Tetrameric c-di-GMP mediates effective transcription factor dimerization to control Streptomyces development.

The cyclic dinucleotide c-di-GMP is a signaling molecule with diverse functions in cellular physiology. Here, we report that c-di-GMP can assemble into a tetramer that mediates the effective dimerization of a transcription factor, BldD, which controls the progression of multicellular differentiation in sporulating actinomycete bacteria. BldD represses expression of sporulation genes during vegetative growth in a manner that depends on c-di-GMP-mediated dimerization. Structural and biochemical analyses show that tetrameric c-di-GMP links two subunits of BldD through their C-terminal domains, which are otherwise separated by ~10 Å and thus cannot effect dimerization directly. Binding of the c-di-GMP tetramer by BldD is selective and requires a bipartite RXD-X8-RXXD signature. The findings indicate a unique mechanism of protein dimerization and the ability of nucleotide signaling molecules to assume alternative oligomeric states to effect different functions.

BldD-based bimolecular fluorescence complementation for in vivo detection of the second messenger cyclic di-GMP.

The widespread bacterial second messenger bis-(3'-5')-cyclic diguanosine monophosphate (c-di-GMP) is an important regulator of biofilm formation, virulence and cell differentiation. C-di-GMP-specific biosensors that allow detection and visualization of c-di-GMP levels in living cells are key to our understanding of how c-di-GMP fluctuations drive cellular responses. Here, we describe a novel c-di-GMP biosensor, CensYBL, that is based on c-di-GMP-induced dimerization of the effector protein BldD from Streptomyces resulting in bimolecular fluorescence complementation of split-YPet fusion proteins. As a proof-of-principle, we demonstrate that CensYBL is functional in detecting fluctuations in intracellular c-di-GMP levels in the Gram-negative model bacteria Escherichia coli and Salmonella enterica serovar Typhimurium. Using deletion mutants of c-di-GMP diguanylate cyclases and phosphodiesterases, we show that c-di-GMP dependent dimerization of CBldD-YPet results in fluorescence complementation reflecting intracellular c-di-GMP levels. Overall, we demonstrate that the CensYBL biosensor is a user-friendly and versatile tool that allows to investigate c-di-GMP variations using single-cell and population-wide experimental set-ups.

c-di-AMP hydrolysis by the phosphodiesterase AtaC promotes differentiation of multicellular bacteria.

Antibiotic-producing Streptomyces use the diadenylate cyclase DisA to synthesize the nucleotide second messenger c-di-AMP, but the mechanism for terminating c-di-AMP signaling and the proteins that bind the molecule to effect signal transduction are unknown. Here, we identify the AtaC protein as a c-di-AMP-specific phosphodiesterase that is also conserved in pathogens such as Streptococcus pneumoniae and Mycobacterium tuberculosis AtaC is monomeric in solution and binds Mn2+ to specifically hydrolyze c-di-AMP to AMP via the intermediate 5'-pApA. As an effector of c-di-AMP signaling, we characterize the RCK_C domain protein CpeA. c-di-AMP promotes interaction between CpeA and the predicted cation/proton antiporter, CpeB, linking c-di-AMP signaling to ion homeostasis in Actinobacteria. Hydrolysis of c-di-AMP is critical for normal growth and differentiation in Streptomyces, connecting ionic stress to development. Thus, we present the discovery of two components of c-di-AMP signaling in bacteria and show that precise control of this second messenger is essential for ion balance and coordinated development in Streptomyces.

Specialized and shared functions of diguanylate cyclases and phosphodiesterases in Streptomyces development.

The second messenger bis-3,5-cyclic di-guanosine monophosphate (c-di-GMP) determines when Streptomyces initiate sporulation. c-di-GMP signals are integrated into the genetic differentiation network by the regulator BldD and the sigma factor σWhiG . However, functions of the development-specific diguanylate cyclases (DGCs) CdgB and CdgC, and the c-di-GMP phosphodiesterases (PDEs) RmdA and RmdB, are poorly understood. Here, we provide biochemical evidence that the GGDEF-EAL domain protein RmdB from S. venezuelae is a monofunctional PDE that hydrolyzes c-di-GMP to 5'pGpG. Despite having an equivalent GGDEF-EAL domain arrangement, RmdA cleaves c-di-GMP to GMP and exhibits residual DGC activity. We show that an intact EAL motif is crucial for the in vivo function of both enzymes since strains expressing protein variants with an AAA motif instead of EAL are delayed in development, similar to null mutants. Transcriptome analysis of ∆cdgB, ∆cdgC, ∆rmdA, and ∆rmdB strains revealed that the c-di-GMP specified by these enzymes has a global regulatory role, with about 20% of all S. venezuelae genes being differentially expressed in the cdgC mutant. Our data suggest that the major c-di-GMP-controlled targets determining the timing and mode of sporulation are genes involved in cell division and the production of the hydrophobic sheath that covers Streptomyces aerial hyphae and spores.

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.

Nucleotide second messengers in Streptomyces.

Antibiotic producing Streptomyces sense and respond to environmental signals by using nucleotide second messengers, including (p)ppGpp, cAMP, c-di-GMP and c-di-AMP. As summarized in this review, these molecules are important message carriers that coordinate the complex Streptomyces morphological transition from filamentous growth to sporulation along with the secondary metabolite production. Here, we provide an overview of the enzymes that make and break these second messengers and suggest candidates for (p)ppGpp and cAMP enzymes to be studied. We highlight the target molecules that bind these signalling molecules and elaborate individual functions that they control in the context of Streptomyces development. Finally, we discuss open questions in the field, which may guide future studies in this exciting research area.

Sensing and responding to diverse extracellular signals: an updated analysis of the sensor kinases and response regulators of Streptomyces species.

Streptomyces venezuelae is a Gram-positive, filamentous actinomycete with a complex developmental life cycle. Genomic analysis revealed that S. venezuelae encodes a large number of two-component systems (TCSs): these consist of a membrane-bound sensor kinase (SK) and a cognate response regulator (RR). These proteins act together to detect and respond to diverse extracellular signals. Some of these systems have been shown to regulate antimicrobial biosynthesis in Streptomyces species, making them very attractive to researchers. The ability of S. venezuelae to sporulate in both liquid and solid cultures has made it an increasingly popular model organism in which to study these industrially and medically important bacteria. Bioinformatic analysis identified 58 TCS operons in S. venezuelae with an additional 27 orphan SK and 18 orphan RR genes. A broader approach identified 15 of the 58 encoded TCSs to be highly conserved in 93 Streptomyces species for which high-quality and complete genome sequences are available. This review attempts to unify the current work on TCS in the streptomycetes, with an emphasis on S. venezuelae.

The Streptomyces master regulator BldD binds c-di-GMP sequentially to create a functional BldD2-(c-di-GMP)4 complex.

Streptomyces are ubiquitous soil bacteria that undergo a complex developmental transition coinciding with their production of antibiotics. This transition is controlled by binding of a novel tetrameric form of the second messenger, 3΄-5΄ cyclic diguanylic acid (c-di-GMP) to the master repressor, BldD. In all domains of life, nucleotide-based second messengers allow a rapid integration of external and internal signals into regulatory pathways that control cellular responses to changing conditions. c-di-GMP can assume alternative oligomeric states to effect different functions, binding to effector proteins as monomers, intercalated dimers or, uniquely in the case of BldD, as a tetramer. However, at physiological concentrations c-di-GMP is a monomer and little is known about how higher oligomeric complexes assemble on effector proteins and if intermediates in assembly pathways have regulatory significance. Here, we show that c-di-GMP binds BldD using an ordered, sequential mechanism and that BldD function necessitates the assembly of the BldD2-(c-di-GMP)4 complex.

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.

Cyclic Dinucleotide-Controlled Regulatory Pathways in Streptomyces Species.

The cyclic dinucleotides cyclic 3',5'-diguanylate (c-di-GMP) and cyclic 3',5'-diadenylate (c-di-AMP) have emerged as key components of bacterial signal transduction networks. These closely related second messengers follow the classical general principles of nucleotide signaling by integrating diverse signals into regulatory pathways that control cellular responses to changing environments. They impact distinct cellular processes, with c-di-GMP having an established role in promoting bacterial adhesion and inhibiting motility and c-di-AMP being involved in cell wall metabolism, potassium homeostasis, and DNA repair. The involvement of c-dinucleotides in the physiology of the filamentous, nonmotile streptomycetes remained obscure until recent discoveries showed that c-di-GMP controls the activity of the developmental master regulator BldD and that c-di-AMP determines the level of the resuscitation-promoting factor A(RpfA) cell wall-remodelling enzyme. Here, I summarize our current knowledge of c-dinucleotide signaling in Streptomyces species and highlight the important roles of c-di-GMP and c-di-AMP in the biology of these antibiotic-producing, multicellular bacteria.

Nucleotide second messenger-mediated regulation of a muralytic enzyme in Streptomyces.

Peptidoglycan degradative enzymes have important roles at many stages during the bacterial life cycle, and it is critical that these enzymes be stringently regulated to avoid compromising the integrity of the cell wall. How this regulation is exerted is of considerable interest: promoter-based control and protein-protein interactions are known to be employed; however, other regulatory mechanisms are almost certainly involved. In the actinobacteria, a class of muralytic enzymes - the 'resuscitation-promoting factors' (Rpfs) - orchestrates the resuscitation of dormant cells. In this study, we have taken a holistic approach to exploring the mechanisms governing RpfA function using the model bacterium Streptomyces coelicolor and have uncovered unprecedented multilevel regulation that is coordinated by three second messengers. Our studies show that RpfA is subject to transcriptional control by the cyclic AMP receptor protein, riboswitch-mediated transcription attenuation in response to cyclic di-AMP, and growth stage-dependent proteolysis in response to ppGpp accumulation. Furthermore, our results suggest that these control mechanisms are likely applicable to cell wall lytic enzymes in other bacteria.

Specialized biopolymers: versatile tools for microbial resilience.

Bacteria produce a wide range of specialized biopolymers that can be classified into polysaccharides, polyamides, and polyesters and are considered to fulfill storage functions. In this review, we highlight recent developments in the field linking metabolism of biopolymers to stress and signaling physiology of the producers and demonstrating that biopolymers contribute to bacterial stress resistance and shape structure and composition of microenvironments. While specialized biopolymers are currently the focus of much attention in biotechnology as innovative and biodegradable materials, our understanding about the regulation and functions of these valuable compounds for the producers, microbial communities, and our environment is still very limited. Addressing open questions about signals, mechanisms, and functions in the area of biopolymers harbors potential for exciting discoveries with high relevance for biotechnology and fundamental research.

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.

Targeting of csgD by the small regulatory RNA RprA links stationary phase, biofilm formation and cell envelope stress in Escherichia coli.

RprA is a small regulatory RNA known to weakly affect the translation of σ(S) (RpoS) in Escherichia coli. Here we demonstrate that csgD, which encodes a stationary phase-induced biofilm regulator, as well as ydaM, which encodes a diguanylate cyclase involved in activating csgD transcription, are novel negatively controlled RprA targets. As shown by extensive mutational analysis, direct binding of RprA to the 5'-untranslated and translational initiation regions of csgD mRNA inhibits translation and reduces csgD mRNA levels. In the case of ydaM mRNA, RprA base-pairs directly downstream of the translational start codon. In a feedforward loop, RprA can thus downregulate > 30 YdaM/CsgD-activated genes including those for adhesive curli fimbriae. However, during early stationary phase, when csgD transcription is strongly activated, the synthesis of csgD mRNA exceeds that of RprA, which allows the accumulation of CsgD protein. This situation is reversed when csgD transcription is shut off - for instance, later in stationary phase or during biofilm formation - or by conditions that further activate RprA expression via the Rcs two-component system. Thus, antagonistic regulation of csgD and RprA at the mRNA level integrates cell envelope stress signals with global gene expression during stationary phase and biofilm formation.

Osmotic stress responses and the biology of the second messenger c-di-AMP in Streptomyces.

Streptomyces are prolific antibiotic producers that thrive in soil, where they encounter diverse environmental cues, including osmotic challenges caused by rainfall and drought. Despite their enormous value in the biotechnology sector, which often relies on ideal growth conditions, how Streptomyces react and adapt to osmotic stress is heavily understudied. This is likely due to their complex developmental biology and an exceptionally broad number of signal transduction systems. With this review, we provide an overview of Streptomyces' responses to osmotic stress signals and draw attention to open questions in this research area. We discuss putative osmolyte transport systems that are likely involved in ion balance control and osmoadaptation and the role of alternative sigma factors and two-component systems (TCS) in osmoregulation. Finally, we highlight the current view on the role of the second messenger c-di-AMP in cell differentiation and the osmotic stress responses with specific emphasis on the two models, S. coelicolor and S. venezuelae.

Recent advances and perspectives in nucleotide second messenger signaling in bacteria.

Nucleotide second messengers act as intracellular 'secondary' signals that represent environmental or cellular cues, i.e. the 'primary' signals. As such, they are linking sensory input with regulatory output in all living cells. The amazing physiological versatility, the mechanistic diversity of second messenger synthesis, degradation, and action as well as the high level of integration of second messenger pathways and networks in prokaryotes has only recently become apparent. In these networks, specific second messengers play conserved general roles. Thus, (p)ppGpp coordinates growth and survival in response to nutrient availability and various stresses, while c-di-GMP is the nucleotide signaling molecule to orchestrate bacterial adhesion and multicellularity. c-di-AMP links osmotic balance and metabolism and that it does so even in Archaea may suggest a very early evolutionary origin of second messenger signaling. Many of the enzymes that make or break second messengers show complex sensory domain architectures, which allow multisignal integration. The multiplicity of c-di-GMP-related enzymes in many species has led to the discovery that bacterial cells are even able to use the same freely diffusible second messenger in local signaling pathways that can act in parallel without cross-talking. On the other hand, signaling pathways operating with different nucleotides can intersect in elaborate signaling networks. Apart from the small number of common signaling nucleotides that bacteria use for controlling their cellular "business," diverse nucleotides were recently found to play very specific roles in phage defense. Furthermore, these systems represent the phylogenetic ancestors of cyclic nucleotide-activated immune signaling in eukaryotes.

Streptomyces development is involved in the efficient containment of viral infections.

The formation of plaques represents the hallmark of phage infection visualizing the clearance of the bacterial lawn in structured environments. In this study, we have addressed the impact of cellular development on phage infection in Streptomyces undergoing a complex developmental life cycle. Analysis of plaque dynamics revealed, after a period of plaque size enlargement, a significant regrowth of transiently phage-resistant Streptomyces mycelium into the lysis zone. Analysis of Streptomyces venezuelae mutant strains defective at different stages of cellular development indicated that this regrowth was dependent on the onset of the formation of aerial hyphae and spores at the infection interface. Mutants restricted to vegetative growth (ΔbldN) featured no significant constriction of plaque area. Fluorescence microscopy further confirmed the emergence of a distinct zone of cells/spores with reduced cell permeability towards propidium iodide staining at the plaque periphery. Mature mycelium was further shown to be significantly less susceptible to phage infection, which is less pronounced in strains defective in cellular development. Transcriptome analysis revealed the repression of cellular development at the early stages of phage infection probably facilitating efficient phage propagation. We further observed an induction of the chloramphenicol biosynthetic gene cluster highlighting phage infection as a trigger of cryptic metabolism in Streptomyces. Altogether, our study emphasizes cellular development and the emergence of transient phage resistance as an important layer of Streptomyces antiviral immunity.

Allosteric regulation of glycogen breakdown by the second messenger cyclic di-GMP.

Streptomyces are our principal source of antibiotics, which they generate concomitant with a complex developmental transition from vegetative hyphae to spores. c-di-GMP acts as a linchpin in this transition by binding and regulating the key developmental regulators, BldD and WhiG. Here we show that c-di-GMP also binds the glycogen-debranching-enzyme, GlgX, uncovering a direct link between c-di-GMP and glycogen metabolism in bacteria. Further, we show c-di-GMP binding is required for GlgX activity. We describe structures of apo and c-di-GMP-bound GlgX and, strikingly, their comparison shows c-di-GMP induces long-range conformational changes, reorganizing the catalytic pocket to an active state. Glycogen is an important glucose storage compound that enables animals to cope with starvation and stress. Our in vivo studies reveal the important biological role of GlgX in Streptomyces glucose availability control. Overall, we identify a function of c-di-GMP in controlling energy storage metabolism in bacteria, which is widespread in Actinobacteria.