Kosońo S, Kataoka M, Seki T, Yoshida T
International Center for Biotechnology, Japan.
Mol Microbiol. 1996 Jan;19(2):397-405. doi: 10.1046/j.1365-2958.1996.379909.x.
The traB gene on the Streptomyces conjugative plasmid pSN22 is required for intermycelial plasmid transfer and the mobilization of chromosomal markers (Cma). The predicted amino acid sequence of TraB contains one Walker type-A and two type-B NTP-binding motifs. Site-directed mutagenesis revealed that the type-A motif and one of the type-B motifs, 109 amino acid residues downstream of the type-A motif, were essential for both plasmid transfer and Cma. The second type-B sequence could be changed without any phenotypic effect. A modified traB gene was constructed, resulting in the production of a functional protein with an amino-terminal c-Myc epitope tag for immunological analysis. This protein was associated with the cytoplasmic membrane, suggesting that TraB is a membrane protein that uses energy from ATP hydrolysis to transport DNA between mycelia. The c-Myc tagging of TraB decreased the efficiency of intramycelial plasmid spread, suggesting that TraB is involved in both inter- and intramycelial transfer processes.
链霉菌接合质粒pSN22上的traB基因对于菌丝间质粒转移和染色体标记物(Cma)的移动是必需的。TraB的预测氨基酸序列包含一个沃克A型和两个B型NTP结合基序。定点诱变显示,A型基序以及位于A型基序下游109个氨基酸残基处的一个B型基序对于质粒转移和Cma都是必不可少的。第二个B型序列可以改变而不产生任何表型效应。构建了一个修饰的traB基因,产生了一种带有氨基末端c-Myc表位标签的功能性蛋白质用于免疫分析。这种蛋白质与细胞质膜相关,表明TraB是一种膜蛋白,它利用ATP水解产生的能量在菌丝间转运DNA。TraB的c-Myc标签降低了菌丝内质粒传播的效率,表明TraB参与了菌丝间和菌丝内的转移过程。