Bacterial cyclic diguanylate signaling networks sense temperature.
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ABSTRACT:
SUBMITTER: Almblad H
PROVIDER: S-EPMC8012707 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cyclic di-GMP (c-di-GMP) is a second messenger molecule that regulates the transition between sessile and motile lifestyles in bacteria. Bacteria often encode multiple diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes that produce and degrade c-di-GMP, respectively. Because of multiple inputs into the c-di-GMP-signaling network, it is unclear whether this system functions via high or low specificity. High-specificity signaling is characterized by individual DGCs or PDEs that are specifically associated with downstream c-di-GMP-mediated responses. In contrast, low-specificity signaling is characterized by DGCs or PDEs that modulate a general signal pool, which, in turn, controls a global c-di-GMP-mediated response. To determine whether c-di-GMP functions via high or low specificity in Vibrio cholerae, we correlated the in vivo c-di-GMP concentration generated by seven DGCs, each expressed at eight different levels, to the c-di-GMP-mediated induction of biofilm formation and transcription. There was no correlation between total intracellular c-di-GMP levels and biofilm formation or gene expression when considering all states. However, individual DGCs showed a significant correlation between c-di-GMP production and c-di-GMP-mediated responses. Moreover, the rate of phenotypic change versus c-di-GMP concentration was significantly different between DGCs, suggesting that bacteria can optimize phenotypic output to c-di-GMP levels via expression or activation of specific DGCs. Our results conclusively demonstrate that c-di-GMP does not function via a simple, low-specificity signaling pathway in V. cholerae.
| S-EPMC3411991 | biostudies-literature
Project description:Proteins that metabolize or bind the nucleotide second messenger cyclic diguanylate regulate a wide variety of important processes in bacteria. These processes include motility, biofilm formation, cell division, differentiation, and virulence. The role of cyclic diguanylate signaling in the lifestyle of Legionella pneumophila, the causative agent of Legionnaires' disease, has not previously been examined. The L. pneumophila genome encodes 22 predicted proteins containing domains related to cyclic diguanylate synthesis, hydrolysis, and recognition. We refer to these genes as cdgS (cyclic diguanylate signaling) genes. Strains of L. pneumophila containing deletions of all individual cdgS genes were created and did not exhibit any observable growth defect in growth medium or inside host cells. However, when overexpressed, several cdgS genes strongly decreased the ability of L. pneumophila to grow inside host cells. Expression of these cdgS genes did not affect the Dot/Icm type IVB secretion system, the major determinant of intracellular growth in L. pneumophila. L. pneumophila strains overexpressing these cdgS genes were less cytotoxic to THP-1 macrophages than wild-type L. pneumophila but retained the ability to resist grazing by amoebae. In many cases, the intracellular-growth inhibition caused by cdgS gene overexpression was independent of diguanylate cyclase or phosphodiesterase activities. Expression of the cdgS genes in a Salmonella enterica serovar Enteritidis strain that lacks all diguanylate cyclase activity indicated that several cdgS genes encode potential cyclases. These results indicate that components of the cyclic diguanylate signaling pathway play an important role in regulating the ability of L. pneumophila to grow in host cells.
| S-EPMC3023162 | biostudies-literature
Project description:The cyclic diguanylate (bis-(3'-5')-cyclic dimeric guanosine monophosphate, c-di-GMP) riboswitch is the first known example of a gene-regulatory RNA that binds a second messenger. c-di-GMP is widely used by bacteria to regulate processes ranging from biofilm formation to the expression of virulence genes. The cocrystal structure of the c-di-GMP responsive GEMM riboswitch upstream of the tfoX gene of Vibrio cholerae reveals the second messenger binding the RNA at a three-helix junction. The two-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch using canonical and noncanonical base-pairing as well as intercalation. These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger. Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.
| S-EPMC2925111 | biostudies-literature
Project description:The second messenger cyclic diguanylate (c-di-GMP) controls diverse cellular processes among bacteria. Diguanylate cyclases synthesize c-di-GMP, whereas it is degraded by c-di-GMP-specific phosphodiesterases (PDEs). Nearly 80% of these PDEs are predicted to depend on the catalytic function of glutamate-alanine-leucine (EAL) domains, which hydrolyze a single phosphodiester group in c-di-GMP to produce 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). However, to degrade pGpG and prevent its accumulation, bacterial cells require an additional nuclease, the identity of which remains unknown. Here we identify oligoribonuclease (Orn)-a 3'→5' exonuclease highly conserved among Actinobacteria, Beta-, Delta- and Gammaproteobacteria-as the primary enzyme responsible for pGpG degradation in Pseudomonas aeruginosa cells. We found that a P. aeruginosa Δorn mutant had high intracellular c-di-GMP levels, causing this strain to overexpress extracellular polymers and overproduce biofilm. Although recombinant Orn degraded small RNAs in vitro, this enzyme had a proclivity for degrading RNA oligomers comprised of two to five nucleotides (nanoRNAs), including pGpG. Corresponding with this activity, Δorn cells possessed highly elevated pGpG levels. We found that pGpG reduced the rate of c-di-GMP degradation in cell lysates and inhibited the activity of EAL-dependent PDEs (PA2133, PvrR, and purified recombinant RocR) from P. aeruginosa. This pGpG-dependent inhibition was alleviated by the addition of Orn. These data suggest that elevated levels of pGpG exert product inhibition on EAL-dependent PDEs, thereby increasing intracellular c-di-GMP in Δorn cells. Thus, we propose that Orn provides homeostatic control of intracellular pGpG under native physiological conditions and that this activity is fundamental to c-di-GMP signal transduction.
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Project description:Cyclic di-nucleotides are important secondary signaling molecules in bacteria that regulate a wide range of processes. In this study, we found that Caenorhabditis elegans can detect and are attracted to multiple signal molecules produced by Vibrio cholerae, specifically the 3',5'-cyclic diguanylate (c-di-GMP), even though this bacterium kills the host at a high rate. C-di-GMP is sensed through C. elegans olfactory AWC neurons, which then evokes a series of signal transduction pathways that lead to reduced activity of two key stress response transcription factors, SKN-1 and HSF-1, and weakened innate immunity. Taken together, our study elucidates the role of c-di-GMP in interkingdom communication. For C. elegans, bacterial c-di-GMP may serve as a cue that they can use to detect food. On the other hand, preexposure to low concentrations of c-di-GMP may impair their immune response, which could facilitate bacterial invasion and survival.
| S-EPMC7679379 | biostudies-literature
Project description:The second messenger cyclic diguanylate (c-di-GMP) is ubiquitously used by bacteria to modulate and shift between different phenotypes including motility, biofilm formation and virulence. Here we show that c-di-GMP-associated genes are widespread on plasmids and that enzymes that synthesize or degrade c-di-GMP are preferentially encoded on transmissible plasmids. Additionally, expression of enzymes that synthesize c-di-GMP was found to increase both biofilm formation and, interestingly, conjugative plasmid transfer rates.
| S-EPMC6092375 | biostudies-literature
Project description:Vibrio cholerae, the causative agent of the disease cholera, can generate rugose variants that have an increased capacity to form biofilms. Rugosity and biofilm formation are critical for the environmental survival and transmission of the pathogen, and these processes are controlled by cyclic diguanylate (c-di-GMP) signaling systems. c-di-GMP is produced by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). Proteins that contain GGDEF domains act as DGCs, whereas proteins that contain EAL or HD-GYP domains act as PDEs. In the V. cholerae genome there are 62 genes that are predicted to encode proteins capable of modulating the cellular c-di-GMP concentration. We previously identified two DGCs, VpvC and CdgA, that can control the switch between smooth and rugose. To identify other c-di-GMP signaling proteins involved in rugosity, we generated in-frame deletion mutants of all genes predicted to encode proteins with GGDEF and EAL domains and then searched for mutants with altered rugosity. In this study, we identified two new genes, cdgG and cdgH, involved in rugosity control. We determined that CdgH acts as a DGC and positively regulates rugosity, whereas CdgG does not have DGC activity and negatively regulates rugosity. In addition, epistasis analysis with CdgG, CdgH, and other DGCs and PDEs controlling rugosity revealed that CdgG and CdgH act in parallel with previously identified c-di-GMP signaling proteins to control rugosity in V. cholerae. We also determined that PilZ domain-containing c-di-GMP binding proteins contribute minimally to rugosity, indicating that there are additional c-di-GMP binding proteins controlling rugosity in V. cholerae.
| S-EPMC2576663 | biostudies-literature
Project description:UnlabelledThe ecological and evolutionary forces that promote and maintain diversity in biofilms are not well understood. To quantify these forces, three Pseudomonas aeruginosa populations were experimentally evolved from strain PA14 in a daily cycle of attachment, assembly, and dispersal for 600 generations. Each biofilm population evolved diverse colony morphologies and mutator genotypes defective in DNA mismatch repair. This diversity enhanced population fitness and biofilm output, owing partly to rare, early colonizing mutants that enhanced attachment of others. Evolved mutants exhibited various levels of the intracellular signal cyclic-di-GMP, which associated with their timing of adherence. Manipulating cyclic-di-GMP levels within individual mutants revealed a network of interactions in the population that depended on various attachment strategies related to this signal. Diversification in biofilms may therefore arise and be reinforced by initial colonists that enable community assembly.ImportanceHow biofilm diversity assembles, evolves, and contributes to community function is largely unknown. This presents a major challenge for understanding evolution during chronic infections and during the growth of all surface-associated microbes. We used experimental evolution to probe these dynamics and found that diversity, partly related to altered cyclic-di-GMP levels, arose and persisted due to the emergence of ecological interdependencies related to attachment patterns. Clonal isolates failed to capture population attributes, which points to the need to account for diversity in infections. More broadly, this study offers an experimental framework for linking phenotypic variation to distinct ecological strategies in biofilms and for studying eco-evolutionary interactions.
| S-EPMC5019052 | biostudies-literature
Project description:Cyclic-diGMP is a bacterial messenger that regulates many physiological processes, including many attributed to pathogenicity. Bacteria synthesize cyclic-diGMP from GTP using diguanylate cyclases; its hydrolysis is catalyzed by phosphodiesterases. Here we report the over-expression and purification of a bi-functional diguanylate cyclase-phosphodiesterase from Agrobacterium vitis S4. Using homology modeling and primary structure alignment, we identify several amino acids predicted to participate in the phosphodiesterase reaction. Upon altering selected residues, we obtain variants of the enzyme that efficiently and quantitatively catalyze the synthesis of cyclic-diGMP from GTP without hydrolysis to pGpG. Additionally, we identify a variant that produces cyclic-diGMP while immobilized to NiNTA beads and can catalyze the conversion of [α-32P]-GTP to [32P]-cyclic-diGMP. In short, we characterize a novel cyclic-diGMP processing enzyme and demonstrate its utility for efficient and cost-effective production of cyclic-diGMP, as well as modified cyclic-diGMP molecules, for use as probes in studying the many important biological processes mediated by cyclic-diGMP.
| S-EPMC5466042 | biostudies-literature
Project description:A series of allosteric ribozymes that respond to the bacterial second messenger cyclic diguanosyl-5'-monophosphate (c-di-GMP) have been created by using in vitro selection. An RNA library was generated by using random-sequence bridges to join a hammerhead self-cleaving ribozyme to an aptamer from a natural c-di-GMP riboswitch. Specific bridge sequences, called communication modules, emerged through two in vitro selection efforts that either activate or inhibit ribozyme self-cleavage upon ligand binding to the aptamer. Representative RNAs were found that exhibit EC(50) (half-maximal effective concentration) values for c-di-GMP as low as 90 nM and IC(50) (half-maximal inhibitory concentration) values as low as 180 nM. The allosteric RNAs display molecular recognition characteristics that mimic the high discriminatory ability of the natural aptamer. Some engineered RNAs operate with ribozyme rate constants approaching that of the parent hammerhead ribozyme. By use of these allosteric ribozymes, cytoplasmic concentrations of c-di-GMP in three mutant strains of Escherichia coli were quantitatively estimated from cell lysates. Our findings demonstrate that engineered c-di-GMP-sensing ribozymes can be used as convenient tools to monitor c-di-GMP levels from complex biological or chemical samples. Moreover, these ribozymes could be employed in high-throughput screens to identify compounds that trigger c-di-GMP riboswitch function.
| S-EPMC4140410 | biostudies-literature