Functional Subsets of the VirB Type IV Transport Complex Proteins Involved in the Capacity of Agrobacterium tumefaciens To Serve as a Recipient in virB-Mediated Conjugal Transfer of Plasmid RSF1010

The virB-encoded type IV transport complex of Agrobacteriumtumefaciens mediates the transfer of DNA and proteins into plantcells, as well as the conjugal transfer of IncQ plasmids, suchas RSF1010, between Agrobacterium strains. While several studieshave indicated that there are physical interactions among the11 VirB proteins, the functional significance of the interactionshas been difficult to establish since all of the proteins arerequired for substrate transfer. Our previous studies, however,indicated that although all of the VirB proteins are requiredfor the capacity of a strain to serve as an RSF1010 donor, onlya subset of these proteins in the recipient is necessary toincrease the conjugal frequency by 3 to 4 logs. The roles ofparticular groups of VirB proteins in this increased recipientactivity were examined in the study reported here. Examinationof the expression of subgroups of virB genes revealed that translationof virB6 is necessary for expression of downstream open readingframes. Expression of limited subsets of the VirB proteins ina recipient strain lacking the Ti plasmid revealed that theVirB7 to VirB10 proteins yield a subcomplex that is functionalin the recipient assay but that the VirB1 to VirB4 proteins,as a group, dramatically increase this activity in strains expressingVirB7 to VirB10. Finally, the membrane distribution and cross-linkingpatterns of VirB10, but not of VirB8 or VirB9, in a strain expressingonly VirB7 to VirB10 are significantly altered compared to thepatterns of the wild type. These characteristics are, however,restored to the wild-type status by coexpression of VirB1 toVirB3. Taken together, these results define subsets of typeIV transport complex proteins that are critical in allowinga strain to participate as a recipient in virB-mediated conjugalRSF1010 transfer.

INTRODUCTION

Top

Introduction

Type IV transport complexes are utilized to transport macromoleculesout of gram-negative bacteria in a wide variety of circumstances,including conjugal transfer of plasmids between bacteria, transferof virulence factors into host cells, and secretion of virulencefactors into the extracellular environment (for reviews seereferences 19, 23, and 83). Included among the pathogens thatutilize type IV secretion systems are Helicobacter pylori, Campylobacterjejuni, Legionella pneumophila, Bordetella pertussis, Brucellasuis, and Bartonella henselae. Agrobacterium tumefaciens, apathogen that causes tumor formation on a wide variety of plants,utilizes the archetypal type IV transporter, the VirB complex(45, 65, 72, 77, 78), to transfer the T-DNA segment of the residentTi (tumor-inducing) plasmid, as well as virulence proteins,into host cells. Specifically, a protein-single-stranded DNAintermediate (VirD2-T strand) and the proteins VirE2 and VirFare substrates for VirB transporter activity (14, 24, 53, 58,66, 74, 85). Once in the plant cells, the T-DNA is ultimatelyintegrated into the nuclear genome and expressed, resultingin the tumorous phenotype of transformed cells (16, 36). BesidesT-DNA and protein transfer to host plant cells, the VirB complexcan direct the conjugal transfer of an IncQ plasmid betweenA. tumefaciens strains or from A. tumefaciens into plant cells(11, 18). The processing of RSF1010 to form a transferable intermediatedepends on its own gene products, the Mob proteins, and theoriT site (18). However, transfer of this plasmid from A. tumefaciensto either plant cells or other bacteria relies on the same VirBtransfer machinery used for T-DNA transfer (11, 17, 33, 81).

The virB operon, along with virD4, encodes 12 membrane-associatedproteins that are postulated to form the macromolecular transferapparatus (for reviews see references 21, 86, and 87). Thisoperon is regulated by a two-component regulatory system, VirAand VirG, that induces vir gene expression in response to plantcompounds such as acetosyringone (AS) (15, 82). Most of theVirB proteins are required for virulence; the only exceptionis the protein encoded by virB1, which, nevertheless, is necessaryfor maximal transfer efficiency (13). The VirB proteins andVirD4 are thought to form a complex that spans the inner andouter membranes. Most models suggest that VirB6, VirB7, VirB8,VirB9, and VirB10 form the core of the VirB complex. VirB6 hasmultiple membrane-spanning domains (27) that suggest that itmay anchor the export apparatus to a specific location in theinner membrane, although recent data suggest that its activitiesare more critical for T-pilus formation than for transport complexintegrity (37). VirB7, an outer membrane lipoprotein, interactswith itself (37, 61) and with VirB9 via disulfide bonds betweenunique reactive cysteine residues present in each protein (3,9, 70). Several lines of evidence indicate that the VirB7-VirB9complex, in turn, is necessary for VirB10 stabilization andcomplex formation (9, 10, 31). Recent evidence indicates thepresence of a high-molecular-weight subassembly containing theseproteins that can be solubilized from isolated membranes (44).In the yeast two-hybrid assay, interactions among VirB8, VirB9,and VirB10 were observed (28), as were interactions betweenVirB proteins that should result in a contiguous complex fromthe cytoplasm to the outer membrane (76). Finally, immunofluorescenceand immunoelectron microscopic methods (46) have shown thatVirB8, VirB9, and VirB10 are concentrated at several foci onthe cell surface, which represent potential sites of complexformation and/or potential activity.

While the VirB7 to VirB10 proteins have been proposed to bethe core of the transport complex, all of the VirB proteinsare required for the formation of the T pilus (34, 48). Thisextracellular structure is primarily composed of processed VirB2proteins (47), but it also includes VirB5 (62) and VirB7 (61)as minor components. Interestingly, a subassembly of these proteins,distinct from the VirB7-VirB8-VirB9-VirB10 subassembly, canbe isolated from membrane preparations of wild-type strain C58(44). The T pilus is probably involved in contact with the hostcell, although it may also provide a conduit for DNA transport(49). VirB4 and VirB11 are membrane localized and have ATP bindingsites that are necessary for virulence (12, 25, 60, 71), andVirD4, also required for virulence, is membrane localized andhas a nucleoside triphosphate binding domain (6, 56). TheseATPases could be involved either directly in the transport processor in construction of the transport apparatus.

The proposed VirB complex is being intensively investigated,particularly with regard to the localization of individual VirBproteins, interactions between VirB proteins, and possible interactionsbetween transported substrates and particular VirB proteins.However, the complex nature of the VirB transporter has madeit difficult to relate physical characteristics to function.Our previous studies demonstrated that the presence of the Tiplasmid in a recipient Agrobacterium cell increases the frequencyof RSF1010 conjugal transfer by as much as 3 to 4 logs (17).Moreover, while all of the VirB proteins are required for astrain to serve as an RSF1010 donor, only a subset of theseproteins is necessary for increased recipient activity. Specifically,several of the VirB proteins are not required for recipientactivity, including VirD4 and VirB11 (17), as well as the ATPaseactivity of VirB4, although the VirB4 protein is required (25).Moreover, point mutations in VirB9 that quantitatively affectthe export activity of the VirB complex have a similar quantitativeeffect on recipient activity (17). The latter finding indicatesthat at least some of the structural requirements of the typeIV complex are the same for donor and recipient activities.

These results are unexpected since previous studies indicatedthat if anything, the presence of conjugal plasmids can inhibita strain from serving efficiently as a recipient by preventingeither mating pair formation or signaling to initiate donorconjugal DNA synthesis (i.e., surface and entry exclusion) (1).However, the properties of bacterial cells that make them efficientrecipients in conjugal transfer are poorly defined, particularlyin systems other than F plasmid transfer (29). Studies of theF plasmid have demonstrated that ompA mutants of Escherichiacoli strains are poor recipients for this plasmid but are notaffected as recipients for other types of plasmids (32, 38,67). Other experiments have indicated that mutations in thelipopolysaccharide genes can have either positive or negativeeffects on recipient activity, depending on the specific typeof lipopolysaccharide that ends up being produced (4, 63). Evenin these cases, however, most of these mutations do not affectmating carried out on solid surfaces rather than in liquid (2).

The VirB-mediated increase in the capacity of an Agrobacteriumstrain to serve as a recipient thus provides a unique opportunityto examine mechanisms involved in conjugal DNA transfer acrossvarious membrane and wall systems. Characterization of the roleplayed by the VirB proteins in this process requires an understandingof the particular proteins involved and whether interactionsamong them are required. Here we describe a study in which varioussubsets of VirB proteins were expressed in a strain lackinga Ti plasmid. Examination of the resultant strains revealedthat (i) expression of genes downstream of virB6 is dependenton translation of this gene and (ii) expression of the VirB7to VirB10 proteins in a strain leads to a 5- to 10-fold increasein the capacity of the strain to serve as a recipient in theconjugal transfer of RSF1010 between Agrobacterium strains,but wild-type activity is observed only when the VirB1 to VirB4proteins are also present. In addition, membrane localizationand/or the cross-linking patterns of the VirB7, VirB8, VirB9,and VirB10 proteins were examined in this study. The data showthat the membrane localization and chemical cross-linking ofVirB10, but not those of VirB7, VirB8, and VirB9, are dramaticallyaltered in cells expressing only VirB7 to VirB10 but are restoredto the wild-type parameters by coexpression of VirB1 to VirB3.

MATERIALS AND METHODS

Top

Materials and Methods

Strains and growth media. The strains and plasmids used in this study are shown in Table1. E. coli strains used for cloning procedures were grown inLuria-Bertani (LB) media at 37°C. A. tumefaciens strainswere routinely grown at 25°C in LB broth or in AB inductionmedium (ABIM) (84) under appropriate antibiotic selection conditions.The antibiotic concentrations used in liquid and solid mediawere as follows: spectinomycin, 50 and 100 µg/ml, respectively;carbenicillin, 30 and 100 µg/ml, respectively; kanamycin,10 and 50 µg/ml, respectively; and tetracycline, 3 and5 µg/ml, respectively.

View this table:
[in this window]
[in a new window]
TABLE 1. Strains and plasmids

Plasmid construction. The IncP plasmid pED33 (79) contains a virB promoter followedby a polylinker and was used in pZL48 and pZL36 expressing virB6to virB10 and virB7 to virB10, respectively (Table 1). The IncWplasmid pJB20 (10) and its derivatives pYW15c and pYW12 (75)served as vectors for virB1 to virB4, virB1 to virB5, and virB1to virB11 (pZL3, pZL42, and pZL51, respectively). pZL5 was constructedby cloning the XhoI virB promoter- and polylinker-containingfragment from pED31 (79) into pJB20 This plasmid was used toexpress virB2 to virB4 in plasmid pZL24 and virB3 and virB4in plasmid pZL12. All plasmids carrying the virB genes weretested for the capacity to complement the appropriate nonpolardeletions in the PC10XX series (13), and all of the plasmidsexcept pZL36 were positive (see below). pAB123 was constructedby cloning the 4.6-kb KpnI virA-containing fragment of pTiA6from pVRA5 (50) into pMutG (52), an IncRi derivative.

Mutagenesis of virB6. Internal in-frame deletions of virB6 were generated by overlapextension PCR mutagenesis as described previously (51). PCRwas carried out with Pfu DNA polymerase (Stratagene) and thefollowing primers: T3 (5'-CCA AGC GCG CAA TTA ACC CTC ACT AAAGGG-3') (from pBS II), B6 (5'-GTT GGG CAG GCT AAC TAC CA-3')(positions 5544 to 5563), AB6 (5'-CCT AGC CCC GTT CAA CCT GAG-3')(positions 6198 to 6177), AB8 (5'-CGT CAT GGT GCG CCC TGG CCTA-3') (positions 7078 to 7100), M12 reverse (5'-GTC GTT CATCGG ACC GAT TCC GGG TGC GAT-3'; underlined bases carry mutations),D1 forward (5'-CTT ATC ACC ATC-GCG CTC ACC CTC ATG CTT GGT-3')(positions 5910 to 5922 and 5982 to 6003), D1 reverse (5'-GAGGGT GAG CGC-GAT GGT GAT AAG TTG CCC GAT-3') (positions 5994to 5982 and 5922 to 5901), D2 forward (5'-CAA GGG GCA CAG-ACGACC GCG GCC AAG ATC AT-3'), D2 reverse (5'-GGC CGC GGT CGT-CTGTGC CCC TTG GAA AGC AAG-3'), D3 forward (5'-GCG GTG AGT GCG-ATCGGT CCG ATG AAC GAC CAG-3'), D3 reverse (5'-CAT CGG ACC GAT-CGCACT CAC CGC CTC CTG GAT-3'), D4 forward (5'-CAG GTC CAA TCG-ATCGGT CCG ATG AAC GAC CAG-3'), and D4 reverse (5'-CAT CGG ACCGAT-CGA TTG GAC CTG AAC TTG GCT-3'). The deletions within virB6were constructed by two rounds of PCR in which two internalmutagenic primers and two primers (B6, AB8) outside the codingregion were used. For example, the internal mutagenic primersD1 forward and D1 reverse were designed to create an in-framedeletion from amino acids 205 to 224 of VirB6. D1 forward andD1 reverse each contain 12 bases that are 5' to the deletionand 21 bases that are 3' to the deletion. In the first roundof amplification PCRs were carried out with primers D1 forwardand AB8 and primers D1 reverse and B6. The product of each reactionwas gel purified with a QIAquick gel extraction kit (Qiagen),the products were mixed, and the 12-base overhangs from theproducts of the first reactions caused the two fragments tobase pair. Primers B6 and AB8 were then added to the mixture,and a second round of PCR amplification resulted in productionof full-length virB6 carrying the deletion. This amplified PCRproduct was then digested with RsrII and NcoI and exchangedwith the wild-type gene fragment of virB6 in pZL48. By usingthe same strategy VirB6 with an in-frame deletion of amino acids151 to 237 was produced with the mutagenic primers D2 forwardand D2 reverse and outside primers B6 and AB8. The final PCRfragment containing the deletion was digested with RsrII andNcoI and exchanged with the wild-type gene fragment of virB6in pZL48, resulting in pZL48-D2. Similarly, VirB6 with an internaldeletion from amino acid 37 to amino acid 134 was created byusing two internal primers, D3 forward and D3 reverse, and outsideprimers T3 and AB6. The PCR fragment carrying the virB6 deletionwas digested by XhoI and RsrII and was exchanged either withthe wild-type fragment of virB6 on pZL48, which resulted inpZL48-D3, or with the same fragment in the incomplete virB6(deletion from amino acid 151 to amino acid 237) of pZL48-D2,which resulted in pZL48-D5. Two point mutations (Ser to Proat position 133, Glu to Gly at position 133) were introducedinto VirB6 by utilizing the mutant primer AB6M12 (5'-GTC GTTCAT CGG ACC GAT TCC GGG TGC GAT-3'; mutated bases underlined).A 0.6-kb PCR product was amplified from pZL48 with primers AB6M12and T3, digested by XhoI and RsrII, and exchanged with the wild-typevirB6 gene on pZL48, yielding pZL48-M12. A frameshift mutationin virB6 was engineered by digesting pZL48 with RsrII, fillingin with the Klenow fragment, adding a 10-bp XbaI linker (NewEngland Biolabs), and ligating to obtain pZL48-XbaI. All mutationswere confirmed by sequencing.

Immunoblot analysis and protein cross-linking. Equal numbers of cells grown at 25°C in ABIM with or withoutthe vir gene inducer AS (Aldrich) were collected, resuspendedin sodium dodecyl sulfate (SDS) sample buffer (12% sucrose,4% SDS, 0.1 M Tris-HCl[pH 6.8], 5 mM EDTA, 0.04% bromophenolblue, 0.1 M dithiothreitol[DTT]), resolved by electrophoresisin SDS-10 to 12% polyacrylamide gel electrophoresis (PAGE) usingpolyacrylamide gels (acrylamide/bisacrylamide ratio, 29:1).The gels were transferred to a polyvinylidene difluoride membrane(Amersham) and probed with antibodies against VirB8, VirB9,and VirB10 as described previously (10, 51). For analysis ofVirB7, samples were electrophoresed on SDS-12% PAGE polyacrylamidegels with an acrylamide/bisacrylamide ratio of 19:1 in Tris-Tricine-SDSbuffer (Bio-Rad). Protein cross-linking was performed with wholecells by using BS³ (Pierce Chemical Co.) as described previously(10).

Expression analysis by RT-PCR. Total RNA isolation was carried out by an acid-phenol method(55), as follows. Portions (30 ml) of cultures of Agrobacteriumstrains were induced by growing them in ABIM with 200 µMAS for 6 h to 8 h (optical density at 600 nm [OD₆₀₀], 0.4 to0.6) and then harvested. Each cell pellet was resuspended in1.6 ml of buffer (20 mM sodium acetate [pH 5.5], 1 mM EDTA)and then added to an SDS-acid phenol solution (110 µlof 10% SDS, 1 ml of phenol; pH 5.5) at 65°C and incubatedfor 7 min; this was followed by centrifugation at 16,060 x gfor 5 min at 25°C. The aqueous phase was extracted with1 ml of phenol-CH₂Cl₂ (1:1) and then with 1 ml of CH₂Cl₂ (preequilibratedin 10 mM Tris [pH 7.0]), and the RNA was precipitated with 2.5volumes of 100% ethanol. The RNA pellet was washed with 70%ethanol, resuspended in diethyl pyrocarbonate-treated H₂O, quantifiedby determining the OD₂₆₀, and used to synthesize the first strandof cDNA. To ensure linearity of the reverse transcription (RT)-PCR,first-strand cDNA synthesis was performed with 0.3, 0.1, and0.03 µg of each pool of RNA. The cDNAs were synthesizedby using a SuperScript first-strand system for RT-PCR kit (Invitrogen)as recommended by the supplier and a total volume of 20 µl.The RT reaction mixture was precipitated with 2.5 volumes of100% ethanol supplemented with 1 µl of a 20-mg/ml glycogensolution (Roche). After centrifugation the pellets were dissolvedin 40 µl of H₂O and subjected to PCR. The PCR mixtures(total volume, 50 µl) contained 1 µl of the RT reactionmixture, virB8 primers (forward primer [starting from the endof virB7 gene], 5'-CAGCTCCGCAATTCGGTGGAC-3'; reverse primer[starting from the beginning of virB9], 5'-TCAGCCTCCGCACCAGTCGC),and virB1 primers for internal control (forward primer, 5'-GCTTCACTGGCAAGATCACA-3';reverse primer, 5'GTGCAATTTGCTTCTCGTCAAT-3'), and the PCR programconsisted of 94°C for 20 s, 60°C for 30 s, and 72°Cfor 40 s for 20 cycles. Twenty-microliter portions of the PCRmixtures were analyzed by electrophoresis in 1.5% agarose gels.

Membrane localization of VirB8, VirB9, and VirB10. VirB protein localization in the inner or outer membranes wasdetermined by sucrose density gradient fractionation as describedpreviously (9, 26, 31). Briefly, cells (ca. 400 ml) were inducedin ABIM with 100 µM AS overnight at 25°C, harvested,washed three times with 50 mM sodium phosphate buffer (pH 7.6),and resuspended in phosphate buffer containing 20% (wt/vol)sucrose (cell concentration, 1 g/10 ml). The cells were lysedby three passes through a French press at 16,000 lb/in², and1 mM EDTA and 0.01% phenylmethylsulfonyl fluoride protease inhibitorwere added just before lysis. Four milligrams of lysozyme wasadded to each sample, and the samples were incubated on icefor 30 min. After an initial low-speed centrifugation (Sorvall-SS34,15 min, 13,000 rpm) 0.2 M (final concentration) KCl was addedto the cleared cell lysates, which were then subjected to ultracentrifugationin a Beckman L7 ultracentrifuge for 90 min at 45,000 rpm. Theinsoluble pellet was dispersed in phosphate buffer containing0.1 M DTT, 5 mM EDTA, and 20% (wt/vol) sucrose, the concentrationwas adjusted to 2 to 3 mg of protein/ml, and the pellet waslayered on top of a two-step sucrose gradient (1 ml of 70% [wt/vol]sucrose, 2.9 ml of 53% [wt/vol] sucrose). After centrifugationat 4°C for 17 h at 33,000 rpm in a Beckman SW 50.1 rotor,fractions (0.3 ml) were collected from the top of the gradient.NADH oxidase was used as a marker for the inner membrane (57).The protein concentration was determined by using the Bio-Rad(Bradford) protein assay reagent and procedures. Five microlitersof each fraction was resolved on an SDS-10 to 12% PAGE gel,which was blotted and subsequently probed with VirB8, VirB9,and VirB10 antibodies (see below).

Conjugation assays. Mating between donor and recipient strainsof A. tumefaciens was performed as described previously (17).Donor strain A348 containing RSF1010 derivative IncQ plasmidpJB31 and recipient strain A136(pAB123) expressing various virBgenes or A348(pAB123) were grown in LB broth overnight and thentransferred into ABIM with 250 µM AS to an OD₆₀₀ of 0.2and grown for 6 h at 25°C. Donor and recipient bacteriawere then mixed at a ratio of 5:1, and 5 µl of each conjugationmixture was spotted on solid (1.5% Bacto Agar) ABIM with 500µM AS in a 24-well plate. After 3 days of incubation at25°C, the cells were resuspended in 1 ml of 0.9% NaCl. Thecell suspensions were diluted as appropriate and plated ontoLB agar plates supplemented with spectinomycin, carbenicillin,and spectinomycin plus carbenicillin to recover donors, recipients,and transconjugants, respectively. Colonies were counted after3 days of growth at 25°C. A statistical analysis was carriedout by using a two-way mixed model analysis of variance withreplication, with date as a random effect and treatments asfixed effects. The Tukey-Kramer test for unplanned comparisons(69) was used to test for differences in the conjugation frequencyby using the software program JMPIn 4.0.4 (SAS Institute; http://www.duxbury.com/statistics_d/).Data were collected from eight different experiments, and eachrecipient strain was tested at least three times. Three separateconjugations were performed with each recipient strain testedin an experiment.

Virulence assays. The virulence of various A. tumefaciens strains was assayedwith Kalanchoe daigremontiana by wounding the youngest expandedleaves with 2-cm needle scratches and inoculating them with $~$ 10^-6 CFU. Tumor formation was monitored 14, 21, and 28 daysafter inoculation. Virulence assays in which tobacco leaf squaretransformation was used were carried out as described previously(7). Briefly, overnight cultures of agrobacteria were dilutedto an OD₆₀₀ of 0.5 and cocultivated with Nicotiana tabacum cv.Havana 425 leaf squares on hormone-free MS medium with 300 µMAS. After 2 days, the leaf squares were transferred to hormone-freeMS medium containing vancomycin (200 µg/ml) and timentin(200 µg/ml). Tumors were scored and photographed 12 daysafter the start of the cocultivation. A total of 14 to 16 leafsquares were tested for each strain in an experiment, and allstrains were tested at least three times.

RESULTS

Top

Results

Construction of plasmids to express subsets of VirB proteins. To characterize the requirements for the different VirB proteinsin recipient activity, we constructed plasmids that expressedsubsets of the various virB genes from the native virB promoter(Table 1). These plasmids were electroporated into strain A136(pAB123)lacking the Ti plasmid, which also carried the virA-virG regulatorysystem necessary for inducible expression from the virB promoter.They were also tested for the capacity to complement nonpolardeletions in each of the appropriate reading frames of the PC10XXstrains (e.g., PC1001 carries a nonpolar deletion of virB1).All plasmids carrying any combination of the virB1, virB2, virB3,virB4, and virB5 genes were capable of restoring virulence tothe corresponding PC10XX strain. For example, pZL3, carryingvirB1 to virB4, restored the capacities of PC1001, PC1002, PC1003,and PC1004 to induce tumors on Kalanchoe leaves (data not shown).Immunoblot analysis indicated that VirB4 is produced by allplasmids carrying the virB4 gene (data not shown). In contrast,however, virulence assays performed with Kalanchoe leaves andtobacco leaf explants demonstrated that pZL36, designed to expressvirB7 to virB10, only weakly complemented PC1007, PC1008, PC1009,and PC1010 (Fig. 1A and data not shown). pZL48, carrying virB6to virB10, was capable of complementing each of these deletionstrains, as well as PC1006 (Fig. 1B and data not shown), suggestingthat the presence of VirB6 might be important in the activitiesor accumulation of VirB7 to VirB10.

View larger version (20K):
[in this window]
[in a new window]
FIG. 1. Maps and complementation activities of various virB constructs. Plasmids were constructed as described in Table 1 and the text. The capacity to complement either the PC1006 (virB6 deletion) or PC1008 (virB8 deletion) strain of A. tumefaciens was tested by utilizing the tobacco leaf explant assay system described in Materials and Methods. A representative leaf explant was photographed in each case. The number of tumors per leaf explant (mean ± standard error; n = 14) is indicated below each photograph. In this experiment wild-type strain A348 yielded 11.7 ± 0.8 tumors/leaf explant, and PC1006 and PC1008 yielded no tumors. Wt. wild type; aa, amino acid.

Translation of virB6 sequences is required for VirB7 to VirB10 accumulation. Strain A136(pZL36) did not accumulate large quantities of VirB8,VirB9, and VirB10, whereas A136(pZL48) did (Fig. 2A and C),suggesting that the VirB6 protein may stabilize these proteins.However, strain PC1008(pZL36) did not accumulate VirB8, whereasPC1008(pZL48) did (data not shown). This indicated that productionof the VirB6 protein by PC1008 could not support accumulationof VirB8 from the virB7-virB8-virB9-virB10 construct that wasin trans. One possible explanation for this is that virB6 neededto be cis to virB7 to virB10 for the latter genes to be properlyexpressed. We tested the hypothesis that virB6 and virB7 tovirB10 must be cotranslated in order to achieve high-level expressionof VirB7 to VirB10. A frameshift mutation was introduced intothe virB6 coding sequence via a 10-bp XbaI linker at the RsrIIsite (amino acid 138) of virB6 in pZL48, resulting in a theoreticalprotein consisting of 188 amino acids, compared to the 295-amino-acidwild-type VirB6 (Fig. 1C). Complementation tests of the virBdeletion strains with the resulting plasmid, pZL48-XbaI, showedthat virulence was not restored in PC1006 and, importantly,was only partially restored in PC1007, PC1008, PC1009, and PC1010(Fig. 1C and data not shown). Importantly, expression of VirB8,VirB9, and VirB10 was reduced when this plasmid was presentin strain A136(pAB123) (Fig. 2D). Similarly, pZL48-D4, fromwhich the sequences encoding the first 134 amino acids, includingthe start codon, were deleted, exhibited poor expression ofVirB8, VirB9, and VirB10, could not complement PC1006, and barelycomplemented PC1008 for virulence (data not shown). In contrast,two point mutations (Ser to Pro at position 133, Glu to Glyat position 134) incorporated into virB6 of pZL48 resulted inplasmid pZL48-M12, which could not complement PC1006 for virulencebut could complement PC1008 (Fig. 1D) and was able to supportVirB8, VirB9, and VirB10 accumulation (Fig. 2F). To furthercharacterize the possible effects of the VirB6 protein on theVirB7 to VirB10 proteins, two separate internal portions ofVirB6 were deleted in frame and tested individually and together.None of the internal deletions resulted in a form of VirB6 thatcould complement the PC1006 mutant strain in virulence testson tobacco leaves (Fig. 1E). However, every in-frame deletiontested could fully restore virulence in the PC1008 mutant strain(Fig. 1E and data not shown), as well as in PC1007, PC1009,and PC1010 (data not shown), and the strains were able to accumulateVirB8, VirB9, and VirB10 (Fig. 2E and data not shown). Thus,the forms of VirB6 having internal deletions had completelylost the virulence function but had no negative effects on downstreamexpression of virB7 to virB10.

View larger version (47K):
[in this window]
[in a new window]
FIG. 2. Immunoblot analysis of various virB constructs expressed in A136(pAB123). Lane A, pZL36 (virB7 to virB10); lane B, pZL36 and pZL3 (virB7 to virB10, virB1 to virB4); lane C, pZL48 (virB6 to virB10); lane D, pZL48-XbaI (virB6 to virB10 with XbaI linker insertion in virB6); lane E, pZL48-D2 (virB6 to virB10 with deletion from amino acid 151 to amino acid 237 of VirB6); lane F, pZL48-m12 (virB6 to virB10 with point mutations at amino acids 133 and 134 of VirB6); lane G, A348 (wild-type Ti plasmid).

The fact that functional VirB6 protein was not required (e.g.,pZL48-D2) for accumulation of VirB8 to VirB10 means that intactVirB6 protein is not required for this cis effect of translatedvirB6. One possible explanation for these results is that accumulationof virB7 to virB10 mRNA is affected by translation of cis virB6sequences. To test this possibility, RNA was prepared from AS-inducedstrains carrying various versions of virB6 to virB10 (see above)and, on a separate plasmid, virB1 to virB4, also expressed froma virB promoter (pZL3), as an internal control. Additionally,RNA preparations were made from the Ti plasmid-free strain A136and from A348, which carries the wild-type Ti plasmid pTiA6.RT-PCR was carried out by using primers that amplified an 800-bpfragment of virB8 or a 226-bp fragment of virB1. The resultsof these experiments indicated that while virB1 was amplifiedequivalently in all RNA samples used except, as expected, fromA136 samples, the amount of the RT-PCR product from virB8 wasdrastically reduced in the samples that did not contain a translatableform of virB6 (Fig. 3). Strains carrying pZL36 and pZL48-XbaI(Fig. 3A and B) both yielded very reduced levels of the virB8RT-PCR product. In contrast, strains carrying the translatablevirB6 gene with either point mutations or deletions (Fig. 3C and D)or wild-type virB6 (Fig. 3E) yielded levels of this productequivalent to the level obtained with the Ti plasmid of A348(Fig. 3F).

View larger version (37K):
[in this window]
[in a new window]
FIG. 3. RT-PCR analysis of virB8 transcript levels of Agrobacterium virB6 mutant strains. RT reactions were performed with 0.3, 0.1, and 0.03 µg of RNA isolated from AS-induced cells as described in Materials and Methods. virB1 was used as an internal control during RT-PCR. (A) A136(pAB123, pZL36, pZL3) (virB7 to virB10, virB1 to virB4); (B) A136(pAB123, pZL48-XbaI, pZL3) (virB6xbaI-10, virB1 to virB4); (C) A136(pAB123, pZL48-m12, pZL3) (virB6mut-10, virB1 to virB4); (D) A136(pAB123, pZL48-D5, pZL3) (virB6 ${Delta}$ 185aa-10, virB1 to virB4); (E) A136(pAB123, pZL48, pZL3) (virB6 to virB10, virB1 to virB4); (F) A348.

VirB proteins required for minimal and maximal increases in recipient activity. After the plasmids capable of expressing various VirB proteinsin strain A136 were characterized, the roles of these proteinsin the recipient assay were examined. Various strains were matedwith wild-type donor strain A348 carrying the IncQ derivativeplasmid pJB31 as described above. In the first set of experiments,individual subsets of the VirB proteins were tested. Data fromeight different experiments, not all of which included eachstrain, were analyzed by a two-way, mixed model analysis ofvariance with replication. With a type I error rate of 5%, thetreatment effect (different VirB proteins in the recipient)was very significant (F = 209; P < 0.0001; df, 7 and 99).The Tukey-Kramer test for unplanned comparisons (69) was usedto test for differences between strains carrying different VirBproteins. These assays demonstrated that VirB1 to VirB11, whenexpressed in A136, resulted in recipient activity equivalentto that of the wild-type Ti plasmid-containing strain A348 (Fig.4), thus demonstrating that no other Ti plasmid genes are requiredfor this activity. Plasmids designed to express VirB1 to VirB4,VirB1 to VirB5, VirB6 to VirB10, and VirB7 to VirB10 were alsotested. Strains carrying pZL48 (encoding VirB6 to VirB10) orpZL48-D5 (encoding VirB7 to VirB10) yielded recipient activitythat was 5- to 10-fold greater than, and significantly differentfrom, the activity of the A136 control (Fig. 4). In contrast,A136 strains carrying plasmids pZL3 (virB1 to virB4), pZL42(virB1 to virB5), and pZL36 (virB7 to virB10, which were poorlyexpressed [see above]) did not exhibit significant differencesin recipient activity compared to A136. Thus, VirB7 to VirB10represent the subset of VirB proteins tested that provides minimalrecipient activity.

View larger version (38K):
[in this window]
[in a new window]
FIG. 4. Recipient activities of strain A136(pAB123) carrying plasmids expressing various subsets of the VirB proteins. The adjusted mean numbers of transconjugants per output recipient (from the Tukey-Kramer analysis [see Materials and Methods]) are indicated along with 2 standard errors. An asterisk indicates that a value is significantly different than the value obtained for A136(pAB123) at a 95% confidence level; a number sign indicates that a value is not significantly different from the value obtained for A136 (see Materials and Methods for details concerning statistics).

Increasingly complex sets of the VirB proteins were then testedin order to determine the minimal group necessary for wild-typerecipient activity. The results demonstrated that wild-typeactivity was present when either VirB1 to VirB5 (pZL42) or VirB1to VirB4 (pZL3) was expressed along with VirB6 to VirB10 (frompZL48) (Fig. 5A). In contrast, when strains carrying pZL48 aswell as VirB2 to VirB4 or VirB3 and VirB4 were tested, no suchstimulation was observed (Fig. 5A). Similarly, no stimulationof recipient activity was obtained when either VirB1 (pTrcB1)(62) or VirB1 to VirB3 (pZL49) were tested in strains expressingVirB6 to VirB10 (data not shown), demonstrating the importanceof the VirB1, VirB2, VirB3, and VirB4 proteins. These resultsare also consistent with the results of a previous study (17)that demonstrated that strains having nonpolar deletions ofVirB2 or VirB3 in an otherwise wild-type Ti plasmid exhibitlow-level recipient activity similar to that observed in thisstudy for strains expressing only VirB7 to VirB10. To test therole of VirB6 in the observed synergistic interaction betweenVirB6 to VirB10 and VirB1 to VirB4, we examined strains carryingpZL48-D2 and strains carrying pZL48-D5 (with 87- and 185-amino-aciddeletions in VirB6, respectively). Results identical to thoseobtained with pZL48 were obtained; the maximal recipient activitywas observed when pZL3 (virB1 to virB4) was present along withpZL48-D2 or pZL48-D5 (Fig. 5B). These results demonstrate thatthe VirB6 protein plays no role in the increased capacity ofa strain to serve as a recipient in conjugal transfer of IncQplasmids between Agrobacterium strains and that VirB1 to VirB4along with VirB7 to VirB10 are required for maximal recipientactivity.

View larger version (40K):
[in this window]
[in a new window]
FIG. 5. Recipient activities of strain A136(pAB123) containing pZL48 (virB6 to virB10) (A) or pZL48-D2 (virB6 ${Delta}$ 87aa-10) or pZL48-D5 (virB6 ${Delta}$ 185aa-10) (B) with or with out other virB genes, as indicated. The mean numbers of transconjugants per output recipient are indicated. The error bars (some of which are not visible due to scale) indicate the standard errors for three separate conjugations performed on the same day. Similar results were observed in two other experiments.

Localization and cross-linking of VirB10 are dramatically affected by VirB1 to VirB3. The results described above indicate that VirB7, VirB8, VirB9,VirB10 comprise the minimal subset of VirB proteins that canincrease the recipient activity of strain A136 but that expressionof VirB1 to VirB4 is necessary to achieve maximal stimulationof this activity. This suggests that the VirB1-VirB2-VirB3-VirB4subset of proteins affects the VirB7-VirB8-VirB9-VirB10 subsetin some fashion (or vice versa), resulting in increased biologicalactivity. To determine whether the VirB1 to VirB4 proteins affectVirB7 to VirB10, we characterized the membrane distributionof these VirB proteins, as well as their capacity to interactwith themselves or each other as revealed by cross-linking studies.In the first set of experiments, membranes were isolated fromstrains grown in ABIM containing AS (to induce expression ofthe vir genes), and sucrose density gradients were used to separatethe inner and outer membranes, as described in Materials andMethods. The sucrose gradient fractions were then subjectedto SDS-PAGE and immunoblot analysis by using anti-VirB8, anti-VirB9,and anti-VirB10 as probes. In the case of wild-type strain A348,VirB8 localized exclusively to the inner membrane, whereas VirB9and VirB10 were found to be distributed in both of these fractions(Fig. 6), a pattern of distribution that has been observed previously(30, 31, 64, 73). In the case of pZL48-D5 (which expressed onlyVirB7 to VirB10) the distribution of VirB8 and VirB9 was unchangedcompared to the wild-type distribution. However, the distributionof VirB10 was dramatically altered in this case, and this proteinwas found predominantly in the inner membrane (Fig. 6). We nextsought to determine which other VirB proteins may be necessaryfor VirB10 to be distributed in a wild-type fashion in the innerand outer membrane systems. In these experiments plasmids carryingvarious combinations of VirB1, VirB2, VirB3, and VirB4 weretested. Analysis of membrane fractions from a strain producingVirB1 to VirB4 as well as VirB7 to VirB10 [A136(pAB123, pZL3,pZL48-D5)] demonstrated that VirB10 was distributed in boththe inner and outer membranes (Fig. 6), similar to the wild-typedistribution. A similar result was obtained when VirB1 to VirB3were expressed along with VirB7 to VirB10 (data not shown).However, if only VirB2 to VirB4 or VirB3 and VirB4 were expressedalong with VirB7 to VirB10, VirB10 was distributed in the membranefractions in the aberrant fashion observed when VirB7 to VirB10were expressed by themselves (data not shown).

View larger version (52K):
[in this window]
[in a new window]
FIG. 6. Membrane localization of VirB8, VirB9, and VirB10 from A348, A136(pAB123, pZL48-D5, pZL3) (virB6 ${Delta}$ 185aa-10, virB1 to virB4) and A136(pAB123, pZL48-D5) (virB6 ${Delta}$ 185aa-10). Membrane fractions were separated by sucrose density gradient as described in Materials and Methods. Membrane fractions 3 to 14 (from the top to the bottom of the gradient) were subjected to SDS-PAGE, blotted, and probed with anti-VirB8, anti-VirB9, or anti-VirB10. Similar results were observed in three other experiments. IM, inner membrane; OM, outer membrane.

Distribution of the VirB9 and VirB10 proteins in both membranefractions despite their predicted localization to the outerand inner membranes, respectively, has been proposed to be theresult of protein interactions within the complex (21, 31, 73).The capacities of the various VirB proteins to interact witheach other or with themselves were therefore examined by usingtwo different methods. First, the interactions of VirB7 andVirB9 through disulfide bond formation were examined by immunoblotanalysis of reduced or nonreduced samples. These experimentsrevealed that the strain expressing only VirB7 to VirB10 accumulatedVirB7-VirB9 heterodimers in a wild-type fashion (Fig. 7A). Ina second series of experiments, the previously established capacityof VirB9 and VirB10 to form high-molecular-weight aggregatesafter cross-linking with BS³ (10, 80) was tested. To examinethe cross-linking patterns of these proteins, whole cells weretreated with BS³ as described above, and samples were then subjectedto SDS-PAGE and immunoblot analysis. As expected, high-molecular-weightforms of VirB9 and VirB10 were found in samples prepared fromcross-linked cells of wild-type strain A348. In the strain expressingonly VirB7 to VirB10 [A136(pAB123, pZL48-D5)] the abundanceof cross-linked VirB9 was similar to that in the wild-type strain(Fig. 7A). In contrast, the levels of high-molecular-weightforms of VirB10 in the cross-linked samples were dramaticallydiminished in this strain (Fig. 7B). While the overall abundanceof VirB10 was decreased, scanning of the blots revealed a disproportionatelylower abundance of the cross-linked forms of this protein; infour separate experiments, there was an average fivefold changein the ratio of monomers to multimers. Interestingly, as wasthe case for its membrane distribution, the abundance of cross-linkedVirB10 was restored in strains expressing VirB7 to VirB10 ifVirB1 to VirB4 (Fig. 7B) or VirB1 to VirB3 (data not shown)were also expressed. However, strains coexpressing the VirB2to VirB4 proteins or the VirB3 and VirB4 proteins along withVirB7 to VirB10 did not restore the cross-linking capacity ofVirB10 to the wild-type situation (data not shown).

View larger version (72K):
[in this window]
[in a new window]
FIG. 7. Capacity of VirB9 and VirB10 to form high-molecular-weight complexes in the presence or absence of VirB1 to VirB4. Samples from AS-induced strains were added to loading buffer with DTT (lanes +), added to loading buffer without DTT (lanes -), or cross-linked with BS³ and added to loading buffer with DTT (lanes x). Extracts from equivalent numbers of cells were then resolved by SDS-10% PAGE, blotted, and probed with anti-VirB10 (A) or anti-VirB9 (B) antibodies. Gel 1, A348; gel 2, A136(pAB123, pZL48-D5, pZL3) (virB6 ${Delta}$ 185aa-10, virB1 to virB4); gel 3, A136(pAB123, pZL48-D5) (virB6 ${Delta}$ 185aa-10).

DISCUSSION

Top

Discussion

The objective of this study was to determine whether particularsubsets of the VirB proteins are required for the virB-mediatedincrease in the capacity of a strain to serve as a recipientin virB-mediated conjugal transfer of plasmid RSF1010 betweenagrobacteria. In particular, we sought to determine whethersubsets that had previously been speculated to be functionallyimportant in the VirB donor complex (e.g., VirB7 to VirB10 [22])were functional in the recipient phenotype. Development of constructsto test the role of the VirB6 to VirB10 proteins in the recipientassay revealed that translation through the virB6 open readingframe to sequences upstream of virB7 is critical to the expressionof downstream virB genes. This is clearly the case, for example,when expression of virB7 to virB10 from constructs that lack(pZL36) or contain (pZL48) such translatable virB6 sequencesis studied (Fig. 1 and 2). When translation of virB6 does notstart (e.g., pZL48-D4 or pZL36) or is disrupted by a nonsensemutation (pZL48-XbaI), then both the levels of the VirB7 toVirB10 proteins and the levels of virB7 to virB10 mRNA are significantlyreduced (Fig. 2 and 3, respectively). In-frame deletions ofvirB6 sequences do not have such an effect on downstream expression,demonstrating that the intact VirB6 protein is not requiredfor accumulation of virB7 to virB10 mRNA or the VirB7 to VirB10proteins. The regulatory mechanisms responsible for the effecton RNA accumulation are not clear. However, the results of ananalysis of strain PC1006, having a precise deletion of theentire coding sequence of virB6 and the nontranslated sequenceupstream of virB7 (13), are consistent with important effectsof the sequences immediately 5' to the virB7 gene. The deletionin this strain results in virB7 translation that uses the ribosomebinding site and upstream sequence 5' to virB6. This has a clearnegative effect on the accumulation of VirB7 and VirB8 comparedto the wild-type accumulation (data not shown), as well as onVirB9 and VirB10 (13). These results suggest that the sequencesupstream of virB7 cannot be completely replaced by those upstreamof virB6 in terms of virB7 to virB10 expression. Thus, translationthrough virB6 and utilization of the intergenic region betweenvirB6 and virB7 are crucial in the accumulation of virB7 tovirB10 mRNA. These findings are consistent with models proposingthat translation of polycistronic messages is necessary forstability of the mRNA molecule (see reference 20 for an analysisof decay models).

Utilizing strains expressing different subsets of the VirB proteins,we tested their activities in the recipient assay. The dataobtained demonstrate that (i) VirB6 is not involved in the functionsnecessary for recipient activity, (ii) the VirB7-VirB8-VirB9-VirB10subset is the minimal subset of VirB proteins tested in therecipient that can increase conjugal DNA transfer, and (iii)a synergistic interaction (either direct or indirect) amongthe VirB1 to VirB4 and VirB7 to VirB10 proteins yields significantbiological activity. The first conclusion indicates that VirB6is involved in a donor-specific function of this type IV transportcomplex. For example, it could be involved in substrate recognitionand/or targeting to the transport complex or in the energy transductionnecessary for substrate movement out of the cell. Examinationof the membrane localization of VirB8, VirB9, and VirB10 indicatedthat these proteins are distributed normally in the cell envelopein cells lacking VirB6 (Fig. 6). As expected from previous studies(70), VirB7-VirB9 heterodimer formation does not require VirB6.We show here that VirB6 is not required for the capacity ofVirB9 or VirB10 to form wild-type high-molecular-weight formsupon chemical cross-linking (Fig. 7). These results, therefore,support a model in which VirB6 has donor-specific activity butis not involved in the structural integrity of the VirB7-VirB8-VirB9-VirB10subassembly that is proposed to be the transport complex core.

The observation that expression of VirB7 to VirB10 by themselvesincreases recipient activity (Fig. 4) provides the first experimentalevidence that this group of proteins, often proposed as thetransenvelope channel of the VirB complex (22), is by itselfcapable of transport or transport-related activity. Previousexperiments (17) demonstrated that nonpolar deletion of virB7,virB8, virB9, or virB10 in an otherwise wild-type Ti plasmidcompletely eliminates recipient activity. This, along with thedata presented in this paper, suggests that VirB7 to VirB10constitute the minimal subset of VirB proteins that can supportrecipient activity. The mechanism of this activity is not yetknown (see below). The biological activity of VirB7 to VirB10is consistent with the recent observation that a high-molecular-weightsubassembly consisting of VirB7 to VirB10 exists in the membraneof wild-type cells (44). Several studies have previously indicatedthat there are specific interactions between members of thisgroup of proteins (3, 9, 10, 28, 70). Mutations that disruptsuch interactions also disrupt recipient activity (17), indicatingthat there is a relationship between core structure and recipientfunction. However, the fact that the VirB1 to VirB4 proteins,as a group, are required along with VirB7 to VirB10 for maximalrecipient activity (Fig. 5) indicates that a synergistic interactionbetween these two subsets of proteins is crucial for the biologicalactivity of the complex.

Several studies have revealed interactions between specificmembers of the VirB1-VirB2-VirB3-VirB4 subset with members ofthe VirB7-VirB8VirB9-VirB10 subset (8, 61, 76). The experimentspresented in this report show that in strains expressing VirB7to VirB10 and VirB1 to VirB3, there is wild-type membrane localizationof VirB10 and there are wild-type levels of high-molecular-weightforms of VirB10, as revealed by chemical cross-linking. Whilethe possible role of VirB2 and VirB3 in this activity is beinginvestigated, the results indicate that at least VirB1 is requiredfor wild-type VirB10 localization and interaction with itselfor other proteins in cross-linking studies. A recent study showedthat VirB1 interacts with VirB10 in a yeast two-hybrid interactionassay (76). One possibility is that the peptidoglycanase activity(54) of VirB1 interacting with VirB10 is necessary to providean appropriate periplasmic environment for VirB10 to assemble.These results are also consistent with a model (21, 31, 73)in which tight associations of VirB10 with other componentsof the VirB complex are necessary for both appropriate localizationand optimal biological activity. Previous studies (10) demonstratedthat VirB7 and VirB9 are necessary for wild-type levels of high-molecular-weightforms of VirB10 in response to chemical cross-linking. Thus,the VirB1-VirB2-VirB3 subset may function through direct effectson VirB10 or indirectly by facilitating interaction of the VirB7-VirB9heterodimer with VirB10.

Interestingly, our results demonstrate that VirB4 is not necessaryfor wild-type membrane localization and cross-linking of theVirB7 to VirB10 proteins but is required, along with VirB1 toVirB3, for maximal recipient activity. This could be the resultof interactions of VirB4 with either of these groups. Previousstudies (25) showed that VirB4 can dimerize and can supportrecipient activity in the absence of its ATPase activity. Additionally,VirB4 interaction with VirB10 has been demonstrated by usingthe yeast two-hybrid system (76). One possibility that emergedfrom the present study is that VirB10 needs to be in an appropriatestructure and location in order to interact with VirB4 in afashion that supports recipient activity. In contrast to interactionsof VirB7 to VirB10 with themselves or other VirB proteins, thereis relatively little evidence concerning interactions amongVirB1, VirB2, VirB3, and VirB4. One candidate protein with whichVirB4 may interact is VirB3; nonpolar mutations in VirB4 resultin lower levels of VirB3 and the loss of VirB3 associated withthe outer membrane (42). The observation that nonpolar deletionsof virB1, virB2, or virB3 result in lower levels of VirB4 (13)is consistent with a model in which these proteins interact,although other possibilities for this result have not been ruledout. VirB1interactions with VirB4 in the yeast two-hybrid systemhave also been observed (76) and may be important in recipientfunction.

Our data provide evidence for the functionality of the VirB1to VirB4 and VirB7 to VirB10 proteins in a transport process,in this case the movement of DNA into the bacterial cell duringconjugation. In general, the role of the recipient cell in conjugalplasmid transfer is poorly understood, except for F plasmidtransfer in liquid mating (29). Thus, there is little precedencefor the remarkable increase in recipient activity (3 to 4 logs)that results from the presence of specific proteins (in thiscase, parts of the VirB complex). Increases in either matingpair formation or DNA transfer efficiency could be responsiblefor the phenotype, and experiments to distinguish between thesepossibilities are in progress. We have, however, recently demonstratedthat the presence of the IncP plasmid RK2 also increases thecapacity of Agrobacterium cells to serve as recipients in thisassay (unpublished observations). This suggests that the VirBcomplex may reveal a transport capacity of type IV systems thatis normally obscured in other conjugal plasmid systems thathave evolved surface and/or entry exclusion as a means of blockingrecipient activity (1). Consistent with this model, Hofreuteret al. (40, 41) recently demonstrated that natural transformationcompetence in Helicobacter pylori is mediated by homologuesto VirB4 and to VirB7 to VirB10, further implicating them ascomponents of a DNA transport channel. Similarly, Bacon et al.(5) found that mutations in comB3 (related to virB10 of theTi plasmid) of Campylobacter jejuni resulted in an 80% decreasein competence for natural transformation, while the VirB11 homologuewas not involved in this activity. These results have led tothe proposal that natural competence in these bacteria is aninverse of the conjugation process (39, 68). The recipient activitymediated by the VirB subsets described here provides a uniqueopportunity to examine how recipient cells are involved in conjugalDNA transfer and how type IV complexes may play a role in thisprocess.

ACKNOWLEDGMENTS

We thank Christian Baron for providing pTrcB1, Anath Das forproviding pAD1287, Pat Zambryski for providing pMutG, PeterChristie for providing anti-VirB7 antibodies, and Arlene Wise,Colleen McCullen, Mark Jacobs, Mecky Pohlschröder, andFevzi Daldal for reading early versions of the manuscript. Specialthanks go to Heidi Kuehne and Warren Ewens for help with thestatistical analysis.

This work was supported by NSF grant MCB 9817149.

FOOTNOTES

* Corresponding author. Mailing address: Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018. Phone: (215) 898-8684. Fax: (215) 898-8780. E-mail: abinns{at}sas.upenn.edu.

REFERENCES

Top