Guo et al. 10.1073/pnas.0701738104. |
Fig. 7. The time course of GFP-VirD2 polar localization. The plasmid pCB1gd2 encoding the GFP-VirD2 fusion under the lac promoter control was introduced into a virD2- mutant WR1715. The bacterial cells were grown in the induction medium containing 200 mM acetosyringone (AS). The cells were photographed under a fluorescent microscope at different time intervals of AS induction (0, 3, 6, and 16 h). Approximately 500 cells were examined to calculate the percentage of the cells showing polar fluorescence at one pole (unipolar) or both poles (bipolar).
Table 1. VBP homologs in bacteria
Species | Proteins (accession no.) | Identities, % | Positives, % |
Agrobacterium tumefaciens str. C58 | Hypothetical protein (VBP1) (AAL45807) | 100 | 100 |
A. tumefaciens str. C58 | Conserved hypothetical protein (VBP2) (AAL45654) | 72 | 80 |
Rhizobium etli CFN42 | Putative nucleotidyltransferase protein (YP_468267) | 65 | 79 |
Sinorhizobium meliloti (strain 1021) megaplasmid pSymB | Conserved hypothetical protein (CAC48978) | 58 | 76 |
S. meliloti (strain 1021) megaplasmid pSymA | Hypothetical protein SMa0967 (AAK65181) | 58 | 76 |
A. tumefaciens str. C58 | Nucleotidyltransferase (VBP3) (AAL45650) | 56 | 71 |
Sphingopyxis alaskensis RB2256 | Hypothetical protein Sala_2595 (YP_617634) | 48 | 68 |
S. alaskensis RB2256 | Hypothetical protein Sala_2515 (YP_617555) | 50 | 63 |
Agrobacterium radiobacter K84 plasmid pAgK84 | Probable nucleotidyltransferase (AAS02134) | 43 | 60 |
Sphingomonas sp. KA1 | Hypothetical protein (BAC56756) | 43 | 62 |
Rickettsia bellii RML369-C | Nucleotidyltransferase (ABE04144) | 36 | 55 |
Rickettsia felis URRWXCal2 | NT (nucleotidyltransferas) domain and HEPN (higher eukarytoes and prokaryotes nucleotide-binding) domain (AAY61791) | 32 | 52 |
R. felis URRWXCal2 | NT (nucleotidyltransferase) domain and HEPN (higher eukarytoes and prokaryotes nucleotide-binding) domain (AAY62013) | 32 | 53 |
Bacteroides thetaiotaomicron VPI-5482 | Hypothetical protein BT4509 (NP_813420) | 32 | 51 |
VBP1 amino acid sequence (accession no. AAL45807) was used to conduct BLAST analysis in the databases. The bacterial protein sequences with more significant homology to VBP1 were compiled in the table.
Table 2. The effect of vbp mutation on the transfer of a RSF1010 derivative pML122 from A. tumefaciens to E. coli
Donor strains (genotype) | Without any helper strain | With the helper strain MT616 | ||||
No. of input donors (× 109) | No. of transconjugants | Frequency ± SD (× 10-8) | No. of input donors (× 106) | No. of transconjugants | Frequency ± SD (× 10-4) | |
A348 (vbp+) | 1.07 | 97 | 9.0 ± 4.0 | 1.2 | 886 | 8.2 ± 6.1 |
AMV2 (vbp2-) | 1.03 | 77 | 7.4 ± 5.0 | 0.9 | 676 | 6.8 ± 3.3 |
AMV23 (vbp2-; vbp3-) | 0.97 | 89 | 9.2 ± 1.2 | 0.9 | 787 | 8.7 ± 4.9 |
GMI9017 (vbp1-) | 0.97 | 55 | 5.7 ± 2.7 | 1.0 | 694 | 6.7 ± 3.5 |
GMV12 (vbp1-; vbp2-) | 0.93 | 52 | 5.5 ± 4.5 | 0.9 | 645 | 6.9 ± 2.3 |
GMV123 (vbp1-; vbp2-; vbp3-) | 0.96 | 0 | <10-9 | 1.1 | 503 | 5.1 ± 4.3 |
At12044 (virB4-) | 1.03 | 81 | 7.8 ± 4.1 | 1.0 | 795 | 7.6 ± 5.4 |
At10011 (virB11-) | 1.03 | 87 | 8.4 ± 8.5 | 1.2 | 499 | 4.5 ± 3.5 |
GMV123(pCBV1) (vbp1+) | 0.93 | 63 | 6.7 ± 4.0 | 1.0 | 814 | 6.9 ± 5.8 |
GMV123(pCBV2) (vbp2+) | 1.01 | 79 | 7.9 ± 4.4 | 0.9 | 389 | 4.4 ± 5.7 |
GMV123(pCBV3) (vbp3+) | 1.13 | 65 | 5.8 ± 2.6 | 0.9 | 626 | 6.4 ± 4.6 |
The plasmid pML122 was introduced into the A. tumefaciens strains listed. The agrobacterial cells harboring pML122 were mated with E. coli cells in the presence or absence of the helper strain MT616, which contains the conjugative plasmid pRK600 carrying the RK2 transfer genes. The numbers of input donors and transconjugants and the conjugation frequency are the means of triplicates from one of the three independent experiments, which generated similar results.
Table 3. The effect of vbp mutation on the transfer of an IncP plasmid derivative pSW172 from A. tumefaciens to E. coli
Donor strains (genotype) | Without any helper strain | With the helper strain MT616 | ||||
No. of input donors (× 109) | No. of transconjugants | Frequency ± SD (× 10-9) | No. of input donors (× 107) | No. of transconjugants | Frequency ± SD (× 10-5) | |
A348 (vbp+) | 1.13 | 0 | <10-9 | 1.09 | 860 | 8.8 ± 5.3 |
AMV2 (vbp2-) | 0.98 | 0 | <10-9 | 0.97 | 783 | 7.9 ± 3.7 |
AMV23 (vbp2-; vbp3-) | 1.21 | 0 | <10-9 | 1.37 | 891 | 7.3 ± 4.1 |
GMI9017 (vbp1-) | 1.19 | 0 | <10-9 | 1.05 | 696 | 7.1 ± 2.9 |
GMV12 (vbp1-; vbp2-) | 0.99 | 0 | <10-9 | 1.21 | 899 | 6.8 ± 4.3 |
GMV123 (vbp1-; vbp2-; vbp3-) | 1.18 | 0 | <10-9 | 1.17 | 836 | 7.4 ± 5.1 |
The plasmid pSW172 was introduced into the A. tumefaciens strains listed. The agrobacterial cells harboring pSW172 were mated with E. coli cells in the presence or absence of the helper strain MT616, which contains the conjugative plasmid pRK600 carrying the RK2 transfer genes. The numbers of input donors and transconjugants and the conjugation frequency are the means of triplicates from one of the three independent experiments, which generated similar results.
Table 4. Bacterial strains and plasmids used in this study
Strain and plasmid | Relevant characteristic(s) | Source or reference | ||
Strains | ||||
Escherichia coli | ||||
DH5a | EndA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 (argF-lacZYA) U169 f80dlacZ | Bethesda Research Laboratories | ||
BL21(DE3) | F- omp T hsdSB(rB-mB-) gal dcm (DE3) | Invitrogen | ||
MT607 | Pro-82 thi-1 hsdR17 supE44 end44 endA1 recA56 | 1 | ||
MT616 | MT607(pKR600), mobilizer | 1 | ||
Agrobacterium tumefaciens | ||||
C58 | Wild type, nopaline-type pTiC58 plasmid | Laboratory collection | ||
A348 | Wild type, A136(pTiA6NC) (octopine-type) | Laboratory collection | ||
LBA4404 | A vir helper; harbors disarmed Ti plasmid pAL4404; a T-DNA deletion derivative of pTiAch5 (octopine type); RfR, SmR | 2 | ||
WR1715 | Harbors a Ti plasmid with 70% of virD2 deleted (aa 94-388) | 3 | ||
At12044 | Derivative of A348; virB4:Tn5virB | 4 | ||
At10011 | Derivative of A348 in which virB11 was deleted, KmR | 4 | ||
At12506 | A348; virD4: tn3hoho1 | 5 | ||
AMV2 | Derivative of A348 in which vbp2 was deleted | 6 | ||
AMV23 | Derivative of A348 in which vbp2 and vbp3 were mutated, CbR | 6 | ||
GMI9017 | C58 cured of pAtC58; lacking vbp1; SmR, SpR, RfR | 7 | ||
GMV12 | Derivative of GMI9017 in which vbp2 was deleted | 6 | ||
GMV123 | Derivative of GMI9017 in which vbp2 and vbp3 were mutated; CbR | 6 | ||
GMV123d2 | Derivative of GMI9017 in which vbp2, vbp3 and virD2 were mutated; CbR | This study | ||
Plasmids | ||||
pIG121Hm | Binary vector plasmid, carrying the b-glucuronidase gene (gusA) between the T-borders; KmR | 8 | ||
pRV1 | pRSET-A carrying at XhoI a 947-bp fragment containing the full length vbp1 ORF fused in-frame with (His)6; AmpR | 6 | ||
pCB301 | A mini binary vector; KmR | 9 | ||
pCBV1 | pCB301 carrying at HindIII a 2,052-bp fragment containing the 939 bp full length vbp1 ORF as well as its 597-bp upstream and 516-bp downstream sequences; KmR | 6 | ||
pCBV2 | pCB301 carrying at HindIII a 2029 bp fragment containing the 933-bp full-length vbp2 ORF as well as its 602-bp upstream and 494-bp downstream sequences; KmR | 6 | ||
pCBV3 | pCB301 carrying at HindIII a 2,025-bp fragment containing the 924-bp full-length vbp3 ORF as well as its 597-bp upstream and 504-bp downstream sequence; KmR | 6 | ||
pCBS52A | A derivative of pCBV1, in which serine 52 (S52) was changed to alanine; KmR | This study | ||
pCBY139A | A derivative of pCBV1, in which tyrosine 139 (Y139) was changed to alanine; KmR | This study | ||
pCBH218A | A derivative of pCBV1, in which histidine 218 (H218) was changed to alanine; KmR | This study | ||
pCBP239A | A derivative of pCBV1, in which proline 239 (P239) was changed to alanine; KmR | This study | ||
pCBR260A | A derivative of pCBV1, in which arginine 260 (R260) was changed to alanine; KmR | This study | ||
pSW172 | Broad-host-range IncP plasmid ; TcR | 10 | ||
pML122 | A derivative of IncQ plasmid RSF1010; GmR | 11 | ||
pUC19 | Cloning vector, ColE1 oriV bla; AmpR | US Biochemical | ||
pUCA19 | pUC19 carrying an agrobacterial replicon | Laboratory collection | ||
pUCAg | pUCA19 carrying a full-length gfp ORF fused onto the lac promoter; CbR | This study | ||
pUCAgv | pUCA19 carrying a full-length gfp-vbp1 ORF fused onto the lac promoter; CbR | This study | ||
pUCAKgv | A derivative of pUCAgv in which the carbenicillin resistance gene was replaced by a kanamycin resistance gene; KmR | This study | ||
pCBlgd2 | A derivative of pCB301 in which the T-border fragment was replaced by the lacZ fragment. A full-length gfp-virD2 ORF fused onto the lac promoter; KmR | This study | ||
pCBgd2v | pCBlgd2 carrying the vbp1 gene; KmR | This study | ||
Amp, ampicillin; Cb, carbenicillin; Gm, gentamicin; Km, kanamycin; Rf, rifampicin; Sm, streptomycin; Sp, spectinomycin.
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Table 5. Primers used in this study
Primers | Sequence | Locus | Purpose |
Ptdna1 | 5'-gcggcggcgataactctcag-3' | Corresponding to the "ipt" gene of T-region in the Ti plasmid of C58 | To amplify a fragment of T-DNA |
Ptdna2 | 5'-ggaattgactatgagcagcttg-3' | Corresponding to the "ipt" gene of T-region in the Ti plasmid of C58 | To amplify a fragment of T-DNA |
Ptibac1 | 5'-gtggctgtgttggttatgagc-3' | Corresponding to the "virB1" gene of Ti plasmid of C58 | To amplify a backbone fragment of Ti plasmid |
Ptibac2 | 5'-gtcttcggatttgcggttgtc-3' | Corresponding to the "virB1" gene of Ti plasmid of C58 | To amplify a backbone fragment of Ti plasmid |
Oligo G-402 | 5'-catacggcgtgacatcg-3' | Corresponding to the T-region of pIG121Hm. | To amplify the T-DNA of pIG121Hm |
Oligo-intron | 5'-acatggatccctacagg-3' | Corresponding to the T-region of pIG121Hm. | To amplify the T-DNA of pIG121Hm |
Prsf1 | 5'-ctgcgctaggctacacaccg-3' | To amplify a fragment of plasmid pML122 | |
Prsf2 | 5'-cgacgaactccggcatgtgc-3' | An RSF1010 fragment in plasmid pML122 | To amplify a fragment of plasmid pML122 |
PvbpCS1 | 5'-gggctgcaggaattcgatatc-3' | Near Hind III site of pCB301 | To amplify a fragment of plasmid pCBV1 |
PvbpCS2 | 5'-gaggtcgacggtatcgataag-3' | Near Hind III site of pCB301 | To amplify a fragment of plasmid pCBV1 |
SI Text
Binding of VBP1 to T-Complex.
A. tumefaciens cells were grown in 5 liters of IB medium containing 200 mM AS; the cells were harvested, washed once with water, and resuspended in 15 ml of lysis buffer A (2.5 mM MgCl2,50 mM NaCl, 2 mM PMSF, 20 mg/ml leupeptins, 30 mM Tris·HCl, pH, 7.4). The cells were disrupted by French press. The crude extracts were centrifuged at 12,000 ´g for 15 min at 4°C, and the supernatants were used immediately to perform the following immunoprecipitation or pull-down assays.For immunoprecipitation assays, the supernatant was diluted 2-fold with lysis buffer B (2.5 mM MgCl2, 50 mM NaCl, 2 mM PMSF, 20 mg/ml leupeptines, 10 mg/ml antipain, 50 mM Tris·HCl, pH 7.4). The extract was then precleared with 50% slurry of protein A-Sepharose (in lysis buffer B) (at a ratio of 1:6) and preimmune serum (at a dilution of 1:150). Then VBP1 antibody was added at a dilution of 1:200 and 50% (wt/vol) slurry of protein A-Sepharose was added at a ratio of 1:8. The mixture was incubated at 4°C for 4 h with rocking, centrifuged, and washed five times with lysis buffer B. The immunoprecipitates were eluted by heating at 96°C for 20 min in 10 mM Tris·HCl, pH 6.8. The T-strand in the samples was detected by PCR amplification using primers Ptdna1 and Ptdna2. Primers Ptibac1 and Ptibac2 were used to amplify a fragment of Ti plasmid backbone. Proteins in the samples were separated by SDS/PAGE and detected by Western blot.
For the pull-down assays, E. coli strain BL21(DE32)(pRV1) was used to produce His-VBP1 as described (1). Refolded His-VBP1 bound to TALON metal affinity resin was incubated with freshly prepared A. tumefaciens crude extracts. After incubation at 4°C for 1 h, the resin was washed four times with lysis buffer A. The bound T-complex was eluted with lysis buffer A containing 150 mM imidazol. VirD2 and VirE2 in the elutes were detected by Western blot. To detect the T-strand, the eluate was treated with proteinase and then extracted with phenol/chloroform immediately; the T-strand was amplified by PCR.
Binding of VBP to T4SS Components.
A. tumefaciens cells were grown in 1 liter of IB, harvested, and resuspended in 4 ml of lysis buffer B; the immunoprecipitation assays were conducted as described for the binding of VBP1 to T-complex, except with the following modifications. After French press, Triton X-100 was added to a final concentration of 0.5%, and the mixture was incubated for 1 h at 4°C with rocking. The mixture was centrifuged at 12,000 ´ g for 20 min at 4°C. The immunoprecipitates bound to Protein A-Sepharose were washed twice with lysis buffer B supplemented with 0.1% Triton X-100 and three times with lysis buffer B.Conjugation assay.
The conjugation assay was conducted under noninducing conditions as described by Zyl et al. (2). Donor A. tumefaciens cells were cultured in MG/L overnight at 28°C with appropriate antibiotics. The recipient E. coli MT607 and the helper MT616 cells were cultured in LB overnight at 37°C. The cells were washed with water twice and resuspended at 1 ´ 109 cells per milliliter. Equal volumes of donor and recipient (and helper) cell suspension were mixed. The cell mixtures were diluted and plated on AB agar plates with appropriate antibiotics to count the number of input donor cells. For the conjugation assay, the mixtures were spotted on LB agar plates and incubated overnight at 28°C. The agar plug was excised, suspended in water, and vigorously shaken to dislodge mating cells. The cells were collected by centrifugation and resuspended in water. They were then plated onto LB agar plates with appropriate antibiotics and grown at 37°C to select for transconjugant E. coli cells.1. Guo M, Hou Q, Hew CL, Pan SQ (2007) Mol Plant-Microbe Interact 20:1201-1212.
2. Grynberg M, Erlandsen H, Godzik A (2003) Trends Bio Sci 28:224-226.