Bacterial Multicellularity: The Biology of Escherichia coli Building Large-Scale Biofilm Communities.

Biofilms are a widespread multicellular form of bacterial life. The spatial structure and emergent properties of these communities depend on a polymeric extracellular matrix architecture that is orders of magnitude larger than the cells that build it. Using as a model the wrinkly macrocolony biofilms of Escherichia coli, which contain amyloid curli fibers and phosphoethanolamine (pEtN)-modified cellulose as matrix components, we summarize here the structure, building, and function of this large-scale matrix architecture. Based on different sigma and other transcription factors as well as second messengers, the underlying regulatory network reflects the fundamental trade-off between growth and survival. It controls matrix production spatially in response to long-range chemical gradients, but it also generates distinct patterns of short-range matrix heterogeneity that are crucial for tissue-like elasticity and macroscopic morphogenesis. Overall, these biofilms confer protection and a potential for homeostasis, thereby reducing maintenance energy, which makes multicellularity an emergent property of life itself.

Non-lethal exposure to H2O2 boosts bacterial survival and evolvability against oxidative stress.

Unicellular organisms have the prevalent challenge to survive under oxidative stress of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). ROS are present as by-products of photosynthesis and aerobic respiration. These reactive species are even employed by multicellular organisms as potent weapons against microbes. Although bacterial defences against lethal and sub-lethal oxidative stress have been studied in model bacteria, the role of fluctuating H2O2 concentrations remains unexplored. It is known that sub-lethal exposure of Escherichia coli to H2O2 results in enhanced survival upon subsequent exposure. Here we investigate the priming response to H2O2 at physiological concentrations. The basis and the duration of the response (memory) were also determined by time-lapse quantitative proteomics. We found that a low level of H2O2 induced several scavenging enzymes showing a long half-life, subsequently protecting cells from future exposure. We then asked if the phenotypic resistance against H2O2 alters the evolution of resistance against oxygen stress. Experimental evolution of H2O2 resistance revealed faster evolution and higher levels of resistance in primed cells. Several mutations were found to be associated with resistance in evolved populations affecting different loci but, counterintuitively, none of them was directly associated with scavenging systems. Our results have important implications for host colonisation and infections where microbes often encounter reactive oxygen species in gradients.

Transmembrane redox control and proteolysis of PdeC, a novel type of c-di-GMP phosphodiesterase.

The nucleotide second messenger c-di-GMP nearly ubiquitously promotes bacterial biofilm formation, with enzymes that synthesize and degrade c-di-GMP being controlled by diverse N-terminal sensor domains. Here, we describe a novel class of widely occurring c-di-GMP phosphodiesterases (PDE) that feature a periplasmic "CSS domain" with two highly conserved cysteines that is flanked by two transmembrane regions (TM1 and TM2) and followed by a cytoplasmic EAL domain with PDE activity. Using PdeC, one of the five CSS domain PDEs of Escherichia coli K-12, we show that DsbA/DsbB-promoted disulfide bond formation in the CSS domain reduces PDE activity. By contrast, the free thiol form is enzymatically highly active, with the TM2 region promoting dimerization. Moreover, this form is processed by periplasmic proteases DegP and DegQ, yielding a highly active TM2 + EAL fragment that is slowly removed by further proteolysis. Similar redox control and proteolysis was also observed for a second CSS domain PDE, PdeB. At the physiological level, CSS domain PDEs modulate production and supracellular architecture of extracellular matrix polymers in the deeper layers of mature E. coli biofilms.

Genetic dissection of Escherichia coli's master diguanylate cyclase DgcE: Role of the N-terminal MASE1 domain and direct signal input from a GTPase partner system.

The ubiquitous second messenger c-di-GMP promotes bacterial biofilm formation by playing diverse roles in the underlying regulatory networks. This is reflected in the multiplicity of diguanylate cyclases (DGC) and phosphodiesterases (PDE) that synthesize and degrade c-di-GMP, respectively, in most bacterial species. One of the 12 DGCs of Escherichia coli, DgcE, serves as the top-level trigger for extracellular matrix production during macrocolony biofilm formation. Its multi-domain architecture-a N-terminal membrane-inserted MASE1 domain followed by three PAS, a GGDEF and a degenerate EAL domain-suggested complex signal integration and transmission through DgcE. Genetic dissection of DgcE revealed activating roles for the MASE1 domain and the dimerization-proficient PAS3 region, whereas the inhibitory EALdeg domain counteracts the formation of DgcE oligomers. The MASE1 domain is directly targeted by the GTPase RdcA (YjdA), a dimer or oligomer that together with its partner protein RdcB (YjcZ) activates DgcE, probably by aligning and promoting dimerization of the PAS3 and GGDEF domains. This activation and RdcA/DgcE interaction depend on GTP hydrolysis by RdcA, suggesting GTP as an inhibitor and the pronounced decrease of the cellular GTP pool during entry into stationary phase, which correlates with DgcE-dependent activation of matrix production, as a possible input signal sensed by RdcA. Furthermore, DgcE exhibits rapid, continuous and processive proteolytic turnover that also depends on the relatively disordered transmembrane MASE1 domain. Overall, our study reveals a novel GTP/c-di-GMP-connecting signaling pathway through the multi-domain DGC DgcE with a dual role for the previously uncharacterized MASE1 signaling domain.

The Escherichia coli MarA protein regulates the ycgZ-ymgABC operon to inhibit biofilm formation.

The Escherichia coli marRAB operon is a paradigm for chromosomally encoded antibiotic resistance. The operon exerts its effect via an encoded transcription factor called MarA that modulates efflux pump and porin expression. In this work, we show that MarA is also a regulator of biofilm formation. Control is mediated by binding of MarA to the intergenic region upstream of the ycgZ-ymgABC operon. The operon, known to influence the formation of curli fibres and colanic acid, is usually expressed during periods of starvation. Hence, the ycgZ-ymgABC promoter is recognised by σ38 (RpoS)-associated RNA polymerase (RNAP). Surprisingly, MarA does not influence σ38 -dependent transcription. Instead, MarA drives transcription by the housekeeping σ70 -associated RNAP. The effects of MarA on ycgZ-ymgABC expression are coupled with biofilm formation by the rcsCDB phosphorelay system, with YcgZ, YmgA and YmgB forming a complex that directly interacts with the histidine kinase domain of RcsC.

Common plant flavonoids prevent the assembly of amyloid curli fibres and can interfere with bacterial biofilm formation.

Like all macroorganisms, plants have to control bacterial biofilm formation on their surfaces. On the other hand, biofilms are highly tolerant against antimicrobial agents and other stresses. Consequently, biofilms are also involved in human chronic infectious diseases, which generates a strong demand for anti-biofilm agents. Therefore, we systematically explored major plant flavonoids as putative anti-biofilm agents using different types of biofilms produced by Gram-negative and Gram-positive bacteria. In Escherichia coli macrocolony biofilms, the flavone luteolin and the flavonols myricetin, morin and quercetin were found to strongly reduce the extracellular matrix. These agents directly inhibit the assembly of amyloid curli fibres by driving CsgA subunits into an off-pathway leading to SDS-insoluble oligomers. In addition, they can interfere with cellulose production by still unknown mechanisms. Submerged biofilm formation, however, is hardly affected. Moreover, the same flavonoids tend to stimulate macrocolony and submerged biofilm formation by Pseudomonas aeruginosa. For Bacillus subtilis, the flavonone naringenin and the chalcone phloretin were found to inhibit growth. Thus, plant flavonoids are not general anti-biofilm compounds but show species-specific effects. However, based on their strong and direct anti-amyloidogenic activities, distinct plant flavonoids may provide an attractive strategy to specifically combat amyloid-based biofilms of some relevant pathogens.

Targeting Bacterial Biofilms by the Green Tea Polyphenol EGCG.

Bacterial biofilms are multicellular aggregates in which cells are embedded in an extracellular matrix of self-produced biopolymers. Being refractory to antibiotic treatment and host immune systems, biofilms are involved in most chronic infections, and anti-biofilm agents are being searched for urgently. Epigallocatechin-3-gallate (EGCG) was recently shown to act against biofilms by strongly interfering with the assembly of amyloid fibres and the production of phosphoethanolamin-modified cellulose fibrils. Mechanistically, this includes a direct inhibition of the fibre assembly, but also triggers a cell envelope stress response that down-regulates the synthesis of these widely occurring biofilm matrix polymers. Based on its anti-amyloidogenic properties, EGCG seems useful against biofilms involved in cariogenesis or chronic wound infection. However, EGCG seems inefficient against or may even sometimes promote biofilms which rely on other types of matrix polymers, suggesting that searching for 'magic bullet' anti-biofilm agents is an unrealistic goal. Combining molecular and ecophysiological aspects in this review also illustrates why plants control the formation of biofilms on their surfaces by producing anti-amyloidogenic compounds such as EGCG. These agents are not only helpful in combating certain biofilms in chronic infections but even seem effective against the toxic amyloids associated with neuropathological diseases.

The green tea polyphenol EGCG inhibits E. coli biofilm formation by impairing amyloid curli fibre assembly and downregulating the biofilm regulator CsgD via the σ(E) -dependent sRNA RybB.

In bacterial biofilms, which are often involved in chronic infections, cells are surrounded by a self-produced extracellular matrix that contains amyloid fibres, exopolysaccharides and other biopolymers. The matrix contributes to the pronounced resistance of biofilms against antibiotics and host immune systems. Being highly inflammatory, matrix amyloids such as curli fibres of Escherichia coli can also play a role in pathogenicity. Using macrocolony biofilms of commensal and pathogenic E. coli as a model system, we demonstrate here that the green tea polyphenol epigallocatachin gallate (EGCG) is a potent antibiofilm agent. EGCG virtually eliminates the biofilm matrix by directly interfering with the assembly of curli subunits into amyloid fibres, and by triggering the σ(E) cell envelope stress response and thereby reducing the expression of CsgD - a crucial activator of curli and cellulose biosynthesis - due to csgD mRNA targeting by the σ(E) -dependent sRNA RybB. These findings highlight EGCG as a potential adjuvant for antibiotic therapy of biofilm-associated infections. Moreover, EGCG may support therapies against pathogenic E. coli that produce inflammatory curli fibres along with Shigatoxin.

The EAL domain protein YciR acts as a trigger enzyme in a c-di-GMP signalling cascade in E. coli biofilm control.

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 BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli.

The blue light using FAD (BLUF)-EAL protein YcgF is a known blue-light sensor of Escherichia coli, but its direct regulatory output and physiological function have remained unknown. Here, we demonstrate that unlike other EAL domain proteins, YcgF does not degrade the signaling molecule c-di-GMP, but directly binds to and releases the MerR-like repressor YcgE from its operator DNA upon blue-light irradiation. As a consequence, a distinct regulon of eight small proteins (of 71-126 amino acids) is strongly induced. These include YmgA and YmgB, which, via the RcsC/RcsD/RcsB two-component phosphorelay system, activate production of the biofilm matrix substance colanic acid as well as acid resistance genes and the biofilm-associated bdm gene and down-regulate adhesive curli fimbriae. Thus, small proteins under YcgF/YcgE control seem to act as "connectors" that provide additional signal input into a two-component signaling pathway. Moreover, we found ycgF and ycgE expression to be strongly activated at low temperature, and we elucidate how blue light, cold, and starvation signals are integrated in the expression and activity of the YcgF/YcgE/small protein signaling pathway. In conclusion, this pathway may modulate biofilm formation via the two-component network when E. coli has to survive in an extrahost aquatic environment.

Genome-Based Comparison of Cyclic Di-GMP Signaling in Pathogenic and Commensal Escherichia coli Strains.

UNLABELLED: The ubiquitous bacterial second messenger cyclic di-GMP (c-di-GMP) has recently become prominent as a trigger for biofilm formation in many bacteria. It is generated by diguanylate cyclases (DGCs; with GGDEF domains) and degraded by specific phosphodiesterases (PDEs; containing either EAL or HD-GYP domains). Most bacterial species contain multiples of these proteins with some having specific functions that are based on direct molecular interactions in addition to their enzymatic activities. Escherichia coli K-12 laboratory strains feature 29 genes encoding GGDEF and/or EAL domains, resulting in a set of 12 DGCs, 13 PDEs, and four enzymatically inactive "degenerate" proteins that act by direct macromolecular interactions. We present here a comparative analysis of GGDEF/EAL domain-encoding genes in 61 genomes of pathogenic, commensal, and probiotic E. coli strains (including enteric pathogens such as enteroaggregative, enterohemorrhagic, enteropathogenic, enterotoxigenic, and adherent and invasive Escherichia coli and the 2011 German outbreak O104:H4 strain, as well as extraintestinal pathogenic E. coli, such as uropathogenic and meningitis-associated E. coli). We describe additional genes for two membrane-associated DGCs (DgcX and DgcY) and four PDEs (the membrane-associated PdeT, as well as the EAL domain-only proteins PdeW, PdeX, and PdeY), thus showing the pangenome of E. coli to contain at least 35 GGDEF/EAL domain proteins. A core set of only eight proteins is absolutely conserved in all 61 strains: DgcC (YaiC), DgcI (YliF), PdeB (YlaB), PdeH (YhjH), PdeK (YhjK), PdeN (Rtn), and the degenerate proteins CsrD and CdgI (YeaI). In all other GGDEF/EAL domain genes, diverse point and frameshift mutations, as well as small or large deletions, were discovered in various strains. IMPORTANCE: Our analysis reveals interesting trends in pathogenic Escherichia coli that could reflect different host cell adherence mechanisms. These may either benefit from or be counteracted by the c-di-GMP-stimulated production of amyloid curli fibers and cellulose. Thus, EAEC, which adhere in a "stacked brick" biofilm mode, have a potential for high c-di-GMP accumulation due to DgcX, a strongly expressed additional DGC. In contrast, EHEC and UPEC, which use alternative adherence mechanisms, tend to have extra PDEs, suggesting that low cellular c-di-GMP levels are crucial for these strains under specific conditions. Overall, our study also indicates that GGDEF/EAL domain proteins evolve rapidly and thereby contribute to adaptation to host-specific and environmental niches of various types of E. coli.

Bacterial Signal Transduction by Cyclic Di-GMP and Other Nucleotide Second Messengers.

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.

Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli.

In recent years, Escherichia coli has served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely used E. coli K-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 "degenerate" enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenic E. coli strains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling in E. coli, we now propose a general and systematic dgc and pde nomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains of E. coli in future studies.

Vertical stratification of matrix production is essential for physical integrity and architecture of macrocolony biofilms of Escherichia coli.

Bacterial macrocolony biofilms grow into intricate three-dimensional structures that depend on self-produced extracellular polymers conferring protection, cohesion and elasticity to the biofilm. In Escherichia 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.

Bacterial regulatory networks--from self-organizing molecules to cell shape and patterns in bacterial communities.

The ESF-EMBO Conference on 'Bacterial Networks' (BacNet13) was held in March 2013, in Pultusk, Poland. It brought together 164 molecular microbiologists, bacterial systems biologists and synthetic biologists to discuss the architecture, function and dynamics of regulatory networks in bacteria.

Stress responses go three dimensional - the spatial order of physiological differentiation in bacterial macrocolony biofilms.

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 Escherichia 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.

Small RNAs in the control of RpoS, CsgD, and biofilm architecture of Escherichia coli.

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, elasticity, and wrinkled morphology to these biofilms. Curli and cellulose synthesis is controlled by a three-level transcription factor (TF) cascade with the RpoS sigma subunit of RNA polymerase at the top, the MerR-like TF MlrA, and the biofilm regulator CsgD, with two c-di-GMP control modules acting as key switching devices. Additional signal input and fine-tuning is provided by an entire series of small RNAs-ArcZ, DsrA, RprA, McaS, OmrA/OmrB, GcvB, and RydC--that differentially control all three TF modules by direct mRNA interaction. This review not only summarizes the mechanisms of action of these sRNAs, but also addresses the question of how these sRNAs and the regulators they target contribute to building the intriguing three-dimensional microarchitecture and macromorphology of these biofilms.

The global repressor FliZ antagonizes gene expression by σS-containing RNA polymerase due to overlapping DNA binding specificity.

FliZ, a global regulatory protein under the control of the flagellar master regulator FlhDC, was shown to antagonize σ(S)-dependent gene expression in Escherichia coli. Thereby it plays a pivotal role in the decision between alternative life-styles, i.e. FlhDC-controlled flagellum-based motility or σ(S)-dependent curli fimbriae-mediated adhesion and biofilm formation. Here, we show that FliZ is an abundant DNA-binding protein that inhibits gene expression mediated by σ(S) by recognizing operator sequences that resemble the -10 region of σ(S)-dependent promoters. FliZ does so with a structural element that is similar to region 3.0 of σ(S). Within this element, R108 in FliZ corresponds to K173 in σ(S), which contacts a conserved cytosine at the -13 promoter position that is specific for σ(S)-dependent promoters. R108 as well as C(-13) are also crucial for DNA binding by FliZ. However, while a number of FliZ binding sites correspond to known σ(S)-dependent promoters, promoter activity is not a prerequisite for FliZ binding and repressor function. Thus, we demonstrate that FliZ also feedback-controls flagellar gene expression by binding to a site in the flhDC control region that shows similarity only to a -10 element of a σ(S)-dependent promoter, but does not function as a promoter.

Cellulose as an architectural element in spatially structured Escherichia coli biofilms.

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.

Molecular function and potential evolution of the biofilm-modulating blue light-signalling pathway of Escherichia coli.

Escherichia coli senses blue light via the BLUF-EAL protein BluF (YcgF). The degenerate EAL domain of BluF does not have cyclic-di-GMP phosphodiesterase activity, but BluF directly antagonizes the MerR-like repressor BluR (YcgE), which leads to expression of the ycgZ-ymgABC operon and activation of the Rcs system (Tschowri et al., 2009; Genes Dev 23: 522-534). While bluR, bluF and ycgZ have individual transcriptional start sites, comparative genome analysis indicates that the bluR-bluF-ycgZ-ymgAB region represents a functional unit in various enteric bacteria that is characterized by bluF alleles encoding degenerate EAL domains. Re-introducing conserved amino acids involved in phosphodiesterase activity of EAL domains did not restore enzymatic activity or c-di-GMP binding of BluF, but weakened its ability to antagonize BluR and improved a residual interaction with the BluR paralogue MlrA, which controls expression of the biofilm regulator CsgD and curli fibres. We identified the BluR binding site in the ycgZ promoter and observed that BluR also has residual affinity for the MlrA-dependent csgD promoter. Altogether, we propose that BluF evolved from a blue light-regulated PDE into a specific antagonist of a duplicate of MlrA that became BluR, which controls not only curli but various biofilm functions via the Ymg/Rcs pathway.