Berger B R, Christie P J
Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston 77030.
J Bacteriol. 1994 Jun;176(12):3646-60. doi: 10.1128/jb.176.12.3646-3660.1994.
The Agrobacterium tumefaciens virB gene products are proposed to assemble into a transport system capable of exporting complexes of DNA and protein across the bacterial envelope en route to plant cells. Nonpolar null mutations were constructed in each of the 11 virB genes of the A. tumefaciens pTiA6NC plasmid. In tumorigenicity assays, delta virB1 mutants exhibited severely attenuated virulence and delta virB2 through delta virB11 mutants exhibited avirulence. NdeI restriction sites introduced at the predicted translational start sites of the virB genes were used to subclone each of the virB genes downstream of the lacZ or virB promoter on broad-host-range plasmids. virB gene expression plasmids were used to define promoter and general sequence requirements for genetic complementation of the deletion mutations. Whereas virB1 and virB2 complemented delta virB1 and delta virB2, respectively, only when expressed in trans from the virB promoter, virB3 through virB11 complemented the corresponding deletion mutations when expressed in trans from either the lacZ or virB promoter. Several virB genes required additional upstream or downstream sequences for complementation: (i) virB2 complemented the delta virB2 mutation only when the complementing plasmid coexpressed virB1 and virB2, (ii) virB6 and virB9 complemented the delta virB6 and delta virB9 mutations only when the complementing plasmids carried at most 55 and 230 bp of sequences residing 5' of these genes, respectively, and (iii) virB7 and virB8 complemented the delta virB7 and delta virB8 mutations only when the complementing plasmid coexpressed virB7 and virB8. These studies established that virB1 is an accessory virulence determinant and virB2 through virB11 are absolutely essential for the A. tumefaciens infection process.
根癌土壤杆菌的virB基因产物被认为可组装成一个转运系统，该系统能够在DNA和蛋白质复合物运往植物细胞的途中，将其跨细菌包膜输出。在根癌土壤杆菌pTiA6NC质粒的11个virB基因中分别构建了非极性无效突变。在致瘤性测定中，ΔvirB1突变体表现出毒力严重减弱，而ΔvirB2至ΔvirB11突变体表现出无毒性。在virB基因预测的翻译起始位点引入的NdeI限制性酶切位点，用于将每个virB基因亚克隆到广宿主范围质粒上lacZ或virB启动子下游。virB基因表达质粒用于确定缺失突变体遗传互补的启动子和一般序列要求。虽然virB1和virB2分别仅在从virB启动子反式表达时才能互补ΔvirB1和ΔvirB2，但virB3至virB11在从lacZ或virB启动子反式表达时可互补相应的缺失突变。几个virB基因需要额外的上游或下游序列来实现互补：（i）只有当互补质粒共表达virB1和virB2时，virB2才能互补ΔvirB2突变；（ii）只有当互补质粒分别携带这些基因5'端最多55和230 bp的序列时，virB6和virB9才能互补ΔvirB6和ΔvirB9突变；（iii）只有当互补质粒共表达virB7和virB8时，virB7和virB8才能互补ΔvirB7和ΔvirB8突变。这些研究表明virB1是一个辅助毒力决定因素，而virB2至virB11对于根癌土壤杆菌的感染过程是绝对必需的。
