From conjugation to T4S systems in Gram‐negative bacteria: a mechanistic biology perspective
2019; Springer Nature; Volume: 20; Issue: 2 Linguagem: Inglês
10.15252/embr.201847012
ISSN1469-3178
Autores Tópico(s)Enzyme Structure and Function
ResumoReview2 January 2019Open Access From conjugation to T4S systems in Gram-negative bacteria: a mechanistic biology perspective Gabriel Waksman Corresponding Author Gabriel Waksman [email protected] orcid.org/0000-0003-0708-2726 Institute of Structural and Molecular Biology, UCL and Birkbeck, London, UK Search for more papers by this author Gabriel Waksman Corresponding Author Gabriel Waksman [email protected] orcid.org/0000-0003-0708-2726 Institute of Structural and Molecular Biology, UCL and Birkbeck, London, UK Search for more papers by this author Author Information Gabriel Waksman *,1 1Institute of Structural and Molecular Biology, UCL and Birkbeck, London, UK *Corresponding author. Tel: +44 207 631 6833; E-mail: [email protected] EMBO Reports (2019)20:e47012https://doi.org/10.15252/embr.201847012 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Conjugation is the process by which bacteria exchange genetic materials in a unidirectional manner from a donor cell to a recipient cell. The discovery of conjugation signalled the dawn of genetics and molecular biology. In Gram-negative bacteria, the process of conjugation is mediated by a large membrane-embedded machinery termed "conjugative type IV secretion (T4S) system", a large injection nanomachine, which together with a DNA-processing machinery termed "the relaxosome" and a large extracellular tube termed "pilus" orchestrates directional DNA transfer. Here, the focus is on past and latest research in the field of conjugation and T4S systems in Gram-negative bacteria, with an emphasis on the various questions and debates that permeate the field from a mechanistic perspective. Glossary CP coupling protein cryo-EM cryo-electron microscopy Cryo-ET cryo-electron tomography dtr DNA transfer and replication ICE integrated conjugative elements IMC inner-membrane complex IM inner membrane IR inverted repeat LPS lipopolysaccharides mpf mating pair formation OMCC outer-membrane core complex OM outer membrane OmpA OM protein A RHH ribbon-helix-helix T4S type IV secretion TrIP transfer DNA immuno-precipitation Introduction Bacterial conjugation is the process by which DNA is transferred unidirectionally from a donor cell to a recipient cell. It plays a crucial role in horizontal gene transfer, the major means by which bacteria evolve and adapt to their environment, and also a process of immense biomedical importance since conjugation is the main vector of propagation of antibiotics resistance genes. It was first described by Lederberg and Tatum in the 1940s 1. Its discovery signalled the dawn of molecular biology once it was demonstrated that the transfer of genetic information was unidirectional and that the entire genome of Escherichia coli could be passed from one cell to another starting at a defined site 2. Indeed, landmark discoveries followed: the mapping of the E. coli genome (mapped in "minutes", i.e. the time taken by a particular gene to be transferred from donor to recipient, with time 0 being the mating start—when donor and recipient cells were put in the presence of each other) or the discovery of gene structure and regulation (please refer to the fascinating account of this research in the Nobel lectures by the founding fathers of the field of molecular biology, Francois Jacob, Andre Lwoff and Jacques Monod in 1965 3). The various machineries utilized during conjugation to execute DNA transfer are usually encoded by conjugative plasmids or other genetic mobile elements such as integrated conjugative elements (ICE). Plasmids are ubiquitous in bacteria and are defined as a collection of genetic modules organized into a stable, usually circular, self-replicating replicon, which does not usually contain genes essential for cell functions (reviewed in ref. 4). Several of these modules contain genes encoding proteins that assemble into large complexes mediating most commonly the plasmid's own transfer to a recipient bacterial cell, but also intriguingly (but rarely) to a eukaryotic cell such as yeast, plant or human cells 5-7. Interestingly, these modules are evolutionary related to clusters found in genomic islands of a restricted number of bacterial pathogens such as Helicobacter pylori, Bordetella pertussis or Legionella pneumophila where they play essential roles in pathogenicity by injecting protein effectors into eukaryotic hosts 8 (Fig 1). Figure 1. The various processes in which T4S systems are involvedT4S systems are involved in DNA transport during conjugation, transformation and A. tumefaciens infection, and in effector transport by a number of bacterial pathogens. This figure was modified from Grohmann et al 108. Download figure Download PowerPoint Conjugation in Gram-negative bacteria is mediated by three large complexes: a DNA-processing machinery called "the relaxosome"; a membrane-embedded transport machinery termed "type IV secretion (T4S) system"; and a pilus 9. Conjugation starts with the assembly of the relaxosome to a particular site on the plasmid DNA called the "origin of transfer" or OriT. The relaxosome includes one key protein called the "relaxase" and a number of accessory proteins. The relaxase plays essential roles: (i) it catalyses a nicking reaction on a single strand of OriT DNA at the so-called nic site and covalently reacts to the 5′-phosphate generated by the nicking reaction; and (ii) it binds to the T4S system through interactions with one of the constituents of the transport machinery, the coupling protein (reviewed in ref. 10). The T4S system is one of six secretion systems embedded in both membranes of Gram-negative bacteria 11. Minimally, they are composed of 12 proteins termed "VirB1-11 and VirD4" (to use the naming nomenclature derived from the Agrobacterium tumefaciens T4S system) 12. Three components, VirB7, VirB9 and VirB10, form the so-called outer-membrane core complex (OMCC), absent in Gram-positive T4S systems where there is no OM 13. The OMCC connect to an inner-membrane complex (IMC) composed of VirD4, VirB4, VirB3, VirB6, VirB8 and part of VirB10. OMCC and IMC are connected through a stalk of unknown composition, perhaps made of VirB2 and VirB5 14 or VirB10 14-16. At least two ATPases (VirB4 and VirD4), or sometimes three (VirB4, VirD4 and VirB11), power the system. Finally, the conjugative pilus of Gram-negative bacteria is an essential element in conjugation. For decades, it was the only feature in conjugating cells that could be observed or purified 17. It is made of a major component, VirB2, and a minor one, VirB5. VirB2 assembles into a large helical filament with perhaps VirB5 at its tip 18. Pili have been hypothesized to either serve as attachment devices mediating recognition of and attachment to recipient cells or serve as a conduit for relaxase/ssDNA transport, or both. Some conjugative pili are capable of retraction, which will bring donor and recipient cells together 19 resulting in close proximity. Indeed, tight conjugative junctions have been observed which have led to the suggestion that cell-to-cell contacts are required for conjugation to take place 20, 21. However, transfer has also been observed when cells are some distance apart (see detailed discussion below) 22. In this review, I will first describe the up-to-date knowledge on each of these complexes and then will discuss the various and sometimes contradictory mechanistic insights that the most recent research shed on the mechanisms of conjugation and type IV secretion. The relaxosome Excellent reviews have been written on the subject 10, 23, 24 and I will here only focus on recent research illuminating relaxase mechanism. Relaxases Relaxases are phosphodiesterases that catalyse the site- and strand-specific cleavage of the plasmid OriT region at a site termed "nic" (Fig 2A, upper and lower panels). Upon cleavage, the enzyme remains covalently attached to the 5′ end of the T-strand through a phosphotyrosyl linkage. It is this covalent ssDNA–protein conjugate/complex that constitutes the T4S secretion substrate that is to be transferred through the transport machinery. The transport of the relaxase alongside the T-DNA is rationalized (and subsequently demonstrated 25) by the requirement to recircularize the single-strand T-DNA once the complete copy of the T-DNA is transferred to the recipient cell. Figure 2. The relaxosome(A) Schematic diagram of relaxosome composition and assembly. Upper panel: Composition of the F plasmid family relaxosome. The F-family relaxosome is composed of the relaxase TraI and three accessory proteins: TraY and TraM encoded by the plasmid and IHF encoded by the bacterial genome. All proteins assemble at the plasmid's origin of transfer (OriT) in a process that affects DNA topology around the OriT region. OriT also contains the nick site (nic). This site is flanked by regions (in orange and yellow for the regions 5′ or 3′ to nic, respectively) that, when single-stranded, would each bind a TraI molecule. Lower panel: Schematic representation of the OriT region of the F plasmid. The binding sites for each relaxosome components are depicted by boxes coloured according to the protein to which they bind using the same protein colour-coding shown in the upper panel. Under each box, the protein which binds to the depicted site and the name of the site are indicated. "TraI A" and "TraI B" indicate the region 5′ and 3′ to nic to which the trans-esterase and helicase domains of two individual TraI molecules bind, respectively (depicted in Fig 2B). (B) Domain structure of TraI and binding to OriT. Upper panel: Domain structure of TraI. TraI is composed of a trans-esterase domain (orange), a vestigial (green) and an active helicase (blue) domain, and a C-terminal domain (grey). Boundary residues are indicated for R1 plasmid TraI. Lower panel: TraI binding to OriT. TraI is indicated schematically with each volume indicating the various TraI domains using the same colour-coding as described above. Two TraI molecules bind OriT, one on each side of the nic site. TraI bound to sequence 5′ (indicated by the orange strip below) to the nic site is bound through its trans-esterase domain and its overall conformation is open (not shown here). TraI bound to sequence 3′ (indicated by the yellow strip above) to the nic site is bound through its helicase domains and its overall conformation is closed (not shown here). (C) Structure of TraI in its helicase-loaded mode. The TraI–ssDNA complex is shown with TraI and the ssDNA in ribbon and stick representation, respectively. The domains are coloured coded as in upper panel (B). The linker between the trans-esterase and vestigial helicase domain is shown in grey. Download figure Download PowerPoint Relaxases are usually (but not always) large multidomain proteins (Fig 2B, upper panel). In all cases, they contain a trans-esterase (also termed "relaxase") domain of about 300 amino acids that executes the phosphodiesterase reaction. This domain locates at the N-terminus. Additional domains at the C-terminus of the protein may support DNA helicase or DNA primase activities, or extra domain of unknown function 26. Relaxases can be classified into eight "MOB" families, MOBF, MOBH, MOBQ, MOBC, MOBP and MOBV, MOBT and MOBB 27, among which MOBF and MOBP family relaxases have been the best studied 23, 24. Here, I will focus on MOBF relaxases as they have been the focus of the most recent research. MOBF relaxases include TraI encoded by the F-family plasmids (F, R1 and pED208 for example) and TrwC encoded by the R388 plasmid. Both include a helicase domain at their C-terminus but F-family plasmid TraI proteins have a more extensive domain structure with an N-terminal trans-esterase domain (residues 1–306) that catalyses the nicking and covalent attachment of the T-strand to the relaxase 28, a vestigial helicase domain (residues 315–828) that operates as a ssDNA-binding domain 29, an active helicase domain (residues 864–1,461) that unwinds DNA in the 5′-to-3′ direction, and a C-terminal domain, the function of which is still unclear but might be used as a recruitment platform for relaxosome components 30, 31 (residue numbers here are for the R1 plasmid TraI; Fig 2B, upper panel). TrwC consists of only two domains, an N-terminal trans-esterase domain and a single (active) helicase C-terminal domain 32 with functions similar to the corresponding domains in F-family relaxases. The functional domains of MOBF relaxases appear to have different DNA-binding requirements. The trans-esterase domains bind with high affinity to the region of OriT immediately 5′ to the nic site containing sequences likely to form an inverted repeat (IR), while, as shown for F-family relaxases, the helicase domains display sequence specificity for the region of OriT immediately 3′ to the nic site 29, 33. Crystal structures of the trans-esterase domain of TrwC of plasmid R388 and of TraI of F plasmid have provided the molecular basis of the interaction between the trans-esterase domain and its IR-containing ssDNA-binding site 28, 34-36. This work dating back from 2003 has been reviewed extensively and will only be described briefly here taking the TrwC trans-esterase domain as a model 36, 37. The protein displays a fold built on a two-layer alpha/beta sandwich, with a deep narrow cleft that forms the active site. Typically, IR repeats on double-stranded DNA have the potential to form extruded cruciform structures, likely important for binding. In the structure, one IR arm of the extruded cruciform was used in complex formation and shown to be firmly embraced by the protein. The IR arm is followed by a ssDNA segment that enters the active site containing two catalytic tyrosines, Tyr18 and Tyr26. At this point, the ssDNA is presented with two potential exit paths 36. Tyr 18 has been shown to be the catalytic residue onto which the 5′-phosphate resulting from the nicking/cleavage reaction in the donor cell would covalently react; Tyr26 has been implicated in a second cleavage reaction occurring, this time, in the recipient cell to create the essential 3′-OH required for end-joining recircularization of the plasmid DNA 38, 39. This second reaction would occur as a second copy of the nic site inevitably appears when a second copy of the T-strand is "pushed" into the recipient cell (more details below). Thus, the two exit paths could be used simultaneously to bring into proximity the Y18 hydroxyl-5′-phosphate adduct and a free 3′-OH resulting from the cleavage at nic of a second copy of the T-strand. It is however important to note that not all relaxases are endowed with two catalytic tyrosines. When only one exists, as is the case for the TraI F plasmid relaxase, a second copy of the relaxase is required to catalyse the production of the free 3′-OH either in the donor cell or in the recipient cell (more details below) 40. As mentioned above, two binding sites on either side of the nic site provide selective binding platforms for, on the one hand, trans-esterase binding 5′ of nic, and, on the other, helicase binding 3′ of nic. Recently, Ilangovan et al 41 have shown that full-length F-family TraI binds the ssDNA 5′ of nic in an open conformation being susceptible to rapid proteolysis degradation, but binds ssDNA 3′ of nic in a closed form being resistant to proteolytic degradation. Moreover, making elegant use of OriT-derived oligonucleotides containing (i) a photoactivatable cleavage site instead of nic and (ii) judiciously positioned fluorophores, Ilangovan et al 41 demonstrated that OriT can simultaneously bind two TraI molecules (Fig 2B, lower panel), one on each side of the nic site, with the TraI bound 5′ of nic being in an open conformation, while that bound 3′ of nic being in a closed conformation, providing the first experimental evidence that, indeed, two TraI molecules can co-occupy OriT, an observation which, as will be explained below, has profound mechanistic implications. Ilangovan et al 41 also determined the structure of the closed form of TraI by cryo-electron microscopy (cryo-EM) to atomic resolution (Fig 2C). This structure is bound to a 22-mer oligonucleotide derived from the sequence 3′ of nic. Regions of TraI for which the structure could be obtained included the trans-esterase domain, the vestigial and the active helicase domains, but not the C-terminal domain for which no electron density was observed, suggesting this domain is either very flexible or disordered. The salient features of the structure are the following. Firstly, while the three domains (trans-esterase (in orange), vestigial helicase (in green) and active helicase (in blue)) are linearly arranged in the primary sequence, they are not adjacent in the three-dimensional structure (Fig 2B and C); instead, the active helicase domain is positioned near the trans-esterase domain, whereas the vestigial helicase domain is distal relative to the latter. Long linkers between domains facilitate such a domain configuration. Secondly, the ssDNA binds longitudinally across the entire structure, with its 5′ half bound to the trans-esterase and active helicase domains, while its 3′ half is bound to the active and vestigial helicase domain. Interestingly, when superimposing the structure of the single IR-bound trans-esterase domain of TrwC with that of TraI full-length bound to ssDNA, the DNAs sterically clash, suggesting that, in TraI, helicase-associated ssDNA binding and trans-esterase-associated IR binding are mutually exclusive, possibly accounting for earlier observations of negative cooperativity between the two sites 29. Thirdly, the ssDNA is almost completely buried within the structure, explaining the remarkably high processivity of this enzyme. Indeed, TraI is one of the most processive monomeric helicases known and exhibits a fast unwinding rate of ~1,100 bp/s 42, 43. Fourthly, the vestigial and active helicase domains have very similar structures, both exhibiting the classical helicase sub-domain organization of the SF1A/B family resembling most the RecD2 helicase, an SF1B family helicase exhibiting the same 5′-to-3′ directionality as TraI. Indeed, like RecD2, each helicase domain of TraI contains four sub-domains, termed N-terminal (N-term), 1A, 2A and 2B. The "N-term" domain forms an α-helical bundle while the 1A and 2A domains both exhibit a RecA-like fold. However, the 2B sub-domain differs markedly from that of RecD2. Similarly to RecD2, the 2B sub-domains of TraI, in both the vestigial and active helicase domains, are formed by sequence insertions within the 2A domain; however, the 2B sub-domains of TraI are much larger, containing additional sequences that themselves form an additional sub-domain termed "2B-like". Thus, the 2B and 2B-like (2B/2B-like) sub-domains form an extended sub-structure that is observed clamping down on top of the ssDNA, resulting in the ssDNA being mostly buried. Interestingly, in each helicase domain, these 2B/2B-like sub-domains are mounted onto two linker sequences that form hinges onto which these sub-domains could pivot between two configurations, open and closed. In the open configuration, the ssDNA-binding site of the helicase domains would be accessible to binding, and thus, the relaxase would load to the ssDNA sequence. Once bound, the 2B/2B-like sub-domains would close, clamping down onto the ssDNA and unwinding would start. These open/closed states of the helicase domains may or may not correspond to the open and closed states of the relaxase characterized biochemically based on the protease sensitivity experiments described above. Finally, the 2B/2B-like domains have an additional role: they may provide the surfaces responsible for recruitment of TraI to the T4S system. As will be described below, VirD4, a T4S system protein, serves as a recruitment platform for the relaxosome. Because of this role, VirD4 is often known as "the coupling protein". Two regions, termed TSA and TSB, of R1 plasmid TraI (an F-family plasmid) were identified to serve as translocation signals, mediating presumably the recruitment of TraI to the T4S system, perhaps through interactions with the VirD4/TraD protein 44. Similar sequences were identified in the R388 TrwC relaxase 45. Remarkably, these translocation signals are not located at either the C- or N-terminus of these proteins as is usually the case for most known translocation signals, but in their middle. TSA and TSB map to the 2B/2B-like sub-domains and the putative VirD4-interacting region within these sub-domains map opposite to the ssDNA-interacting region, suggesting that these regions are available for binding to the T4S system, even when bound to ssDNA 41, 46. Accessory proteins The relaxase is part of a bigger complex, the relaxosome, which contains 2–3 additional proteins, termed "accessory" proteins (Fig 2A, upper panel). Much has been written about these proteins (reviewed in refs. 10, 41), and thus, only a short description will be given here for the F plasmid relaxosome accessory proteins. In this case, the relaxosome is formed of the relaxase TraI, two plasmid-encoded proteins, TraM and TraY, the genome-encoded IHF (integrated host factor) heterodimeric protein, and OriT. IHF consists of two ~10-kDa subunits, about 30% identical in sequence. The structure of IHF bound to a double-stranded DNA shows that IHF induces a 160° bend in the DNA 47. TraY is a small protein (131 residues), structurally related to the ribbon-helix-helix (RHH) family, which bends the DNA by 50–55° 48. F TraM is a 127-residue protein with an N-terminal domain that binds DNA, and a C-terminal domain responsible for tetramerization. Its N-terminal DNA-binding domain homodimerizes to form a RHH, and two TraM tetramers are required to cooperatively bind a minimal DNA-binding site 49. These proteins together with the relaxase bind multiple sites within OriT (summarized in ref. 50 and in Fig 2A, lower panel). In the OriT of the F plasmid (F OriT), there are two IHF-binding sites (IHF A and B), two TraY-binding sites (sbyA and sbyC), three TraM-binding sites (sbmA-C), and as mentioned above, two TraI-loading sites on each side of nic. The site sequence is the following: sbmA, sbmB, IHF B, sbmC, sbyA, sbyC, IHF A, TraI-binding IR (TraI-A), nic and TraI helicase loading site (TraI-B). The relaxosome proteins assemble on the OriT DNA in a defined order 51, 52 and distort its topology severely and locally, leading to disruption in its supercoiled and double-stranded states. Although the interactions of these proteins with DNA has been extensively studied (reviewed in refs. 10, 24, 37), very little is known about how these proteins interact with each other. However, multiple reports have shown that relaxase activity is stimulated in the presence of accessory proteins, indicating direct interactions between these proteins 33, 52-54. As a matter of fact, everything points to the relaxosome being an extremely complex structure: (i) some of these proteins have stable oligomeric states, but some others appear to adopt various oligomerization states upon binding DNA; and (ii) some of these proteins are able to bend DNA quite severely, implying that DNAs and proteins apparently distant from each other based on the linear organization of the various binding sites in OriT might in fact be within proximity. Solving the three-dimensional structure of a relaxosome is one of the greatest challenges of conjugation research. How the relaxosome is recruited to the T4S system apparatus itself has been investigated extensively (reviewed in ref. 55). All interactions are with VirD4, the coupling protein, itself an integral part of the T4S system 56. TraM of F interacts with the C-terminal tail of VirD4/TraD, an interaction that was visualized crystallographically 57. TraI exhibits two translocation signal sequences, but whether these sequences bind VirD4 directly has not been demonstrated (see above). Finally, accessory proteins TrwA (a potential homologue of TraM) and TrwC (the relaxase) from the R388 plasmid interact with VirD4/TrwB 58. Recently, more details of the interaction between a VirD4 protein, that of Legionella pneumophila, with accessory proteins and chaperones have been revealed 59. These details are reminiscent of the TraM/TraD complex by Lu et al 57 as the interactions are also with the C-terminal tail of VirD4. The T4S system Early in conjugation research, it became apparent that the genes involved in conjugation could be divided in two sets: the mating pair formation (mpf) genes responsible for pilus biogenesis and mating junctions, and DNA-transfer replication (dtr) genes responsible for processing the DNA at OriT 60, 61. The MPF complex is now known as the type IV secretion system while the DTR components are known as the relaxosome. Linking the two complexes is the VirD4 coupling protein (CP), which recruits the relaxosome and presents it to the T4S system. As it seems that VirD4 might be an integral and constitutive part of the T4S system 56, I will include it in the description of the system, instead of treating it separately. Also, because each individual VirB1-11/VirD4 protein has been the subject of exhaustive reviews 62, 63, I will focus on describing the large subassemblies and assemblies that the Gram-negative bacterial T4S systems form, the structures of which have been unravelled recently. T4S systems are unique among secretion systems in being able to transport both proteins and DNAs. Functionally, they cumulate many transport and assembly functions: (i) they function as pilus biogenesis machines able to construct and retract pili made of thousands of pilus subunits; (ii) they act as DNA transporters; and (iii) they act as protein transporters. Conjugative T4S systems are remarkable as being able to cumulate all three functions in one apparatus. No wonder the T4S system architecture is immensely more complex than any other secretion systems in Gram-negative bacteria (see a review by Costa et al 11 for an extensive review of secretion systems in these bacteria and another by Galan and Waksman 64 on injection machines, a more focused review targeted to a larger audience). The first large subassembly of a T4S system (that encoded by the pKM101 plasmid) was described in 2009 when Fronzes et al 13 published the purification and subsequent characterization of its structure by cryo-EM. This complex, named "the core complex" was 1.05 megadaltons in size and composed of 14 copies of three of the VirB proteins, VirB9, VirB10 and the lipoprotein VirB7. Being embedded in the two membranes of Escherichia coli (VirB10 is indeed seen making a channel in the outer membrane 65 and has a trans-membrane helix inserted in the inner membrane), this complex was thought to form the T4S system channel. This notion prevailed for a number of years until Low et al 14 published a larger subassembly of the T4S system, this time encoded by the R388 system (a system closely homologous to that of pKM101). This larger complex is composed of eight VirB components, VirB3-10. It contains the same core complex of VirB7, VirB9 and VirB10 (renamed outer-membrane (OM) core complex (OMCC) since it is primarily directed towards the OM) but this complex is observed mounted via a stalk structure on an IMC made of 12 copies each of VirB3, VirB4, VirB5, VirB6, VirB8 and the 14 N-terminal trans-membrane helices of VirB10 emanating from the OMCC. Remarkably, the VirB4 ATPase forms two hexameric barrels protruding in the cytoplasm. This architecture of a head (the OMCC) mounted on two legs (the two VirB4 hexameric barrels) via a stalk/neck was unprecedented among bacterial secretion systems since all others were best described as concentric stacks of rings extending from the cytosol to the extracellular milieu. When describing T4S systems, it is convenient (albeit imperfect) to categorize them in two classes: A and B. T4AS systems broadly consist of the 12 VirB1-11/VirD4 proteins (one exception is the F-family T4AS system which contains more). T4BS systems are generally much larger, consisting of many more proteins, for example 27 for the Dot/Icm T4S system from L. pneumophila 8, 66. Although the cag T4S system from H. pylori was initially classed as a T4AS system, it seems more related to the T4BS systems class. Nevertheless, T4BS systems include most of the VirB/VirD4 proteins of T4AS systems; for example, all three archetypal T4AS ATPases, VirB4, VirD4 and VirB11, have homologues in the Dot/Icm system (DotO, DotL and DotB, respectively) or the cag system (CagE, Cag5 and Cagα). Interestingly, in the T4AS system encoded by the F plasmid, VirB11 is absent. Thus, overall, T4AS systems can be seen as "minimal" T4S systems with T4BS systems being expanded and more elaborated versions. Below, I will review the structures of OMCCs and IMCs of T4AS and T4BS systems, pointing to the differences between the two classes and also their common features. The outer-membrane core complex (OMCC) In most T4AS systems investigated so far, the OMCC forms a cage with two layers, the O- and I-layers. It is made of VirB7, VirB9 and VirB10, with VirB10 lining the interior of the cage and forming a channel in the OM while also being inserted in the IM (Fig 3A). The VirB10 ring is buttressed on the exterior by binary complexes of VirB7 and VirB9. Most OMCC structures (pKM101, R388, A. tumefaciens, H. pylori cag and Xanthomonas citri) exhibit 14-fold symmetry 13, 16, 65, 67, 68; the only exception is that of L. pneumophila, which exhibits 13-fold symmetry 15, 69. Figure 3. The arch
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