Tetracyclic homoisoflavanoid (+)-brazilin: a natural product inhibits c-di-AMP-producing enzyme and Streptococcus mutans biofilms.

The tenacious biofilms formed by Streptococcus mutans are resistant to conventional antibiotics and current treatments. There is a growing need for novel therapeutics that selectively inhibit S. mutans biofilms while preserving the normal oral microenvironment. Previous studies have shown that increased levels of cyclic di-AMP, an important secondary messenger synthesized by diadenylate cyclase (DAC), favored biofilm formation in S. mutans. Thus, targeting S. mutans DAC is a novel strategy to inhibit S. mutans biofilms. We screened a small NCI library of natural products using a fluorescence detection assay. (+)-Brazilin, a tetracyclic homoisoflavanoid found in the heartwood of Caesalpinia sappan, was identified as one of the 11 "hits," with the greatest reduction (>99%) in fluorescence at 100 µM. The smDAC inhibitory profiles of the 11 "hits" established by a quantitative high-performance liquid chromatography assay revealed that (+)-brazilin had the most enzymatic inhibitory activity (87% at 100 µM) and was further studied to determine its half maximal inhibitory concentration (IC50 = 25.1 ± 0.98 µM). (+)-Brazilin non-competitively inhibits smDAC's enzymatic activity (Ki = 140.0 ± 27.13 µM), as determined by a steady-state Michaelis-Menten kinetics assay. In addition, (+)-brazilin's binding profile with smDAC (Kd = 11.87 µM) was illustrated by a tyrosine intrinsic fluorescence quenching assay. Furthermore, at low micromolar concentrations, (+)-brazilin selectively inhibited the biofilm of S. mutans (IC50 = 21.0 ± 0.60 µM) and other oral bacteria. S. mutans biofilms were inhibited by a factor of 105 in colony-forming units when treated with 50 µM (+)-brazilin. In addition, a significant dose-dependent reduction in extracellular DNA and glucan levels was evident by fluorescence microscopy imaging of S. mutans biofilms exposed to different concentrations of (+)-brazilin. Furthermore, colonization of S. mutans on a representative model of enamel using suspended hydroxyapatite discs showed a >90% reduction with 50 µM (+)-brazilin. In summary, we have identified a drug-like natural product inhibitor of S. mutans biofilm that not only binds to smDAC but can also inhibit the function of smDAC. (+)-Brazilin could be a good candidate for further development as a potent therapeutic for the prevention and treatment of dental caries.IMPORTANCEThis study represents a significant advancement in our understanding of potential therapeutic options for combating cariogenic biofilms produced by Streptococcus mutans. The research delves into the use of (+)-brazilin, a natural product, as a potent inhibitor of Streptococcus mutans' diadenylate cyclase (smDAC), an enzyme crucial in the formation of biofilms. The study establishes (+)-brazilin as a non-competitive inhibitor of smDAC while providing initial insights into its binding mechanism. What makes this finding even more promising is that (+)-brazilin does not limit its inhibitory effects to S. mutans alone. Instead, it demonstrates efficacy in hindering biofilms in other oral bacteria as well. The broader spectrum of anti-biofilm activity suggests that (+)-brazilin could potentially serve as a versatile tool in a natural product-based treatment for combating a range of conditions caused by resilient biofilms.

A small step towards an important goal: fragment screen of the c-di-AMP-synthesizing enzyme CdaA.

CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.

Mitochondrial transcription factor A (TFAM) has 5'-deoxyribose phosphate lyase activity in vitro.

Mitochondrial DNA (mtDNA) plays a key role in mitochondrial and cellular functions. mtDNA is maintained by active DNA turnover and base excision repair (BER). In BER, one of the toxic repair intermediates is 5'-deoxyribose phosphate (5'dRp). Human mitochondrial DNA polymerase γ has weak dRp lyase activities, and another known dRp lyase in the nucleus, human DNA polymerase ß, can also localize to mitochondria in certain cell and tissue types. Nonetheless, whether additional proteins have the ability to remove 5'dRp in mitochondria remains unknown. Our prior work on the AP lyase activity of mitochondrial transcription factor A (TFAM) has prompted us to examine its ability to remove 5'dRp residues in vitro. TFAM is the primary DNA-packaging factor in human mitochondria and interacts with mitochondrial DNA extensively. Our data demonstrate that TFAM has the dRp lyase activity with different DNA substrates. Under single-turnover conditions, TFAM removes 5'dRp residues at a rate comparable to that of DNA polymerase (pol) ß, albeit slower than that of pol λ. Among the three proteins examined, pol λ shows the highest single-turnover rates in dRp lyase reactions. The catalytic effect of TFAM is facilitated by lysine residues of TFAM via Schiff base chemistry, as evidenced by the observation of dRp-lysine adducts in mass spectrometry experiments. The catalytic effect of TFAM observed here is analogous to the AP lyase activity of TFAM reported previously. Together, these results suggest a potential role of TFAM in preventing the accumulation of toxic DNA repair intermediates.

The dual GGDEF/EAL domain enzyme PA0285 is a Pseudomonas species housekeeping phosphodiesterase regulating early attachment and biofilm architecture.

Bacterial lifestyles depend on conditions encountered during colonization. The transition between planktonic and biofilm growth is dependent on the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclases (DGCs) activity favor biofilm formation, while low levels were maintained by phosphodiesterases (PDE) encourage planktonic lifestyle. The activity of these enzymes can be modulated by stimuli-sensing domains such as Per-ARNT-Sim (PAS). In Pseudomonas aeruginosa, more than 40 PDE/DGC are involved in c-di-GMP homeostasis, including 16 dual proteins possessing both canonical DGC and PDE motifs, that is, GGDEF and EAL, respectively. It was reported that deletion of the EAL/GGDEF dual enzyme PA0285, one of five c-di-GMP-related enzymes conserved across all Pseudomonas species, impacts biofilms. PA0285 is anchored in the membrane and carries two PAS domains. Here, we confirm that its role is conserved in various P. aeruginosa strains and in Pseudomonas putida. Deletion of PA0285 impacts the early stage of colonization, and RNA-seq analysis suggests that expression of cupA fimbrial genes is involved. We demonstrate that the C-terminal portion of PA0285 encompassing the GGDEF and EAL domains binds GTP and c-di-GMP, respectively, but only exhibits PDE activity in vitro. However, both GGDEF and EAL domains are important for PA0285 PDE activity in vivo. Complementation of the PA0285 mutant strain with a copy of the gene encoding the C-terminal GGDEF/EAL portion in trans was not as effective as complementation with the full-length gene. This suggests the N-terminal transmembrane and PAS domains influence the PDE activity in vivo, through modulating the protein conformation.

Oxygen-selective regulation of cyclic di-GMP synthesis by a globin coupled sensor with a shortened linking domain modulates Shewanella sp. ANA-3 biofilm.

Bacteria utilize heme proteins, such as globin coupled sensors (GCSs), to sense and respond to oxygen levels. GCSs are predicted in almost 2000 bacterial species and consist of a globin domain linked by a central domain to a variety of output domains, including diguanylate cyclase domains that synthesize c-di-GMP, a major regulator of biofilm formation. To investigate the effects of middle domain length and heme edge residues on GCS diguanylate cyclase activity and cellular function, a putative diguanylate cyclase-containing GCS from Shewanella sp. ANA-3 (SA3GCS) was characterized. Binding of O2 to the heme resulted in activation of diguanylate cyclase activity, while NO and CO binding had minimal effects on catalysis, demonstrating that SA3GCS exhibits greater ligand selectivity for cyclase activation than many other diguanylate cyclase-containing GCSs. Small angle X-ray scattering analysis of dimeric SA3GCS identified movement of the cyclase domains away from each other, while maintaining the globin dimer interface, as a potential mechanism for regulating cyclase activity. Comparison of the Shewanella ANA-3 wild type and SA3GCS deletion (ΔSA3GCS) strains identified changes in biofilm formation, demonstrating that SA3GCS diguanylate cyclase activity modulates Shewanella phenotypes.

FlhF affects the subcellular clustering of WspR through HsbR in Pseudomonas aeruginosa.

In bacteria, the second messenger cyclic di-GMP (c-di-GMP) is synthesized and degraded by multiple diguanylate cyclases (DGCs) and phosphodiesterases. A high level of c-di-GMP induces biofilm formation and represses motility. WspR, a hybrid response regulator DGC, produces c-di-GMP when it is phosphorylated. FlhF, a signal recognition particle-type GTPase, is initially localized to the cell poles and is indispensable for polar flagellar localization in Pseudomonas aeruginosa. In this study, we report that deletion of flhF affected biofilm formation and the c-di-GMP level in P. aeruginosa. Phenotypic analysis of a flhF knockout mutant revealed increased biofilm formation, wrinkled colonies on Congo red agar, and an elevated c-di-GMP level compared to the wild-type strain, PAO1. Yeast and bacterial two-hybrid systems showed that FlhF binds to the response regulator HsbR, and HsbR binds to WspR. Deletion of hsbR or wspR in the ΔflhF background abolished the phenotype of ΔflhF. In addition, confocal microscopy demonstrated that WspR-GFP was distributed throughout the cytoplasm and formed a visible cluster at one cell pole in PAO1 and ΔhsbR, but it was mainly distributed as visible clusters at the lateral side of the periplasm and with visible clusters at both cell poles in ΔflhF. These findings suggest that FlhF influences the subcellular cluster and localization of WspR and negatively modulates WspR DGC activity in a manner dependent on HsbR. Together, our findings demonstrate a novel mechanism for FlhF modulating the lifestyle transition between motility and biofilm via HsbR to regulate the DGC activity of WspR.IMPORTANCECyclic di-GMP (c-di-GMP) is a second messenger that controls flagellum biosynthesis, adhesion, virulence, motility, exopolysaccharide production, and biofilm formation in bacteria. Recent research has shown that distinct diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) produce highly specific outputs. Some DGCs and PDEs contribute to the total global c-di-GMP concentration, but others only affect local c-di-GMP in a microenvironment. However, the underlying mechanisms are unclear. Here, we report that FlhF affects the localization and DGC activity of WspR via HsbR and is implicated in local c-di-GMP signaling in Pseudomonas aeruginosa. This study establishes the link between the c-di-GMP signaling system and the flagellar localization and provides insight for understanding the complex regulatory network of c-di-GMP signaling.

Emerging roles for diguanylate cyclase during the evolution of soma in dictyostelia.

BACKGROUND: Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid social amoebas acquired diguanylate cyclase (dgcA) by horizontal gene transfer. Dictyostelium discoideum (Ddis) in taxon group 4 uses c-di-GMP as a secreted signal to induce differentiation of stalk cells, the ancestral somatic cell type that supports the propagating spores. We here investigated how this role for c-di-GMP evolved in Dictyostelia by exploring dgcA function in the group 2 species Polysphondylium pallidum (Ppal) and in Polysphondylium violaceum (Pvio), which resides in a small sister clade to group 4. RESULTS: Similar to Ddis, dgcA is upregulated after aggregation in Ppal and Pvio and predominantly expressed in the anterior region and stalks of emerging fruiting bodies. DgcA null mutants in Ppal and Pvio made fruiting bodies with very long and thin stalks and only few spores and showed delayed aggregation and larger aggregates, respectively. Ddis dgcA- cells cannot form stalks at all, but showed no aggregation defects. The long, thin stalks of Ppal and Pvio dgcA- mutants were also observed in acaA- mutants in these species. AcaA encodes adenylate cyclase A, which mediates the effects of c-di-GMP on stalk induction in Ddis. Other factors that promote stalk formation in Ddis are DIF-1, produced by the polyketide synthase StlB, low ammonia, facilitated by the ammonia transporter AmtC, and high oxygen, detected by the oxygen sensor PhyA (prolyl 4-hydroxylase). We deleted the single stlB, amtC and phyA genes in Pvio wild-type and dgcA- cells. Neither of these interventions affected stalk formation in Pvio wild-type and not or very mildly exacerbated the long thin stalk phenotype of Pvio dgcA- cells. CONCLUSIONS: The study reveals a novel role for c-di-GMP in aggregation, while the reduced spore number in Pvio and Ppal dgcA- is likely an indirect effect, due to depletion of the cell pool by the extended stalk formation. The results indicate that in addition to c-di-GMP, Dictyostelia ancestrally used an as yet unknown factor for induction of stalk formation. The activation of AcaA by c-di-GMP is likely conserved throughout Dictyostelia.

Phylogenetic Analysis and Characterization of Diguanylate Cyclase and Phosphodiesterase in Planktonic Filamentous Cyanobacterium Arthrospira sp.

Cyclic di-GMP (c-di-GMP) is a second messenger of intracellular communication in bacterial species, which widely modulates diverse cellular processes. However, little is known about the c-di-GMP network in filamentous multicellular cyanobacteria. In this study, we preliminarily investigated the c-di-GMP turnover proteins in Arthrospira based on published protein data. Bioinformatics results indicate the presence of at least 149 potential turnover proteins in five Arthrospira subspecies. Some proteins are highly conserved in all tested Arthrospira, whereas others are specifically found only in certain subspecies. To further validate the protein catalytic activity, we constructed a riboswitch-based c-di-GMP expression assay system in Escherichia coli and confirmed that a GGDEF domain protein, Adc11, exhibits potential diguanylate cyclase activity. Moreover, we also evaluated a protein with a conserved HD-GYP domain, Ahd1, the expression of which significantly improved the swimming ability of E. coli. Enzyme-linked immunosorbent assay also showed that overexpression of Ahd1 reduced the intracellular concentration of c-di-GMP, which is presumed to exhibit phosphodiesterase activity. Notably, meta-analyses of transcriptomes suggest that Adc11 and Ahd1 are invariable. Overall, this work confirms the possible existence of a functional c-di-GMP network in Arthrospira, which will provide support for the revelation of the biological function of the c-di-GMP system in Arthrospira.

Illuminating the inner workings of a natural protein switch: Blue-light sensing in LOV-activated diguanylate cyclases.

Regulatory proteins play a crucial role in adaptation to environmental cues. Especially for lifestyle transitions, such as cell proliferation or apoptosis, switch-like characteristics are desirable. While nature frequently uses regulatory circuits to amplify or dampen signals, stand-alone protein switches are interesting for applications like biosensors, diagnostic tools, or optogenetics. However, such stand-alone systems frequently feature limited dynamic and operational ranges and suffer from slow response times. Here, we characterize a LOV-activated diguanylate cyclase (LadC) that offers precise temporal and spatial control of enzymatic activity with an exceptionally high dynamic range over four orders of magnitude. To establish this pronounced activation, the enzyme exhibits a two-stage activation process in which its activity is inhibited in the dark by caging its effector domains and stimulated upon illumination by the formation of an extended coiled-coil. These switch-like characteristics of the LadC system can be used to develop new optogenetic tools with tight regulation.

A Second Role for the Second Messenger Cyclic-di-GMP in E. coli: Arresting Cell Growth by Altering Metabolic Flow.

c-di-GMP primarily controls motile to sessile transitions in bacteria. Diguanylate cyclases (DGCs) catalyze the synthesis of c-di-GMP from two GTP molecules. Typically, bacteria encode multiple DGCs that are activated by specific environmental signals. Their catalytic activity is modulated by c-di-GMP binding to autoinhibitory sites (I-sites). YfiN is a conserved inner membrane DGC that lacks these sites. Instead, YfiN activity is directly repressed by periplasmic YfiR, which is inactivated by redox stress. In Escherichia coli, an additional envelope stress causes YfiN to relocate to the mid-cell to inhibit cell division by interacting with the division machinery. Here, we report a third activity for YfiN in E. coli, where cell growth is inhibited without YfiN relocating to the division site. This action of YfiN is only observed when the bacteria are cultured on gluconeogenic carbon sources, and is dependent on absence of the autoinhibitory sites. Restoration of I-site function relieves the growth-arrest phenotype, and disabling this function in a heterologous DGC causes acquisition of this phenotype. Arrested cells are tolerant to a wide range of antibiotics. We show that the likely cause of growth arrest is depletion of cellular GTP from run-away synthesis of c-di-GMP, explaining the dependence of growth arrest on gluconeogenic carbon sources that exhaust more GTP during production of glucose. This is the first report of c-di-GMP-mediated growth arrest by altering metabolic flow. IMPORTANCE The c-di-GMP signaling network in bacteria not only controls a variety of cellular processes such as motility, biofilms, cell development, and virulence, but does so by a dizzying array of mechanisms. The DGC YfiN singularly represents the versatility of this network in that it not only inhibits motility and promotes biofilms, but also arrests growth in Escherichia coli by relocating to the mid-cell and blocking cell division. The work described here reveals that YfiN arrests growth by yet another mechanism in E. coli, changing metabolic flow. This function of YfiN, or of DGCs without autoinhibitory I-sites, may contribute to antibiotic tolerant persisters in relevant niches such as the gut where gluconeogenic sugars are found.

All DACs in a Row: Domain Architectures of Bacterial and Archaeal Diadenylate Cyclases.

Cyclic dimeric AMP (c-di-AMP) is a widespread second messenger that controls such key functions as osmotic homeostasis, peptidoglycan biosynthesis, and response to various stresses. C-di-AMP is synthesized by diadenylate cyclases that contain the DAC (DisA_N) domain, which was originally characterized as the N-terminal domain in the DNA integrity scanning protein DisA. In other experimentally studied diadenylate cyclases, DAC domain is typically located at the protein C termini and its enzymatic activity is controlled by one or more N-terminal domains. As in other bacterial signal transduction proteins, these N-terminal modules appear to sense environmental or intracellular signals through ligand binding and/or protein-protein interactions. Studies of bacterial and archaeal diadenylate cyclases also revealed numerous sequences with uncharacterized N-terminal regions. This work provides a comprehensive review of the N-terminal domains of bacterial and archaeal diadenylate cyclases, including the description of five previously undefined domains and three PK_C-related domains of the DacZ_N superfamily. These data are used to classify diadenylate cyclases into 22 families, based on their conserved domain architectures and the phylogeny of their DAC domains. Although the nature of the regulatory signals remains obscure, the association of certain dac genes with anti-phage defense CBASS systems and other phage-resistance genes suggests that c-di-AMP might also be involved in the signaling of phage infection.

Two cyanobacterial response regulators with diguanylate cyclase activity, Rre2 and Rre8, participate in biofilm formation.

Phototrophic bacteria face diurnal variations of environmental conditions such as light and osmolarity that affect their carbon metabolism and ability to generate organic compounds. The model cyanobacterium, Synechocystis sp. PCC 6803 forms a biofilm when it encounters extreme conditions like high salt stress, but the molecular mechanisms involved in perception of environmental changes that lead to biofilm formation are unknown. Here, we studied two two-component regulatory systems (TCSs) that contain diguanylate cyclases (DGCs), which produce the second messenger c-di-GMP, as potential components of the biofilm-inducing signaling pathway in Synechocystis. Analysis of single mutants provided evidence for involvement of the response regulators, Rre2 and Rre8 in biofilm formation. A bacterial two-hybrid assay showed that Rre2 and Rre8 each formed a TCS with a specific histidine kinase, Hik12 and Hik14, respectively. The in vitro assay showed that Rre2 had DGC activity regardless of its de/phosphorylation status, whereas Rre8 required phosphorylation for DGC activity. Hik14-Rre8 likely functioned as an inducible sensing system in response to environmental change. Biofilm assays with Synechocystis mutants suggested that pairs of hik12-rre2 and hik14-rre8 responded to high salinity-induced biofilm formation. Inactivation of hik12-rre2 and hik14-rre8 did not affect the performance of the light reactions of photosynthesis. These data suggest that Hik12-Rre2 and Hik14-Rre8 participate in biofilm formation in Synechocystis by regulating c-di-GMP production via the DGC activity of Rre2 and Rre8.

H-NOX Regulates Biofilm Formation in Agrobacterium Vitis in Response to NO.

Transitions between motile and biofilm lifestyles are highly regulated and fundamental to microbial pathogenesis. H-NOX (heme-nitric oxide/oxygen-binding domain) is a key regulator of bacterial communal behaviors, such as biofilm formation. A predicted bifunctional cyclic di-GMP metabolizing enzyme, composed of diguanylate cyclase and phosphodiesterase (PDE) domains (avi_3097), is annotated downstream of an hnoX gene in Agrobacterium vitis S4. Here, we demonstrate that avH-NOX is a nitric oxide (NO)-binding hemoprotein that binds to and regulates the activity of avi_3097 (avHaCE; H-NOX-associated cyclic di-GMP processing enzyme). Kinetic analysis of avHaCE indicates a ∼four-fold increase in PDE activity in the presence of NO-bound avH-NOX. Biofilm analysis with crystal violet staining reveals that low concentrations of NO reduce biofilm growth in the wild-type A. vitis S4 strain, but the mutant ΔhnoX strain has no NO phenotype, suggesting that H-NOX is responsible for the NO biofilm phenotype in A. vitis. Together, these data indicate that avH-NOX enhances cyclic di-GMP degradation to reduce biofilm formation in response to NO in A. vitis.

Elongation factor P modulates Acinetobacter baumannii physiology and virulence as a cyclic dimeric guanosine monophosphate effector.

Cyclic diguanosine monophosphate (c-di-GMP) is widely used by bacteria to control biological functions in response to diverse signals or cues. A previous study showed that potential c-di-GMP metabolic enzymes play a role in the regulation of biofilm formation and motility in Acinetobacter baumannii. However, it was unclear whether and how A. baumannii cells use c-di-GMP signaling to modulate biological functions. Here, we report that c-di-GMP is an important intracellular signal in the modulation of biofilm formation, motility, and virulence in A. baumannii. The intracellular level of c-di-GMP is principally controlled by the diguanylate cyclases (DGCs) A1S_1695, A1S_2506, and A1S_3296 and the phosphodiesterase (PDE) A1S_1254. Intriguingly, we revealed that A1S_2419 (an elongation factor P [EF-P]), is a novel c-di-GMP effector in A. baumannii. Response to a c-di-GMP signal boosted A1S_2419 activity to rescue ribosomes from stalling during synthesis of proteins containing consecutive prolines and thus regulate A. baumannii physiology and pathogenesis. Our study presents a unique and widely conserved effector that controls bacterial physiology and virulence by sensing the second messenger c-di-GMP.

A complete twelve-gene deletion null mutant reveals that cyclic di-GMP is a global regulator of phase-transition and host colonization in Erwinia amylovora.

Cyclic-di-GMP (c-di-GMP) is an essential bacterial second messenger that regulates biofilm formation and pathogenicity. To study the global regulatory effect of individual components of the c-di-GMP metabolic system, we deleted all 12 diguanylate cyclase (dgc) and phosphodiesterase (pde)-encoding genes in E. amylovora Ea1189 (Ea1189Δ12). Ea1189Δ12 was impaired in surface attachment due to a transcriptional dysregulation of the type IV pilus and the flagellar filament. A transcriptomic analysis of surface-exposed WT Ea1189 and Ea1189Δ12 cells indicated that genes involved in metabolism, appendage generation and global transcriptional/post-transcriptional regulation were differentially regulated in Ea1189Δ12. Biofilm formation was regulated by all 5 Dgcs, whereas type III secretion and disease development were differentially regulated by specific Dgcs. A comparative transcriptomic analysis of Ea1189Δ8 (lacks all five enzymatically active dgc and 3 pde genes) against Ea1189Δ8 expressing specific dgcs, revealed the presence of a dual modality of spatial and global regulatory frameworks in the c-di-GMP signaling network.

Characterisation of sequence-structure-function space in sensor-effector integrators of phytochrome-regulated diguanylate cyclases.

Understanding the relationship between protein sequence, structure and function is one of the fundamental challenges in biochemistry. A direct correlation, however, is often not trivial since protein dynamics also play an important functional role-especially in signal transduction processes. In a subfamily of bacterial light sensors, phytochrome-activated diguanylate cyclases (PadCs), a characteristic coiled-coil linker element connects photoreceptor and output module, playing an essential role in signal integration. Combining phylogenetic analyses with biochemical characterisations, we were able to show that length and composition of this linker determine sensor-effector function and as such are under considerable evolutionary pressure. The linker length, together with the upstream PHY-specific domain, influences the dynamic range of effector activation and can even cause light-induced enzyme inhibition. We demonstrate phylogenetic clustering according to linker length, and the development of new linker lengths as well as new protein function within linker families. The biochemical characterisation of PadC homologs revealed that the functional coupling of PHY dimer interface and linker element defines signal integration and regulation of output functionality. A small subfamily of PadCs, characterised by a linker length breaking the coiled-coil pattern, shows a markedly different behaviour from other homologs. The effect of the central helical spine on PadC function highlights its essential role in signal integration as well as direct regulation of diguanylate cyclase activity. Appreciation of sensor-effector linkers as integrator elements and their coevolution with sensory modules is a further step towards the use of functionally diverse homologs as building blocks for rationally designed optogenetic tools.

Antisense Oligonucleotide Rescue of Deep-Intronic Variants Activating Pseudoexons in the 6-Pyruvoyl-Tetrahydropterin Synthase Gene.

We report two new 6-pyruvoyl-tetrahydropterin synthase splicing variants identified through genomic sequencing and transcript analysis in a patient with tetrahydrobiopterin deficiency, presenting with hyperphenylalaninemia and monoamine neurotransmitter deficiency. Variant c.243 + 3A>G causes exon 4 skipping. The deep-intronic c.164-672C>T variant creates a potential 5' splice site that leads to the inclusion of four overlapping pseudoexons, corresponding to exonizations of an antisense short interspersed nuclear element AluSq repeat sequence. Two of the identified pseudoexons have been reported previously, activated by different deep-intronic variants, and were also detected at residual levels in control cells. Interestingly, the predominant pseudoexon is nearly identical to a disease causing activated pseudoexon in the F8 gene, with the same 3' and 5' splice sites. Splice switching antisense oligonucleotides (SSOs) were designed to hybridize with splice sites and/or predicted binding sites for regulatory splice factors. Different SSOs corrected the aberrant pseudoexon inclusion, both in minigenes and in fibroblasts from patients carrying the new variant c.164-672C>T or the previously described c.164-716A>T. With SSO treatment PTPS protein was recovered, illustrating the therapeutic potential of the approach, for patients with different pseudoexon activating variants in the region. In addition, the natural presence of pseudoexons in the wild type context suggests the possibility of applying the antisense strategy in patients with hypomorphic PTS variants with the purpose of upregulating their expression to increase overall protein and activity.

Adaptation of Listeria monocytogenes to perturbation of c-di-AMP metabolism underpins its role in osmoadaptation and identifies a fosfomycin uptake system.

The human pathogen Listeria monocytogenes synthesizes and degrades c-di-AMP using the diadenylate cyclase CdaA and the phosphodiesterases PdeA and PgpH respectively. c-di-AMP is essential because it prevents the uncontrolled uptake of osmolytes. Here, we studied the phenotypes of cdaA, pdeA, pgpH and pdeA pgpH mutants with defects in c-di-AMP metabolism and characterized suppressor mutants restoring their growth defects. The characterization of the pdeA pgpH mutant revealed that the bacteria show growth defects in defined medium, a phenotype that is invariably suppressed by mutations in cdaA. The previously reported growth defect of the cdaA mutant in rich medium is suppressed by mutations that osmotically stabilize the c-di-AMP-free strain. We also found that the cdaA mutant has an increased sensitivity against isoleucine. The isoleucine-dependent growth inhibition of the cdaA mutant is suppressed by codY mutations that likely reduce the DNA-binding activity of encoded CodY variants. Moreover, the characterization of the cdaA suppressor mutants revealed that the Opp oligopeptide transport system is involved in the uptake of the antibiotic fosfomycin. In conclusion, the suppressor analysis corroborates a key function of c-di-AMP in controlling osmolyte homeostasis in L. monocytogenes.

Diguanylate Cyclase (DGC) Implicated in the Synthesis of Multiple Bacteriocins via the Flagellar-Type III Secretion System Produced by Pectobacterium carotovorum subsp. carotovorum.

The plant pathogen Pectobacterium carotovorum subsp. carotovorum (previously Erwinia carotovora subsp. carotovora) causes soft rot and stem rot diseases in a variety of crops, including Chinese cabbage, potato, and tomato. The flagellar-type III secretion systems were used by Pcc's virulence mechanism to export proteins or bacteriocins to the outside of the cell. DGC, a virulence factor that cyclizes c-di-GMP, a common secondary signal in physiological processes and toxin control systems of many bacteria, was discovered in Pcc's genomic DNA. The dgc gene in Pcc was blocked using the method of homologous recombination in our study. In the in vivo setting, the results demonstrated that the dgc knockout strain does not release low molecular weight bacteriocins. The bacteriocin gene (carocin S2, carocin S3, carocin S4) and the flagellar-type III secretion system genes were also unable to be transcribed by the dgc knockout strain in the transcription experiment. We also observed that the amount of bacteriocin expressed changed when the amount of L-glutamine in the environment exceeded a particular level. These data suggested that L-glutamine influenced physiological processes in Pcc strains in some way. We hypothesized a relationship between dgc and the genes involved in Pcc LMWB external export via the flagellar-type secretion system based on these findings. In this study, the current findings led us to propose a mechanism in which DGC's cyclic di-GMP might bind to receptor proteins and positively regulate bacteriocin transcription as well as the synthesis, mobility, and transport of toxins.

The c-di-GMP Phosphodiesterase PipA (PA0285) Regulates Autoaggregation and Pf4 Bacteriophage Production in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa PAO1, 41 genes encode proteins predicted to be involved in the production or degradation of c-di-GMP, a ubiquitous secondary messenger that regulates a variety of physiological behaviors closely related to biofilm and aggregate formation. Despite extensive effort, the entire picture of this important signaling network is still unclear, with one-third of these proteins remaining uncharacterized. Here, we show that the deletion of pipA, which produces a protein containing two PAS domains upstream of a GGDEF-EAL tandem, significantly increased the intracellular c-di-GMP level and promoted the formation of aggregates both on surfaces and in planktonic cultures. However, this regulatory effect was not contributed by either of the two classic pathways modulating biofilm formation, exopolysaccharide (EPS) overproduction or motility inhibition. Transcriptome sequencing (RNA-Seq) data revealed that the expression levels of 361 genes were significantly altered in a ΔpipA mutant strain compared to the wild type (WT), indicating the critical role of PipA in PAO1. The most remarkably downregulated genes were located on the Pf4 bacteriophage gene cluster, which corresponded to a 2-log reduction in the Pf4 phage production in the ΔpipA mutant. The sizes of aggregates in ΔpipA cultures were affected by exogenously added Pf4 phage in a concentration-dependent manner, suggesting the quantity of phage plays a part in regulating the formation of aggregates. Further analysis demonstrated that PipA is highly conserved across 83 P. aeruginosa strains. Our work therefore for the first time showed that a c-di-GMP phosphodiesterase can regulate bacteriophage production and provided new insights into the relationship between bacteriophage and bacterial aggregation. IMPORTANCE The c-di-GMP signaling pathways in P. aeruginosa are highly organized and well coordinated, with different diguanylate cyclases and phosphodiesterases playing distinct roles in a complex network. Understanding the function of each enzyme and the underlying regulatory mechanisms not only is crucial for revealing how bacteria decide the transition between motile and sessile lifestyles, but also greatly facilitates the development of new antibiofilm strategies. This work identified bacteriophage production as a novel phenotypic output controlled transcriptionally by a phosphodiesterase, PipA. Further analysis suggested that the quantity of phage may be important in regulating autoaggregation, as either a lack of phage or overproduction was associated with higher levels of aggregation. Our study therefore extended the scope of c-di-GMP-controlled phenotypes and discovered a potential signaling circuit that can be target for biofilm treatment.