Role of Agrobacterium VirB11 ATPase in T-pilus assembly and substrate selection

Abstract

The VirB11 ATPase is a subunit of the Agrobacterium tumefaciens transfer DNA (T-DNA) transfer system, a type IV secretion pathway required for delivery of T-DNA and effector proteins to plant cells during infection. In this study, we examined the effects of virB11 mutations on VirB protein accumulation, T-pilus production, and substrate translocation. Strains synthesizing VirB11 derivatives with mutations in the nucleoside triphosphate binding site (Walker A motif) accumulated wild-type levels of VirB proteins but failed to produce the T-pilus or export substrates at detectable levels, establishing the importance of nucleoside triphosphate binding or hydrolysis for T-pilus biogenesis. Similar findings were obtained for VirB4, a second ATPase of this transfer system. Analyses of strains expressing virB11 dominant alleles in general showed that T-pilus production is correlated with substrate translocation. Notably, strains expressing dominant alleles previously designated class II (dominant and nonfunctional) neither transferred T-DNA nor elaborated detectable levels of the T-pilus. By contrast, strains expressing most dominant alleles designated class III (dominant and functional) efficiently translocated T-DNA and synthesized abundant levels of T pilus. We did, however, identify four types of virB11 mutations or strain genotypes that selectively disrupted substrate translocation or T-pilus production: (i) virB11/virB11* merodiploid strains expressing all class II and III dominant alleles were strongly suppressed for T-DNA translocation but efficiently mobilized an IncQ plasmid to agrobacterial recipients and also elaborated abundant levels of T pilus; (ii) strains synthesizing two class III mutant proteins, VirB11, V258G and VirB11.I265T, efficiently transferred both DNA substrates but produced low and undetectable levels of T pilus, respectively; (iii) a strain synthesizing the class II mutant protein VirB11.I103T/M301L efficiently exported VirE2 but produced undetectable levels of T pilus; (iv) strains synthesizing three VirB11 derivatives with a four-residue (HMVD) insertion (L75.i4, C168.i4, and L302.i4) neither transferred T-DNA nor produced detectable levels of T pilus but efficiently transferred VirE2 to plants and the IncQ plasmid to agrobacterial recipient cells. Together, our findings support a model in which the VirB11 ATPase contributes at two levels to type IV secretion, T-pilus morphogenesis, and substrate selection. Furthermore, the contributions of VirB11 to machine assembly and substrate transfer can be uncoupled by mutagenesis.

FIG. 1

Effects of VirB11 Walker A mutations on accumulation of T-pilus proteins and other VirB proteins in exocellular (A) and cellular (B) fractions obtained as described in the text. Strains: ΔB11(B11), strain PC1011(pSRB1) expressing virB11 from an IncP replicon; ΔB11, strain PC1011; ΔB11(ΔGKT), PC1011(pPCB7112) expressing virB11ΔGKT174-176; A348(ΔGKT), A348(pPCB7112) coexpressing virB11 and virB11ΔGKT174-176; ΔB11(K/Q), PC1011(pPCB7113) expressing virB11K175Q; A348(K/Q), A348(pPCB7113) coexpressing virB11 and virB11K175Q. M, molecular mass markers, with sizes in kilodaltons indicated on the right. The blots were developed with antisera to the VirB proteins listed on the left.

FIG. 2

Effects of VirB4 Walker A mutations on accumulation of T-pilus-associated proteins and other VirB proteins in exocellular (A) and cellular (B) fractions obtained as described in the text. Strains: ΔB4(B4), strain PC1004(pZDH10) expressing virB4 from an IncP replicon; ΔB4, strain PC1004; ΔB4(ΔGKT), PC1004(pBB17) expressing virB4ΔGKT174–176; A348(ΔGKT), A348(pBB17) coexpressing virB4 and virB4ΔGKT174–176; ΔB4(K/Q), PC1004(pBB15) expressing virB4K175Q; A348(K/Q), A348(pBB15) coexpressing virB4 and virB4K175Q. M, molecular mass markers, with sizes in kilodaltons indicated on the right. The blots were developed with antisera to the VirB proteins listed on the left.

FIG. 3

Effects of virB11 dominant mutations on T-pilus production. Exocellular fractions from PC1011 (designated ΔB11 or Δ) and A348 (designated A348 or A) strains expressing wild-type virB11 and alleles 1 to 12 were analyzed for accumulation of T-pilus-associated proteins, VirB2, VirB5, and VirB7, as listed on the left of each immunoblot. Strain Δ(B11) is pC1011(pSRB1) expressing PvirB::virB11, and strain Δ(PlacZ::B11) is PC1011(pPCB117) expressing PlacZ::virB11; alleles 1 to 12 expressed from PvirB are carried on plasmids pXZB101 to pXZB112. Plasmid pXZB104 was shown previously to encode a wild-type virB11 gene (69); however, we identified a mutation in the PvirB promoter of pXZ104 that results in a reduction in VirB11 production levels. M, molecular mass markers, with sizes in kilodaltons indicated on the right.

FIG. 4

Sucrose density gradient distribution profiles of T pili from various virB11 mutant strains. Exocellular proteins from the strains listed on the left were centrifuged through identically prepared sucrose density gradients, and the fractions were analyzed for the presence of VirB2 pilin. Strains: ΔB11(B11), PC1011(pXZB100); ΔB11(7), PC1011(pXZB107); A348(7), A348(pXZB107); A348(3), A348(pXZB103); ΔB2(B2C64S), PC1002(pVSB10) that synthesizes mutant pilin (53).

FIG. 5

Effects of virB11.i4 mutations on T-pilus production. Exocellular fractions from PC1011 (designated ΔB11) expressing wild-type virB11 (designated B11; from plasmid pJCB903) and alleles for the i4 mutant proteins indicated were analyzed for accumulation of T-pilus proteins, VirB2, VirB5, and VirB7. Corresponding cellular levels of native and mutant forms of VirB11 are shown at the bottom. M, molecular mass markers with sizes in kilodaltons indicated on the right.

FIG. 6

Effects of IncQ plasmid on accumulation of VirB proteins and T-pilus proteins. (A) VirB and VirE2 protein levels in total-cell extracts of A348 and A348(pML122), with VirB proteins listed on the left of each immunoblot. M, molecular mass markers, with sizes in kilodaltons indicated on the right. (B) VirB2 pilin levels in exocellular fractions from A348 and merodiploid strains expressing the dominant alleles 1 through 12 and pML122ΔKm (designated pML). Also shown are the pilin levels in PC1011 (designated Δ11) expressing virB11 (B11; from plasmid pXZB100) and class III alleles.

FIG. 7

Positions of VirB11 mutations grouped according to their effects on pilus production and substrate transfer. (A) Substitution mutations with allele numbers in parantheses as defined by Zhou et al. (69). Phenotypic descriptions are provided for mutations of special interest. Note that all virB11/virB11∗ merodiploid strains expressing the dominant alleles exhibit a T-pilus⁺ IncQ plasmid Tra⁺ T-DNA Tra-deficient phenotype. (B) VirB11 (343 residues) with conserved Walker A and B domains and the Asp and His boxes denoted. The shading identifies the two regions of VirB11 in which dominant mutations were predominantly located. Below the VirB11 representation is the HP0525 secondary structure with β-sheets and α-helices as presented by Yeo et al. (67). The junction between the N- and C-terminal domains, shown by the HP0525 crystal structure to assemble as independent hexameric rings, is indicated. (C) i4 mutations with insertion sites indicated.