journal ID (nlm-ta): PLoS Biol
Journal ID (iso-abbrev): PLoS Biol
Journal ID (publisher-id): plos
Journal ID (pmc): plosbiol
Title:
PLoS Biology
Publisher:
Public Library of Science
(San Francisco, USA
)
ISSN
(Print):
1544-9173
ISSN
(Electronic):
1545-7885
Publication date Collection:
December
2013
Publication date
(Print):
December
2013
Publication date
(Electronic):
10
December
2013
Volume: 11
Issue: 12
Electronic Location Identifier: e1001728
Affiliations
[1
]Laboratoire de Chimie Bactérienne, CNRS UMR 7283, Aix-Marseille Université, Institut
de Microbiologie de la Méditerranée, Marseille, France
Author notes
* E-mail:
tmignot@
123456imm.cnrs.fr
The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: MW FC AD ST EM PB JS TM. Performed the experiments: MW FC AD ST AVLG EM PB. Analyzed the data: MW FC AD ST EM TM JWS. Contributed reagents/materials/analysis tools: AD YVB. Wrote the paper: MW TM.
Article
Publisher ID: PBIOLOGY-D-13-02765DOI: 10.1371/journal.pbio.1001728
PMC ID: 3858216
PubMed ID: 24339744
SO-VID: 21d86147-4183-4219-bf9f-4732e8e7fb7f
Copyright statement:
Copyright @
2013
License:
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
History
Date
received
: 13
July
2013
Date
accepted
: 23
October
2013
Related
Agl, The Multitasking Motor Protein
Page count
Pages: 12
Funding
MW is supported by the Fondation ARC (Dossier n° DOC20120604801). AD is supported
by an NIH grant to Yves Brun (NIH GM51986). ST acknowledges support from the Human
Frontier Science Program under a Cross Disciplinary Fellowship. JWS acknowledges the
Pew Charitable Trust and the Human Frontier Science Program (RGY0075/2008). This work
was mostly supported by an ERC starting grant (DOME-261105) to TM. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Categories
Subject:
Research Article
ScienceOpen disciplines: Life sciences
Data availability:
ScienceOpen disciplines: Life sciences