How Quorum Sensing Connects Sporulation to Necrotrophism in Bacillus thuringiensis

Bacteria use quorum sensing to coordinate adaptation properties, cell fate or commitment to sporulation. The infectious cycle of Bacillus thuringiensis in the insect host is a powerful model to investigate the role of quorum sensing in natural conditions. It is tuned by communication systems regulators belonging to the RNPP family and directly regulated by re-internalized signaling peptides. One such RNPP regulator, NprR, acts in the presence of its cognate signaling peptide NprX as a transcription factor, regulating a set of genes involved in the survival of these bacteria in the insect cadaver. Here, we demonstrate that, in the absence of NprX and independently of its transcriptional activator function, NprR negatively controls sporulation. NprR inhibits expression of Spo0A-regulated genes by preventing the KinA-dependent phosphorylation of the phosphotransferase Spo0F, thus delaying initiation of the sporulation process. This NprR function displays striking similarities with the Rap proteins, which also belong to the RNPP family, but are devoid of DNA-binding domain and indirectly control gene expression via protein-protein interactions in Bacilli. Conservation of the Rap residues directly interacting with Spo0F further suggests a common inhibition of the sporulation phosphorelay. The crystal structure of apo NprR confirms that NprR displays a highly flexible Rap-like structure. We propose a molecular regulatory mechanism in which key residues of the bifunctional regulator NprR are directly and alternatively involved in its two functions. NprX binding switches NprR from a dimeric inhibitor of sporulation to a tetrameric transcriptional activator involved in the necrotrophic lifestyle of B. thuringiensis. NprR thus tightly coordinates sporulation and necrotrophism, ensuring survival and dissemination of the bacteria during host infection.细菌借助群体感应（quorum sensing）协调适应性性状、细胞命运或芽孢形成（sporulation）的定向发生。苏云金芽孢杆菌（Bacillus thuringiensis）在昆虫宿主中的侵染周期，是探究群体感应在自然条件下功能的理想模型体系。该过程受隶属于RNPP家族（RNPP family）的通讯系统调控因子所调控，并通过被重新内化的信号肽（signaling peptide）实现直接调控。其中一类RNPP调控因子NprR，可在其同源信号肽NprX存在时作为转录因子（transcription factor）发挥作用，调控一系列参与该细菌在昆虫尸体内存活的基因表达。 本研究证实，在缺失NprX且不依赖其转录激活功能的情况下，NprR可负向调控芽孢形成。NprR通过阻断磷酸转移酶（phosphotransferase）Spo0F的KinA依赖性磷酸化，抑制Spo0A调控基因的表达，进而延缓芽孢形成过程的起始。这一NprR功能与同样隶属于RNPP家族的Rap蛋白存在显著相似性：后者虽不具备DNA结合结构域（DNA-binding domain），却可通过芽孢杆菌中的蛋白质-蛋白质相互作用间接调控基因表达。对Rap蛋白中直接与Spo0F相互作用的残基进行保守性分析，进一步提示二者对芽孢形成磷酸接力系统存在共同的抑制机制。空载NprR（apo NprR）的晶体结构（crystal structure）证实，NprR具备高度柔性的Rap样结构。 我们提出一种分子调控机制：双功能调控因子（bifunctional regulator）NprR的关键残基可直接且交替参与其两项功能。NprX的结合可将NprR从芽孢形成的二聚体抑制剂，转变为参与苏云金芽孢杆菌坏死营养型生活方式（necrotrophic lifestyle）的四聚体转录激活因子。由此，NprR可紧密协调芽孢形成与坏死营养过程，保障该细菌在宿主侵染过程中的存活与传播。