Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
Environ Microbiol. 2019 Jul;21(7):2248-2260. doi: 10.1111/1462-2920.14594. Epub 2019 Apr 2.
Type VI secretion systems (T6SSs) are widespread, tightly regulated, protein delivery apparatuses used by Gram-negative bacteria to outcompete their neighbours. The pathogen, Vibrio parahaemolyticus, encodes two T6SSs. These T6SSs are differentially regulated by external conditions. T6SS1, an antibacterial system predominantly found in pathogenic isolates, requires warm marine-like conditions and surface sensing for activation. The regulatory network that governs this activation is not well understood. In this work, we devised a screening methodology that allows us to easily monitor the outcome of bacterial competitions and thus to identify mutants that are defective in T6SS1-mediated bacterial killing. The methodology, termed Bacterial Competition Fluorescence (BaCoF), relies on detection of a fluorescent signal as an indicator of the survival and growth of a T6SS-sensitive, GFP-expressing prey that has been co-cultured with mutants derived from a T6SS attacker of interest. Using BaCoF, we screened a random transposon insertion mutant library and identified genes required for V. parahaemolyticus T6SS1 activation, among them TfoY and Tmk. We used epistasis experiments to determine the relationships between the newly identified components and other regulators that were previously described. Thus, we present here a detailed biological understanding of the T6SS1 regulatory network.
VI 型分泌系统（T6SS）是一种广泛存在、调控严格的蛋白输送装置，革兰氏阴性菌利用它来与邻近细菌竞争。病原体副溶血弧菌编码了两个 T6SS。这些 T6SS 受到外部条件的差异调控。T6SS1 是一种主要存在于致病性分离株中的抗细菌系统，需要温暖的海洋样条件和表面感应来激活。管理这种激活的调控网络尚未得到很好的理解。在这项工作中，我们设计了一种筛选方法，使我们能够轻松监测细菌竞争的结果，从而识别出在 T6SS1 介导的细菌杀伤中缺陷的突变体。该方法称为细菌竞争荧光（BaCoF），依赖于荧光信号的检测，作为与 T6SS 攻击菌共培养的 GFP 表达的敏感猎物的存活和生长的指标。使用 BaCoF，我们筛选了随机转座子插入突变体文库，并鉴定了副溶血弧菌 T6SS1 激活所需的基因，其中包括 TfoY 和 Tmk。我们利用上位性实验来确定新鉴定的成分与先前描述的其他调节剂之间的关系。因此，我们在这里提出了对 T6SS1 调控网络的详细生物学理解。
