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Biochemistry of type IV secretion
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Biochemistry of type IV secretion Drusilla L Burns In the past year, our knowledge of type IV transporters of Gram-negative bacteria has further expanded. Advances include the discovery of additional members of this family of proteins, increased knowledge of the morphologies of type IV transporters, and a better understanding of the mechanisms by which macromolecules are exported by these systems. Addresses CBER, US Food and Drug Administration, HFM-434, Building 29, Room 418, 8800 Rockville Pike, Bethesda, MD 20892, USA; e-mail: [email protected] Current Opinion in Microbiology 1999, 2:25–29 http://biomednet.com/elecref/1369527400200025 © Elsevier Science Ltd ISSN 1369-5274
Introduction In recent years, much has been learned about the molecular mechanisms involved in the secretion of macromolecules from Gram-negative bacteria. It is now clear that a few distinct families of macromolecular transporters exist, classified primarily on sequence similarities. One of the more recently discovered of these families is the family of type IV transporters, most members of which function primarily to mobilize DNA, either from bacteria to bacteria or from bacteria to eukaryotic cells. In the past few years, additional members of this group have been discovered that have alternate functions including facilitating the transport of multi-subunit proteins across bacterial membrane barriers. Recent studies have increased our knowledge of this interesting family of transporters including the architecture of the transport apparatus and mechanisms of export, which are the focus of this review.
Members of the type IV family of transporters Perhaps the prototypic member of the type IV transporter family is the VirB system of Agrobacterium tumefaciens that exports a large, single-stranded DNA, known as T-DNA, across the bacterial membranes and into plant cells, where the T-DNA integrates into the plant genome. Expression of oncogenes carried by the T-DNA results in uncontrolled cell division and formation of crown gall tumors (reviewed in [1,2]). The virB locus consists of 11 genes (Figure 1), ten of which (virB2 through virB11) are critical for DNA transfer [3,4]. Although virB1 is not absolutely essential, deletion of this gene severely attenuates virulence and leads to a lower level of efficiency of DNA transfer [3,4]. Several macromolecules can be transported by the VirB system including the single-stranded T-DNA, VirE2, which is a single-stranded DNA binding protein that is believed to coat the T-DNA, and VirD2 that binds covalently to the 5′ end of the T-DNA [5,6]. In addition, VirF, a protein critical for infection of certain plant species, is also transported by this complex [7]. At present, the
structure of the transported substrate is not well defined; however, because successful T-DNA transmission does not require that the DNA and VirE2 enter plant cells as a complex, VirE2 and T-DNA complexed to VirD2 may be transported individually [5,8]. The identity of the transported DNA is not critical since the VirB proteins along with VirD4, a protein that may link protein complexes required for DNA processing with the transport machinery, can direct the conjugal transfer of an IncQ plasmid between bacteria in addition to directing T-DNA transfer [9]. Thus this transport system may actually recognize protein substrates, with DNA passively exported due to the fact that it is bound to an actively transported protein. Interestingly, it has recently been reported that conjugal transfer efficiency of an IncQ plasmid is increased dramatically if the recipient strain, as well as the donor strain, expresses a subset of the VirB proteins [10•]. The finding that the VirB system can direct conjugal transfer of DNA between bacteria suggested that this system might be closely related to other bacterial conjugation systems. Sequence analysis of several conjugal transfer systems added credence to this idea since proteins comprising these systems were found to exhibit high levels of homology to the VirB proteins (Figure 1). These proteins include those encoded within the Tra2 region of the IncP plasmid RP4 that are required for pilus formation and conjugal transfer [11,12], as well as conjugal transfer proteins of the IncN plasmid pKM101 [13]. Surprising homologies between these DNA transport systems and a toxin transport system of Bordetella pertussis have been noted. As shown in Figure 1, the nine proteins of the Ptl system of B. pertussis, required for the secretion of pertussis toxin (a multi-subunit protein) across bacterial membranes, exhibit striking homologies to members of the type IV family of transporters [14,15]. Thus for the first time, a type IV system that solely transports proteins was described. Additional type IV homologues were recently discovered in several other pathogenic bacteria including Helicobacter pylori [16••,17] and Legionella pneumophila [18••,19••] (Figure 2). The functions of these proteins have not been well defined. It is known that the VirB4/PtlC homologue of H. pylori, PicB, may play a role in the induction of interleukin-8 (IL-8) expression in gastric epithelial cells since mutations in picB markedly reduce the ability of the bacteria to induce IL-8 expression [17]. The type IV-related genes of L. pneumophila are required for alteration of the endocytic pathway of infected macrophages in a way that is essential for intracellular replication of the bacteria and subsequent killing of the macrophages by bacteria [18••,19••]. The current
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Figure 1
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Alignment of genes encoding type IV transporter systems. These systems include the VirB system of A. tumefaciens, the Ptl system of B. pertussis, the Tra system of the IncN plasmid pKM101 and the Trb genes of the Tra2 region of the IncP plasmid RP4. Arrows of the same color represent homologous genes. Unfilled arrows represent genes that have no homologue.
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niques such as sucrose gradient density centrifugation and differential detergent solubility. In addition, analysis of gene fusions with phoA (encoding alkaline phosphatase) as well as studies of the protease susceptibility of VirB proteins have been conducted. When all of the data are viewed together, a picture emerges of the localization of the individual proteins of a transporter. VirB1 is processed followed by export of its carboxy-terminal portion to the exterior of the cell [20••]. VirB2 and VirB3 are exported [21,22], with VirB2 localizing to the surface of the cell [23,24••]. VirB4 is believed to be an integral cytoplasmic membrane protein with two periplasmic domains [25•]. VirB5, VirB7, VirB8, VirB9, and VirB10 are membraneassociated. Of these proteins, VirB7, VirB8, VirB9 and VirB10 fractionate with both inner and outer membranes [22,26] and extend into the periplasmic space [21,22,27,28•]. VirB11 is believed to be located on the inner side of the cytoplasmic membrane [29]. The finding that many of the VirB proteins were found in both inner and outer membrane fractions is consistent with the idea that the VirB proteins form a transport complex that spans both membranes.
thought in the field is that these genes (along with additional genes) encode a transport system that is essential for transfer of effector molecules into the host cell, although the molecular nature of these effectors has not yet been defined. Although it has been demonstrated that this system can transfer plasmid DNA from one bacterial cell to another [18••,19••], the question of whether the major role of this system is to transport DNA or effector protein molecules remains unanswered. Because the endocytic pathway is altered within minutes of uptake of L. pneumophila, it has been deemed unlikely that DNA represents the effector molecule in this system [18••]. The possibility exists that the system evolved from conjugal DNA transfer systems and has retained DNA transfer mechanisms, yet functions primarily to transport a protein that is capable of modifying the endocytic pathway of the eukaryotic cell.
Molecular architecture of type IV transporters Localization of the proteins that comprise type IV transporters, especially the proteins of the VirB system, has been accomplished using standard cell fractionation techFigure 2
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The genes encoding the VirB system of A. tumefaciens aligned with genes encoding type IV transporter homologues of H. pylori and L. pneumophila.
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Extensive work has been done to elucidate interactions between VirB proteins. VirB7, which is known to be a lipoprotein [22,30•], appears to be critical for the structural integrity of the VirB apparatus in that in-frame deletions in virB7 result in destabilization of many of the VirB proteins including VirB4, VirB5, VirB8, VirB9, VirB10, and VirB11 [31]. Direct interactions between VirB proteins have been visualized for VirB7–VirB7 [32], VirB7–VirB9 [30 •,32,33], VirB9–VirB9 [33] and VirB1–VirB9 [20••]. Interactions between VirB9 and VirB10 have been postulated on the basis of the ability of VirB9 to influence the ability of VirB10 to form higher molecular weight complexes [34•]. An interaction of VirB4 and VirB3 has been hypothesized on the basis of the finding that stabilization and proper localization of VirB3 depends on the presence of VirB4 [35]. Finally, the VirB transporter is thought to contain more than one VirB11 subunit [36•]. Thus a number of the VirB proteins interact with each other, consistent with the hypothesis that these proteins form a transport apparatus.
The transport process
Of particular note is the recent finding that proteins of the VirB transport system form pili [23]. These pili appear to be approximately 4 nm in diameter [23] and vary in length, however, pili longer than the length of the bacterium have been observed. A 7.2 kDa processed form of VirB2 appears to be the major component of this structure [24••,37]. It remains unknown at this time whether the pili simply mediate contact between the bacterium and the plant cell or whether the pilus might actually serve as a conduit through which proteins and DNA may pass.
At the present time, very little is known about the series of events that occur during the transport process. We do not as yet know whether transport occurs as a one-step process (across both bacterial membranes simultaneously) or as a two-step process (across inner and outer membranes individually). In fact, evidence might suggest that different members of the type IV family may differ in this regard. Two proteins that are known to be transported by the VirB system, VirE2 and VirD2, lack canonical signal sequences. Moreover, the T-DNA strand that originates in the cytoplasm of the cell must be transported by a mechanism whereby it remains intact. Because the periplasm contains many nucleases that could potentially degrade singlestranded DNA, the periplasm would be a hostile environment for the T-DNA unless it were protected by some unknown mechanism. For these reasons, it has been postulated that the VirB system (and, by analogy, probably other conjugal DNA transfer systems) utilize a one-step transport process whereby proteins and the associated DNA cross both bacterial membranes simultaneously [26,47], possibly through a channel formed by VirB proteins.
Available evidence indicates that related transporters may have architectures similar to the VirB transporter. Studies of fusions of alkaline phosphatase with proteins comprising an IncN conjugal transfer system indicates that those proteins have membrane topologies similar to those of the corresponding VirB proteins [13]. At least certain aspects of the architecture of the Ptl system of B. pertussis are very similar to that of the VirB system in that Ptl proteins homologous to VirB7 and VirB9 form homodimers and heterodimers similar to those described in the VirB system ([38]; KM Farizo, DL Burns, unpublished data). Although a pilus-like structure, similar to the one described for the VirB system, has not been reported for the Ptl system, it is interesting to note that the Ptl protein transporter system contains a protein that is homologous to the VirB2 pilin protein. Pili structures have been associated with the Tra2 region of the IncP plasmid RP4 [11] and the Tra region of the IncN plasmid pKM101 contains a pilin-like protein [39]. Thus, while much remains to be learned about the architecture of type IV transporters, the structures of these complexes are beginning to be unraveled and intriguing similarities between transporters of very different macromolecular substrates have emerged. The architectural similarities that have been discovered among members of this family of transporters seems to justify classification on the basis of sequence similarities only.
In general, type IV transporters contain two proteins with nucleotide-binding motifs that are potential candidates for the motor behind the transport process. Alternatively, these proteins might serve to signal the opening of a gate or channel via kinase activity, or act as molecular chaperones in the assembly of the transporter or during the transport process itself. The putative nucleotide-binding motifs of these homologues of the VirB and Ptl systems, VirB4, VirB11, PtlC and PtlH, have been shown to be critical for transport ([40–44]; DM Cook, DL Burns, unpublished data). VirB4 has been determined to have ATPase activity [45] and VirB11 has been reported to have weak ATPase and autophosphorylating activity [29]. ATP appears to be critical for the integrity of the type IV transport complex of plasmid RP4 since the apparatus is rapidly disassembled when intracellular ATP concentration is decreased [46], suggesting the possibility that at least one of the nucleotide-binding homologues of type IV systems may be involved in complex assembly/disassembly.
The Ptl system exhibits some striking contrasts to the VirB system in this regard and evidence suggests that pertussis toxin is secreted by a two-step process rather than a onestep process, despite the fact that Ptl homologues exist for each of the VirB proteins except VirB1 (a nonessential transport protein) and VirB5. Each of the individual subunits of pertussis toxin (S1–S5) that are transported by the Ptl system are synthesized with their own signal sequence [48,49] suggesting that they may cross the inner membrane via a Sec-like system. Each of these subunits contains a number of disulfide bonds [50] that must be correctly formed, a process that normally occurs in the periplasm, and the S2, S3, S4, and S5 subunits of the toxin are only inefficiently exported in the absence of the S1 subunit [51] suggesting that the holotoxin is the form of the protein that
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is transported across the outer membrane. Finally, in the absence of an intact Ptl system, biologically active pertussis toxin accumulates in the cell [14]. These findings are most consistent with a two-step model in which the toxin subunits first individually cross the cytoplasmic membrane followed by assembly of the holotoxin in the periplasmic space and finally transport of the holotoxin via the Ptl apparatus across the final bacterial membrane barrier.
Evolution of type IV transporters The striking homologies between members of type IV transporters that carry out vastly different functions are intriguing and bring up the question of how this family of transporters evolved. Bacterial conjugation most likely predated pathogenesis [52], suggesting that the conjugation systems may be the precursors of the VirB and Ptl systems. Evolution of a strictly protein transport system (e.g. Ptl) from nucleoprotein transport systems (e.g. VirB) may have been a small evolutionary jump since the nucleoprotein transporters most probably recognize and transport the protein portion of the nucleoprotein complex. The fact that pertussis toxin is a nucleotide-binding protein [53] brings up the intriguing possibility that it may be distantly related to the proteins of DNA transfer systems that bind single-stranded DNA.
Conclusions Although much has been learned about this interesting family of transport proteins, we still have much to discover concerning the mechanistic details of the transport process. What is the architecture of an intact type IV transport apparatus? What molecular structure of the substrate is recognized by the transport machinery? What are the series of events that occur during export? No doubt research in the next few years will yield exciting discoveries concerning this family of exporters.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest
8.
Citovsky V, Zupan J, Warnick D, Zambryski P: Nuclear localization of Agrobacterium VirE2 protein in plant cells. Science 1992, 256:1802-1805.
9.
Beijersbergen A, Den Dulk-Ras A, Schilperoort RA, Hooykaas PJJ: Conjugative transfer by the virulence system of Agrobacterium tumefaciens. Science 1992, 256:1324-1327.
10. Bohne J, Yim A, Binns AN: The Ti plasmid increases the efficiency • of Agrobacterium tumefaciens as a recipient in virB-mediated conjugal transfer of an IncQ plasmid. Proc Natl Acad Sci USA 1998, 95:7057-7062. This report demonstrates that Agrobacterium-to-Agrobacterium transfer efficiencies of the IncQ plasmid RSF1010 increase if the recipient strain, in addition to the donor strain, produces a subset of the VirB proteins. Because only a subset of the VirB proteins are required to increase the efficiency of import of DNA, it may be possible to study functional aspects of a simplified version of the transport complex. 11. Haase J, Lurz R, Grahn AM, Bamford DH, Lanka E: Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific pahge propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex. J Bacteriol 1995, 177:4779-4791. 12. Lessl M, Balzer D, Pansegrau W, Lanka E: Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer. J Biol Chem 1992, 267:20471-20480. 13. Pohlman RF, Genetti HD, Winans SC: Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens. Mol Microbiol 1994, 14:655-668. 14. Weiss AA, Johnson FD, Burns DL: Molecular characterization of an operon required for pertussis toxin secretion. Proc Natl Acad Sci USA 1993, 90:2970-2974. 15. Covacci A, Rappuoli R: Pertussis toxin export requires accessory genes located downstream from the pertussis toxin operon. Mol Microbiol 1993, 8:429-434. 16. Covacci A, Falkow S, Berg DE, Rappuoli R: Did the inheritance of a •• pathogenicity island modify the virulence of Helicobacter pylori? Trends Microbiol 1997, 5:205-208. The authors note that the H. pylori chromosome contains four genes that are predicted to encode proteins that exhibit striking homology to members of type IV transport systems extending the type IV family of proteins to another human pathogen. 17.
Tummuru MKR, Sharma SA, Blaser MJ: Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells. Mol Microbiol 1995, 18:867-876.
18. Vogel JP, Andrews HL, Wong SK, Isberg RR: Conjugative transfer •• by the virulence system of Legionella pneumophila. Science 1998, 279:873-876. The type IV family of proteins was further expanded when genes encoding type IV homologues were discovered in L. pneumophila. Although the function of these homologues remains unknown, they were shown to be able to play a role in the transfer of DNA between bacteria. 19. Segal G, Purcell M, Shuman HA: Host cell killing and bacterial •• conjugation require overlapping sets of genes within a 22-kb region of the Legionella pnumophila genome. Proc Natl Acad Sci USA 1998, 95:1669-1674. Simultaneously with Vogel et al. [18••], these authors reported that L. pneumophila contained genes encoding type IV transporter homologues that appeared to be part of a complex that was capable of transporting DNA from bacteria to bacteria. The actual function of this system of proteins remains obscure.
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Zambryski P: Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annu Rev Genet 1988, 22:1-30.
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Zambryski P: Chronicles from the Agrobacterium-plant cell DNA transfer story. Annu Rev Plant Physiol Plant Mol Biol 1992, 43:465-490.
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Berger BR, Christie PJ: Genetic complementation analysis of the Agrobacterium tumefaciens virB2 through virB11 are essential virulence genes. J Bacteriol 1994, 176:3646-3660.
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Fullner KJ: Role of Agrobacterium virB genes in transfer of T complexes and RSF1010. J Bacteriol 1998, 180:430-434.
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20. Baron C, Llosa M, Zhou S, Zambryski PC: VirB1, a component of •• the T-complex transfer machinery of Agrobacterium tumefaciens, is processed to a C-terminal secreted product, VirB1*. J Bacteriol 1997, 179:1203-1210. Our knowledge of the architecture of type IV transporters was extended when it was found that a processed from of VirB1 is exported from the bacterial cell and associates with the VirB9–VirB7 heterodimer.
6.
Binns AN, Beaupre CE, Dale EM: Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010. J Bacteriol 1995, 177:4890-4899.
21. Beijersbergen A, Smith SJ, Hooykaas PJJ: Localization and topology of VirB proteins of Agrobacterium tumefaciens. Plasmid 1993, 32:212-218.
7.
Regensburg-Tuink AJG, Hooykaas PJJ: Transgenic N. glauca plants expressing bacterial virulence gene virF are converted into hosts for nopaline strains of A. tumefaciens. Nature 1993, 363:69-71.
22. Fernandez D, Dang TAT, Spudich GM, Zhou X-R, Berger B, Christie PJ: The Agrobacterium tumefaciens virB7 gene product, a proposed component of the T-complex transport apparatus, is a
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membrane-associated lipoprotein exposed at the periplasmic surface. J Bacteriol 1996, 178:3156-3167. 23. Fullner KJ, Lara JC, Nester EW: Pilus assembly by Agrobacterium TDNA transfer genes. Science 1996, 273:1107-1109. 24. Lai E-M, Kado CI: Processed VirB2 is the major subunit of the •• promiscuous pilus of Agrobacterium tumefaciens. J Bacteriol 1998, 180:2711-2727. The earlier exciting finding that a pilus is associated with the VirB transport machinery was further extended with this report in which the authors identified the major subunit of the pilus as being VirB2. 25. Dang TAT, Christie PJ: The VirB4 ATPase of Agrobacterium • tumefaciens is a cytoplasmic membrane protein exposed at the periplasmic surface. J Bacteriol 1997, 179:453-462. VirB4 is likely to play a critical role in the transport process and therefore knowledge of its membrane morphology is an important step in discerning its function. By fusing VirB4 to alkaline phosphatase lacking a signal sequence, the authors were able to elucidate the membrane topology of this protein. From these data, VirB4 appears to be an integral cytopalsmic membrane protein with two periplasmic domains. 26. Thorstenson YR, Kuldau GA, Zambryski PC: Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure. J Bacteriol 1993, 176:5233-5241. 27.
Ward JE, Dale EM, Nester EW, BinnsAN: Identification of a VirB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens. J Bacteriol 1990, 172:5200-5210.
28. Das A, Xie Y-H: Construction of transposon Tn3phoA: its • application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins. Mol Microbiol 1998, 27:405-414. The authors use VirB/alkaline phosphatase fusions to discern the membrane topology of a number of the VirB proteins. Previous work has studied the topology of these proteins when expressed in E. coli. This work, for the first time, examines topology of these proteins in Agrobacterium. 29. Christie PJ, Ward JE, Gordon MP, Nester EW: A gene required for tranfer of T-DNA to plants encodes an ATPase with autophosphorylating activity. Proc Natl Acad Sci USA 1989, 86:9677-9681. 30. Baron C, Thorstenson YR, Zambryski PC The lipoprotein VirB7 • interacts with VirB9 in the membranes of Agrobacterium tumefaciens. J Bacteriol 1997, 179:1211-1218. The yeast two-hybrid system was used to visualize the interaction between VirB9 and VirB7, which represents one of the first successful uses of this assay system to define the interactions between membrane proteins. 31. Fernandez D, Spucich GM, Zhou X-R, Christie PJ: The Agrobacterium tumefaciens VirB7 lipoprotein is required for stabilization of VirB proteins during assembly of the T-complex transport apparatus. J Bacteriol 1996, 178:3168-3176. 32. Spudich GM, Fernandez D, Zhou Z-R, Christie PJ; Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatus. Proc Natl Acad Sci USA 1996, 93:7512-7517. 33. Anderson LB, Hertzel AV, Das A: Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex. Proc Natl Acad Sci USA 1996, 93:8889-8894. 34. Beaupre CE, Bohne J, Dale EM, Binns AN: Interactions between • VirB9 and VirB10 membrane proteins involved in movement of DNA from Agrobacterium tumefaciens into plant cells. J Bacteriol 1997, 179:78-89. VirB9 is essential for VirB10 to participate in higher molecular weight complexes suggesting that VirB9 may either directly or indirectly interact with VirB10, providing additional evidence for the existence of a relatively large transport apparatus. 35. Jones AL, Shirasu K, Kado CI: The prouduct of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein. J Bacteriol 1994, 176:5255-5261.
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36. Zhou Z-R, Christie PJ: Suppression of mutant phenotypes of the • Agrobacterium tumefaciens VirB11 ATPase by overproduction of VirB proteins. J Bacteriol 1997, 179:5835-5842. This report demonstrates that the VirB transporter contains more than one VirB11 subunit and that VirB11 interacts with VirB9 and VirB10 during transporter biogenesis, providing additional evidence that the transporter may be a large apparatus. 37.
Jones AL, Lai E-M, Shirasu K, Kado CI: VirB2 is a processed pilinlike protein encoded by the Agrobacterium tumefaciens Ti plasmid. J Bacteriol 1996, 178:5706-5711.
38. Farizo KM, Cafarella TG, Burns DL: Evidence for a ninth gene, ptlI, in the locus encoding the pertussis toxin secretion system of Bordetella pertussis and formation of a PtlI-PtlF complex. J Biol Chem 1996, 271:31643-31649. 39. Cellini C, Kalogeraki S, Winans SC: The hydrophobic TraM protein of pKM101 is required for conjugal transfer and sensitivity to donor-specific bacteriophage. Plasmid 1997, 37:181-188. 40. Rashkova S, Spudich GM, Christie PJ: Characterization of membrane and protein interaction determinants of the Agrobacterium tumefaciens VirB11 ATPase. J Bacteriol 1997, 179:583-591. 41. Stephens KM, Roush C, Nester E: Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence. J Bacteriol 1995, 177:27-36. 42. Fullner KJ, Stephens KM, Nester EW: An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA. Mol Gen Genet 1994, 245:704-715. 43. Berger BR, Christie PJ: The Agrobacterium tumefaciens virB4 gene product is an essential virulence protein requiring an intact nucleoside triphosphate-binding domain. J Bacteriol 1993, 175:1723-1734. 44. Kotob SI, Burns DL: Essential role of the consensus nucleotidebinding site of PtlH in secretion of pertussis toxin from Bordetella pertussis. J Bacteriol 1997, 179:7577-7580. 45. Shirasu K, Koukolikova-Nicola Z, Hohn B, Kado CI: An innermembrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes. Mol Microbiol 1994, 11:581-588. 46. Daugelavicius R, Bamford JKH, Grahn AM, Lanka E, Bamford DH: The IncP plasmid-encoded cell envelope-associated DNA transfer complex increases cell permeability. J Bacteriol 1997, 179:5195-5202. 47.
Christie PJ: Agrobacterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional tranporters in eubacteria. J Bacteriol 1997, 179:3085-3094.
48. Locht C, Keith JM: Pertussis toxin gene: nucleotide sequence and genetic organization. Science 1986, 232:1258-1264. 49. Nicosia A, Perugini M, Franzini C, Casagli MC, Borri MG, Antoni G, Almoni M, Neri P, Ratti G, Rappuoli R: Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication. Proc Natl Acad Sci USA 1986, 83:4631-4635. 50. Stein PE, Boodhoo A, Armstrong GD, Cockle SA, Klein MH, Read RJ: The crystal structure of pertussis toxin. Structure 1994, 2:45-57. 51. Pizza M, Bugnoli M, Manetti R, Covacci A, Rappuoli R: The subunit S1 is important for pertussis toxin secretion. J Biol Chem 1990, 265:17759-17763. 52. Winans SC, Burns DL, Christie PJ: Adaptation of a conjugal transfer system for the export of pathogenic macromolecules. Trends Microbiol 1996, 4:64-68. 53. Burns DL, Manclark CR: Adenine nucleotides promote dissociation of pertussis toxin subunits. J Biol Chem 1986, 261:4324-4327.
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Biochemistry of type IV secretion Drusilla L Burns In the past year, our knowledge of type IV transporters of Gram-negative bacteria has further expanded. Advances include the discovery of additional members of this family of proteins, increased knowledge of the morphologies of type IV transporters, and a better understanding of the mechanisms by which macromolecules are exported by these systems. Addresses CBER, US Food and Drug Administration, HFM-434, Building 29, Room 418, 8800 Rockville Pike, Bethesda, MD 20892, USA; e-mail: [email protected] Current Opinion in Microbiology 1999, 2:25–29 http://biomednet.com/elecref/1369527400200025 © Elsevier Science Ltd ISSN 1369-5274
Introduction In recent years, much has been learned about the molecular mechanisms involved in the secretion of macromolecules from Gram-negative bacteria. It is now clear that a few distinct families of macromolecular transporters exist, classified primarily on sequence similarities. One of the more recently discovered of these families is the family of type IV transporters, most members of which function primarily to mobilize DNA, either from bacteria to bacteria or from bacteria to eukaryotic cells. In the past few years, additional members of this group have been discovered that have alternate functions including facilitating the transport of multi-subunit proteins across bacterial membrane barriers. Recent studies have increased our knowledge of this interesting family of transporters including the architecture of the transport apparatus and mechanisms of export, which are the focus of this review.
Members of the type IV family of transporters Perhaps the prototypic member of the type IV transporter family is the VirB system of Agrobacterium tumefaciens that exports a large, single-stranded DNA, known as T-DNA, across the bacterial membranes and into plant cells, where the T-DNA integrates into the plant genome. Expression of oncogenes carried by the T-DNA results in uncontrolled cell division and formation of crown gall tumors (reviewed in [1,2]). The virB locus consists of 11 genes (Figure 1), ten of which (virB2 through virB11) are critical for DNA transfer [3,4]. Although virB1 is not absolutely essential, deletion of this gene severely attenuates virulence and leads to a lower level of efficiency of DNA transfer [3,4]. Several macromolecules can be transported by the VirB system including the single-stranded T-DNA, VirE2, which is a single-stranded DNA binding protein that is believed to coat the T-DNA, and VirD2 that binds covalently to the 5′ end of the T-DNA [5,6]. In addition, VirF, a protein critical for infection of certain plant species, is also transported by this complex [7]. At present, the
structure of the transported substrate is not well defined; however, because successful T-DNA transmission does not require that the DNA and VirE2 enter plant cells as a complex, VirE2 and T-DNA complexed to VirD2 may be transported individually [5,8]. The identity of the transported DNA is not critical since the VirB proteins along with VirD4, a protein that may link protein complexes required for DNA processing with the transport machinery, can direct the conjugal transfer of an IncQ plasmid between bacteria in addition to directing T-DNA transfer [9]. Thus this transport system may actually recognize protein substrates, with DNA passively exported due to the fact that it is bound to an actively transported protein. Interestingly, it has recently been reported that conjugal transfer efficiency of an IncQ plasmid is increased dramatically if the recipient strain, as well as the donor strain, expresses a subset of the VirB proteins [10•]. The finding that the VirB system can direct conjugal transfer of DNA between bacteria suggested that this system might be closely related to other bacterial conjugation systems. Sequence analysis of several conjugal transfer systems added credence to this idea since proteins comprising these systems were found to exhibit high levels of homology to the VirB proteins (Figure 1). These proteins include those encoded within the Tra2 region of the IncP plasmid RP4 that are required for pilus formation and conjugal transfer [11,12], as well as conjugal transfer proteins of the IncN plasmid pKM101 [13]. Surprising homologies between these DNA transport systems and a toxin transport system of Bordetella pertussis have been noted. As shown in Figure 1, the nine proteins of the Ptl system of B. pertussis, required for the secretion of pertussis toxin (a multi-subunit protein) across bacterial membranes, exhibit striking homologies to members of the type IV family of transporters [14,15]. Thus for the first time, a type IV system that solely transports proteins was described. Additional type IV homologues were recently discovered in several other pathogenic bacteria including Helicobacter pylori [16••,17] and Legionella pneumophila [18••,19••] (Figure 2). The functions of these proteins have not been well defined. It is known that the VirB4/PtlC homologue of H. pylori, PicB, may play a role in the induction of interleukin-8 (IL-8) expression in gastric epithelial cells since mutations in picB markedly reduce the ability of the bacteria to induce IL-8 expression [17]. The type IV-related genes of L. pneumophila are required for alteration of the endocytic pathway of infected macrophages in a way that is essential for intracellular replication of the bacteria and subsequent killing of the macrophages by bacteria [18••,19••]. The current
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Figure 1
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Alignment of genes encoding type IV transporter systems. These systems include the VirB system of A. tumefaciens, the Ptl system of B. pertussis, the Tra system of the IncN plasmid pKM101 and the Trb genes of the Tra2 region of the IncP plasmid RP4. Arrows of the same color represent homologous genes. Unfilled arrows represent genes that have no homologue.
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niques such as sucrose gradient density centrifugation and differential detergent solubility. In addition, analysis of gene fusions with phoA (encoding alkaline phosphatase) as well as studies of the protease susceptibility of VirB proteins have been conducted. When all of the data are viewed together, a picture emerges of the localization of the individual proteins of a transporter. VirB1 is processed followed by export of its carboxy-terminal portion to the exterior of the cell [20••]. VirB2 and VirB3 are exported [21,22], with VirB2 localizing to the surface of the cell [23,24••]. VirB4 is believed to be an integral cytoplasmic membrane protein with two periplasmic domains [25•]. VirB5, VirB7, VirB8, VirB9, and VirB10 are membraneassociated. Of these proteins, VirB7, VirB8, VirB9 and VirB10 fractionate with both inner and outer membranes [22,26] and extend into the periplasmic space [21,22,27,28•]. VirB11 is believed to be located on the inner side of the cytoplasmic membrane [29]. The finding that many of the VirB proteins were found in both inner and outer membrane fractions is consistent with the idea that the VirB proteins form a transport complex that spans both membranes.
thought in the field is that these genes (along with additional genes) encode a transport system that is essential for transfer of effector molecules into the host cell, although the molecular nature of these effectors has not yet been defined. Although it has been demonstrated that this system can transfer plasmid DNA from one bacterial cell to another [18••,19••], the question of whether the major role of this system is to transport DNA or effector protein molecules remains unanswered. Because the endocytic pathway is altered within minutes of uptake of L. pneumophila, it has been deemed unlikely that DNA represents the effector molecule in this system [18••]. The possibility exists that the system evolved from conjugal DNA transfer systems and has retained DNA transfer mechanisms, yet functions primarily to transport a protein that is capable of modifying the endocytic pathway of the eukaryotic cell.
Molecular architecture of type IV transporters Localization of the proteins that comprise type IV transporters, especially the proteins of the VirB system, has been accomplished using standard cell fractionation techFigure 2
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The genes encoding the VirB system of A. tumefaciens aligned with genes encoding type IV transporter homologues of H. pylori and L. pneumophila.
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Extensive work has been done to elucidate interactions between VirB proteins. VirB7, which is known to be a lipoprotein [22,30•], appears to be critical for the structural integrity of the VirB apparatus in that in-frame deletions in virB7 result in destabilization of many of the VirB proteins including VirB4, VirB5, VirB8, VirB9, VirB10, and VirB11 [31]. Direct interactions between VirB proteins have been visualized for VirB7–VirB7 [32], VirB7–VirB9 [30 •,32,33], VirB9–VirB9 [33] and VirB1–VirB9 [20••]. Interactions between VirB9 and VirB10 have been postulated on the basis of the ability of VirB9 to influence the ability of VirB10 to form higher molecular weight complexes [34•]. An interaction of VirB4 and VirB3 has been hypothesized on the basis of the finding that stabilization and proper localization of VirB3 depends on the presence of VirB4 [35]. Finally, the VirB transporter is thought to contain more than one VirB11 subunit [36•]. Thus a number of the VirB proteins interact with each other, consistent with the hypothesis that these proteins form a transport apparatus.
The transport process
Of particular note is the recent finding that proteins of the VirB transport system form pili [23]. These pili appear to be approximately 4 nm in diameter [23] and vary in length, however, pili longer than the length of the bacterium have been observed. A 7.2 kDa processed form of VirB2 appears to be the major component of this structure [24••,37]. It remains unknown at this time whether the pili simply mediate contact between the bacterium and the plant cell or whether the pilus might actually serve as a conduit through which proteins and DNA may pass.
At the present time, very little is known about the series of events that occur during the transport process. We do not as yet know whether transport occurs as a one-step process (across both bacterial membranes simultaneously) or as a two-step process (across inner and outer membranes individually). In fact, evidence might suggest that different members of the type IV family may differ in this regard. Two proteins that are known to be transported by the VirB system, VirE2 and VirD2, lack canonical signal sequences. Moreover, the T-DNA strand that originates in the cytoplasm of the cell must be transported by a mechanism whereby it remains intact. Because the periplasm contains many nucleases that could potentially degrade singlestranded DNA, the periplasm would be a hostile environment for the T-DNA unless it were protected by some unknown mechanism. For these reasons, it has been postulated that the VirB system (and, by analogy, probably other conjugal DNA transfer systems) utilize a one-step transport process whereby proteins and the associated DNA cross both bacterial membranes simultaneously [26,47], possibly through a channel formed by VirB proteins.
Available evidence indicates that related transporters may have architectures similar to the VirB transporter. Studies of fusions of alkaline phosphatase with proteins comprising an IncN conjugal transfer system indicates that those proteins have membrane topologies similar to those of the corresponding VirB proteins [13]. At least certain aspects of the architecture of the Ptl system of B. pertussis are very similar to that of the VirB system in that Ptl proteins homologous to VirB7 and VirB9 form homodimers and heterodimers similar to those described in the VirB system ([38]; KM Farizo, DL Burns, unpublished data). Although a pilus-like structure, similar to the one described for the VirB system, has not been reported for the Ptl system, it is interesting to note that the Ptl protein transporter system contains a protein that is homologous to the VirB2 pilin protein. Pili structures have been associated with the Tra2 region of the IncP plasmid RP4 [11] and the Tra region of the IncN plasmid pKM101 contains a pilin-like protein [39]. Thus, while much remains to be learned about the architecture of type IV transporters, the structures of these complexes are beginning to be unraveled and intriguing similarities between transporters of very different macromolecular substrates have emerged. The architectural similarities that have been discovered among members of this family of transporters seems to justify classification on the basis of sequence similarities only.
In general, type IV transporters contain two proteins with nucleotide-binding motifs that are potential candidates for the motor behind the transport process. Alternatively, these proteins might serve to signal the opening of a gate or channel via kinase activity, or act as molecular chaperones in the assembly of the transporter or during the transport process itself. The putative nucleotide-binding motifs of these homologues of the VirB and Ptl systems, VirB4, VirB11, PtlC and PtlH, have been shown to be critical for transport ([40–44]; DM Cook, DL Burns, unpublished data). VirB4 has been determined to have ATPase activity [45] and VirB11 has been reported to have weak ATPase and autophosphorylating activity [29]. ATP appears to be critical for the integrity of the type IV transport complex of plasmid RP4 since the apparatus is rapidly disassembled when intracellular ATP concentration is decreased [46], suggesting the possibility that at least one of the nucleotide-binding homologues of type IV systems may be involved in complex assembly/disassembly.
The Ptl system exhibits some striking contrasts to the VirB system in this regard and evidence suggests that pertussis toxin is secreted by a two-step process rather than a onestep process, despite the fact that Ptl homologues exist for each of the VirB proteins except VirB1 (a nonessential transport protein) and VirB5. Each of the individual subunits of pertussis toxin (S1–S5) that are transported by the Ptl system are synthesized with their own signal sequence [48,49] suggesting that they may cross the inner membrane via a Sec-like system. Each of these subunits contains a number of disulfide bonds [50] that must be correctly formed, a process that normally occurs in the periplasm, and the S2, S3, S4, and S5 subunits of the toxin are only inefficiently exported in the absence of the S1 subunit [51] suggesting that the holotoxin is the form of the protein that
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is transported across the outer membrane. Finally, in the absence of an intact Ptl system, biologically active pertussis toxin accumulates in the cell [14]. These findings are most consistent with a two-step model in which the toxin subunits first individually cross the cytoplasmic membrane followed by assembly of the holotoxin in the periplasmic space and finally transport of the holotoxin via the Ptl apparatus across the final bacterial membrane barrier.
Evolution of type IV transporters The striking homologies between members of type IV transporters that carry out vastly different functions are intriguing and bring up the question of how this family of transporters evolved. Bacterial conjugation most likely predated pathogenesis [52], suggesting that the conjugation systems may be the precursors of the VirB and Ptl systems. Evolution of a strictly protein transport system (e.g. Ptl) from nucleoprotein transport systems (e.g. VirB) may have been a small evolutionary jump since the nucleoprotein transporters most probably recognize and transport the protein portion of the nucleoprotein complex. The fact that pertussis toxin is a nucleotide-binding protein [53] brings up the intriguing possibility that it may be distantly related to the proteins of DNA transfer systems that bind single-stranded DNA.
Conclusions Although much has been learned about this interesting family of transport proteins, we still have much to discover concerning the mechanistic details of the transport process. What is the architecture of an intact type IV transport apparatus? What molecular structure of the substrate is recognized by the transport machinery? What are the series of events that occur during export? No doubt research in the next few years will yield exciting discoveries concerning this family of exporters.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest
8.
Citovsky V, Zupan J, Warnick D, Zambryski P: Nuclear localization of Agrobacterium VirE2 protein in plant cells. Science 1992, 256:1802-1805.
9.
Beijersbergen A, Den Dulk-Ras A, Schilperoort RA, Hooykaas PJJ: Conjugative transfer by the virulence system of Agrobacterium tumefaciens. Science 1992, 256:1324-1327.
10. Bohne J, Yim A, Binns AN: The Ti plasmid increases the efficiency • of Agrobacterium tumefaciens as a recipient in virB-mediated conjugal transfer of an IncQ plasmid. Proc Natl Acad Sci USA 1998, 95:7057-7062. This report demonstrates that Agrobacterium-to-Agrobacterium transfer efficiencies of the IncQ plasmid RSF1010 increase if the recipient strain, in addition to the donor strain, produces a subset of the VirB proteins. Because only a subset of the VirB proteins are required to increase the efficiency of import of DNA, it may be possible to study functional aspects of a simplified version of the transport complex. 11. Haase J, Lurz R, Grahn AM, Bamford DH, Lanka E: Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific pahge propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex. J Bacteriol 1995, 177:4779-4791. 12. Lessl M, Balzer D, Pansegrau W, Lanka E: Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer. J Biol Chem 1992, 267:20471-20480. 13. Pohlman RF, Genetti HD, Winans SC: Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens. Mol Microbiol 1994, 14:655-668. 14. Weiss AA, Johnson FD, Burns DL: Molecular characterization of an operon required for pertussis toxin secretion. Proc Natl Acad Sci USA 1993, 90:2970-2974. 15. Covacci A, Rappuoli R: Pertussis toxin export requires accessory genes located downstream from the pertussis toxin operon. Mol Microbiol 1993, 8:429-434. 16. Covacci A, Falkow S, Berg DE, Rappuoli R: Did the inheritance of a •• pathogenicity island modify the virulence of Helicobacter pylori? Trends Microbiol 1997, 5:205-208. The authors note that the H. pylori chromosome contains four genes that are predicted to encode proteins that exhibit striking homology to members of type IV transport systems extending the type IV family of proteins to another human pathogen. 17.
Tummuru MKR, Sharma SA, Blaser MJ: Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells. Mol Microbiol 1995, 18:867-876.
18. Vogel JP, Andrews HL, Wong SK, Isberg RR: Conjugative transfer •• by the virulence system of Legionella pneumophila. Science 1998, 279:873-876. The type IV family of proteins was further expanded when genes encoding type IV homologues were discovered in L. pneumophila. Although the function of these homologues remains unknown, they were shown to be able to play a role in the transfer of DNA between bacteria. 19. Segal G, Purcell M, Shuman HA: Host cell killing and bacterial •• conjugation require overlapping sets of genes within a 22-kb region of the Legionella pnumophila genome. Proc Natl Acad Sci USA 1998, 95:1669-1674. Simultaneously with Vogel et al. [18••], these authors reported that L. pneumophila contained genes encoding type IV transporter homologues that appeared to be part of a complex that was capable of transporting DNA from bacteria to bacteria. The actual function of this system of proteins remains obscure.
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20. Baron C, Llosa M, Zhou S, Zambryski PC: VirB1, a component of •• the T-complex transfer machinery of Agrobacterium tumefaciens, is processed to a C-terminal secreted product, VirB1*. J Bacteriol 1997, 179:1203-1210. Our knowledge of the architecture of type IV transporters was extended when it was found that a processed from of VirB1 is exported from the bacterial cell and associates with the VirB9–VirB7 heterodimer.
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Binns AN, Beaupre CE, Dale EM: Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010. J Bacteriol 1995, 177:4890-4899.
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22. Fernandez D, Dang TAT, Spudich GM, Zhou X-R, Berger B, Christie PJ: The Agrobacterium tumefaciens virB7 gene product, a proposed component of the T-complex transport apparatus, is a
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membrane-associated lipoprotein exposed at the periplasmic surface. J Bacteriol 1996, 178:3156-3167. 23. Fullner KJ, Lara JC, Nester EW: Pilus assembly by Agrobacterium TDNA transfer genes. Science 1996, 273:1107-1109. 24. Lai E-M, Kado CI: Processed VirB2 is the major subunit of the •• promiscuous pilus of Agrobacterium tumefaciens. J Bacteriol 1998, 180:2711-2727. The earlier exciting finding that a pilus is associated with the VirB transport machinery was further extended with this report in which the authors identified the major subunit of the pilus as being VirB2. 25. Dang TAT, Christie PJ: The VirB4 ATPase of Agrobacterium • tumefaciens is a cytoplasmic membrane protein exposed at the periplasmic surface. J Bacteriol 1997, 179:453-462. VirB4 is likely to play a critical role in the transport process and therefore knowledge of its membrane morphology is an important step in discerning its function. By fusing VirB4 to alkaline phosphatase lacking a signal sequence, the authors were able to elucidate the membrane topology of this protein. From these data, VirB4 appears to be an integral cytopalsmic membrane protein with two periplasmic domains. 26. Thorstenson YR, Kuldau GA, Zambryski PC: Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure. J Bacteriol 1993, 176:5233-5241. 27.
Ward JE, Dale EM, Nester EW, BinnsAN: Identification of a VirB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens. J Bacteriol 1990, 172:5200-5210.
28. Das A, Xie Y-H: Construction of transposon Tn3phoA: its • application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins. Mol Microbiol 1998, 27:405-414. The authors use VirB/alkaline phosphatase fusions to discern the membrane topology of a number of the VirB proteins. Previous work has studied the topology of these proteins when expressed in E. coli. This work, for the first time, examines topology of these proteins in Agrobacterium. 29. Christie PJ, Ward JE, Gordon MP, Nester EW: A gene required for tranfer of T-DNA to plants encodes an ATPase with autophosphorylating activity. Proc Natl Acad Sci USA 1989, 86:9677-9681. 30. Baron C, Thorstenson YR, Zambryski PC The lipoprotein VirB7 • interacts with VirB9 in the membranes of Agrobacterium tumefaciens. J Bacteriol 1997, 179:1211-1218. The yeast two-hybrid system was used to visualize the interaction between VirB9 and VirB7, which represents one of the first successful uses of this assay system to define the interactions between membrane proteins. 31. Fernandez D, Spucich GM, Zhou X-R, Christie PJ: The Agrobacterium tumefaciens VirB7 lipoprotein is required for stabilization of VirB proteins during assembly of the T-complex transport apparatus. J Bacteriol 1996, 178:3168-3176. 32. Spudich GM, Fernandez D, Zhou Z-R, Christie PJ; Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatus. Proc Natl Acad Sci USA 1996, 93:7512-7517. 33. Anderson LB, Hertzel AV, Das A: Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex. Proc Natl Acad Sci USA 1996, 93:8889-8894. 34. Beaupre CE, Bohne J, Dale EM, Binns AN: Interactions between • VirB9 and VirB10 membrane proteins involved in movement of DNA from Agrobacterium tumefaciens into plant cells. J Bacteriol 1997, 179:78-89. VirB9 is essential for VirB10 to participate in higher molecular weight complexes suggesting that VirB9 may either directly or indirectly interact with VirB10, providing additional evidence for the existence of a relatively large transport apparatus. 35. Jones AL, Shirasu K, Kado CI: The prouduct of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein. J Bacteriol 1994, 176:5255-5261.
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36. Zhou Z-R, Christie PJ: Suppression of mutant phenotypes of the • Agrobacterium tumefaciens VirB11 ATPase by overproduction of VirB proteins. J Bacteriol 1997, 179:5835-5842. This report demonstrates that the VirB transporter contains more than one VirB11 subunit and that VirB11 interacts with VirB9 and VirB10 during transporter biogenesis, providing additional evidence that the transporter may be a large apparatus. 37.
Jones AL, Lai E-M, Shirasu K, Kado CI: VirB2 is a processed pilinlike protein encoded by the Agrobacterium tumefaciens Ti plasmid. J Bacteriol 1996, 178:5706-5711.
38. Farizo KM, Cafarella TG, Burns DL: Evidence for a ninth gene, ptlI, in the locus encoding the pertussis toxin secretion system of Bordetella pertussis and formation of a PtlI-PtlF complex. J Biol Chem 1996, 271:31643-31649. 39. Cellini C, Kalogeraki S, Winans SC: The hydrophobic TraM protein of pKM101 is required for conjugal transfer and sensitivity to donor-specific bacteriophage. Plasmid 1997, 37:181-188. 40. Rashkova S, Spudich GM, Christie PJ: Characterization of membrane and protein interaction determinants of the Agrobacterium tumefaciens VirB11 ATPase. J Bacteriol 1997, 179:583-591. 41. Stephens KM, Roush C, Nester E: Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence. J Bacteriol 1995, 177:27-36. 42. Fullner KJ, Stephens KM, Nester EW: An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA. Mol Gen Genet 1994, 245:704-715. 43. Berger BR, Christie PJ: The Agrobacterium tumefaciens virB4 gene product is an essential virulence protein requiring an intact nucleoside triphosphate-binding domain. J Bacteriol 1993, 175:1723-1734. 44. Kotob SI, Burns DL: Essential role of the consensus nucleotidebinding site of PtlH in secretion of pertussis toxin from Bordetella pertussis. J Bacteriol 1997, 179:7577-7580. 45. Shirasu K, Koukolikova-Nicola Z, Hohn B, Kado CI: An innermembrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes. Mol Microbiol 1994, 11:581-588. 46. Daugelavicius R, Bamford JKH, Grahn AM, Lanka E, Bamford DH: The IncP plasmid-encoded cell envelope-associated DNA transfer complex increases cell permeability. J Bacteriol 1997, 179:5195-5202. 47.
Christie PJ: Agrobacterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional tranporters in eubacteria. J Bacteriol 1997, 179:3085-3094.
48. Locht C, Keith JM: Pertussis toxin gene: nucleotide sequence and genetic organization. Science 1986, 232:1258-1264. 49. Nicosia A, Perugini M, Franzini C, Casagli MC, Borri MG, Antoni G, Almoni M, Neri P, Ratti G, Rappuoli R: Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication. Proc Natl Acad Sci USA 1986, 83:4631-4635. 50. Stein PE, Boodhoo A, Armstrong GD, Cockle SA, Klein MH, Read RJ: The crystal structure of pertussis toxin. Structure 1994, 2:45-57. 51. Pizza M, Bugnoli M, Manetti R, Covacci A, Rappuoli R: The subunit S1 is important for pertussis toxin secretion. J Biol Chem 1990, 265:17759-17763. 52. Winans SC, Burns DL, Christie PJ: Adaptation of a conjugal transfer system for the export of pathogenic macromolecules. Trends Microbiol 1996, 4:64-68. 53. Burns DL, Manclark CR: Adenine nucleotides promote dissociation of pertussis toxin subunits. J Biol Chem 1986, 261:4324-4327.