Peng W T, Banta L M, Charles T C, Nester E W
Department of Microbiology, University of Washington, Seattle, Washington 98195-7242, USA.
J Bacteriol. 2001 Jan;183(1):36-45. doi: 10.1128/JB.183.1.36-45.2001.
The virulence of Agrobacterium tumefaciens depends on both chromosome- and Ti plasmid-encoded gene products. In this study, we characterize a chromosomal locus, chvH, previously identified by TnphoA mutagenesis and shown to be required for tumor formation. Through DNA sequencing and comparison of the sequence with identified sequences in the database, we show that this locus encodes a protein similar in sequence to elongation factor P, a protein thought to be involved in peptide bond synthesis in Escherichia coli. The analysis of vir-lacZ and vir-phoA translational fusions as well as Western immunoblotting revealed that the expression of Vir proteins such as VirE2 was significantly reduced in the chvH mutant compared with the wild-type strain. The E. coli efp gene complemented detergent sensitivity, virulence, and expression of VirE2 in the chvH mutant, suggesting that chvH and efp are functionally homologous. As expected, ChvH exerts its activity at the posttranscriptional level. Southern analysis suggests that the gene encoding this elongation factor is present as a single copy in A. tumefaciens. We constructed a chvH deletion mutant in which a 445-bp fragment within its coding sequence was deleted and replaced with an omega fragment. On complex medium, this mutant grew more slowly than the wild-type strain, indicating that elongation factor P is important but not essential for the growth of Agrobacterium.
根癌土壤杆菌的毒力取决于染色体和Ti质粒编码的基因产物。在本研究中，我们对一个染色体位点chvH进行了表征，该位点先前通过TnphoA诱变鉴定，并且被证明是肿瘤形成所必需的。通过DNA测序以及将该序列与数据库中已鉴定的序列进行比较，我们发现该位点编码一种与延伸因子P序列相似的蛋白质，延伸因子P是一种被认为参与大肠杆菌肽键合成的蛋白质。对vir-lacZ和vir-phoA翻译融合体以及蛋白质免疫印迹分析表明，与野生型菌株相比，chvH突变体中VirE2等Vir蛋白的表达显著降低。大肠杆菌efp基因可弥补chvH突变体对去污剂的敏感性、毒力以及VirE2的表达，这表明chvH和efp在功能上是同源的。正如预期的那样，ChvH在转录后水平发挥其活性作用。Southern分析表明，编码这种延伸因子的基因在根癌土壤杆菌中以单拷贝形式存在。我们构建了一个chvH缺失突变体，其中其编码序列内的一个445 bp片段被删除，并用一个ω片段取代。在复合培养基上，该突变体的生长速度比野生型菌株慢，这表明延伸因子P对根癌土壤杆菌的生长很重要，但并非必不可少。
