Zhang L H, Fath M J, Mahanty H K, Tai P C, Kolter R
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Genetics. 1995 Sep;141(1):25-32. doi: 10.1093/genetics/141.1.25.
Colicin V (ColV) is peptide antibiotic secreted by Escherichia coli through a dedicated exporter composed of three proteins, CvaA, CvaB, and TolC. ColV secretion is independent of the E. coli general secretory pathway (Sec) but requires an N-terminal export signal specific for the CvaAB/TolC exporter. ColV secretion was characterized using genetic and biochemical methods. When the ColV N-terminal extension is replaced with the OmpA signal sequence, the Sec system can localize ColV to the periplasm. Periplasmic ColV is lethal to cells lacking the ColV immunity protein, Cvi. Based on this result, a genetic assay was designed to monitor for the presence of periplasmic ColV during normal CvaAB/TolC mediated secretion. Results indicate that low levels of ColV may be present in the periplasm during secretion. Precursor and mature ColV were also characterized from the wild-type system and in various exporter mutant backgrounds using immunoprecipitation. ColV processing is rapid in wild-type cells, and CvaA and CvaB are critical for processing to occur. In contrast, processing occurs normally, albeit more slowly, in a TolC mutant.
大肠杆菌素V（ColV）是由大肠杆菌通过一种由三种蛋白质CvaA、CvaB和TolC组成的专用输出蛋白分泌的肽抗生素。ColV的分泌不依赖于大肠杆菌的一般分泌途径（Sec），但需要一个对CvaAB/TolC输出蛋白特异的N端输出信号。使用遗传和生化方法对ColV的分泌进行了表征。当ColV的N端延伸序列被OmpA信号序列取代时，Sec系统可将ColV定位到周质中。周质中的ColV对缺乏ColV免疫蛋白Cvi的细胞具有致死性。基于这一结果，设计了一种遗传检测方法来监测在正常的CvaAB/TolC介导的分泌过程中周质中ColV的存在情况。结果表明，在分泌过程中周质中可能存在低水平的ColV。还使用免疫沉淀法从野生型系统和各种输出蛋白突变背景中对前体ColV和成熟ColV进行了表征。在野生型细胞中，ColV的加工过程很快，并且CvaA和CvaB对加工过程的发生至关重要。相比之下，在TolC突变体中，加工过程正常发生，尽管速度较慢。
