Morphological form in multicellular aggregates emerges from the interplay of genetic constitution and environmental signals. Bacterial macrocolony biofilms, which form intricate three-dimensional structures, such as large and often radially oriented ridges, concentric rings, and elaborate wrinkles, provide a unique opportunity to understand this interplay of “nature and nurture” in morphogenesis at the molecular level. Macrocolony morphology depends on self-produced extracellular matrix components. In Escherichia coli , these are stationary phase-induced amyloid curli fibers and cellulose. While the widely used “domesticated” E. coli K-12 laboratory strains are unable to generate cellulose, we could restore cellulose production and macrocolony morphology of E. coli K-12 strain W3110 by “repairing” a single chromosomal SNP in the bcs operon. Using scanning electron and fluorescence microscopy, cellulose filaments, sheets and nanocomposites with curli fibers were localized in situ at cellular resolution within the physiologically two-layered macrocolony biofilms of this “de-domesticated” strain. As an architectural element, cellulose confers cohesion and elasticity, i.e., tissue-like properties that—together with the cell-encasing curli fiber network and geometrical constraints in a growing colony—explain the formation of long and high ridges and elaborate wrinkles of wild-type macrocolonies. In contrast, a biofilm matrix consisting of the curli fiber network only is brittle and breaks into a pattern of concentric dome-shaped rings separated by deep crevices. These studies now set the stage for clarifying how regulatory networks and in particular c-di-GMP signaling operate in the three-dimensional space of highly structured and “tissue-like” bacterial biofilms.

A naturally modified cellulose: Cellulose is the most abundant biopolymer on Earth and an important component of bacterial biofilms. Thongsomboon et al. used solid-state nuclear magnetic resonance spectroscopy to identify a naturally derived, chemically modified cellulose, phosphoethanolamine cellulose (see the Perspective by Galperin and Shalaeva). They went on to identify the genetic basis and molecular signaling involved in introducing this modification in bacteria, which regulates biofilm matrix architecture and function. This discovery has implications for understanding bacterial biofilms and for the generation of new cellulosic materials. Science , this issue p. 334 ; see also p. 276

Summary: In natural habitats, bacteria often occur in multicellular communities characterized by a robust extracellular matrix of proteins, amyloid fibres, exopolysaccharides and extracellular DNA . These biofilms show pronounced stress resistance including a resilience against antibiotics that causes serious medical and technical problems. This review summarizes recent studies that have revealed clear spatial physiological differentiation, complex supracellular architecture and striking morphology in macrocolony biofilms. By responding to gradients of nutrients, oxygen, waste products and signalling compounds that build up in growing biofilms, various stress responses determine whether bacteria grow and proliferate or whether they enter into stationary phase and use their remaining resources for maintenance and survival. As a consequence, biofilms differentiate into at least two distinct layers of vegetatively growing and stationary phase cells that exhibit very different cellular physiology. This includes a stratification of matrix production with a major impact on microscopic architecture, biophysical properties and directly visible morphology of macrocolony biofilms. Using E scherichia coli as a model system, this review also describes our detailed current knowledge about the underlying molecular control networks – prominently featuring sigma factors, transcriptional cascades and second messengers – that drive this spatial differentiation and points out directions for future research.

Amyloid curli fibers and cellulose are extracellular matrix components produced in the stationary phase top layer of E. coli macrocolonies, which confer physical protection, strong cohesion, elasti...

The first International Symposium on c-Di-GMP Signaling in Bacteria (22 to 25 March 2015, Harnack-Haus, Berlin, Germany) brought together 131 molecular microbiologists from 17 countries to discuss recent progress in our knowledge of bacterial nucleotide second messenger signaling. While the focus was on signal input, synthesis, degradation, and the striking diversity of the modes of action of the current second messenger paradigm, i.e., cyclic di-GMP (c-di-GMP), “classics” like cAMP and (p)ppGpp were also presented, in novel facets, and more recent “newcomers,” such as c-di-AMP and c-AMP-GMP, made an impressive appearance. A number of clear trends emerged during the 30 talks, on the 71 posters, and in the lively discussions, including (i) c-di-GMP control of the activities of various ATPases and phosphorylation cascades, (ii) extensive cross talk between c-di-GMP and other nucleotide second messenger signaling pathways, and (iii) a stunning number of novel effectors for nucleotide second messengers that surprisingly include some long-known master regulators of developmental pathways. Overall, the conference made it amply clear that second messenger signaling is currently one of the most dynamic fields within molecular microbiology, with major impacts in research fields ranging from human health to microbial ecology.

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. IMPORTANCE c-di-GMP signaling in bacteria is believed to occur via changes in cellular c-di-GMP levels controlled by antagonistic and potentially interacting pairs of diguanylate cyclases (DGCs) and c-di-GMP phosphodiesterases (PDEs). Our systematic analysis of protein-protein interaction patterns of all 29 GGDEF/EAL domain proteins of E. coli , together with our measurements of cellular c-di-GMP levels, challenges both aspects of this current concept. Knocking out distinct DGCs and PDEs has drastic effects on E. coli biofilm formation without changing the cellular c-di-GMP level. In addition, rather than generally coming in interacting DGC/PDE pairs, a subset of DGCs and PDEs operates as central interaction hubs in a larger "supermodule," with other DGCs and PDEs behaving as “lonely players” without contacts to other c-di-GMP-related enzymes. On the basis of these data, we propose a novel concept of “local” c-di-GMP signaling in bacteria with multiple enzymes that make or break the second messenger c-di-GMP.