Heterogeneity of Matrix Production in Bacterial Biofilm Formation

细菌生物膜显示基本上遵循营养物和/或氧梯度的空间生理分化。大肠杆菌的大菌落生物膜的冷冻切片和原位荧光和扫描EM显示出明显的分层，在底层和外部菌落边缘中具有生长的鞭毛细胞，并且在顶层中具有产生生物膜基质（由淀粉样卷曲纤维和纤维素组成）的小的静止期细胞。在这些层之间，过渡区显示出明显的异质性，其中基质产生细胞和无基质细胞彼此相邻。matrixON状态在后代中稳定地维持，这导致在大菌落生物膜的过渡区中随机取向的小链（如果仅产生卷曲纤维）或基质包围的细胞的垂直“柱”（如果产生卷曲和纤维素）。卷曲和纤维素的产生受到关键生物膜调节剂CsgD的控制，该调节剂在大菌落内显示出相同的异质表达模式。csgD的转录受转录因子级联（RpoS，MlrA）和通过第二信使c-di-GMP输入的复杂信号调节。此外，csgD mRNA被几种小的调节RNA靶向。该控制网络具有三个相互抑制的图案以及正反馈回路，即具有产生输出双稳态的潜力的调节模式。初步遗传分析和数学建模（与K.尤瑟夫和M博士Kleist，FU柏林）指出c-di-GMP控制模块是双峰CsgD表达的主要来源，因此是基质产生的主要来源。总的来说，拟议的研究解决了细菌生物膜的不同区域中细胞外基质生产的异质性，这（根据我们的初步数据）是发展中生物膜的结构完整性所需的劳动分工的一个例子。由于这种异质性是在直接相邻的细胞中观察到的，因此它不是由不同的外部条件引起的，而是由潜在的调节网络产生输出双稳态的潜力引起的，这得到了网络关键电路的初步数学建模的支持。我们项目的总体目标是阐明精确的分子机制和生理功能，并进一步完善和测试高度结构化细菌群落中这种复杂平衡和精确定位异质性的数学模型。

Bacterial biofilms show spatial physiological differentiation that essentially follows nutrients and/or oxygen gradients. Cryomicrotomy and in-situ fluorescence and scanning EM of macrocolony biofilms of Escherichia coli revealed a clear stratification, with growing flagellated cells in the bottom layer and the outer colony rims and small stationary phase cells that produce biofilm matrix (consisting of amyloid curli fibres and cellulose) in the top layer. Between these layers, a transition zone shows pronounced heterogeneity with matrix-producing and matrix-free cells located right next to each other. The matrixON state is stably maintained in progeny, which results in randomly oriented small chains (if only curli fibres are produced) or vertical 'pillars' (if curli and cellulose are produced) of matrix-surrounded cells in the transition zones of macrocolony biofilms. Curli and cellulose production is under the control of the key biofilm regulator CsgD, which shows the same pattern of heterogeneous expression within macrocolonies. Transcription of csgD is regulated by a transcription factor cascade (RpoS, MlrA) and complex signal input via the second messenger c-di-GMP. Moreover, csgD mRNA is targeted by several small regulatory RNAs. This control network features three motifs of mutual inhibition as well as a positive feedback loop, i.e. regulatory patterns that have the potential to generate output bistability. Preliminary genetic analyses and mathematical modeling (in collaboration with Dr. K. Yousef and Dr. M. v. Kleist, FU Berlin) point to the c-di-GMP control module as a major source of bimodal CsgD expression and therefore matrix production. Overall, the proposed research addresses the heterogeneity of extracellular matrix production in distinct zones of a bacterial biofilm, which (according to our preliminary data) is an example of division of labor that is required for structural integrity of a developing biofilm. Since this heterogeneity is observed in directly adjacent cells, it is not caused by different external conditions, but rather arises from the potential of the underlying regulatory network to produce output bistability, which is supported by preliminary mathematical modeling of the key circuits of the network. The overall goal of our project is to clarify the precise molecular mechanisms and the physiological function and to further refine and test the mathematical model of this intricately balanced and precisely localized heterogeneity in a highly structured bacterial community.