Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 321 Church St SE, Minneapolis, MN 55455, USA
Correspondence
Anath Das
anath{at}cbs.umn.edu
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ABSTRACT |
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INTRODUCTION |
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Functional analysis of the VirB proteins showed that VirB1, a putative transglycosylase, is the only non-essential VirB protein but is required for a high efficiency of DNA transfer (Berger & Christie, 1994; Mushegian et al., 1996
). VirB2 is a major component of the T-pilus, a structure essential for DNA transfer (Fullner et al., 1996
; Lai & Kado, 1998
). VirB5 is a minor component of the T-pilus (Schmidt-Eisenlohr et al., 1999
). VirB4 and VirB11 are two ATP-binding proteins that provide energy essential for the function of the transport apparatus (Atmakuri et al., 2004
; Judd et al., 2005b
). VirB6, VirB7, VirB8, VirB9 and VirB10 are postulated to assemble a T4S apparatus for substrate delivery (Das & Xie, 2000
; Judd et al., 2005a
, b
). Many interactions likely to be required for the assembly of the T4S apparatus have been identified (Finberg et al., 1995
; Anderson et al., 1996
; Beaupre et al., 1997
; Das & Xie, 2000
; Ward et al., 2002
; Krall et al., 2002
; Jakubowski et al., 2004
; Atmakuri et al., 2004
; Judd et al., 2005a
, b
). VirB7 forms a disulfide-linked VirB7-VirB7 homodimer and a disulfide linked VirB7-VirB9 heterodimer. The VirB7 homodimer is postulated to function in T-pilus biogenesis and the VirB7-VirB9 heterodimer in the assembly of the T4S apparatus (Hapfelmeier et al., 2000
; Das & Xie, 2000
). VirB7VirB10 form a protein complex that localizes to the bacterial cell pole (Judd et al., 2005b
).
VirB6 is a polytopic integral membrane protein with four or five transmembrane (TM) segments (Jakubowski et al., 2004; Judd et al., 2005a
). VirB6 affects stability of two VirB proteins, viz. VirB3 and VirB5, and the formation of the VirB7-VirB7 dimer (Hapfelmeier et al., 2000
). Since these proteins and probably the VirB7 homodimer are involved in T-pilus biogenesis, VirB6 was postulated to regulate T-pilus formation. Several recent studies suggest an essential role of VirB6 in T4S apparatus assembly and function as well. VirB6 forms complexes with the core T4S apparatus components, and small insertions at random sites within the coding region blocked various steps in the T-DNA translocation process (Cascales & Christie, 2004
; Jakubowski et al., 2004
). VirB6, like the coupling protein VirD4, localized to the cell pole, and five VirB proteins are required for its polar localization (Judd et al., 2005a
). A conserved tryptophan at position 197 and the C-terminal end of VirB6 are required for polar localization of the protein (Judd et al., 2005a
). Mutants defective in polar targeting failed to transfer DNA to plant cells. In a separate study a VirB6-GFP fusion was reported to localize to a cell pole in the absence of the other VirB proteins (Jakubowski et al., 2004
).
In the present study we report the identification of individual amino acids that are essential for VirB6 function. We demonstrate that the primary sequence of one of the TM segments plays an important role in VirB6 function and the sequence of this domain is conserved in the VirB6 family of proteins. VirB6 colocalizes with two core constituents of the T4S apparatus and VirB6-GFP does not localize to a cell pole.
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METHODS |
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VirB6-GFP fusion.
A virB6-gfp (green fluorescent protein) fusion gene where the last codon of virB6 was fused to codon 3 of gfp mut3 (Cormack et al., 1996) was constructed as follows. Plasmid pGFP2X is a pUC118 derivative with the mutant gfp mut3 gene. A unique XhoI site at codon 3 of gfp was introduced during PCR-mediated cloning of the structural gene. A 1·4 kb DNA fragment containing the virDp-virB6 gene lacking the translation stop codon was obtained by PCR and cloned as an Acc65IXhoI fragment into plasmid pGFP2X to construct plasmid pAD1759. Plasmid pAD1759 containing the virDp-virB6-gfp chimeric gene was fused to plasmid pAD1412 to construct the wide-host-range plasmid pB6GFP.
Mutagenesis of virB6.
Random mutations in virB6 were introduced by error-prone PCR using Taq DNA polymerase (Vogel & Das, 1994). The regions encoding amino acids 1168 and 168295 were amplified by PCR and purified with the QIAquick PCR purification kit (Qiagen). The PCR fragments were cloned as a SphIEcoRI or EcoRIBglII (encoding residues 1168 and 168295, respectively) fragment into pAD1482 DNA to create a chimeric virB6 gene encoding a mutagenized N- or C-terminal region. Other mutations were introduced by deoxyoligonucleotide-directed site-specific mutagenesis using uracil-containing single-stranded pAD1426 as a template (Judd et al., 2005a
). All mutations were identified by DNA sequence analysis. All plasmids were fused to the wide-host-range plasmid pAD1412 for analysis in A. tumefaciens.
Anti-VirB antibodies.
Polyclonal antibodies against a His-tagged TrpE-VirB6 fusion protein were raised in rabbits and purified by affinity chromatography (Judd et al., 2005a). Other VirB antibodies used in this study have been described previously (Kumar et al., 2000
; Judd et al., 2005b
). For analysis by Western blotting, proteins were denatured for 30 min at 37 °C, separated by electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Judd et al., 2005a
). Antigens were detected by the chemiluminescence method using peroxidase-conjugated secondary antibodies (Amersham).
Immunofluorescence microscopy (IFM).
The procedure for IFM was as described previously (Kumar & Das, 2002; Judd et al., 2005a
). Briefly, A. tumefaciens was grown overnight in induction medium or uninduced conditions to mid-exponential phase (OD600
0·5) at 20 °C. At this growth temperature the VirB proteins accumulate at a similar level and no defect in either the assembly of the T-pilus or virulence is observed (Baron et al., 2001
). Cells were collected by low-speed centrifugation, washed in PBS, fixed in a 4 % paraformaldehyde solution and permeabilized by treatment with lysozyme (5 mg ml1, final concentration) for 10 min at 4 °C. Samples were placed on a polylysine-coated multi-well slide, washed and treated overnight with affinity-purified anti-VirB6 antibodies (1 : 600 dilution) at 4 °C followed by Alexa-fluor 488 (green) conjugated secondary antibodies (Molecular Probes). Samples were viewed in a Nikon E800 fluorescence microscope equipped with a CoolCam 2000, liquid-cooled, three-chip colour video camera. Images were processed with Adobe Photoshop version 6.0 for presentation.
A. tumefaciens AD1068, which expresses a His-tagged VirB6 protein, was used for colocalization experiments. AD1068 is a derivative of PC1006 containing plasmid phisB6 (Judd et al., 2005a). For colocalization analysis, the slides were first probed with anti-VirBn antibodies followed by the cognate secondary antibodies, and then with monoclonal anti-His antibodies and the cognate secondary antibodies. Changing the order of addition of the two primary antibodies had no effect on final results. Only those bacteria that were labelled with both probes were used for quantitative analysis. Foci were considered coincident when at least one half of each focus overlapped with the other (Lachmanovich et al., 2003
).
Other methods.
Plasmid DNA was introduced into A. tumefaciens by electroporation (Mersereau et al., 1990). Bacteria were grown at 30 °C in AB minimal medium (Chilton et al., 1974
) supplemented with the appropriate antibiotic. For induction, cells were grown in AB Mes pH 5·8 medium at 20 °C in the absence or presence of the inducer acetosyringone (100 µM, final concentration) where appropriate. Virulence was monitored by tumour formation assays on Kalanchöe diagremontiana leaves (Judd et al., 2005a
). All infections were performed a minimum of four times and tumours were scored 23 weeks post-infection.
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RESULTS |
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Screening of several hundred transformants led to the isolation of 21 mutants with an avirulent phenotype (Fig. 1). DNA sequence analysis of the putative virB6 mutant genes identified thirteen unique mutants (Table 1
). Eleven mutants had a single nucleotide change that led to a change in the amino acid sequence, one was a double mutant with changes in two amino acids, and the remaining one was a triple mutant with mutations in three amino acids. Several mutations mapped to two regions of the protein: four to the large periplasmic loop P1 (aa 56165) including two at adjacent residues (D118G, S133P, E134G, Q150A) and three to the transmembrane domain TM3 (L210P, L213P, I217T). Of the remaining four, two mapped to the second periplasmic domain (T224I, L249R), one to the TM4 domain (A267T), and one near the N-terminal end of the protein (F9S). The double and triple mutants had one mutation at the N-terminal end (F9S, A7V). Since the F9S mutant is avirulent (mutant 2, Table 1
), the phenotype of these two mutants may be due to the defect near the N-terminus of the protein. In protein transfer assays, none of the mutants supported VirE2 transfer in complementation in planta (Otten et al., 1984
).
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The pTiA6 VirB6 TM3 has an unusual primary amino acid sequence in that it is rich in leucine (Fig. 2). Within an eight-residue segment (aa 206213), seven residues are leucine and six of these are in tandem. This region is preceded by an essential tryptophan at position 197 that is invariant in the VirB6 family of proteins (Judd et al., 2005a
). A comparison of sequences among the VirB6 homologues showed a high degree of sequence conservation in this region. The TM3 homologous domain is rich in the three bulky, nonpolar amino acids, leucine, isoleucine and valine, which occupy seven positions within a thirteen-residue segment. The conservation of L/I/V sequences and the mapping of several mutations to the TM3 domain suggest that the primary sequence of TM3 plays an important role in VirB6 function.
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In mutational studies, impairment of protein function can result from an undesired effect of the mutation on protein stability. Unfortunately, quantitative analysis on the VirB6 mutants could not be performed using Western blot assays because we were unable to detect the wild-type protein and the mutant proteins in such an assay (Judd et al., 2005a and data not shown). The anti-VirB6 antibodies used in these studies, however, did recognize a His-tagged VirB6, four other VirB6 mutants, and VirB6-PhoA fusions in Western blot assays, and wild-type VirB6 and its mutants in IFM (Judd et al., 2005a
). The failure to detect VirB6 and most of its mutants in Western blot assays is probably due to the formation of SDS-resistant oligomer that fails to enter the gel. IFM was used to examine whether the point mutants reported here have a major effect on the accumulation of the mutant proteins. All mutants did express VirB6 at a level comparable to that of the wild-type strain, suggesting that the null phenotype is not due to instability of a mutant protein, but due to an effect on protein function (see Fig. 5
).
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VirB6 colocalizes with a subset of VirB proteins
We recently reported that VirB6 colocalized with two components essential for T4S, VirD4 and VirB8 (Judd et al., 2005a, b
). Those studies and others suggested that VirB6 is a core component of the T4S apparatus (Jakubowski et al., 2003
). To gain additional support for this hypothesis, we studied colocalization of VirB6 and other VirB proteins. It is expected that proteins that are components of a protein complex will colocalize with one another. In another study we observed that VirB8 colocalized with a subset of the VirB proteins and postulated that these proteins are the principal components that function in the assembly of the T4S apparatus (Judd et al., 2005b
). VirB8 did not colocalize with three of the VirB proteins, VirB4, VirB5 and VirB11. To determine whether VirB6 exhibits a similar behaviour, colocalization analysis of a His-tagged VirB6 and other VirB proteins was performed. Since colocalization analysis requires the use of two primary antibodies from different sources and all of our antibodies were raised in rabbits, the wild-type strain could not be used for this analysis. The His-tagged VirB6 was used instead because it is fully functional in DNA transfer and complemented the virB6 deletion mutant (Judd et al., 2005a
), and monoclonal antibodies against the tag are commercially available. His-tagged VirB6 probed with monoclonal anti-His antibodies formed red foci (Fig. 7
). The other VirB proteins probed with the cognate rabbit anti-VirB antibodies formed green foci. In a superimposed image, foci of proteins that colocalize had a yellow colour resulting from the combination of red and green. VirB6, like VirB8, colocalized with VirB3 and VirB9, and did not colocalize with VirB4, VirB5 and VirB11 (Fig. 7
, Table 2
).
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DISCUSSION |
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Three mutants mapped to an 8 aa segment in the TM3 domain while only one mutant was found within the remaining TM domains. The richness of three bulky hydrophobic residues, leucine, isoleucine and valine, in this domain and the conservation of this feature in the VirB6 homologues suggest that both hydrophobicity and the primary sequence of TM3 are important for VirB6 function. This domain is preceded by another conserved region that includes an invariant tryptophan at position 197, and a conserved threonine at 189, a basic residue at 190 and an acidic residue at 195 (Fig. 2). High sequence conservation in this region suggests functional importance. Tryptophan 197 is required for polar localization of VirB6 (Judd et al., 2005a
). Insertion mutations around this region, the class II mutants of Jakubowski et al. (2004)
, failed in a subsequent step in T-DNA delivery, the transfer of DNA from VirB6 to VirB8.
Deletion of the pTiA6 virB6 gene had an effect on VirB7 and its complexes. Contrary to the observations with the pTiC58 VirB6 (Hapfelmeier et al., 2000), we did not observe a destabilizing effect of pTiA6 VirB6 on the T-pilus-associated proteins VirB3 and VirB5. In agreement with earlier reports, we observed that a deletion in virB6 abolished the formation of the VirB7 homodimer, but not that of the VirB7-VirB9 heterodimer (Hapfelmeier et al., 2000
; Jakubowski et al., 2003
). The later is in agreement with our previous report that demonstrated that VirB7 and VirB9 are sufficient to form the VirB7-VirB9 dimer (Anderson et al., 1996
). The absence of the VirB7 homodimer, and not that of the VirB7-VirB9 heterodimer, in the virB6 deletion mutant confirms an earlier conclusion that the two dimers form through independent pathways.
The effect of VirB6 on VirB7-VirB7 dimer formation may also be an indirect one. The VirB7-VirB7 dimer may be a dead-end complex to sequester excess VirB7 that could not participate in the formation of the preferred VirB7-VirB9 complex. Since VirB6 stabilizes VirB7, the absence of VirB6 significantly reduces the amount of VirB7 in the cell. All of the VirB7 interacts with the preferred partner VirB9 to form the VirB7-VirB9 complex, leaving no excess protein to form the VirB7 homodimer. The undetectable level of the VirB7 homodimer in the virB6 deletion mutant indicates that that VirB7 has a much higher affinity for VirB9 than for itself. In the presence of VirB6, the bacterium accumulates more VirB7. After all available VirB9 is used up in the formation of the VirB7-VirB9 complex, the excess VirB7 forms homodimer to sequester its reactive cysteine. This property was noted previously in the VirB9 protein (Anderson et al., 1996). In the absence of VirB7, VirB9 forms a disulfide-linked homodimer as well as other non-specific disulfide-linked complexes with cellular proteins. When VirB7 was co-expressed with VirB9, formation of only the preferred VirB7VirB9 complex was observed.
Subcellular localization studies presented here demonstrate that VirB6 colocalized with at least two core components of the T4S apparatus. It colocalized with VirB8 and VirB9, and not with VirB4, VirB5 and VirB11 (Fig. 6 and Judd et al., 2005b
). These results, in conjunction with colocalization studies with VirB8 presented elsewhere (Judd et al., 2005b
), support the conclusion that VirB6 is a core component of the T4S apparatus. Localization of VirB6 to a cell pole required the presence of VirB7VirB11, suggesting a requirement for an interaction of VirB6 with a polar VirB7VirB10 complex (Judd et al., 2005a
, b
). While VirB11 is required for this process, the VirB6 complex and VirB11 occupy different regions of the cell pole, as neither VirB6 nor VirB8 colocalized with VirB11. This situation is analogous to the polar location of replicons in A. tumefaciens in which four replicons (those of linear chromosome, circular chromosome, pTiC58 and pAtC58) localized to the cell pole but neither plasmid replicon colocalized with another replicon (Kahng & Shapiro, 2003
). Compatible plasmids in bacteria are also found to occupy non-overlapping sites within the same subregion of a cell, e.g. mid-cell, quarter-cell and cell pole (Ho et al., 2002
). Occupation of non-overlapping sites by the VirB6VirB10 complex and VirB4/VirB5/VirB11 suggests that the latter group of VirB proteins is positioned at the cell pole for the proper functioning of the T4S apparatus.
According to the current model of DNA transfer, the A. tumefaciens T-DNA is delivered sequentially from VirD4 to VirB11, VirB6, VirB8 and VirB2/VirB9 (Cascales & Christie, 2004). Our colocalization results are in apparent disagreement with this model, which predicts VirB11 to colocalize with the other VirB proteins. A likely explanation for the difference is that subcellular localization of VirB11 (and VirB4) is dynamic in nature. Dynamic localization of bacterial proteins and origins of replications is well documented (Gitai et al., 2004
). We postulate that under specific conditions, e.g. in the presence of a substrate, recipient or both, VirB4 and VirB11 (transiently) associate with the VirB6VirB10 complex to form an export-competent complex. This association may represent the last step in the assembly of the T4S apparatus or the conversion of an export-incompetent fully assembled apparatus into an export-competent apparatus.
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ACKNOWLEDGEMENTS |
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Received 12 July 2005;
revised 15 September 2005;
accepted 19 September 2005.
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