3.A.7 The Type IV (Conjugal DNA-Protein Transfer or VirB) Secretory Pathway (IVSP) Family
Protein complexes of the IVSP family consist of multiple subunits that span the two membranes of the Gram-negative bacterial cell envelope and export proteins or DNA-protein complexes out of the cell and into the cytoplasm of a recipient cell (Saier, 2007). These systems are very promiscuous, being capable of transporting DNA-protein complexes into other bacteria, yeast and plants. The VirB system of agrobacterial species is specifically designed to transfer T-DNA into plant cells, but it can also transfer the IncQ plasmid RSF1010 into plant cells and into other agrobacterial cells. The various Inc systems are designed to mediate plasmid transfer from the donor bacterium to a recipient bacterium. While additional proteins may be required for the complete transfer process, proteins of the VirB system (VirB2-11 and VirD4) appear to be the primary ones involved in export from the cytoplasm across the two membranes of the agrobacterial envelope. VirB1 is a lytic transglycosylase involved in the local remodeling of the peptidoglycan. A type IV secretion system transport signal is present at the C terminus of VirD2 and it is necessary for virulence (van Kregten et al., 2009). This suggests a role for VirD2 as a pilot protein driving translocation of the T-strand. The overall archetecture of the Type IVa pilus has been determined (Chang et al. 2016).
T4SSs have undergone extensive modular evolution through recombination, and the resulting mosaicism complicates phylogeny-based classification. Alvarez-Martinez and Christie (2009) classify T4SSs on the basis of function as conjugation machines, effector translocators, or DNA release/uptake systems, but this approach has its limitations, because conjugation systems also translocate protein substrates independently of DNA, and some effector translocator systems also conjugatively transfer DNA to target cells. Recent work suggests that all prokaryotic T4SSs possess several common mechanistic features, and many have also acquired novel properties for specialized purposes (Alvarez-Martinez and Christie, 2009). Interestingly, conjugation in the cytoplasm and vacuoles of eukaryotic cells has been demonstrated (Lim et al., 2008).
VirB1 consists of two domains. The C-terminal third of the protein, VirB1*, is cleaved from VirB1 and secreted to the outside of the bacterial cell. Both domains are required for wild-type virulence. VirB1* is essential for the formation of the T pilus, a subassembly of the IVSP complex, composed of processed and cyclized VirB2 (major subunit) and VirB5 (minor subunit) (Zupan et al., 2007). Thus, VirB1 is a bifunctional protein required for IVSP assembly. The N-terminal lytic transglycosylase domain provides localized lysis of the peptidoglycan cell wall to allow insertion of the IVSP complex while the C-terminal VirB1* promotes T-pilus assembly through protein-protein interactions with T-pilus subunits.
The export process appears to be driven by ATP hydrolysis. Substrates transferred to the plant cell cytoplasm include VirE2, VirF and the VirD2-T-DNA complex. Two ATPases, VirB4, and VirB11, localized to the cytoplasmic side of the inner membrane, may energize the process. VirB4 together with VirB6 and VirB10 may comprise the integral membrane transport pore. VirB8, VirB9 and VirB10 may also span the cytoplasmic membrane as well as the periplasm and the outer membrane. The latter three proteins may connect the inner membrane complex with the outer membrane complex consisting of disulfide linked VirB7 and VirB9. Thus, a minimum of six proteins comprise the structural and catalytic elements of the bimembrane channel complex. Proteins covalently linked to the DNA may be recognized as a prelude to nucleoprotein complex transport. The VirE2 and VirF proteins of Agrobacterium tumefaciens have been shown to be transformed with the DNA to plants. VirB4, B8, B5 and B2 have been shown to interact. VirB4 interacts and stabilizes VirB8 and mediates incorporation of VirB5 and VirB2 into external pili (Yuan et al., 2005). VirB6 may function in T-pilus biogenesis and as a component of the IV SP apparatus. It interacts with VirB3 and VirB9 but not VirB4, 5 or 11. (Judd et al., 2005).
The twelve proteins of the vir-encoded T4SS can be divided into several subgroups: (1) the transfer pilus (T pilus) composed of VirB2 (the major pilin subunit), VirB5 (a minor pilin subunit), and VirB7 (a lipoprotein that may help anchor the pilus to the outer membrane); (2) the trans envelope proteins VirB6-VirB10; (3) three inner membrane-associated proteins with nucleotide binding domains (VirD4, VirB4, and VirB11), which likely energize transporter assembly or substrate translocation; and (4) proteins with additional specific or unknown functions such as (a) VirB1 (a two-domain protein with two proposed functions, peptidoglycan lysis to assemble the trans envelope core structure and plant interaction via the secreted VirB1 fragment VirB1*), (b) VirD4 (thought to couple the DNA substrate to its cognate T4SS transporter), and (c) VirB3, a pilin-like protein required for the assembly of the T-pilus and for T-DNA secretion. VirB3 has 2 TMSs with both ends in the cytoplasm (Mossey et al., 2010). Stabilization of VirB3 requires VirB4, VirB7 and VirB8 (Mossey et al., 2010).
The Agrobacterium tumefaciens VirB/D4 T4SS transfers oncogenic T-DNA to plant cells, transferred as a nucleoprotein T-complex with VirD2 as the pilot protein. As a derivative of plasmid conjugation systems, the VirB/D4 T4SS can also transfer certain mobilizable plasmids and bacterial proteins like VirE2 and VirF. A cytoplasmic VirD2-binding protein (VBP) is involved in the recruitment of the T-complex to the energizing components of the T4SS, including VirD4, VirB4, and VirB11 (Guo et al., 2007). VBP is also important for the recruitment of a conjugative plasmid to a different transfer system independent of VirB/D4. Thus, VBP functions as a recruiting protein that helps couple nucleoprotein substrates to the appropriate transport sites for conjugative DNA transfers (Guo et al., 2007).
Draper et al., 2006 have studied the VirB4 ATPase and its protein interactions. Both VirB4 and VirB11 are probably hexamers, and the C-terminus of VirB4 interacts with the N-terminus of VirB11 such that the VirB4 C-terminus stacks above VirB11 in the periplasm. VirB4 localizes to the membrane and periplasm, not cytoplasm. The C-terminus of VirB4 also interacts with VirB1, B8 and B10. These observations and others allowed Draper et al., 2006 to propose a structural model for the complex. Interestingly, VirB3-VirB6 and VirB8-VirB11, but not VirB7, are essential for mediating persistence of Brucella in the reticuloendothelial system (den Hartigh et al., 2008).
Agrobacterium tumefaciens VirB10 couples ATP consumption to substrate transfer through the VirB/D4 type IV secretion channel (TVSP) and also mediates biogenesis of the virB-encoded T pilus. Jakubowski et al., 2009 determined the functional importance of VirB10 domains denoted as the: (i) N-terminal cytoplasmic region, (ii) transmembrane (TM) alpha-helix, (iii) proline-rich region (PRR) and (iv) C-terminal beta-barrel domain.Their results indicated that VirB10 stably integrates into the IM, extends via its PRR across the periplasm, and interacts via its beta-barrel domain with the VirB7-VirB9 channel complex. Distinct domains of VirB10 regulate formation of the secretion channel or the T pilus (Jakubowski et al., 2009 ).
The VirB system of A. tumefaciens is related to (1) a natural competence (CAG; ComB) system in Helicobacter pylori, which may also be involved in transfer of virulence factors including the CagA antigen into host animal cells by a type IV secretion system, (2) the TraS/TraB system of the Pseudomonas aeruginosa conjugative plasmid, RP1, (3) the Ptl system, involved in secretion of pertussis toxin of Bordetella pertussis, (4) the trb system from plasmid pTiC58, one of three loci required for conjugal transfer of this Ti plasmid, (5) the Tra system of plasmid F in E. coli, and the Dot conjugative transfer/virulence system of Legionella pneumophila. Although members of the type IV secretion family share many characteristics, not all systems contain the same sets of genes. Thus, the VirB system of Ti plasmids and the trb system of RP4 have only six genes in common. The distantly related CAG system of H. pylori contains only 4 trb homologues, and the Dot system of L. pneumophila contains only 2 recognizable VirB homologues. Homologues of only one VirB protein, VirB10 (TrbI), are present in all known type IV secretion systems characterized.
Distant homologues of many components of the VirB type IV protein secretion system of Agrobacteria are involved in natural competence in Helicobacter pylori (Hofreuter et al., 2003). These proteins are the ComB2-B4 and ComB6-B10 proteins, distantly related to VirB2-B4 and VirB6-B10 of Agrobacteria. Other VirB constituents of Agrobacteria have not yet been identified or shown to be involved in competence (Karnholz et al., 2006).
Helicobacter pylori use a type IV secretion system to deliver CagA into eukaryotic cells to induce interleukin-8 secretion. This system has a filamentous surface organelle with a rigid needle covered by CagY, a VirB10 homologue. CagY also clusters in the outer membrane while a VirB7 homologue is found at the base of the organelle (Rohde et al., 2003). The structure resembles the needle-like structures of type III secretion systems (TC #3.A.6).
VirD4 of Agrobacteria and TrwB of the E. coli R388 conjugative system are known to play a role in DNA transfer. TrwB is an integral membrane protein (507 aas) with two N-terminal TMSs and a large C-terminal cytoplasmic domain of about 450 residues. It forms a hexameric structure with six equivalent monomers forming an almost spherical quaternary structure like the F1-ATPase (TC# 3.A.2) and a central channel, 20 Å in diameter (Gomis-Rüth et al., 2001). It has two domains, an α/β nucleotide-binding domain reminiscent of RecA and DNA ring helicases, and an all-α domain. It resembles the SpoIIIE family FtsK protein (TC# 3.A.12). Like these proteins, TrwB and VirD4 might energize (or contribute to the energization of transfer by 'pushing' the DNA through the conjugation pore). These proteins are very distantly related to the VirB4 ATPase of Agrobacteria.
The TraG (RP4), TraG or VirD4 (A. tumefaciens Ti plasmid), TraD(F) and HP0524 (H. pylori) components of type IV protein secretion systems have been shown to form oligomers and bind DNA without sequence specificity (Schröder et al., 2002). Although they exhibit NTP binding motifs, attempts to demonstrate NTP hydrolysis activity have been unsuccessful. TraG and the TraI relaxase of plasmid RP4 have been shown to interact. TraG of RP4 is a 650 residue transmembrane protein with 4 N-terminal TMSs in the first 150 residues of the protein and possibly as many as two additional TMSs in the large C-terminal, hydrophilic domain. The N- and C-termini are in the cytoplasm. Schröder et al. (2002) propose that these proteins form a multimeric transmembrane pore to which the relaxosome binds via TraG-DNA and TraG-TraI interactions. The complete system consists of sub-complexes (Krall et al., 2002; Ward et al., 2002).
The two pathogenic γ-proteobacteria, Legionella pneumophila and Coxiello burnetii, use a functionally homologous secretion system for pathogenesis. The prototype of this so-called type IVB secretion system is the L. pneumophila Icm/Dot system. The IncI plasmid Tra/Trb system (such as that of plasmid R64) is also of this IVB type. They are very sequence divergent from the type IVA system (Lammertyn and Anne, 2004; Segal et al., 2005). Twenty-five constituents of the Icm/Dot system have been identified although not all have been shown to be important for secretion.
The best characterized T4SS are the type IVA systems, which exhibit extensive similarity to the Agrobacterium VirB T4SS. In contrast, type IVB secretion systems share almost no sequence homology to the type IVA systems, are composed of approximately twice as many proteins, and remain largely uncharacterized. Type IVB systems include the Dot/Icm systems found in Legionella and Coxiella and the conjugative apparatus of IncI plasmids. Vincent et al., (2006) have reported the first extensive characterization of a type IVB system, the Legionella Dot/Icm secretion apparatus. A critical five-protein subassembly spans both bacterial membranes and comprises the core of the secretion complex. This transmembrane connection is mediated by protein dimer pairs consisting of two inner membrane proteins, DotF and DotG, which are able to independently associate with DotH/DotC/DotD in the outer membrane. The Legionella core subcomplex appears to be functionally analogous to the Agrobacterium VirB7-10 subcomplex, suggesting a conservation of the core subassembly in these evolutionarily distant type IV secretion machines.
Conjugative plasmids use a type IV ssDNA-protein translocase coupled to nucleoprotein complex (the relaxosome). TrwB is a membrane-bound homohexamer with a central pore. It hydrolyzes ATP with positive cooperativity with respect to ATP concentration and is a DNA-dependent ATPase (Tato et al., 2005). It may be the DNA-translocating motor.
Neisseria gonorrhoeae secretes chromosomal DNA via a modified type IV secretion system to the growth medium without a migration partner (Hamilton et al., 2005). The process of DNA donation for natural transformation of bacteria has been assumed to involve bacterial cell death. However, in Neisseria gonorrhoeae, mutations in three genes in the gonococcal genetic island (GGI) reduced the ability of a strain to act as a donor in transformation and to release DNA into the culture. The GGI has characteristics of a horizontally acquired genomic island and encodes homologues of type IV secretion system proteins. This model island could be integrated in strains not carrying the GGI and spontaneously excises from that site. The GGI was likely acquired and integrated into the gonococcal chromosome by site-specific recombination and may be lost by site-specific recombination or natural transformation. Mutations in six putative type IV secretion system genes were assayed for the ability to secrete DNA. Five of the mutations greatly reduced or completely eliminated DNA secretion. Thus, N. gonorrhoeae secretes DNA via a specific process. Donated DNA may be used in natural transformation, contributing to antigenic variation and the spread of antibiotic resistance (Hamilton et al., 2005).
As noted in the previous paragraphs, the Neisseria gonorrhoeae type IV secretion system secretes chromosomal DNA for natural transformation. Salgado-Pabon et al. showed that Tyr93 in TraI is required for DNA secretion while Asp120 is required for wild-type levels of DNA secretion. The TraI N-terminal region promotes membrane interaction. Possibly Tyr93 initiates DNA processing. Disruption of an inverted-repeat sequence eliminated DNA secretion, suggesting that this sequence may serve as the origin of transfer for chromosomal DNA secretion (Salgado-Pabon et al., 2007). The neisserial DNA binding components are PilQ and PilG (Lang et al., 2009).
Fronzes et al. (2009) reported the 15Å resolution cryo-electon microscopy structure of the core complex of a T4SS (IVSP) system. The core complex is composed of three proteins, VirB7, VirB9 and VirB10, each present in 14 copies and forming an approximately 1.1-megadalton two-chambered, double membrane-spanning channel. The structure is doubled-walled, with each compenent apparently spanning a large part of the channel. The complex is open on the cytoplasmic side and constricted on the extracellular side. Overall, the T4SS core complex structure is different in both architecture and compostion from the other known double membrane-spanning secretion system that has been structurally characterized.
The I and O layers insert in the inner and outer membrane, respectively. Chandran et al., 2009 solved the crystal structure of an approximately 0.6 MDa outer-membrane complex containing the entire O layer, the largest determined for an outer-membrane channel. VirB10 is the outer-membrane channel with a hydrophobic double-helical transmembrane region. This structure establishes VirB10 as the only known protein crossing both membranes of Gram-negative bacteria. Comparison of the cryo-electron microscopy (cryo-EM) and crystallographic structures points to conformational changes regulating channel opening and closing (Chandran et al., 2009).
Clostridium perfringen causes fatal human infections, such as gas gangrene, as well as gastrointestinal diseases in both humans and animals. Detailed molecular analysis of the tetracycline resistance plasmid pCW3 from C. perfringens has shown that it represents the prototype of a unique family of conjugative antibiotic resistance and virulence plasmids. Bannam et al. (2006) have identified the pCW3 replication region by deletion and transposon mutagenesis and showed that the essential rep gene encodes a basic protein with no similarity to any known plasmid replication proteins. An 11-gene conjugation locus containing 5 genes that encode putative proteins with similarity to proteins from the conjugative transposon Tn916 was identified, although the genes' genetic arrangements were different. Functional genetic studies demonstrated that two of the genes in this transfer clostridial plasmid (tcp) locus, tcpF and tcpH, were essential for the conjugative transfer of pCW3, and comparative analysis confirmed that the tcp locus was not confined to pCW3. The conjugation region was present on all known conjugative plasmids from C. perfringens, including an enterotoxin plasmid and other toxin plasmids. It seems that nonreplicating Tn916-like elements can evolve to become conjugation loci of replicating plasmids that carry major virulence genes or antibiotic resistance determinants.
In C. perfringens conjugative plasmids encode important virulence factors, such as toxins and resistance determinants. All of these plasmids carry a conjugation loci that consists of 11 genes: intP, tcpA to tcpJ (Bannam et al., 2008). Three proteins, TcpA, a potential coupling protein, TcpF, a putative ATPase that is similar to ORF15 from Tn916, and TcpH, which contains VirB6-like domains, are essential for conjugation in the prototype conjugative plasmid pCW3. To analyze the functional domains of TcpH, a putative structural component of the mating pair formation complex, deletion and site-directed mutants were constructed and analyzed (Teng et al., 2008). The N-terminal 581 residues and the conserved 242VQQPW246 motif were required for conjugative transfer. TcpH interactes with itself and TcpC. Analysis of tcpH mutants demonstrated that the region required for these interactions was also localized to the N-terminal 581 residues and that the function of the C-terminal region of TcpH was independent of protein-protein interactions. TcpH and TcpF were located at both cell poles of donor C. perfringens cells. The results provide evidence that TcpH is located in the cell membrane where it oligomerizes and interacts with TcpC to form part of the mating-pair formation complex, which is located at the cell poles and is closely associated with TcpF. TcpA, TcpC, TcpG and TcpH interact to form an essential part of the conjugation complex (Steen et al., 2009).
The ESAT-6 secretion pathway was first described for M. tuberculosis (see TC# 9.A.25). It has been proposed that at least two virulence factors, ESAT-6 (early secreted antigen target, 6 kDa) and CFP-10 (culture filtrate protein, 10 kDa), are secreted via this pathway in a Sec-independent manner (Berthet et al. 1998; Sørensen et al. 1995). Since this pathway was discovered in mycobacteria, it has become known as the Snm pathway (secretion in mycobacteria) (Converse and Cox 2005). The genes for ESAT-6 and CFP-10 are located in conserved gene clusters, which also encode proteins with domains that are conserved in FtsK- and SpoIIIE-like transporters. These conserved FtsK/SpoIIIE domains have been termed FSDs (Burts et al. 2005).
In other gram-positive bacteria, including S. aureus, B. subtilis, B. anthracis, C. acetobutylicum, and L. monocytogenes, homologues of ESAT-6 have been identified (Pallen 2002). The genes for these ESAT-6 homologues are also found in gene clusters that contain at least one gene for a membrane protein with an FSD. In S. aureus, two proteins, named EsxA and EsxB, have been identified that seem to be secreted via the ESAT-6 pathway (Burts et al. 2005). The esxA and esxB genes are part of a cluster containing six other genes for proteins that have been implicated in the translocation of EsxA and EsxB. These include the EsaB and EsaC proteins, with a predicted cytoplasmic location, as well as the predicted membrane proteins EsaA, EssA, EssB, and EssC, among which EssC contains an FSD. Mutations in essA, essB, or essC result in a loss of EsxA and EsxB production, which may be related to inhibition of the synthesis of these proteins or their folding into a protease-resistant conformation. All sequenced S. aureus strains contain this cluster of esa, ess, and esx genes, but it seems to be absent from S. epidermidis. Interestingly, the genes for EsxB and EsaC appear to be absent from the S. aureus MRSA252 strain. This implies that the ESAT-6 machinery of this strain may be required for the transport of only EsxA and perhaps a few other unidentified proteins. If so, EsaC would be dispensable for an active ESAT-6 pathway and might be specifically involved in the export of EsxB. Alternatively, the ESAT-6 pathway could be inactive in the S. aureus MRSA252 strain due to the absence of EsaC (Sibbald et al. 2006).
TC Blast searches revealed that only EssC shows considerable sequence identity with mycobacterial protein secretion systems (3.A.24). However EssA, B and C all show low sequence similarity with IVSP family members (3.A.7). Therefore, this secretion system is considered to belong to family 3.A.7. However, it is likely that 3.A.7 and 3.A.24 are distantly related. EssB, required for secretion of EsxA and EsxB via the ESAT-6 (WXG100-specific translocon) secretion system (COG4499), associates with the membrane in S. aureus but remains soluble when expressed in E. coli. It may oligomerize and interact with other membrane constituents of the secretion system (Chen et al. 2012). EsaD may be a constituent of the ESAT-6 secretory system (Anderson et al. 2011). The two homologous secreted dimeric proteins, EsxA and EsxB, are of known structure and may function as extracellular chaperones or adaptors, facilitating interactions with host cell receptor proteins (Sundaramoorthy et al. 2008).
Bacterial type IV coupling proteins (T4CPs) bind and mediate the delivery of DNA substrates through associated type IV secretion systems (T4SSs) (Whitaker et al. 2015). T4CPs consist of a transmembrane domain, a conserved nucleotide-binding domain (NBD) and a sequence-variable helical bundle called the all-alpha-domain (AAD). In the T4CP structural prototype, plasmid R388-encoded TrwB, the NBD assembles as a homohexamer resembling RecA and DNA ring helicases, and the AAD, which sits at the channel entrance of the homohexamer, is structurally similar to N-terminal domain 1 of recombinase XerD. Whitaker et al. 2015 defined the contributions of AADs from the Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC T4CPs to DNA substrate binding. AAD deletions abolished DNA transfer whereas production of the AADin otherwise wild-type donor strains diminished transfer of cognate, but not heterologous, substrates. Reciprocal swaps of AADs between PcfC and VirD4 abolished transfer of cognate DNA substrates, although strikingly, the VirD4-AADPcfC chimera supported transfer of a mobilizable MOBQ plasmid. Purified AADs from both T4CPs bound DNA substrates without sequence preference, but specifically bound cognate processing proteins required for cleavage at origin-of-transfer sequences. The soluble domains of VirD4 or PcfC lacking their AADs neither exerted negative dominance in vivo nor specifically bound cognate processing proteins in vitro. Probably the T4CP AADs contribute to DNA substrate selection through binding of associated processing proteins. Furthermore, MOBQ plasmids have evolved a docking mechanism that bypasses the AAD substrate discrimination checkpoint, which might account for their capacity to promiscuously transfer through many different T4SSs. It seems that a helical bundle (the all-alpha-domain (AAD) of T4SS receptors functions as a substrate specificity determinant. AADs from two substrate receptors, Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC, bind DNA without sequence or strand preference but specifically bind the cognate relaxases responsible for nicking and piloting the transferred strand through the T4SS. Possibly interactions of receptor AADs with DNA processing factors constitutes the basis for selective coupling of mobile DNA elements with type IV secretion channels (Whitaker et al. 2015).
Coupling proteins are present in all conjugative systems, but are also parts of many T4SSs involved in bacterial virulence, where they are required for protein translocation only. They may play a major role in substrate recruitment. Increasing evidence supports also a role in signal transmission leading to activation of secretion (Llosa and Alkorta 2017). Many conjugative coupling proteins are of the VirD4 protein family. Their conserved features include a nucleotide-binding domain, essential for substrate translocation, a C-terminal domain involved in substrate interactions, and a transmembrane domain, anchoring them to the inner membrane. Purified soluble deletion mutants display ATP hydrolysis activity and non-specific DNA binding.
The overall transport reaction catalyzed by IVSP family complexes is thus believed to be:
Protein or nucleoprotein (donor cytoplasm) + ATP (in) → Protein or nucleoprotein
(recipient cytoplasm or medium) + ADP (in) + Pi (in).
Conjugal DNA-protein transfer complexes. The VirB4 ATPase may mediate dislocation of the membrane-integrated VirB2 pilin (N- and C-termini out; cytoplasmic loop= residues 90-94 inside) during biogenesis of the Agrobacterium VirB/VirD4 type IV secretion system. VirB6 domains direct the ordered export of the DNA substrate through a type IV secretion System (Jakubowski et al. 2004).
VirB complex of Agrobacterium tumefaciens
The type IV β-proteobacterial DNA secretion system (non contact dependent) for natural transformation)
DNA secretion system of Neisseria gonorrhoeae
The Cag pathogenicity island (PAI) proteins (Kutter et al., 2008). CagD (Cag24) is essential for CagA translocation and maximal induction of interleukin-8 secretion (Cendron et al., 2009). It is required for a pro-inflammatory response and multiple cytoskeletal and gene regulatory effects in gastric epithelial cells. Its molecular properties are described in a review by Fischer (2011). Cag16 (CagM) (of unknown function and not dermonstrably homologous to proteins of type IV secretion systems in other bacteria) has 3-4 TMSs and is essential for CagA export. It may therefore be a core protein of the Cag complex (Ling et al. 2013).
The Cag PAI proteins of Helicobacter pylori:
Cag4 (like VirD4) O25259
Cag6 (like VirB10) O25261
Cag7 (like VirB9) O25262
Cag9 (like VirB4) O25264
The putative type IV secretion system of Bacillus anthracis (most genes are encoded on the two virulience plasmids pXO1 and pXO2; Grynberg et al., 2007).
Gram-positive bacteria (Firmicutes)
Type IV secretion system of Bacillus anthracis
pXO1-42 (like VirD4; BXA0064; Q8KYQ0)
pXO1-57 (like VB4; BXA0083; Q8KYP1)
pXO1-58 (like CpaE; BXA0084; Q8KYP0)
pXO1-59 (like CpaF, VirB11; BXA0085; Q7CMH0)
pXO1-60 (like TadB, TadC; BXA0086; Q7CMG9)
pXO1-61 (like TadB, TadC; BXA0087; Q8KYN9)
pXO1-63 (like PilA; BXA0089; Q8KYN8)
pXO1-64 (like PilA; BXA0090; Q7CMG7)
pXO1-66 (like PapD; BXA0092; Q7CMG6)
pXO1-67 (like CheW; BXA0093; Q7CMG5)
pXO1-68 (like CpaA; BXA0068; Q8KYP9)
pXO1-77 (like VirB4; BXA0077; Q7CMH3)
pXO1-78 (like VB4; BXA0107; Q8R8B3)
pXO1-79 (like VB6; BXA0108; Q7CMF7)
pXO2-09 (like VirB4-C; BXB0008; Q6F059)
pXO2-10 (like VirB4-N; BXB0009; Q9RN22)
pXO2-15 (like VirD4-C; BXB0014; Q9RN17)
pXO2-16 (like VirD4-N; BXB0015; Q7CMB1)
pXO2-23/24 (like TadB, TadC; BXB0022; Q9RN08)
pXO2-25 (like CpaF, TadA, VirB11; BXB0023; Q6F054)
BA4072 (like VD4; Q81WB0)
The plasmid pLS20 conjugation system. Several conserved conjugation proteins carried on the Bacillus subtilis plasmid pLS20, (VirB1, VirB4, VirB11, VirD2, and VirD4 homologs) assemble at a single cell pole, but also at other sites along the cell membrane, in cells during the lag phase of growth (Bauer et al., 2011).
Gram-positive bacteria (Firmicutes)
Plasmid pLS20 conjugation system of Bacillus subtilis
VirB1 (Orf30) (E9RJ18)
VirB4 (Orf24) (E9RJ16)
VirB11 (Orf13) (E9RJ00)
VirD2 (Orf34) (E9RJ23)
VirD4 (Orf24) (E9RJ12)
Gram-positive bacteria (Firmicutes)
Conjugation system, IntP/TcpA-J (Bannam et al., 2008; Teng et al., 2008). TcpA but not TcpB is essential for transfer of the conjugative plasmid pCW3 (Parsons et al., 2007). TcpA interacts with other pCW3-encoded proteins to form an essential part of the conjugation complex (Steen et al., 2009).
Gram positive bacteria (Firmicutes)
IntP/TcpA-J of Clostridium perfringens
IntP (251aas;0TMSs) (Q15HV4)
TcpA (532aas;3TMSs) (Shows similarity to proteins in 3.A.12; e.g. SpoIIIE) (Q15HV3)
TcpB (302aas;0TMSs) (Shows similarity to proteins in 3.A.12; e.g. SpoIIIE) (Q2L5Y2)
TcpC (359aas;1TMS) (Q15HV1)
TcpD (115aas;3TMSs) (Q15HV0)
TcpE (125aas;2TMSs) (Q15HU9)
TcpF (840aas;0TMSs) (shows limited sequence similarity to 3.A.7.13.1 Q8R8B3) (Q15HU7)
TcpG (334aas;1TMS) (Q15HU6)
TcpH (831aas;8TMSs(4+4)) (Contains UirB6-like domains and shows sequence similarity in the same regions to 3.A.1.14.18 (Q5HGV2) and 3.A.7.12.1 (O25262)) (Q15HU5)
TcpI (164aas;4TMSs) (Q15HU4)
TcpH (258aas;1TMS) (Q15HU3)
The nonspecific tight adherence fibril (pilus) secretion septum (Flp-1, Flp-2, TadV-RcpCAB Tad ZA-G) (Kachlany et al., 2000; Bhattacharjee et al., 2001; Haase et al., 2003; Schreiner et al., 2003; Wang and Chen, 2005; Tomich et al. 2007). The outer membrane components of the Tad (tight adherence) secreton have been identified (Clock et al. 2008). The tad locus is required for tenacious, nonspecific adherence to surfaces and formation of extremely strong biofilms. This locus is dedicated to the biogenesis of Flp pili, which are required for colonization and virulence (Perez et al. 2006).
TadA-G, V, Z of Acinobacillus (Aggregatibacter) actinomycetemcomitans
Flp-1 (75aas) (Q9ANW5)
Flp-2 (76aas) (O66150)
RcpA secretin (460aas) (Q8GD00)
RcpB (167aas) (Q9X6J2)
RcpC (shows 25% identity, 41% similarity with 3.A.1.122.2; O31712)(274aas) (Q8GD01)
TadA (ATPase; Most like LvhB11; 3.A.7.5.1; Q9RLR3) (426aas) (Q9XC06)
TadB (295aas) (Q9XC05)
TadC (288aas) (Q9S4A8)
TadD (Most like 8.A.11.1.1; Q9LDC0 peptidyl prolyl isomerase) (253aas) (Q9S4A7)
TadE (191aas) (Q9S4A6)
TadF (Most like 3.A.7.14.1; Q52001) (200aas) (Q9S4A5)
TadG (Most similar to 1.A.13.1.2; Q9UNF7; Ca2+-activated Cl- channel)
(Q9S4A4) TadV (142aas) (Q8GD02)
TadZ (374aas) (Q8GCZ9)
F-like conjugation DNA transfer system (Greub et al., 2004).
The F-type conjugation system of Protochlamydia amoebophila
TraA; TrbB; RecD (721aas) (Q6MDD6)
TrbC (198aas) (Q6MB92)
TraB (408aas) (Q6MBA0)
TraC (828aas) (Q6MB95)
TraD (556aas) (Q6MB84)
TraE (189aas) (Q6MBA2)
TraFa (160aas) (Q6MB94)
TraFb (263aas) (Q6MB87)
TraG (927aas) (Q6MB85)
TraH (452aas) (Q6MB86)
TraN (184aas) (Q6MB88)
TraU (311aas) (Q6MB91)
TraW (210aas) (Q6MB93)
The EsxA/B protein secretion pathway system (EPSP) (Anderson et al. 2011; Burts et al. 2005; Chen et al. 2012; Sundaramoorthy et al. 2008). EssC is an integral component of the bacterial """"Type VII"""" secretion system (T7SS). The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop containing ATPase-type domains, D1-3, forming the C-terminal intracellular segment. Zoltner et al. 2016 presented crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution but the full-length recombinant protein, with molecular weight about 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key component of pore formation required for secretion. The extreme C-terminal ATPase domain appears essential for EssC activity as a key part of the T7SS whilst D2 and FHA domains are required for the production of a stable and functional protein (Zoltner et al. 2016). TheTMSs of EssA and EssB interact, regulating secretion (Ahmed et al. 2018). This system resembles the ESX systems of Mycobacterium tuberculosis (TC# 3.A.24).
Low G+C Gram-postive bacteria
EPSP of Staphylococcus aureus (EsaA-C/EssA-C)
EsaA (1009 aas) (Q99WU3)
EsaB (80 aas) (Q99WU1)
EsaC (130 aas) (Q99WT8)
EssA (152 aas) (Q99WU2)
EssB (444 aas) (Q99WU0)
EssC (1479 aas) (Q932J9)
EsaD (617aas) (A6QDR8)
The Cpa protein secretion system, PilA-CpaA-F. PilA (58aas) is similar to Flp pilin (1.E.52.3.2) (Tomich et al. 2007).
The Cpa system of Caulobacter crescentus
PilA (pilin) (Q9L720)
CpaA (Prepilin peptidase) (Q9L719)
CpaB (Pilus assembly protein) (Q9L718)
CpaC Secretin (Q9L717)
CpaD (Pilus assembly protein) (Q9L716)
CpaE (Pilus assembly ATPase) (Q9L715)
CpaF (Pilus assembly ATPase) (Q9L714)
The putative Pcf type IV protein/DNA secretion (conjugation) system of the Enterococcus faecalis plasmid pCF10. All Pcf proteins with more than 100 aas were included (Hirt et al. 2005).
The Pfc system of Enterococcus faecalis pCF10 plasmid. All Pcf proteins with more than 100 aas were included (Hirt et al. 2005).
PcfY, 206 aas, Q5G3L7
PcfC, 609 aas, 3 TMSs, Q5G3N9
PcfD, 719 aas, Q5G3N8
PcfG, 561 aas, Q7X5P3
PcfK, 258 aas, Q5G3N1
PcfJ, 445 aas, Q5G3N2
PcfV, 341 aas, 10 TMSs, Q5G3M0
PcfB, 161 aas, Q5G3P0
PcfF, 118 aas, Q5G3N6
PcfO, 149 aas, 1 TMS, Q5G3M7
PcfA, 202 aas, Q5G3P1
PcfX, 219 aas, Q5G3L8
PcfR, 164 aas, 2 TMSs, Q5G3M4
PcfU, 109 aas, Q5G3M1
PcfL, 182 aas, Q5G3N0
PcfH, 116 aas, 3 TMSs, Q5G3N4
PcfS, 150 aas, ssDNA binding protein, Q5G3M3
PcfT, 192 aas, Q5G3M2
Trw conjugation system of E. coli plasmid IncP. The hexameric form of TrwK is a catalytically active ATPase, much like the structurally related protein TrwB, the conjugative coupling protein (Arechaga et al, 2008), which is a structure-specific DNA-binding protein (Matilla et al., 2010)
Trw conjugation system of E. coli plasmid R388
TrwL1 (like VirB2)
TrwM (like VirB3)
TrwK (like VirB4)
TrwJ (like VirB5)
TrwI (like VirB6)
TrwH (like VirB7)
TrwG (like VirB8)
TrwF (like VirB9)
TrwE (like VirB10)
TrwD (like VirB11)
TrwB (like TraG and VirD4)
P-type conjugal transfer system
Conjugal transfer system of Ilyobacter polytropus
TraG (620 aas; E3HE20)
TrbB (310 aas; E3HE22)
TrbC (106 aas; E3HE16)
TrbD (85 aas; E3HE23)
TrbE (804 aas; E3HE24)
TrbF (227 aas; E3HE17)
TrbG (274 aas; E3HE18)
TrbI (364 aas; E3HE19)
TrbJ (251 aas; E3HE25)
TrbL (350 aas; E3HE26)
Uncharacterized protein (117 aas; E3HE21)
LvhB proteins of Legionella pneumophila (De Buck et al., 2007)
LvhB proteins of Legionella pneumophila
LvhB2 (like VirB2)
LvhB3 (like VirB3)
LvhB4 (like VirB4)
LvhB5 (like VirB5)
LvhB6 (like VirB6)
LvhB7 (like VirB7)
LvhB8 (like VirB8)
LvhB9 (like VirB9)
LvhB10 (like VirB10)
LvhD4 (like VirD4) (Q5ZW42)
Gram-positive bacteria (Firmicutes)
Competence system for DNA uptake (ComB) (Hofreuter et al., 2003; Karnholz et al., 2006). DNA uptake into the periplasm occurs preferentially at the poles. Transport into the cytoplasm depends on ComEC. Thus, DNA uptake may occur in a two step process (Stingl et al., 2010).
ComB1-4 and ComB6-10 of Helicobacter pylori
ComB6 (hp0037; Hp26695 genome)
ComB7 (hp0038; Hp26695 genome)
The Icm/Dot or Dot/Icm multicomponent protein secretion system. IcmS and IcmW form a complex that interacts with and may translocate substrate proteins (Ninio et al., 2005; De Buck et al., 2007; Cambronne and Roy, 2007). The crystal structure of the IcmR-IcmQ complex has been solved (Raychaudhury et al., 2009). Legionella pneumophila survives and replicates inside host cells by secreting ~300 effectors through the defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IVB secretion system (T4BSS). Ghosal et al. 2019, using electron cryotomography mapped the location of the core and accessory components of the Legionella core transmembrane subcomplex, revealing a well-ordered central channel that opens into a large, windowed secretion chamber with an unusual 13-fold symmetry. Immunofluorescence microscopy deciphered an early-stage assembly process that begins with the targeting of Dot/Icm components to the bacterial poles. Polar targeting of this T4BSS is mediated by two Dot/Icm proteins, DotU and IcmF, that are homologues of the T6SS membrane complex components TssL and TssM, suggesting that the Dot/Icm T4BSS is a hybrid system. Thus, the Dot/Icm complex assembles in an 'axial-to-peripheral' pattern (Ghosal et al. 2019).
The Icm/Dot system of Legionella pneumophila
IcmT1 (IM; like TraK) (AAU26538)
IcmS (Cyt) (AAU26539)
IcmR (Cyt) (AAU26540)
IcmQ (Cyt) (AAU26541)
IcmP/DotM (L Trb) (IM; like TrbA) (AAU26542)
IcmO/DotL (IM; like TrbC and VirD4) (AAU26543)
IcmN/DotK/LphA (peri) (AAU26544)
IcmM/DotJ (IM) (AAU26545)
IcmL/DotI (IM; like TraM) (AAU26546)
IcmK/DotH (peri; like TraN) (AAU26547)
IcmE/DotG (IM; like TraO and VirB10) (AAU26548)
IcmG/DotF (IM; like TraP) (AAU26549)
IcmC/DotE (IM; like TraQ) (AAU26550)
IcmD/DotP (IM; like TraR) (AAU26551)
IcmJ/DotN (Cyt; like TraT) (AAU26552)
IcmB/DotB (Cyt; like TraU and VirB4) (AAU26553)
IcmF (IM) (AAU26554)
IcmH (IM) (AAU26555)
IcmW (Cyt) (AAU28746)
IcmX (peri; like TraW) (AAU28747)
IcmV (IM; like TraX) (AAU28745)
DotA (IM; like TraY) (AAK63144)
DotB (Cyt; like TraJ and VirB11) (AAU28734)
DotC (peri; like TraI) (AAU28733)
DotD (peri; like TraH) (AAU28732)