Bacterial biofilms are highly structured multicellular communities whose formation involves flagella and an extracellular matrix of adhesins, amyloid fibers, and exopolysaccharides. Flagella are produced by still-dividing rod-shaped Escherichia coli cells during postexponential growth when nutrients become suboptimal. Upon entry into stationary phase, however, cells stop producing flagella, become ovoid, and generate amyloid curli fibers. These morphological changes, as well as accompanying global changes in gene expression and cellular physiology, depend on the induction of the stationary-phase sigma subunit of RNA polymerase, σS (RpoS), the nucleotide second messengers cyclic AMP (cAMP), ppGpp, and cyclic-di-GMP, and a biofilm-controlling transcription factor, CsgD. Using flagella, curli fibers, a CsgD::GFP reporter, and cell morphology as “anatomical” hallmarks in fluorescence and scanning electron microscopy, different physiological zones in macrocolony biofilms of E. coli K-12 can be distinguished at cellular resolution. Small ovoid cells encased in a network of curli fibers form the outer biofilm layer. Inner regions are characterized by heterogeneous CsgD::GFP and curli expression. The bottom zone of the macrocolonies features elongated dividing cells and a tight mesh of entangled flagella, the formation of which requires flagellar motor function. Also, the cells in the outer-rim growth zone produce flagella, which wrap around and tether cells together. Adjacent to this growth zone, small chains and patches of shorter curli-surrounded cells appear side by side with flagellated curli-free cells before curli coverage finally becomes confluent, with essentially all cells in the surface layer being encased in “curli baskets.”

C-di-GMP—which is produced by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases (PDEs)—is a ubiquitous second messenger in bacterial biofilm formation. In Escherichia coli, several DGCs (YegE, YdaM) and PDEs (YhjH, YciR) and the MerR-like transcription factor MlrA regulate the transcription of csgD, which encodes a biofilm regulator essential for producing amyloid curli fibres of the biofilm matrix. Here, we demonstrate that this system operates as a signalling cascade, in which c-di-GMP controlled by the DGC/PDE pair YegE/YhjH (module I) regulates the activity of the YdaM/YciR pair (module II). Via multiple direct interactions, the two module II proteins form a signalling complex with MlrA. YciR acts as a connector between modules I and II and functions as a trigger enzyme: its direct inhibition of the DGC YdaM is relieved when it binds and degrades c-di-GMP generated by module I. As a consequence, YdaM then generates c-di-GMP and—by direct and specific interaction—activates MlrA to stimulate csgD transcription. Trigger enzymes may represent a general principle in local c-di-GMP signalling.

The bacterial second messenger bis-(3′-5′)-cyclic diguanosine monophosphate (c-di-GMP) ubiquitously promotes bacterial biofilm formation. Intracellular pools of c-di-GMP seem to be dynamically negotiated by diguanylate cyclases (DGCs, with GGDEF domains) and specific phosphodiesterases (PDEs, with EAL or HD-GYP domains). Most bacterial species possess multiple DGCs and PDEs, often with surprisingly distinct and specific output functions. One explanation for such specificity is “local” c-di-GMP signaling, which is believed to involve direct interactions between specific DGC/PDE pairs and c-di-GMP-binding effector/target systems. Here we present a systematic analysis of direct protein interactions among all 29 GGDEF/EAL domain proteins of Escherichia coli. Since the effects of interactions depend on coexpression and stoichiometries, cellular levels of all GGDEF/EAL domain proteins were also quantified and found to vary dynamically along the growth cycle. Instead of detecting specific pairs of interacting DGCs and PDEs, we discovered a tightly interconnected protein network of a specific subset or “supermodule” of DGCs and PDEs with a coregulated core of five hyperconnected hub proteins. These include the DGC/PDE proteins representing the c-di-GMP switch that turns on biofilm matrix production in E. coli. Mutants lacking these core hub proteins show drastic biofilm-related phenotypes but no changes in cellular c-di-GMP levels. Overall, our results provide the basis for a novel model of local c-di-GMP signaling in which a single strongly expressed master PDE, PdeH, dynamically eradicates global effects of several DGCs by strongly draining the global c-di-GMP pool and thereby restricting these DGCs to serving as local c-di-GMP sources that activate specific colocalized effector/target systems.

Bacterial biofilms are large aggregates of cells embedded in an extracellular matrix of self-produced polymers. In macrocolony biofilms of Escherichia coli, this matrix is generated in the upper biofilm layer only and shows a surprisingly complex supracellular architecture. Stratified matrix production follows the vertical nutrient gradient and requires the stationary phase σS (RpoS) subunit of RNA polymerase and the second messenger c-di-GMP. By visualizing global gene expression patterns with a newly designed fingerprint set of Gfp reporter fusions, our study reveals the spatial order of differential sigma factor activities, stringent control of ribosomal gene expression and c-di-GMP signalling in vertically cryosectioned macrocolony biofilms. Long-range physiological stratification shows a duplication of the growth-to-stationary phase pattern that integrates nutrient and oxygen gradients. In addition, distinct short-range heterogeneity occurs within specific biofilm strata and correlates with visually different zones of the refined matrix architecture. These results introduce a new conceptual framework for the control of biofilm formation and demonstrate that the intriguing extracellular matrix architecture, which determines the emergent physiological and biomechanical properties of biofilms, results from the spatial interplay of global gene regulation and microenvironmental conditions. Overall, mature bacterial macrocolony biofilms thus resemble the highly organized tissues of multicellular organisms.

SummaryBacterial macrocolony biofilms grow into intricate three‐dimensional structures that depend on self‐produced extracellular polymers conferring protection, cohesion and elasticity to the biofilm. In E scherichia coli, synthesis of this matrix – consisting of amyloid curli fibres and cellulose – requires CsgD, a transcription factor regulated by the stationary phase sigma factor RpoS, and occurs in the nutrient‐deprived cells of the upper layer of macrocolonies. Is this asymmetric matrix distribution functionally important or is it just a fortuitous by‐product of an unavoidable nutrient gradient? In order to address this question, the RpoS‐dependent csgD promoter was replaced by a vegetative promoter. This re‐wiring of csgD led to CsgD and matrix production in both strata of macrocolonies, with the lower layer transforming into a rigid ‘base plate’ of growing yet curli‐connected cells. As a result, the two strata broke apart followed by desiccation and exfoliation of the top layer. By contrast, matrix‐free cells at the bottom of wild‐type macrocolonies maintain colony contact with the humid agar support by flexibly filling the space that opens up under buckling areas of the macrocolony. Precisely regulated stratification in matrix‐free and matrix‐producing cell layers is thus essential for the physical integrity and architecture of E . coli macrocolony biofilms.