Sagulenko E, Sagulenko V, Chen J, Christie P J
Department of Microbiology and Molecular Genetics, The University of Texas-Houston Medical School, Houston, Texas 77030, USA.
J Bacteriol. 2001 Oct;183(20):5813-25. doi: 10.1128/JB.183.20.5813-5825.2001.
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.
VirB11 ATP酶是根癌土壤杆菌转移DNA（T-DNA）转移系统的一个亚基，该转移系统是一种IV型分泌途径，在感染过程中用于将T-DNA和效应蛋白传递到植物细胞中。在本研究中，我们检测了virB11突变对VirB蛋白积累、T菌毛产生和底物转运的影响。合成在核苷三磷酸结合位点（沃克A基序）发生突变的VirB11衍生物的菌株积累了野生型水平的VirB蛋白，但未能产生T菌毛或在可检测水平输出底物，这确立了核苷三磷酸结合或水解对T菌毛生物合成的重要性。对于该转移系统的另一个ATP酶VirB4，也获得了类似的结果。对表达virB11显性等位基因的菌株的分析总体表明，T菌毛的产生与底物转运相关。值得注意的是，表达先前指定为II类（显性且无功能）的显性等位基因的菌株既不转移T-DNA，也未产生可检测水平的T菌毛。相比之下，表达大多数指定为III类（显性且有功能）的显性等位基因的菌株有效地转运T-DNA并合成大量的T菌毛。然而，我们确实鉴定出了四种类型的virB11突变或菌株基因型，它们选择性地破坏了底物转运或T菌毛产生：（i）表达所有II类和III类显性等位基因的virB11/virB11*部分二倍体菌株在T-DNA转运方面受到强烈抑制，但能有效地将一个IncQ质粒转移到农杆菌受体中，并且也能产生大量的T菌毛；（ii）合成两种III类突变蛋白VirB11、V258G和VirB11.I265T的菌株分别有效地转移了两种DNA底物，但产生的T菌毛水平较低且不可检测；（iii）合成II类突变蛋白VirB11.I103T/MI301L的菌株有效地输出了VirE2，但产生的T菌毛水平不可检测；（iv）合成三种带有四个残基（HMVD）插入（L75.i4、C168.i4和L302.i4）的VirB11衍生物的菌株既不转移T-DNA，也不产生可检测水平的T菌毛，但能有效地将VirE2转移到植物中，并将IncQ质粒转移到农杆菌受体细胞中。总之，我们的研究结果支持了一个模型，其中VirB11 ATP酶在两个层面上对IV型分泌、T菌毛形态发生和底物选择有贡献。此外，通过诱变可以将VirB11对机器组装和底物转移的贡献分开。
