Heterogeneity of Matrix Production in Bacterial Biofilm Formation

细菌生物膜表现出空间生理分化，基本上遵循营养和/或氧气梯度。冰冻切片、大菌落生物膜的原位荧光和扫描电子显微镜观察表明，大菌落生物膜具有明显的层次性，底层有鞭毛细胞生长，顶层有外菌落边缘和形成生物膜基质(由淀粉样卷曲纤维和纤维素组成)的小的静止相细胞。在这些层之间，过渡区表现出明显的异质性，产生基质的细胞和无基质的细胞紧挨着。在后代中稳定地保持matrixON状态，这导致在大菌落生物膜的过渡区中，在基质周围的细胞形成随机定向的小链(如果只产生卷曲的纤维)或垂直的“柱状”(如果产生卷曲和纤维素)。卷曲和纤维素的生产受到关键的生物膜调节因子CsgD的控制，CsgD在大菌落中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输入的复杂信号的调控。此外，csgD mRNA是几个小的调控RNA的靶标。这个控制网络具有三个相互抑制的基序以及一个正反馈回路，即有可能产生输出双稳的调节模式。初步的遗传分析和数学模型(与K.Yousef博士和M.v.Kleist博士，Fu柏林合作)表明，c-di-GMP控制模块是CsgD双峰表达的主要来源，因此产生基质。总体而言，拟议的研究解决了细菌生物膜不同区域细胞外基质产生的异质性，这(根据我们的初步数据)是正在发育的生物膜结构完整性所需的分工的一个例子。由于这种异质性是在直接相邻的单元中观察到的，所以它不是由不同的外部条件引起的，而是由底层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这种高度结构的细菌群落中复杂平衡和精确定位的异质性的数学模型。

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.