Lohrke S M, Nechaev S, Yang H, Severinov K, Jin S J
Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
J Bacteriol. 1999 Aug;181(15):4533-9. doi: 10.1128/JB.181.15.4533-4539.1999.
The two-component regulatory system, composed of virA and virG, is indispensable for transcription of virulence genes within Agrobacterium tumefaciens. However, virA and virG are insufficient to activate transcription from virulence gene promoters within Escherichia coli cells, indicating a requirement for additional A. tumefaciens genes. In a search for these additional genes, we have identified the rpoA gene, encoding the alpha subunit of RNA polymerase (RNAP), which confers significant expression of a virB promoter (virBp)::lacZ fusion in E. coli in the presence of an active transcriptional regulator virG gene. We conducted in vitro transcription assays using either reconstituted E. coli RNAP or hybrid RNAP in which the alpha subunit was derived from A. tumefaciens. The two forms of RNAP were equally efficient in transcription from a sigma(70)-dependent E. coli galP1 promoter; however, only the hybrid RNAP was able to transcribe virBp in a virG-dependent manner. In addition, we provide evidence that the alpha subunit from A. tumefaciens, but not from E. coli, is able to interact with the VirG protein. These data suggest that transcription of virulence genes requires specific interaction between VirG and the alpha subunit of A. tumefaciens and that the alpha subunit from E. coli is unable to effectively interact with the VirG protein. This work provides the basis for future studies designed to examine vir gene expression as well as the T-DNA transfer process in E. coli.
由virA和virG组成的双组分调节系统对于根癌土壤杆菌毒力基因的转录是必不可少的。然而，virA和virG不足以激活大肠杆菌细胞中毒力基因启动子的转录，这表明需要根癌土壤杆菌的其他基因。在寻找这些其他基因的过程中，我们鉴定出了rpoA基因，它编码RNA聚合酶（RNAP）的α亚基，在存在活性转录调节因子virG基因的情况下，该亚基能使大肠杆菌中virB启动子（virBp）::lacZ融合基因有显著表达。我们使用重组的大肠杆菌RNAP或α亚基来自根癌土壤杆菌的杂交RNAP进行了体外转录分析。这两种形式的RNAP在从依赖σ⁷⁰的大肠杆菌galP1启动子转录时效率相同；然而，只有杂交RNAP能够以依赖virG的方式转录virBp。此外，我们提供的证据表明，来自根癌土壤杆菌而非大肠杆菌的α亚基能够与VirG蛋白相互作用。这些数据表明，毒力基因的转录需要VirG与根癌土壤杆菌的α亚基之间的特异性相互作用，并且大肠杆菌的α亚基无法有效地与VirG蛋白相互作用。这项工作为未来旨在研究大肠杆菌中毒力基因表达以及T-DNA转移过程的研究奠定了基础。
