High Levels of DegU-P Activate an Esat-6-Like Secretion System in Bacillus subtilis

The recently discovered Type VII/Esat-6 secretion systems seem to be widespread among bacteria of the phyla Actinobacteria and Firmicutes. In some species they play an important role in pathogenic interactions with eukaryotic hosts. Several studies have predicted that the locus yukEDCByueBC of the non-pathogenic, Gram-positive bacterium Bacillus subtilis would encode an Esat-6-like secretion system (Ess). We provide here for the first time evidences for the functioning of this secretion pathway in an undomesticated B. subtilis strain. We show that YukE, a small protein with the typical features of the secretion substrates from the WXG100 superfamily is actively secreted to culture media. YukE secretion depends on intact yukDCByueBC genes, whose products share sequence or structural homology with known components of the S. aureus Ess. Biochemical characterization of YukE indicates that it exists as a dimer both in vitro and in vivo. We also show that the B. subtilis Ess essentially operates in late stationary growth phase in absolute dependence of phosphorylated DegU, the response regulator of the two-component system DegS-DegU. We present possible reasons that eventually have precluded the study of this secretion system in the B. subtilis laboratory strain 168.

近期发现的VII型/Esat-6分泌系统（Type VII/Esat-6 secretion system）广泛分布于放线菌门（Actinobacteria）和厚壁菌门（Firmicutes）的细菌中。在部分物种内，该系统在与真核宿主的致病互作中发挥关键作用。多项研究此前预测，无致病性革兰氏阳性细菌枯草芽孢杆菌（Bacillus subtilis）的基因座yukEDCByueBC可编码Esat-6样分泌系统（Esat-6-like secretion system, Ess）。本文首次提供了该分泌通路在未驯化枯草芽孢杆菌菌株中发挥功能的实验证据。研究发现，具备WXG100超家族分泌底物典型特征的小分子蛋白YukE可被主动分泌至培养基中。YukE的分泌依赖完整的yukDCByueBC基因，其编码产物与金黄色葡萄球菌（Staphylococcus aureus）Ess系统的已知组分存在序列或结构同源性。对YukE的生化表征显示，其在体外与体内均以二聚体形式存在。本文还证实，枯草芽孢杆菌的Ess系统主要在稳定生长期晚期发挥功能，且完全依赖双组分系统DegS-DegU的响应调节因子磷酸化DegU。最后，本文阐明了此前阻碍针对枯草芽孢杆菌实验室菌株168开展该分泌系统研究的潜在原因。