DNA Substrate-Induced Activation of the Agrobacterium VirB… · DNA Substrate-Induced Activation of the Agrobacterium VirB/VirD4 Type IV Secretion System Eric Cascales,* Krishnamohan

DNA Substrate-Induced Activation of the Agrobacterium VirB/VirD4Type IV Secretion System

Eric Cascales,* Krishnamohan Atmakuri,* Mayukh K. Sarkar, Peter J. Christie

Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, Texas, USA

The bitopic membrane protein VirB10 of the Agrobacterium VirB/VirD4 type IV secretion system (T4SS) undergoes a structuraltransition in response to sensing of ATP binding or hydrolysis by the channel ATPases VirD4 and VirB11. This transition, de-tectable as a change in protease susceptibility, is required for DNA substrate passage through the translocation channel. Here, wepresent evidence that DNA substrate engagement with VirD4 and VirB11 also is required for activation of VirB10. Several DNAsubstrates (oncogenic T-DNA and plasmids RSF1010 and pCloDF13) induced the VirB10 conformational change, each by mech-anisms requiring relaxase processing at cognate oriT sequences. VirD2 relaxase deleted of its translocation signal or any of thecharacterized relaxases produced in the absence of cognate DNA substrates did not induce the structural transition. Translo-cated effector proteins, e.g., VirE2, VirE3, and VirF, also did not induce the transition. By mutational analyses, we supplied evi-dence that the N-terminal periplasmic loop of VirD4, in addition to its catalytic site, is essential for early-stage DNA substratetransfer and the VirB10 conformational change. Further studies of VirB11 mutants established that three T4SS-mediated pro-cesses, DNA transfer, protein transfer, and pilus production, can be uncoupled and that the latter two processes proceed inde-pendently of the VirB10 conformational change. Our findings support a general model whereby DNA ligand binding with VirD4and VirB11 stimulates ATP binding/hydrolysis, which in turn activates VirB10 through a structural transition. This transitionconfers an open-channel configuration enabling passage of the DNA substrate to the cell surface.

The type IV secretion systems (T4SSs) mediate the transfer ofDNA and protein substrates across the envelopes of many

Gram-negative and -positive bacterial species (1). The conjuga-tion systems comprise a large subfamily of the T4SSs. These med-ically important systems are responsible for widespread transmis-sion of antibiotic resistance genes and virulence genes carried byconjugative plasmids and chromosomally integrated elements.Overall, conjugation can be depicted as three distinct biochemicalreactions. The DNA transfer and replication (Dtr) proteins bind acognate origin-of-transfer (oriT) sequence and initiate processingof the DNA substrate for transfer (2–4). Next, the Dtr-oriT com-plex, termed the relaxosome, engages with the type IV couplingprotein (T4CP) (5). Finally, the T4CP delivers the DNA substrateto a transenvelope channel comprised of the mating pair forma-tion (Mpf) proteins for translocation across the cell envelope (6,7). Studies in recent years have begun to define mechanistic andstructural details of conjugation systems. Additionally, an accu-mulating body of evidence suggests that a combination of intra-and extracellular signals act to coordinate the conjugative DNAtransfer reactions in space and time. The present investigationsadvance our understanding of a signaling mechanism required forDNA substrate transfer through the Agrobacterium tumefaciensVirB/VirD4 T4SS.

Formally, conjugation initiates when the relaxase and one ormore accessory factors bind a cognate origin-of-transfer (oriT)sequence (2, 3). The relaxase nicks the DNA strand destined fortransfer (T-strand) and remains covalently bound via an active-site tyrosine to the 5= end of the T-strand. The relaxase and acces-sory factors carry the translocation signals for substrate dockingwith the T4CP, and the relaxase mediates passage of the boundT-strand through the channel. T4CPs typically possess an N-ter-minal transmembrane (TM) domain and a large C-terminal cyto-plasmic domain with a nucleoside triphosphate binding domain(NBD) (1, 5). A combination of X-ray crystallography and elec-

tron microscopy studies of the plasmid R388-encoded TrwBT4CP established that these ATPases adopt a homohexameric,F1-F0-like structure (8, 9). T4CPs share sequence and structuralsimilarities with the FtsK and SpoIIIE DNA translocases (10), giv-ing rise to a proposal that, in addition to functioning as the sub-strate receptors, the T4CPs translocate DNA substrates throughtheir central channels across the cytoplasmic membrane.

T4CPs interact with Mpf channels, which in Gram-negativebacteria are highly complex structures composed of one or twoother ATPases, a cytoplasmic membrane translocon, and the en-velope-spanning translocation channel (1, 11). Recent electronmicroscopy studies have advanced our understanding of Mpfchannel architecture with the isolation and structural character-ization of a machine subassembly termed the core complex (12,13). This core complex, derived from the Escherichia coli pKM101-encoded T4SS, is a large, �1.05-MDa structure of 185 Å in widthand height composed of 14 copies each of three subunits desig-nated TraN, TraO, and TraF. The core complex is configured intwo layers (I and O layers) that form a double-walled ringlikestructure. The I layer, composed of the N-terminal domains of

Received 25 January 2013 Accepted 29 March 2013

Published ahead of print 5 April 2013

Address correspondence to Peter J. Christie, [email protected].

* Present address: Eric Cascales, Laboratoire d’Ingénierie des SystèmesMacromoléculaires, IBSM—CNRS, 31 Ch. Joseph Aiguier, Marseille, France;Krishnamohan Atmakuri, Vaccine and Infectious Disease Research Center,Translational Health Science and Technology Institute 496, Phase III, Udyog ViharGurgaon, Haryana, India.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00114-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.00114-13

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TraO and TraF, is anchored in the cytoplasmic membrane and isopened at the base by a 55-Å-diameter hole. The O layer, com-posed of TraN and the C-terminal domains of TraO and TraF, hasa main body and a narrower cap on the outermost side of thecomplex that is inserted in the outer membrane. A narrow hole(10 Å) exists in the cap, resulting in a channel extending throughthe entire cylindrical structure. In addition to its membrane-span-ning ring-shaped complexes at the cytoplasmic and outer mem-branes, the core has a large chamber which is thought to house thetranslocation channel. Another noteworthy feature is that theTraF subunit spans the entire cell envelope such that an �-helicaldomain termed the antennae projection (AP) extends from the Olayer to form an outer membrane pore (13).

Most T4SS loci in Gram-negative bacteria code for homologsof TraN, TraO, and TraF, and biochemical evidence from studiesof various systems support the notion that core complexes arecommon structural features of T4SSs. In the A. tumefaciens VirB/VirD4 T4SS, the TraN, TraO, and TraF homologs are VirB7,VirB9, and VirB10, respectively (1, 14). We have isolated and vi-sualized ring-shaped complexes from A. tumefaciens that are com-posed minimally of these three subunits (15), and ongoing effortsare aimed at generating a high-resolution structure. This pre-sumptive VirB core complex is envisioned to carry out two func-tions in relation to substrate transfer (16). First, it comprises astructural scaffold for assembly of the envelope-spanning translo-cation channel. By use of a chromatin immunoprecipitation(ChIP)-based, formaldehyde (FA) cross-linking assay termedtransfer DNA immunoprecipitation (TrIP), we identified closecontacts between the translocating T-DNA substrate and theVirD4 T4CP, VirB11 ATPase, polytopic VirB6, bitopic VirB8,core complex subunit VirB9, and VirB2 pilin (17–19). Integratingthese findings with those from the pKM101 structural studies, wehave proposed a general architectural model whereby VirD4T4CP, VirB11, and VirB4 (a third ATPase also required for sub-strate transfer) are located at the entrance of the translocationchannel and function to deliver docked substrate into the channel.The cytoplasmic membrane translocon composed minimally ofVirB6 and VirB8 sits within the 55-Å ring formed by the VirB10TM helices. Finally, VirB6 and VirB8 together with VirB9 andVirB2 pilin form the translocation channel within the chamber ofthe VirB7/VirB9/VirB10 core complex to mediate substrate trans-fer from the cytoplasmic membrane to the cell surface (20).

Previously, we reported that VirB10 undergoes a structuraltransition in response to an intracellular signal generated throughthe coordinated activities of the VirD4 and VirB11 ATPases (21).The structural transition, detectable as a change in protease sus-ceptibility, correlates with the capacity of DNA substrates to formFA-cross-linkable contacts with VirB2 and VirB9. VirB10 thusserves not only as a structural scaffold but also functions dynam-ically through a conformational switch to couple cytoplasmicmembrane ATP energy to gating of the translocation channel nearor at the outer membrane. Consistent with this mode of action, amutation (G272R) in a region of VirB10 located near the outermembrane was shown to lock VirB10 in the activated conforma-tion, resulting in leakage of secretion substrates to the cell surfaceindependently of target cell contact (22).

In the present study, we sought to further define signaling re-quirements for activation of VirB10. We report our discovery that,in addition to catalytically active VirD4 and VirB11, activationrequires DNA substrate processing and engagement of the trans-

fer intermediate with both ATPases. Strikingly, DNA, but not pro-tein, substrates activate VirB10, suggesting that translocation ofDNA and protein substrates through this T4SS are mechanisti-cally distinct processes. Mutational analyses of the channelATPases identified additional mechanistic features of DNA-li-gand-induced channel activation. We discuss our findings in abroader context of known or postulated signals required for acti-vation of conjugative DNA transfer.

MATERIALS AND METHODSBacterial strains and growth conditions. Bacterial strains used in thisstudy are listed in Table 1. Strain KA2002 (LBA4404 deleted of virD4) wasconstructed by deletion of the virD4 gene in A. tumefaciens strainLBA4404 by marker exchange-eviction mutagenesis using plasmidpKA126 (18) as previously described (29). A. tumefaciens strains weregrown in LB supplemented with mannitol and glutamate at 28°C (42).Conditions for induction of the A. tumefaciens vir genes in AB-inducingmedium (ABIM; glucose-containing minimal medium [pH 5.5], 1 mMphosphate, 200 �M acetosyringone [AS]) have been previously described(42). When necessary, medium was supplemented with antibiotics (in �g ·ml�1) as follows: gentamicin (100), kanamycin (100), tetracycline (5),carbenicillin (100 or 5 for strain LBA4404), spectinomycin (500), rifam-pin (75), erythromycin (100), streptomycin (100).

Bacterial plasmids. Plasmids used in this study are listed in Table 1.Plasmid pKA171 expresses the full-length virD2 relaxase gene expressedfrom the PvirB promoter. It was constructed by PCR amplification using5=-AAATTCCATGGCCGATCG (the NcoI site is underlined) and 5=-CCACTCGAGCGTATCGTGAAC-3= (the XhoI site is underlined) as for-ward and reverse primers, respectively, and pMY1153 as a template. ThePCR product was digested with NcoI and XhoI, and the resulting frag-ment was introduced into pPC914KS.NcoI, substituting for the virB1 andvirB2= sequences. Plasmid pKA172 expresses virD2 deleted of 35 codonsfrom its 3= end. It was constructed by PCR amplification using 5=-CTGCAGCCCCATATGCCTGATCGCGCTCAA-3= (the NdeI site is under-lined) and 5=-AGCTTCCGGTTGCTCGAGTCACTACTATTCTAGATGCTCGGTACCGAT-3= (the XhoI site is underlined) as forward andreverse primers, respectively, and pMY1153 as a template. The PCR prod-uct was digested with NdeI and XhoI, and the resulting fragment wasintroduced into pPC914KS, resulting in expression of virD2�C35 fromthe PvirB promoter. Plasmid pKA21 expresses PvirB-virD4 from the IncPplasmid pXZ153. It was constructed by introducing PvirB-virD4 as anXbaI-KpnI fragment from pKA9 (see below) into similarly digestedpXZ153. Plasmid pKA250 expresses PvirB-virD2 and PvirB-virD4 from theIncP plasmid pXZ153. It was constructed by ligating XhoI-digestedpKA171 with similarly digested pKA21. Plasmid pKA251 expressingPvirB-virD2�C35 and PvirB-virD4 was constructed by joining XhoI-di-gested pKA172 and pKA21.

Plasmid pKA9 expressing PvirB-virD4 served as a template for con-struction of virD4 deletion and substitution mutations by QuikChangemutagenesis as described previously (18). The following plasmids wereconstructed with the following forward primers in parentheses and cor-responding complementary primers (not shown), and the mutations ofinterest were confirmed by sequencing across the entire virD4 gene frag-ment: pKA84 deleted of N-terminal codons 2 to 10 (�2-10; 5=-ACTTCGCCCCAGCATATGACCCTGAGCATC-3=), pKA85 (�2-67; 5=-ACCGTCTTCTGGCATATGTTATCTGTTTGTC-3=), pKA155 (�42-48; 5=-GGTCTCATACCAAAAGATCGCTTCACCATTGAAGCCCCGGCG-3=), pKA158(�52-58; 5=-GCTTTTTGGTATGAGACGTCGACCGTCTTCTGGCGTGGTTTATCT-3=), pKA157 (F49C; 5=-TTTGACGTTTTCGCATGCTGGTATGAGACCCCGCTT-3=), pKA156 (Y51A; 5=-GACGTTTTCGCCTTTTGGGCTGAGACCCCGCTTTACTTGGGT-3=).

TrIP assay. The TrIP assay was performed essentially as previouslydescribed (15, 17). Briefly, cells from a 6-ml vir-induced culture wereharvested, washed, and resuspended in 20 mM sodium phosphate buffer(pH 6.8) with formaldehyde (FA) at a final concentration of 0.1% and

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TABLE 1 Bacterial strains and plasmids

Bacterial strain or plasmid Relevant characteristic(s) Source or reference

StrainsE. coli

DH5� ��80dlacZ�M15 �(lacZYA-argF)U169 recA1 endA1 hsdR17(rK� mK

�) supE44 thi-1gyrA relA1

Gibco-BRL

A. tumefaciensA348 Strain C58 containing octopine-type Ti plasmid pTiA6NC 23C58C1RS Strain C58, Strr Rifr 24KE1 �virE operon 25At12516 virE2� 26Mx219 A348 with a virF::Tn3HoHo1 mutation 27Mx311 A348 with a virD2::Tn3HoHo1 mutation 27LBA4404 T-DNA deletion strain 28KA2000 A348 with a �virD4 mutation 18KA2002 LBA4404 with a �virD4 mutation generated by marker exchange-eviction mutagenesis This studyPC1006 A348 with a nonpolar �virB6 mutation 29PC1010 A348 with a nonpolar �virB10 mutation 29PC1011 A348 with a nonpolar �virB11 mutation 29

PlasmidsVectors

pXZ153 Kanr; IncP plasmid with Kanr gene from pUC4K 30pBin19 Kanr; IncP plasmid with T-DNA border repeats 31pBSBBR Carbr Kanr; pBSIISK� (Crbr) ligated to pBBR1MCS2 (Kanr) 32pBSIISK�NdeI Carbr; pBSIISK� containing an NdeI site at the translational start site of lacZ 29pPC914KS 29pPC914KS.NcoI 33

IncQ/CloDF plasmidspML122�Kanr Genr; mobilizable RSF1010 (IncQ) derivative 34pJB31 Spcr; RSF1010 derivative 35pAJ1 pJB31 lacking mobA 36pAJ6 pJB31 lacking oriT 36pClo-Leu pBin19 derivative carrying the mob region of pCloDF13; Kanr Gift from J. Escudero, A.

Vergunst, and P. J. J.Hooykaas

p�Clo-LEU pJClo deleted of CloDF13 mob region 37pClo�EB-LEU pJClo deleted of nonessential mobE and mobB T4CP gene 37pClo�EC-LEU pJClo deleted of nonessential mobE and mobC relaxase gene 37

virD2 plasmidspMY1153 pUC18CM expressing the virD operon from the Ptac promoter 38pKA171 Carbr; pPC914KS expressing PvirB-virD2 This studypKA172 Carbr; pPC914KS expressing PvirB-virD2�C35 This studypKA250 Kanr Carbr; pXZ153 expressing PvirB-virD4 and PvirB-virD2 This studypKA251 Kanr Carbr; pXZ153 expressing PvirB-virD4 and PvirB-virD2�C35 This study

virD4 plasmidspKA9 Carbr; pPC914KS expressing PvirB-virD4 18pKA21 Kanr; pXZ153 expressing PvirB-virD4 18pKA42 Kanr; pBBR1MCS2 expressing PvirB-virD4 18pKA84 Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4�2–10 This studypKA85 Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4�2–67 This studypKA43 Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4�2-86 39pKA155 Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4�42-48 This studypKA158 Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4�54-60 This studypKA157 Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4F49C This studypKA156 Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4Y51A This study

virB6 plasmidspSJB964 Kanr Carbr; pXZ151 expressing PvirB-virB6 40pSJB5XXX Kanr Carbr; pXZ151 expressing virB6.i4 alleles; XXX is the position of the residue relative to the

beginning of the protein that immediately precedes the i4 mutation40

virB11 plasmidspXZB100 Kanr Carbr; pXZ151 expressing PvirB-virB11 30pXZB102 I265T 30pXZB105 L75F 30

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incubated for 20 min at 18°C with shaking. FA was then added in 0.2%increments to reach a final concentration of 1% over a 15-min period.Cells were incubated for 40 min at room temperature (RT) without shak-ing and then pelleted and solubilized by resuspension in 200 �l in TESbuffer (50 mM Tris-Cl [pH 6.8], 2 mM EDTA, 1% -mercaptoethanol,1% SDS). For immunoprecipitation, protein A-Sepharose CL-4B (Phar-macia) (30-�l bed volume) was incubated with 1.1 ml of the detergent-solubilized material for 60 min at RT and centrifuged at 5,000 g toremove protein A-Sepharose and nonspecifically bound proteins. Thesupernatant was incubated overnight at 4°C with anti-VirD4 or anti-VirBantibodies (Christie laboratory collection) coupled to protein A-Sephar-ose CL-4B. The beads were washed twice with NP1 buffer supplementedwith 1% Triton X-100 and once with NP1 buffer supplemented with 0.1%Triton X-100. Immunoprecipitates were eluted by incubation for 20 minat 96°C in 20 �l of 10 mM Tris-Cl (pH 6.8). Soluble fractions (S) andimmunoprecipitates (IP) were analyzed for the presence of T-DNA,pML122, or Ti plasmid by PCR amplification with primers 5=-GGGCGATTATGGCATCCAGAAAGCC and 5=-GTCGGCGGCCCACTTGGCACACAG for gene 7 present on the TLDNA, 5=-CCTGCGGATGTCAGGGCTCTCGT and 5=-TGTCCGTGCTTGCCAATCCCCG for the ophDClocus on pTiA6NC (control), 5=-CTCAGTGGTTCAAGCGGTACA and5=-TGATAGTTCTTCGGGCTGGTT for a fragment of mobA carried onRSF1010 derivatives used in this study, and 5=-GTGAGCAAAGCCGCTGCC and 5=-AGCCAATTGATCCTGCA for a fragment of the repABlocus on pTiAch5 (control). To compare levels of T-strand by quantitativeTrIP (QTrIP), immunoprecipitates were subjected to 20 cycles of PCRamplification using the primers specific for gene 7 of the T-DNA. On the21st cycle, a single round of PCR amplification was performed with addi-tion of 1.0 mCi of [32P]dGTP (Amersham Biosciences), as described pre-viously (17, 18). PCR products were column purified with the QIAquickPCR purification kit (Qiagen) to remove unincorporated nucleotides. Ali-quots of the eluted material were mixed with 3.5 ml of scintillation liquid(Ecolite, ICN) and counted with a Beckman Coulter. The entire TrIPprotocol was repeated three times in triplicate, and the average value froma single experiment was reported.

Quantitation of Ti plasmid copy number and T-strands. Relativenumbers of T-strands generated per Ti plasmid were determined as pre-viously described (43). Briefly, Ti plasmid copy numbers and T-strandlevels were determined by subjecting equal amounts of total DNA to 20cycles of PCR amplification using primers specific for T-DNA (gene 7 ofTL-DNA) and Ti plasmid (ophDC locus) (17). As described above, on the21st cycle, 1.0 mCi of [�-32P]dGTP was added for a single round of PCRamplification and PCR products were purified. The number of T-strandsgenerated per molecule of Ti plasmid was obtained as a ratio of cpm ofradioactivity incorporated into a T-DNA-specific amplicon (gene 7) tothat obtained for the Ti plasmid-specific amplicon (ophDC), multipliedby the factor 3.76 (973/259; obtained as a ratio of number of G�C bases inPCR products of ophDC locus [973] to gene 7 [259]).

Protease susceptibility assay for VirB10 conformational change. A.tumefaciens spheroplasts were treated with Streptomyces griseus protease(Sigma) for detection of the VirB10* proteolytic degradation product aspreviously described (21). Briefly, vir-induced cells were either treatedwith sodium arsenate (25 mM) (Allied Chemical, Morristown, NJ) for 30min or left untreated. Cells were harvested by centrifugation, and the cell

pellets were resuspended at 1:30 volume of the inducing culture in 20 mMTris-HCl (pH 8.0), 20% (wt/vol) sucrose. Cells were converted to sphero-plasts by addition of 100 �g/ml of lysozyme (Sigma), incubation for 5 minon ice, addition of 2 mM EDTA, and further incubation for 45 min on ice.Cells were then centrifuged at 5,000 g for 5 min, and spheroplasts weregently resuspended in 20 mM Tris-HCl (pH 8.0), 10 mM MgSO4, and20% sucrose. Spheroplasts were incubated with S. griseus protease(Sigma) at a final concentration of 100 �g/ml for 15 min on ice, harvestedby mixing with an equal volume of 2 � Laemmli’s buffer, and then imme-diately boiled for 10 min.

Protein analyses. Protein samples were suspended in Laemmli’s buf-fer and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis (PAGE) using glycine or tricine buffer systems as previ-ously described (40). For detection by immunostaining, proteins weretransferred onto nitrocellulose membranes, and immunoblots were de-veloped with antibodies to the VirD2, VirD4, and VirB proteins from ourlaboratory collection (18, 39, 40) and goat anti-rabbit antibodies conju-gated to alkaline phosphatase (Bio-Rad). Proteins were loaded on SDS-polyacrylamide gels on a per-cell (CFU) equivalent basis. Molecular massmarkers were obtained from GIBCO-BRL (Grand Island, NY).

Conjugation assays. Derivatives of RSF1010 and pCloDF13 were in-troduced into various A. tumefaciens donor strains by electroporation,and the resulting strains were mated with an A348Spcr on a nitrocellulosefilter on an ABIM agar plate containing AS as previously described (18).Frequencies of transfer were estimated as transconjugants recovered perdonor.

Virulence assays. A. tumefaciens strains were tested for virulence byinoculating wound sites of Kalanchoe daigremontiana leaves (44). Eachexperiment was repeated at least three times for each strain on separateleaves.

RESULTSProcessed T-DNA but not protein substrates is required forVirB10 conformational change. VirB10 adopts alternative pro-tease-susceptible and -resistant conformations as a function ofcellular energetic status and production of the VirD4 and VirB11ATPases (21). As shown previously (21) and in Fig. 1, the nativeprotein migrates as an �48-kDa species in SDS-polyacrylamidegels, but upon treatment of spheroplasts from the wild-type (WT)strain A348 with the S. griseus serine protease, anti-VirB10 anti-bodies also react with an �40-kDa species designated VirB10*.Neither full-length VirB10 nor the VirB10* species are detected inextracts of the similarly treated �virB10 mutant, confirming theidentity of the latter as a proteolytic product of VirB10. Arsenatetreatment of A. tumefaciens cells, which dissipates cellular ATP,prior to conversion to spheroplasts and proteolysis abolishes for-mation of VirB10*. This conformational transition also requiresproduction of catalytically active forms of the VirD4 T4CP andVirB11 ATPases, suggesting that VirB10 couples ATP energy tosubstrate translocation through its interactions at or near the cy-toplasmic membrane with a VirD4/VirB11 complex.

Besides energizing VirB10, VirD4 and VirB11 bind the relaxo-

TABLE 1 (Continued)

Bacterial strain or plasmid Relevant characteristic(s) Source or reference

pXZB108 L11P/D56G/E335G 30pXZB101 I88T/I103T 30pXZB103 H269R 30pXZB109 I103T/M301T 30pJCB8XXX Kanr Carbr; pXZ151 expressing virB11.i4 alleles; XXX is the position of the residue relative to

the beginning of the protein that immediately precedes the i4 mutation41

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some or T-DNA transfer intermediate and deliver the substrateinto the translocation channel (17, 18, 43). To examine whetherthese early DNA substrate docking reactions might be coupled toactivation of VirB10, we assayed strains lacking T-DNA for gen-eration of the VirB10* species. Strikingly, upon protease treat-ment of spheroplasts from the �T-DNA strain LBA4404, we de-tected only the protease-resistant, 48-kDa species (Fig. 1).Introduction of pBin19, an IncP plasmid that carries oriT-likeT-DNA border sequences, restored the VirB10 protease suscepti-bility profile of LBA4404 to the WT pattern, whereas introductionof an IncP plasmid lacking the border sequence did not regeneratethis pattern (Fig. 1). We also did not detect the VirB10* species byprotease treatment of a virD2 mutant carrying T-DNA or pBin19substrates (Fig. 1). Previously, we showed that the T-DNA sub-strate does not form an FA-cross-linkable contact with VirD4 inthe absence of relaxase, which indicates that generation of therelaxosome (VirD2 and accessory factor VirD1 bound at the T-DNA border) or production of the VirD2–T-strand intermediateobligatorily precedes substrate docking with the T4CP (17). Ourpresent findings further suggest that docking of the relaxosome orprocessed T-DNA with VirD4 is also required for activation ofVirB10.

Strain LBA4404 (�T-DNA) produces the VirD2 relaxase,which is itself a substrate of the VirB/VirD4 channel in the absenceof associated T-strand (45, 46). LBA4404 also produces severaleffector proteins, such as VirE2, VirE3, and VirF, that are translo-cated through the VirB/VirD4 T4SS independently of DNA (47,48). The finding that protease treatment of LBA4404 spheroplastsdoes not generate VirB10* suggests that protein substrates do notinduce the observed VirB10 conformational switch. We furtherassayed virE2, virE (deleted of virE1, virE2, virE3), or virF mutantstrains, each of which are competent for translocation of T-DNAto plants, for generation of the VirB10* species. Treatment ofspheroplasts from these strains resulted in appearance of VirB10*(Fig. 1), providing additional evidence that activation of VirB10requires engagement of DNA, but not protein, substrates with theT4CP.

C terminus of VirD2 relaxase is required for substrate trans-fer and the VirB10 conformational change. The VirD2 C termi-nus contains two motifs that are required for delivery of theVirD2–T-strand intermediate to plants, a bipartite nuclear local-

ization sequence (NLS) that mediates delivery of the transfer in-termediate to the plant nuclear pore and the omega (�) sequence(DGRGG) that contributes to efficient T-DNA transfer throughthe VirB/VirD4 T4SS (Fig. 2A) (49). A VirD2 mutant deleted ofthese C-terminal motifs is completely deficient for DNA substratetransfer (Fig. 2A) (46). Conversely, fusion of a 40-residue, C-ter-minal fragment carrying these motifs to a reporter protein sufficesfor delivery of the reporter to plant cells, implying that this frag-ment carries the necessary information for substrate docking andtranslocation through the VirB/VirD4 T4SS (46). To evaluate theimportance of the DNA substrate docking reaction for channelactivation, we asked whether deletion of the VirD2 C-terminaltranslocation signal disrupted VirB10 activation. In the initialstudies, we confirmed that a �C35 mutation did not affect relax-ase-mediated processing of the T-strand. As shown previously forwild-type strain A348 (43), a �virD2 mutant expressing nativevirD2 in trans accumulated multiple copies of processed T-strandsupon induction of the vir genes (Fig. 2B). Within 8 to 12 h postin-duction, cells accumulated �8 to 10 free T-strands per Ti plasmid,and this number increased to as many as 15 T-strands per Tiplasmid over the next 4 to 8 h. The kinetics of T-strand accumu-lation matched profiles observed for Vir protein accumulation asshown for VirD2 and VirB9, whereas the constitutively synthe-

FIG 1 Detection of the VirB10 conformational switch by protease treat-ment. Strains in the left blot were pretreated with arsenate (Ars; �) or H2O(�); those in the center and right blots were pretreated with H2O only.Spheroplasts were treated with S. griseus protease (Prot; �) or left un-treated (�). B10, VirB10 (�48-kDa) B10*, VirB10* proteolytic degrada-tion product (�40 kDa) detected by immunostaining with anti-VirB10antibodies. Strains, left to right: WT, A348; �B10, �virB10 mutant PC1010;�D2, Mx355(pKA42) virD2::Tn3HoHo1 mutant expressing virD4 from apBBR plasmid; �T-DNA, �T-DNA mutant LBA4404; �T-DNA, LBA4404carrying IncP plasmids with (pBin19) or without (pXZ151) a T-DNA bor-der sequence; �D2, Mx355(pKA42, pBin19); �E2, virE2 mutant At12516;�E, �virE operon mutant KE1; �F, virF mutant Mx219 (virF::Tn3HoHo1).

FIG 2 Contribution of a VirD2 C-terminal translocation signal to T-DNAtransfer and VirB10 activation. (A) D2, VirD2 full length; �C35, C-termi-nal truncation mutant. Domains: relaxase, domain of unknown function(DUF), and C-terminal nuclear localization sequence (NLS), � sequence.Numbers correspond to residues relative to the beginning of the protein.Plant leaf was inoculated with A348, wild-type strain; �D2 mutant, Mx355(virD2::Tn3HoHo1 mutant) with pKA42 expressing virD4; �D2(D2), Mx355 withpKA250 expressing virD2 and virD4; �D2(D2�C35), Mx355 with pKA251 ex-pressing virD2�C35 and virD4. (B) Numbers of T-strands per Ti plasmid in strains�D2, Mx355(pKA42); �D2(D2), Mx355(pKA250); and �D2(D2�C35),Mx355(pKA251) (see the text for details). Ind. time, T-strand levels determined at0, 8, 12, and 16 h following vir gene induction with AS. Protease, spheroplasts weretreated with S. griseus protease (�) or left untreated (�). B10, VirB10 (�48-kDa)B10*, VirB10* proteolytic degradation product (�40 kDa) detected by immuno-staining with anti-VirB10 antibodies. Bottom, steady-state levels of D2 (VirD2),ChvE, B9 (VirB9) at the postinduction times induced as assessed by immunostain-ing with the respective antibodies.




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sized sugar-binding protein ChvE accumulated at comparablelevels throughout the AS induction period (Fig. 2B). The virD2mutant strain failed to generate free T-strands, and this strainexpressing virD2�C35 generated multiple copies of free T-strandsover the 16-h induction period, establishing that the �C35 trun-cation did not affect substrate processing (Fig. 2B).

Protease treatment of spheroplasts from the �virD2 mutantexpressing native virD2 in trans generated the VirB10* species,whereas similar treatments of spheroplasts from both the �virD2mutant and VirD2�C35-producing strains failed to generate thisVirB10 species (Fig. 2B). Both VirD2 and VirD2�C35 producedfrom the PvirB promoter accumulated at similar levels followingAS induction. We conclude from these findings that VirD2-me-diated docking of the relaxosomal complex or the transfer inter-mediate with VirD4 is essential for activation of VirB10.

Mobilizable IncQ and pCloDF13 substrates induce VirB10conformational change. Next, we asked whether other DNA sub-strates of the VirB/VirD4 system induce the VirB10 conforma-tional switch. This T4SS mobilizes transfer of non-self-transmis-sible plasmids, RSF1010 (IncQ) and pCloDF13, to recipient cells(32, 36, 37, 50). RSF1010 engages with the VirD4 T4CP and trans-locates along the same pathway as the T-DNA substrate throughthe VirB channel, although substrate specificity checkpoints havebeen identified by mutational analyses that allow translocation ofone but not both substrates (see below) (32, 40, 41, 44). We firstestablished that strain LBA4404 (�T-DNA) mobilizes transfer ofthe RSF1010 derivative pML122 at a frequency of �10�5

transconjugants (Tc)/donor, which is comparable to previouslyreported values (Fig. 3A) (44). LBA4404 also mobilized transfer ofanother RSF1010 derivative (pJB31) but not mutant forms of thisplasmid lacking an intact mobA relaxase gene (pAJ1) or functionaloriT sequence (pAJ6) (Fig. 3A). pCloDF13 codes for Mob func-tions required for nicking at oriT, but in contrast to RSF1010 and

other mobilizable plasmids, pCloDF13 also codes for a VirD4-likeT4CP. This T4CP interfaces with the A. tumefaciens VirB Mpfchannel, as shown by the capacity of chimeric machines composedof the A. tumefaciens VirB Mpf subunits and pCloDF13-encodedMobB to mobilize plasmid transfer in the presence (32) or absenceof a T-DNA substrate (Fig. 3B). As shown for RSF1010, pCloDF13derivatives bearing mutations of the entire mob region, the mobCrelaxase gene, or the mobB T4CP gene failed to translocatethrough the VirB T4SS (Fig. 3B).

Protease treatment of spheroplasts from LBA4404 carrying theIncQ plasmids pML122 or pJB31 generated the VirB10* species,establishing that these DNA substrates activate VirB10. Similartreatments of spheroplasts from LBA4404(pJB31) cells depleted ofcellular ATP by treatment with arsenate or cells carrying the oriTor mobA mutant plasmids did not yield the VirB10* species. Ad-ditionally, protease treatment of spheroplasts from LBA4404bearing a �virD4 mutation (strain KA2002) did not generate theVirB10* species even when this strain carried the pML122 sub-strate, establishing the importance of VirD4 synthesis for the ob-served pML122-induced conformational switch (Fig. 3B). Finally,protease treatment of spheroplasts from strains carrying pClo(pCloDF13 derivative) generated the VirB10* species, indicatingthat pClo engages with its own T4CP to activate VirB10. As shownfor the T-DNA and RSF1010 substrates, depletion of cellular ATPlevels with arsenate abolished pClo-mediated activation ofVirB10, as did mutations of the mob region or the mobC relaxaseor mobB T4CP genes (Fig. 3B).

The VirD4 periplasmic domain is necessary for substratetransfer and the VirB10 conformational change. Our findingsthus far suggest that DNA substrates induce a conformationalchange in VirB10 through engagement with the VirD4 T4CP. Tofurther explore the requirements for energy coupling, we deter-mined effects of VirD4 mutations on the VirB10 conformational

FIG 3 Requirement of DNA substrate processing for VirB10 activation. (A) Top, histogram shows transfer frequencies (Tc/donor, transconjugants per donor)of A348(pML122) and LBA4404 (�T-DNA) lacking (�) or carrying pML122 or pJB31 (IncQ plasmids) or pJB31 derivatives pAJ1 (mobA relaxase mutant) orpAJ6 (mutated oriT) to A348Spcr recipient cells. Data from a representative assay are shown along with standard deviations from replicate experiments. (A)Detection of the VirB10 conformational switch by protease treatment. Ars, arsenate (�) or H2O (�) pretreatment. Prot, S. griseus protease treatment (�) oruntreated (�). B10, VirB10 (�48-kDa) B10*, VirB10* proteolytic degradation product (�40 kDa) detected by immunostaining with anti-VirB10 antibodies. (B)A348(pML122) and KA2002 (�T-DNA, �virD4) carrying the plasmids indicated were assayed for plasmid mobilization and generation of the VirB10* speciesas described for panel A. Strains: A348(pML122); KA2002 lacking (�) or carrying pClo (pClo-Leu) or derivatives pClo�mob (lacks entire mob region),pClo�mobB (relaxase minus), or pClo�mobC (T4CP minus).

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status. The importance of VirD4 ATP binding or hydrolysis foractivation of VirB10 was shown previously through our studies ofa Walker A mutant (K152Q) (21). In view of the topological mod-els experimentally generated for VirD4 and VirB10 (Fig. 4A) (51,52) and evidence that homologs of these proteins interact via theirN-terminal regions (53, 54), we sought to test whether the N-ter-minal transmembrane (TM) domain of VirD4 might participatein the activation reaction. A VirD4 mutant deleted of the TMdomain (�2-86) interacts with T-DNA but fails to deliver thesubstrate to the VirB11 ATPase (Fig. 4B) (18), likely becausethis domain fails to interact productively with VirB10 or othermachine subunits. As expected, protease treatment of theVirD4�2-86-producing strain failed to generate the VirB10*species (Fig. 4C).

We next examined the importance of subdomains within theVirD4 TM domain to substrate transfer and VirB10 activation.Accordingly, we analyzed the effects of deletion mutations com-prising (i) the extreme N-terminal cytoplasmic region (�2-10),(ii) the cytoplasmic region plus the first transmembrane helix andthe periplasmic loop (�2-67), and (iii) two 7-residue deletions inthe first and second halves of the periplasmic loop (�42-48, �52-58) (Fig. 4). The latter deletions were constructed to test the func-tional importance of two regions of the periplasmic loop, one(�52-58) comprised mainly of highly conserved residues amongclose VirB10 homologs associated with other Rhizobial speciesT4SSs and a less-well-conserved sequence (�42-48) (see Fig. S1 in

the supplemental material). We also constructed two substitutionmutations of invariant aromatic residues (F49C, Y51C).

All of the mutant proteins accumulated at levels comparable tothe native protein, suggesting that observed phenotypes are notlikely due to problems of protein instability (Fig. 4C). We assayedmutant strains for the capacity to incite virulence on plants and todeliver the T-DNA through the VirB/VirD4 channel by use ofthe TrIP assay (17). Interestingly, the mutations grouped intothree classes. One mutation (�2-10) designated class I was per-missive for substrate transfer, as defined both by restoration ofvirulence of a �virD4 mutant strain and formation of FA-cross-linked contacts between the T-DNA and the VirD4/VirB channelsubunits. Class II mutations phenocopied the �2-86 mutation inrendering the mutant proteins competent for binding of the DNAsubstrate but not for substrate transfer to VirB11. Mutations inthis class included the two large TMD deletions (�2-67, �2-86)and deletion of highly conserved residues (�52-58) in theperiplasmic domain. The class III mutations phenocopied theK152Q Walker A mutation; the mutant proteins interacted withthe T-DNA and transferred the substrate to VirB11 but not to thecytoplasmic membrane proteins VirB6 and VirB8. Mutations inthis class included three periplasmic domain mutations, �42-48,F49C, and Y51C. Strikingly, therefore, the VirD4 periplasmic do-main contributes to substrate transfer reactions between theT4CP, VirB11, and the VirB6 and VirB8 channel subunits. Addi-

FIG 4 Effects of VirD4 mutations on DNA transfer and VirB10 activation. (A) Schematic showing VirD4, VirB11, and VirB10 membrane topologies. TheN-terminal 85 residues of VirD4 are depicted, with residues that are not conserved (black), similar (gray), or invariant (gray with black outline) among homologsassociated with T4SSs in related rhizobial species. Underlined residues denote deletion mutations �42-48 and �52-58; residues F49 and Y51 were substitutedwith Cys, and K152 in the Walker A motif was substituted with Gln. (B) A348 (WT) or K2001 (�virD4) expressing variant forms of VirD4 were assayed forT-DNA transfer through the VirB/VirD4 T4SS. Table lists: strains, VirD4 variants; D4-B9, VirD4 and VirB subunits with a detectable (�) or undetectable (�)T-DNA interaction as monitored by TrIP; Vir, T-DNA transfer to plants as monitored by virulence (�, virulent; �, avirulent). Mutant strains were grouped into3 classes: (i) permissive, (ii) blocked T-DNA transfer from VirD4 to VirB11, (iii) blocked T-DNA transfer from VirB11 to VirB6. (C) A348 and KA2000 (�virD4)strains lacking (�) or producing D4 (native VirD4) or the VirD4 mutant derivatives shown were assayed for steady-state accumulation of the VirD4 variants(top) and B10 (native VirB10) and B10* (VirB10*) upon protease treatment of spheroplasts (bottom) by immunostaining with the anti-VirD4 or -VirB10antibodies.




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tionally, distinct regions of the periplasmic domain differentiallymodulate these early transfer reactions.

We assayed for effects of the NTD mutations on the VirB10conformational status and found that all mutations that disruptedT-DNA transfer abolished generation of the VirB10* species(Fig. 4C). These findings indicate that the VirD4 periplasmic do-main contributes not only to early-stage transfer reactions in thecytoplasm but also to activation of VirB10 and, therefore, a later-stage reaction required for substrate transfer through the distalportion of the channel.

VirB11 mutations exert correlative effects on substratetransfer and VirB10 conformation. Next, we sought to furtherdefine the contribution of VirB11 to VirB10 activation by charac-terizing two sets of previously isolated mutations. The first set wasisolated originally as dominant negative suppressors of WT virB11gene function (30, 41). These mutations fall into two classes, thosethat block WT gene function in merodiploid strains and are non-functional when expressed in the absence of the WT gene (domi-nant, nonfunctional) and those that retain functionality when ex-pressed in the absence of the WT gene (dominant, functional).

As expected, �virB11 strains producing the dominant, func-tional mutant proteins displayed a WT DNA transfer profile asmonitored with the TrIP assay (Fig. 5A). We also showed with asemiquantitative variation of the TrIP assay termed QTrIP (17)that complementation of a �virB11 mutant with wild-type virB11fully restored T-DNA transfer to levels detected in the WT strainA348, and strains producing the dominant, functional mutantproteins similarly efficiently transferred the substrate through thechannel (Fig. 5A). Treatment of spheroplasts from strains produc-ing the dominant, functional mutants also generated the VirB10*species, indicating that the mutant proteins coordinate withVirD4 both for DNA transfer and VirB10 activation (Fig. 5B).

In contrast, the �virB11 strains producing two dominant, non-functional mutant proteins (I88T/I103T, H269R) were blocked inthe earliest step of T-DNA transfer from VirD4 to VirB11, asshown with the TrIP and QTrIP assays (Fig. 5A). Protease treat-ment of these strains also failed to generate the VirB10* species(Fig. 5B). These mutant proteins thus fail to interact productivelywith VirD4 both for DNA transfer and activation of VirB10. In-terestingly, however, one dominant, nonfunctional mutant pro-tein (L11P/D56G/E335G) did support DNA transfer from VirD4to the membrane proteins VirB6 and VirB8 but not to VirB2 andVirB9 (Fig. 5A). Yet, the corresponding mutant strain did notgenerate the VirB10* species, indicative of a failure to induce theconformational switch (Fig. 5B). This mutant protein thereforeappears to engage productively with VirD4 to support the earlysubstrate transfer reactions but nonproductively with the T4CPfor activation of VirB10, which is required for later-stage transferfrom VirB6 and VirB8 to VirB2 and VirB9 (17, 21).

Finally, we determined that the I265T mutation as well as asecond mutation (L18.i4, see below) conferred the interestingphenotype of inducing the VirB10 structural transition and sup-porting T-DNA transfer but not pilus biogenesis (Fig. 5B) (41).Conversely, as shown previously, a virD4 mutant fails to activateVirB10 or transfer DNA but elaborates pili (18, 55). These findingsestablish that the observed DNA ligand- and ATP-driven confor-mational change of VirB10 is not required for pilus production.

Differential effects of VirB11 insertion mutations on sub-strate transfer and VirB10 activation. The second set of muta-tions was generated by insertion of a 4-residue epitope (HMVD;i4) along the length of VirB11. In contrast to that described above,these mutations were generally recessive and classified as permis-sive or loss-of-function according to their effects on T-DNAtransfer to plants (41). As expected, strains producing the permis-

FIG 5 Effects of VirB11 substitution mutations on T-DNA transfer and VirB10 activation. (A) Top, transfer of T-DNA to native and mutant forms of VirB11, as assessedwith the TrIP assay. Strains: PC1011 (�virB11) lacking (�) or producing B11 (native VirB11) or the mutant derivatives shown. S, supernatant from the IP reaction; IP,immunoprecipitated material recovered with anti-VirB11 antibodies (�B11); Ct, Ti plasmid control amplicon; T-DNA, T-DNA amplicon. Bottom, results of QTrIPassays showing levels of processed T-DNA (T-strand) recovered by precipitation with anti-VirD4, -VirB11, -VirB6, and -VirB9 antibodies relative to strain A348 (WTstrain), as described previously (17). Results are presented as a percentage of the T-DNA detected in the immunoprecipitates from WT A348. Schematics summarizeeffects of dominant, nonfunctional mutations in blocking DNA transfer at specific stages of the translocation pathway. WT, no stage-specific block (conferred by nativeVirB11 and the dominant, functional mutant proteins shown). A �virB10 mutation (�B10) blocks later-stage transfer from VirB6/VirB8 to VirB2/VirB9 (17). (B)PC1011 (�virB11) lacking (�) or producing B11 (native VirB11) or the mutant derivatives shown were assayed for accumulation of B10 (native VirB10) and B10*(VirB10*) upon protease treatment of spheroplasts by immunostaining with the anti-VirB10 antibodies. Tra, T-DNA transfer to plants as monitored by virulence (�,virulent; �, avirulent,); Pil, production of detectable (�) or undetectable (�) levels of pili.

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sive mutations displayed the WT pattern of T-DNA channel sub-unit interactions as monitored with the TrIP assay (Fig. 6). Cor-respondingly, protease treatment of these strains generated theVirB10* species indicative of VirB10 activation. In contrast,strains producing the loss-of-function mutations showed a blockin T-DNA transfer from VirD4 to VirB11, and these strains failedto generate the VirB10* species (Fig. 6).

Interestingly, strains producing a subset of the loss-of-functionmutations retained the ability to deliver VirE2 to plant cells de-spite the block in T-DNA transfer (Fig. 6) (41). The mutant pro-teins (L75.i4, C168.i4, L302.i4) conferring this substrate discrim-ination phenotype failed to interact with the T-DNA butnevertheless supported transfer of VirE2 to plant cells as deter-mined with a mixed-infection assay (41). Thus, protein substratessuch as VirE2 neither activate VirB10 (see Fig. 1) nor requireVirB10 activation for translocation through the VirB/VirD4 T4SS(Fig. 6).

The T-DNA Tra� mutations could exert their effects by dis-rupting VirB11 interactions with VirD4 or with the T-DNA sub-strate. To distinguish between these possibilities, we testedwhether these mutations also disrupted translocation of anothersubstrate, the RSF1010 derivative pML122. Interestingly, the threemutations that were permissive for VirE2 translocation (L75.i4,C168.i4, L302.i4) also were permissive for transfer of the IncQplasmid (Fig. 6 and 7). In strains carrying both DNA substrates,these discrimination mutations still selectively arrested T-DNAtransfer at the VirD4 to VirB11 step without disrupting IncQ plas-

mid translocation (Fig. 7). In these strains, engagement of theIncQ plasmid substrate with VirD4 and VirB11 also resulted inactivation of VirB10, as evidenced by generation of the VirB10*species upon protease treatment (Fig. 7). In contrast, strains car-rying both DNA substrates and virB11 alleles with mutations thatcompletely blocked transfer of all tested DNA substrates (E115.i4,F281.i4) at the VirD4 to VirB11 step also failed to activate VirB10(Fig. 7).

Together, these findings establish that the VirB11 mutant pro-teins conferring the T-DNA Tra�, IncQ Tra� phenotype are infact capable of engaging productively with VirD4 to mediate IncQplasmid translocation and VirB10 activation. These mutant pro-teins thus appear to block T-DNA transfer through a lack of aproductive interaction with the T-DNA transfer intermediate.The data further suggest that activation of VirB10 requires notonly coordination of the VirD4 and VirB11 ATPases but also pro-ductive engagement of a DNA substrate with both VirD4 andVirB11. The VirB11 mutant proteins conferring the substrate dis-crimination phenotype are capable of activating VirB10, but onlyif they establish a productive interaction with the IncQ plasmidsubstrate.

VirB6 channel blocking mutations do not affect VirB10 con-formational change. Finally, we asked whether channel subunitsacting downstream of the ATPases in the translocation pathwaymight also regulate the activation reaction. VirB6 mutations alsohave been isolated that either block substrate transfer altogether or

FIG 6 Effects of VirB11.i4 mutations on T-DNA transfer and VirB10 activa-tion. Top, transfer of T-DNA to native and mutant forms of VirB11 and toVirB6 and VirB9, as assessed with the TrIP assay. Strains: PC1011 (�virB11)lacking (�) or producing B11 (native VirB11) or the i4 mutant proteinsshown. S, supernatant from the IP reaction; IP, immunoprecipitated materialrecovered with anti-VirB11, -VirB6, or -VirB9 antibodies (�B11, �B6, �B9).For PC1011 strains producing native or mutant forms of VirB11 (the i4 mu-tation is inserted after the residue listed), amplicons from the immunoprecipi-tate (IP) fractions are shown. Ct, control, chromosomal DNA amplicon; T-DNA, T-DNA amplicon. Bottom, PC1011 (�virB11) lacking (�) or producingB11 (native VirB11) or the mutant derivatives were assayed for accumulationof B10 (native VirB10) and B10* (VirB10*) upon protease treatment ofspheroplasts by immunostaining with the anti-VirB10 antibodies. T-DNA,T-DNA transfer to plants as monitored by virulence (�, virulent; �, aviru-lent); VirE2, transfer of VirE2 to plant cells as monitored with a mixed infec-tion assay (�, transfer; �, no transfer). Pil, production of detectable (�) orundetectable (�) levels of pili. Schematics summarize effects of mutations inblocking DNA transfer at specific stages of the translocation pathway. WT, nostage-specific block.

FIG 7 Effects of VirB11 substrate discrimination mutations on DNA transferand VirB10 activation. Upper, transfer of T-DNA and IncQ plasmid pML122to VirD4, native and mutant forms of VirB11, and VirB6 as assessed with theTrIP assay. Strains: A348 (WT strain) without and with pML122 (IncQ plas-mid); PC1011(pML122) (�virB11 mutant carrying pML122) without (�) orwith plasmids expressing B11 (native VirB11) or i4 mutant proteins (the i4mutation is inserted after the residue listed). S, supernatant from the IP reac-tion; IP, immunoprecipitated material recovered with anti-VirD4, -VirB11, or-VirB6 antibodies (�D4, �B11, �B6). For PC1011 strains producing native ormutant forms of VirB11, amplicons from the immunoprecipitate (IP) frac-tions are shown. Ct, control, Ti plasmid amplicon; IncQ, pML122 plasmidamplicon; T-DNA, T-DNA amplicon. Lower, A348(pML122) andPC1011(pML122) strains were assayed for accumulation of B10 (nativeVirB10) and B10* (VirB10*) upon protease treatment of spheroplasts by im-munostaining with the anti-VirB10 antibodies. T-DNA, T-DNA transfer toplants as monitored by virulence (�, virulent; �, avirulent); IncQ, transfer ofIncQ plasmid pML122 to agrobacterial recipients.




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selectively inhibit T-DNA transfer at specific stages in the translo-cation pathway while permitting translocation of other substrates(19, 40). We tested for effects of these mutations on activation ofVirB10 and found that none blocked generation of the VirB10*species (Fig. 8B). These findings strongly indicate that the early-stage reactions involving DNA substrate engagement with VirD4and VirB11 and productive contacts between the two ATPases

are necessary and sufficient for inducing the VirB10 conforma-tional switch.

DISCUSSION

The A. tumefaciens VirB7/VirB9/VirB10 core complex serves as astructural scaffold for biogenesis of the VirB/VirD4 translocationchannel and pilus (11, 12, 15). In the assembled core complex,VirB10 adopts a topology that is unique among bacterial proteinsby spanning the entire cell envelope. This makes VirB10 ideallysuited for sensing of both intra- and extracellular signals requiredfor activation of the translocation channel (13, 52). Previously, wegained evidence that VirB10 undergoes a structural transition inresponse to sensing of ATP utilization by the VirD4 and VirB11ATPases (21), and in the present study we now have shown that asecond type of intracellular signal, docking of a DNA substratewith the channel ATPases, also is required for activation ofVirB10. As a framework for this discussion, Fig. 9 presents a modeldepicting the requirements for activation of VirB10 and DNAtranslocation. The DNA transfer pathway through the VirB/VirD4 channel, as defined by TrIP, involves substrate dockingwith the VirD4 T4CP and delivery to VirB11 and then passageacross the cytoplasmic membrane via a presumptive VirB6/VirB8translocon and the periplasm and outer membrane via a channelformed at least in part by VirB2 and VirB9 (17). We propose herethat two intracellular signals, DNA ligand binding and ATP en-ergy utilization, induce the VirB10 structural transition, whichopens a channel gate at or near the outer membrane enablingsubstrate passage to the cell surface. Although ligand- and energy-induced channel gating has been described for small-moleculeand macromolecular transport systems (56–59), this is the firstreport indicating that the similar signals activate gating of theT4SS channel.

Our initial studies established that engagement of DNA, butnot protein, substrates with the VirD4 T4CP is a prerequisite for

FIG 8 Effects of i4 mutations in VirB6 on T-DNA transfer and VirB10activation. (A) Schematic showing steps in the T-DNA transfer pathwayblocked by VirB6.i4 mutant proteins (the i4 mutation is inserted at theresidue listed) when produced in the nonpolar �virB6 mutant PC1006.�B6(B6) (PC1006 producing native VirB6) and A290 (A290.i4 mutant)display a WT T-DNA translocation phenotype (see reference 19). (B)PC1006 (�virB6 mutant) lacking (�) or producing native VirB6 (B6) orthe i4 mutant proteins were assayed for accumulation of B10 (nativeVirB10) and B10* (VirB10*) upon protease treatment of spheroplasts byimmunostaining with the anti-VirB10 antibodies. T-DNA, T-DNA trans-fer to plants as monitored by virulence (�, virulent; �, avirulent); IncQ/E2, transfer of IncQ plasmid pML122 to agrobacterial recipients or VirE2to plant cells as monitored by a mixed infection assay; Pil, production ofdetectable (�) or undetectable (�) levels of pili (see reference 40).

FIG 9 Model depicting the DNA substrate translocation pathway and activating intracellular signals. Transfer stages: (1) DNA transfer initiates by engagementof the relaxosome or the relaxase–T-strand transfer intermediate with the VirD4 T4CP, (2) the VirD4 T4CP delivers the substrate to VirB11, (3) VirB11 deliversthe substrate to the cytoplasmic membrane translocon comprised of VirB6 and VirB8, (4) the translocon delivers the substrate across the cytoplasmic membrane,and (5) substrate passes through the distal portion of the transfer channel comprised of VirB2 and VirB9 (17). Intracellular activating signals: (A) DNA substratedocking with VirD4 and delivery to VirB11 and (B) VirD4 and VirB11 ATP hydrolysis activities. These signals stimulate a conformational switch in VirB10 thatis required for gating or assembly of the distal portion of the translocation channel.

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VirB10 activation. We supplied evidence that processing anddocking of three different DNA substrates, T-DNA, RSF1010, andpCloDF13, with VirD4 (or MobB in the case of pCloDF13) wererequired for inducing VirB10’s structural transition (Fig. 1 and 3).Furthermore, both components of the DNA substrate, the relax-ase and the DNA, were essential for inducing the transition. Al-though we did not definitively establish that a direct relaxase-T4CP interaction is required for channel activation, severalstudies have supplied strong evidence that relaxases interact withcognate T4CPs and that such an interaction is required for DNAtranslocation. With our TrIP assay, for example, we previouslydetermined that VirD2 synthesis is essential for establishment of adetectable VirD4 –T-DNA cross-link (17). Here, we furthershowed that a catalytically active form of VirD2 did not suffice forestablishment of the VirD4 –T-DNA interaction, but that an intactC terminus was also required (Fig. 2). The C-terminal region ofVirD2 carries an NLS and an � sequence, the latter of which re-sembles charged signals carried by T4SS effector proteins (45–48).This C-terminal region has been shown to mediate the delivery ofa fused reporter protein to plant cells, confirming that it carriessufficient information for docking with and translocation throughthe VirB/VirD4 T4SS (45, 46). Finally, studies of several conjuga-tion systems have provided compelling evidence that relaxasesinteract directly with their cognate T4CPs (60–62). Taken to-gether, therefore, our data strongly indicate that VirD2 mediatesDNA substrate docking with VirD4 via its C terminus and that thisinteraction is essential for activation of VirB10.

In general, relaxosome docking with the T4CP is consideredimportant for coupling of the oriT nicking, T-strand-unwinding,and translocation reactions in space and time (3). In A. tumefa-ciens, it is conceivable that relaxosomes of the RSF1010 andCloDF13 plasmid substrates engage with VirD4 for coupled pro-cessing and translocation. However, the findings that A. tumefa-ciens cells accumulate multiple copies of the VirD2–T-strandtransfer intermediate per Ti plasmid (Fig. 2) (43) and, further-more, that the ParA-like accessory factor VirC1 contributes to therecruitment of free VirD2–T-strand complexes to VirD4 (43) sug-gest that the initiating signal for T-DNA transfer is probably notthe relaxosome docking reaction per se but rather engagement ofthe transfer intermediate with VirD4. Whether other accessoryfactors involved in the substrate recruitment reaction, e.g., ParA-like VirC1 or factors designated Vbp’s (43, 63), might also con-tribute to substrate activation of the VirB/VirD4 channel remainsan intriguing question for further investigation.

We were initially surprised that ligand-induced activation ofVirB10 is a property of DNA and not protein transfer through theT4SS. In fact, it is possible that both substrates induce structuralchanges in the channel but that our protease susceptibility assaydetects only a DNA ligand-induced change. However, monomericproteins differ appreciably as substrates compared with relaxase–single-stranded DNA (ssDNA) particles, and it is reasonable toexpect that different mechanisms exist for their translocation.With regard to DNA translocation, recently it was reported thatthe R388-encoded TrwB is a DNA-dependent ATPase whose cat-alytic activity and oligomeric state are also modulated by the Dtraccessory factor TrwA (64, 65). These findings raise the intriguingpossibility of an ordered reaction whereby docking of the relaxo-some or transfer intermediate stimulates ATP hydrolysis activity,which induces a change in conformation or oligomeric state in theT4CP and, in turn, a conformational change in VirB10. The con-

formational switch in VirB10 might confer an open-channel con-figuration required for passage of long ssDNA polymers. It shouldalso be noted that, whereas the DNA transfer pathway was definedby TrIP (17), the route of protein translocation through the T4SSis completely unknown, and different models have been proposed(18, 19, 66). Of particular interest is the shoot-and-pump model,which posits that DNA and protein substrates are delivered acrossthe membrane via two different translocases, DNA through thelumen of the T4CP hexamer and protein monomers through anMpf-encoded translocase, e.g., VirB6/VirB8 (66).

Our mutational analyses established the importance of VirD4’speriplasmic domain for both early-stage DNA transfer reactionsand VirB10 activation (Fig. 4). VirD4 interacts with VirB10 (18,52), and homologs of these subunits encoded by the R388 and R27(IncH) plasmid transfer systems also have been shown to interactvia their N-terminal regions (53, 54, 67). These findings support aproposal that periplasmic domains of VirD4 and VirB10 interactdirectly for coupling of ATP energy with the conformationalswitch; if so, the periplasmic domain mutations might exert theireffects on energy activation by disrupting this interaction. Whydisruption of a VirD4-VirB10 interaction would impact early-stage DNA transfer from VirD4 to VirB11 or the VirB6/VirB8channel subunits is not immediately obvious. However, previ-ously we showed that synthesis of VirB10 and other core complexsubunits is necessary even for the VirD4 to VirB11 transfer step(18), implying that core complex interactions with VirD4 and/orVirB11 are essential for establishment of productive contacts be-tween the ATPases themselves. The VirD4 periplasmic domainmutations might disrupt a T4CP-VirB10 interaction necessary forformation or stabilization of the presumptive core/VirD4/VirB11ternary complex. Finally, although we favor the notion that theperiplasmic domain mutations disrupt a VirD4-VirB10 bindingpartner interface, we cannot exclude the possibility that the mu-tations exert their effects indirectly through global disruption ofVirD4’s conformational or oligomeric state. Extensive biochemi-cal studies by the de la Cruz and Alkorta groups showing theimportance of TrwB’s N-terminal TMD to its activity, structure,and stability are consistent with such a possibility (68–72).

The VirB11 family members are structurally dynamic hexam-ers that undergo major conformational changes upon ATP bind-ing (73–75). Such ATP-induced conformational changes do notseem to play a role in DNA substrate transfer from VirD4 toVirB11, since a VirB11 Walker A mutant does not disrupt thistransfer step (18). However, phenotypes of the ATP binding sitemutations clearly establish that ATP binding/hydrolysis is re-quired for the VirB10 structural transition (Fig. 5) (21). Addition-ally, we found that the capacity of VirB11 to interact with substrateDNA was strictly correlated with activation of VirB10 (Fig. 5 to 7).This correlation held true in our characterization of a large collec-tion of substitution and i4 mutations but was best illustrated byanalyses of the substrate discrimination mutations (Fig. 7). In theabsence of the IncQ plasmid substrate, the discrimination muta-tions blocked both T-DNA transfer from VirD4 to VirB11 and theVirB10 structural transition. In the presence of the IncQ plasmidsubstrate, the mutations still blocked T-DNA transfer but permit-ted translocation of the IncQ plasmid, and in this genetic contextthe VirB11 mutant proteins supported activation of VirB10. Thesephenotypes strongly indicate that engagement of the DNA sub-strate with VirB11 is essential for activation of VirB10 and that thesubstrate discrimination mutations selectively block VirB11 from




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interacting productively with the T-DNA substrate. How VirB11discriminates between substrates is unknown, but the mechanismcould involve reported differences in the translocation signals car-ried by the VirD2 and MobA relaxase components of the T-DNAand RSF1010 transfer intermediates (46, 76) or accessory factorsshown to be important for substrate processing or recruitment tothe T4SS, e.g., VirD1 or ParA-like VirC1 for T-DNA or MobB forRSF1010 (43, 76).

We located the VirB11 mutations on a structural model de-rived from the Brucella suis VirB11 crystal structure (74). B. suisVirB11 is considered a reliable structural prototype for the VirB11family of ATPases (see Fig. S2 in the supplemental material) (74).The B. suis VirB11 monomer consists of N-terminal and C-termi-nal domains (designated NTD and CTD) joined by a linker region.The protein assembles as a hexameric double-ring structure, witheach ring formed by extensive interactions among adjacent NTDsand CTDs, respectively. The hexamer is further stabilized by adomain swap in which the linker region of one monomer fits intoa groove of the CTD in an adjacent monomer. Not surprisingly,the permissive mutations generally mapped within nonstructuredloops, whereas the nonpermissive mutations mapped in the ATPbinding pocket or in structural folds in the NTD or CTD or linkerregion (see Fig. S3A and B in the supplemental material). Most ofthe nonpermissive mutations can therefore be expected to disruptassembly or conformational flexibility of the hexamer, whereasmutations on the surface or in the lumen of the hexamer coulddirectly interfere with VirB11 binding to VirD4, another partnerprotein, or substrate. The substrate discrimination mutations arelocated near the Walker A motif (C168.i4), within the lumen ofthe hexamer (L302.i4), or within the NTD (see Fig. S3B). In viewof these structural assignments, it is intriguing to speculate that acombination of ATP-mediated conformational changes and thecentral lumen of the VirB11 hexamer contribute directly to therecognition and processing of DNA substrates for deliverythrough the inner membrane translocase.

Finally, it is interesting to note that the DNA ligand and ATPenergy signals characterized in this study, while necessary, are notsufficient for channel activation and substrate transfer. In the1970s, studies of the F transfer system supplied evidence that anextracellular signal, generated upon formation of pilus-mediatedcontact with a recipient cell, stimulated early DNA processingreactions in the donor cell (77). The nature of this contact-medi-ated signal remains unidentified, but in view of our present find-ings and evidence that VirB10-like proteins span the entire cellenvelope, it is enticing to suggest that such a signal is transmittedacross the donor cell envelope via the VirB10 sensor subunit.Zechner and her colleagues also recently determined that bacte-riophage R17, whose receptor is the F-like R1-encoded pilus, en-ters the cell only if the relaxosome is docked with a catalyticallyactive form of the TraD T4CP (78). Phage R17 binding appears tosupply a requisite extracellular signal, possibly by mimickingrecipient cell contact, which together with the DNA substrate-T4CP docking signal activates the channel to allow phage up-take. Thus, a combination of intracellular signals and a con-tact-mediated extracellular signal might generally activateT4SS channels via conformational effects on VirB10-like sub-units for transmission of nucleic acid substrates in either direc-tion across the cell envelope.

ACKNOWLEDGMENTS

We thank members of the Christie laboratory for helpful discussions.These studies were supported by NIH GM48476.

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