Heterogeneity of Matrix Production in Bacterial Biofilm Formation

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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.