Profiling Tel1 Signaling Reveals a Non-Canonical Motif Targeting DNA Repair and Telomere Control Machineries.

The stability of the genome relies on Phosphatidyl Inositol 3-Kinase-related Kinases (PIKKs) that sense DNA damage and trigger elaborate downstream signaling responses. In S. cerevisiae, the Tel1 kinase (ortholog of human ATM) is activated at DNA double strand breaks (DSBs) and short telomeres. Despite the well-established roles of Tel1 in the control of telomere maintenance, suppression of chromosomal rearrangements, activation of cell cycle checkpoints, and repair of DSBs, the substrates through which Tel1 controls these processes remain incompletely understood. Here we performed an in depth phosphoproteomic screen for Tel1-dependent phosphorylation events. To achieve maximal coverage of the phosphoproteome, we developed a scaled-up approach that accommodates large amounts of protein extracts and chromatographic fractions. Compared to previous reports, we expanded the number of detected Tel1-dependent phosphorylation events by over 10-fold. Surprisingly, in addition to the identification of phosphorylation sites featuring the canonical motif for Tel1 phosphorylation (S/T-Q), the results revealed a novel motif (D/E-S/T) highly prevalent and enriched in the set of Tel1-dependent events. This motif is unique to Tel1 signaling and not shared with the Mec1 kinase, providing clues to how Tel1 plays specialized roles in DNA repair and telomere length control. Overall, these findings define a Tel1-signaling network targeting numerous proteins involved in DNA repair, chromatin regulation, and telomere maintenance that represents a framework for dissecting the molecular mechanisms of Tel1 action.

Human gut microbes express functionally distinct endoglycosidases to metabolize the same N-glycan substrate.

Bacteroidales (syn. Bacteroidetes) are prominent members of the human gastrointestinal ecosystem mainly due to their efficient glycan-degrading machinery, organized into gene clusters known as polysaccharide utilization loci (PULs). A single PUL was reported for catabolism of high-mannose (HM) N-glycan glyco-polypeptides in the gut symbiont Bacteroides thetaiotaomicron, encoding a surface endo-ß-N-acetylglucosaminidase (ENGase), BT3987. Here, we discover an ENGase from the GH18 family in B. thetaiotaomicron, BT1285, encoded in a distinct PUL with its own repertoire of proteins for catabolism of the same HM N-glycan substrate as that of BT3987. We employ X-ray crystallography, electron microscopy, mass spectrometry-based activity measurements, alanine scanning mutagenesis and a broad range of biophysical methods to comprehensively define the molecular mechanism by which BT1285 recognizes and hydrolyzes HM N-glycans, revealing that the stabilities and activities of BT1285 and BT3987 were optimal in markedly different conditions. BT1285 exhibits significantly higher affinity and faster hydrolysis of poorly accessible HM N-glycans than does BT3987. We also find that two HM-processing endoglycosidases from the human gut-resident Alistipes finegoldii display condition-specific functional properties. Altogether, our data suggest that human gut microbes employ evolutionary strategies to express distinct ENGases in order to optimally metabolize the same N-glycan substrate in the gastroinstestinal tract.

In-depth mapping of DNA-PKcs signaling uncovers noncanonical features of its kinase specificity.

DNA-PKcs is a DNA damage sensor kinase with established roles in DNA double-strand break repair via nonhomologous end joining. Recent studies have revealed additional roles of DNA-PKcs in the regulation of transcription, translation, and DNA replication. However, the substrates through which DNA-PKcs regulates these processes remain largely undefined. Here, we utilized quantitative phosphoproteomics to generate a high coverage map of DNA-PKcs signaling in response to ionizing radiation and mapped its interplay with the ATM kinase. Beyond the detection of the canonical S/T-Q phosphorylation motif, we uncovered a noncanonical mode of DNA-PKcs signaling targeting S/T-ψ-D/E motifs. Sequence and structural analyses of the DNA-PKcs substrate recognition pocket revealed unique features compared to closely related phosphatidylinositol 3-kinase-related kinases that may explain its broader substrate preference. These findings expand the repertoire of DNA-PKcs and ATM substrates while establishing a novel preferential phosphorylation motif for DNA-PKcs.

IN-DEPTH MAPPING OF DNA-PKcs SIGNALING UNCOVERS CONSERVED FEATURES OF ITS KINASE SPECIFICITY.

DNA-PKcs is a DNA damage sensor kinase with established roles in DNA double-strand break repair via non-homologous end joining. Recent studies have revealed additional roles of DNA-PKcs in the regulation of transcription, translation and DNA replication. However, the substrates through which DNA-PKcs regulates these processes remain largely undefined. Here we utilized quantitative phosphoproteomics to generate a high coverage map of DNA-PKcs signaling in response to ionizing radiation and mapped its interplay with the ATM kinase. Beyond the detection of the canonical S/T-Q phosphorylation motif, we uncovered a non-canonical mode of DNA-PKcs signaling targeting S/T-ψ-D/E motifs. Cross-species analysis in mouse pre-B and human HCT116 cell lines revealed splicing factors and transcriptional regulators phosphorylated at this novel motif, several of which contain SAP domains. These findings expand the list of DNA-PKcs and ATM substrates and establish a novel preferential phosphorylation motif for DNA-PKcs that connects it to proteins involved in nucleotide processes and interactions.

Rediscovery of Histiotusalienus Thomas, 1916 a century after its description (Chiroptera, Vespertilionidae): distribution extension and redescription.

Histiotus is a Neotropical genus of bat that currently includes 11 species. The systematics of Histiotus has been the focus of several studies over the last decades. However, no broad systematic revision has been made, and taxonomic issues such as synonymies, use of subspecies, and specimens that do not fit the description of valid species still persist, as pointed out by several authors. Histiotusalienus was described in 1916 and is known only by the holotype. Here we present a second record of H.alienus and an amended diagnosis of this species. We use qualitative, quantitative, and morphometric analyses based on data from 184 specimens of Histiotus and almost all valid species. Our amended diagnosis establishes the taxonomic limits of H.alienus, as well as a comprehensive comparison with congeners. We also explore new diagnostic characters for H.alienus and provide a few notes on the natural history of this species. Our results highlight skull similarities among Histiotus species and reinforce the usefulness of external morphology for their correct identification. Despite our new insights into the taxonomy of the genus, several taxonomic issues remain, and a comprehensive revision of the genus is needed.

Evaluation of different ensiling methods for Saccharina latissima preservation: influence on chemical composition and in vitro ruminal fermentation.

Saccharina latissima is a brown seaweed that could be used in ruminant feeding, but its fast deteriorating and seasonal growth nature limit their utilisation in the practice. Ensiling could be used as a preservation method, but information of its effects on the nutritional value of the seaweed is limited. This study evaluated the in vitro ruminal fermentation of different S. latissima silages using ruminal inoculum either from goats fed a mixed diet (60:40 oat hay:concentrate) or from sheep fed a high-forage diet (90:10 alfalfa hay:concentrate) to simulate different small ruminant production systems. S. latissima was ensiled in vacuum bags without additives (Control), with formic acid (4 g/kg seaweed; FA), with lactic acid bacteria (LAB) or with LAB after a pre-wilting treatment to reach a seaweed dry matter (DM) content of 30% (30LAB). Ensiling S. latissima decreased (p < 0.05) the content in DM, neutral detergent fibre and total extractable polyphenols, but nitrogen and fat content were unaffected. For both ruminal inoculums, ensiling decreased (p < 0.05) the asymptotic gas production after 120 h of fermentation (excepting for FA silage with goats' inoculum), but the total volatile fatty acid (VFA) production was unaffected. The VFA profile shifted towards greater (p < 0.05) acetate and lower (p < 0.05) propionate proportions in all silages compared with the pre-ensiling S. latissima. When goats inoculum was used, greater (p < 0.05) CH4 production compared with pre-ensiling S. latissima was observed in all silages, except Control one, which led to greater (p < 0.05) CH4/total VFA ratio. In contrast, no differences among samples (p > 0.05) in either CH4 production or CH4/total VFA ratio were observed when sheep' inoculum was used. Fermentation of all samples started earlier with goats' inoculum than with sheep' inoculum, which was attributed to the different diet fed to the animals. These results suggest that ensiling S. latissima with either formic acid or lactic acid bacteria could be a viable conservation method to preserve the nutritive value.

GPR-160 Receptor Signaling in the Dorsal Vagal Complex of Male Rats Modulates Meal Microstructure and CART-Mediated Hypophagia.

The g-protein coupled receptor GPR-160, recently identified as a putative receptor for the cocaine and amphetamine-regulated transcript (CART) peptide, shows abundant expression in the energy-balance control nuclei, including the dorsal vagal complex (DVC). However, its physiological role in the control of food intake has yet to be fully explored. Here, we performed a virally mediated, targeted knockdown (KD) of Gpr160 in the DVC of male rats to evaluate its physiological role in control of feeding. Our results indicate that DVC Gpr160 KD affects meal microstructure. Specifically, DVC Gpr160 KD animals consumed more frequent, but shorter meals during the dark phase and showed decreased caloric intake and duration of meals during the light phase. Cumulatively, however, these bidirectional effects on feeding resulted in no difference in body weight gain. We next tested the role of DVC GPR-160 in mediating the anorexigenic effects of exogenous CART. Our results show that DVC Gpr160 KD partially attenuates CART's anorexigenic effects. To further characterize Gpr160+ cells in the DVC, we utilized single-nucleus RNA sequencing data to uncover abundant GPR-160 expression in DVC microglia and only minimal expression in neurons. Altogether, our results suggest that DVC CART signaling may be mediated by Gpr160+ microglia, which in turn may be modulating DVC neuronal activity to control food intake.

Direito ao lazer nas praças públicas: percepção dos usuários das praças da poligonal histórica de São Carlos-SP / Leisure in public squares: perception of users of the squares of the historic polygonal of São Carlos ­SP

O lazer, como uma das funções sociais da cidade, desempenha papel importante na saúde da população, porém sofreu interferências ao longo das modificações do urbano pós-moderno, especialmente com o esvaziamento do centro da cidade em horários que excedam os comerciais, afastando a população da permanência espaços abertos dessas regiões. Nesse sentido, a partir da combinação de levantamento bibliográfico, estudo de caso e análise quali-quantitativa, buscou-se avaliar a apropriação das praças da Poligonal Histórica de São Carlos, trazendo à luz a importância do lazer como função social da cidade e como direito fundamental dos cidadãos. Por meio dos resultados obtidos, foi possível observar que pouco tem sido feito para que os espaços sejam atualizados às necessidades humanas. Viu-se que os equipamentos disponíveis não correspondem às expectativas da população no exercício do direito ao lazer na região, caracterizada por grandes trechos de circulação e poucas oportunidades de atividades estacionárias ou de lazer ativo. Espera-se que a metodologia abordada sirva de mote para estudos futuros, tanto em diferentes regiões do município, quanto em outras cidades médias, com vistas a diminuir as diferenças socioambientais e garantir o desenvolvimento ordenado e sustentável.

Leisure, as one of the social functions of the city, plays an important role in the health of the population, but has suffered interference along the modifications of the postmodern urban layout, especially because of the emptying of the city ́s center at times that exceed commercial hours, keeps the population away from staying in places intended for leisure. In this sense, the pesent study, based on the combination of bibliographical research, case study and quali-quantitative analysis model, we sought to assess the appropriation of the squares in the Historical Polygonal of São Carlos, bringing to light the importance of leisure as a social function of the city and as a fundamental right to the citizens. Through the results obtained, it was possible to observe that little has been done so that the spaces aren't updated to human needs. It was seen that the available equipment does not correspond to the expectations of the population in the exercise of the right to leisure in the region, characterized by large stretches of circulation and few opportunities for stationary activities or active leisure. It is expected that the methodology addressed will serve as a motto for future studies, both in different regions of the municipalityand in other medium-sized cities, with a view to reducing socio-environmental differences and ensuring orderly and sustainable development.

A Novel Insular/Orbital-Prelimbic Circuit That Prevents Persistent Avoidance in a Rodent Model of Compulsive Behavior.

BACKGROUND: A common symptom of obsessive-compulsive disorder is the persistent avoidance of cues incorrectly associated with negative outcomes. This maladaptation becomes increasingly evident as subjects fail to respond to extinction-based treatments such as exposure-with-response prevention therapy. While previous studies have highlighted the role of the insular-orbital cortex in fine-tuning avoidance-based decisions, little is known about the projections from this area that might modulate compulsive-like avoidance. METHODS: Here, we used anatomical tract-tracing, single-unit recording, and optogenetics to characterize the projections from the insular-orbital cortex. To model exposure-with-response prevention and persistent avoidance in rats, we used the platform-mediated avoidance task followed by extinction-with-response prevention training. RESULTS: Using tract-tracing and unit recording, we found that projections from the agranular insular/lateral orbital (AI/LO) cortex to the prefrontal cortex predominantly target the rostral portion of the prelimbic (rPL) cortex and excite rPL neurons. Photoinhibiting this projection induced persistent avoidance after extinction-with-response prevention training, an effect that was still present 1 week later. Consistent with this, photoexcitation of this projection prevented persistent avoidance in overtrained rats. This projection to rPL appears to be key for AI/LO's effects, considering that there was no effect of photoinhibiting AI/LO projections to the ventral striatum or basolateral amygdala. CONCLUSIONS: Our findings suggest that projections from the AI/LO to the rPL decreases the likelihood of avoidance behavior following extinction. In humans, this connectivity may share some homology of projections from lateral prefrontal cortices (i.e., ventrolateral prefrontal cortex, orbitofrontal cortex, and insula) to other prefrontal areas and the anterior cingulate cortex, suggesting that reduced activity in these pathways may contribute to obsessive-compulsive disorder.

Mitigation of ruminal methane production with enhancing the fermentation by supplementation of different tropical forage legumes.

The aim of this research was to evaluate the influence of forage species adapted to the tropical region of Ecuador on gas production, enteric methane, digestion, and ruminal fermentation. The tree forage evaluated were C. arborea, E. fusca, B. forficata, E. poeppigiana, C. argentea, G. sepium, C. tora, and F. macrophylla. Ruminal fluid of four adult sheep fistulated with permanent cannulas in the rumen was used in the in vitro gas production technique. The in vitro gas production parameters were lower (P < 0.05) in the C. arborea (A = 41.68 mL gas/g DM, c = 0.044%/h and Lag = 1.654 h) and the average gas production rate for B. forficata was 1.017 mL/h (P < 0.05). C. arborea presented higher (P = 0.0001) effective degradation and real DM digestibility (40.461 g/kg and 82.51 mg/g, respectively). With respect to VFA, the highest (P < 0.05) proportion of acetic, propionic, and butyric was observed in C. arborea, G. sepium, and E. poeppigiana (72.52, 23.09, and 7.44 mol/100 mol, respectively) and the lowest (P = 0.0001) ratio: acetic/propionic was observed in G. sepium (2.92 mol/100 mol). The content of NH3-N (mg/L) showed no difference. The lowest (P = 0.0001) methane production was observed in C. arborea (1.23 mL CH4/g DM). The use of forage species of tropical climate rich in secondary metabolites in ruminant diets has the capacity to reduce the gas production and enteric methane; however, this is at the expense of the reduction of the fermentation of organic matter in the rumen.

GIP Receptor Agonism Attenuates GLP-1 Receptor Agonist-Induced Nausea and Emesis in Preclinical Models.

Glucagon-like peptide 1 receptor (GLP-1R) agonists decrease body weight and improve glycemic control in obesity and diabetes. Patient compliance and maximal efficacy of GLP-1 therapeutics are limited by adverse side effects, including nausea and emesis. In three different species (i.e., mice, rats, and musk shrews), we show that glucose-dependent insulinotropic polypeptide receptor (GIPR) signaling blocks emesis and attenuates illness behaviors elicited by GLP-1R activation, while maintaining reduced food intake, body weight loss, and improved glucose tolerance. The area postrema and nucleus tractus solitarius (AP/NTS) of the hindbrain are required for food intake and body weight suppression by GLP-1R ligands and processing of emetic stimuli. Using single-nuclei RNA sequencing, we identified the cellular phenotypes of AP/NTS cells expressing GIPR and GLP-1R on distinct populations of inhibitory and excitatory neurons, with the greatest expression of GIPR in γ-aminobutyric acid-ergic neurons. This work suggests that combinatorial pharmaceutical targeting of GLP-1R and GIPR will increase efficacy in treating obesity and diabetes by reducing nausea and vomiting.

Modeling the spread of COVID-19 in New York City.

This paper proposes an ensemble predictor for the weekly increase in the number of confirmed COVID-19 cases in the city of New York at zip code level. Within a Bayesian model averaging framework, the baseline is a Poisson regression for count data. The set of covariates includes autoregressive terms, spatial effects, and demographic and socioeconomic variables. Our results for the second wave of the coronavirus pandemic show that these regressors are more significant to predict the number of new confirmed cases as the pandemic unfolds. Both pointwise and interval forecasts exhibit strong predictive ability in-sample and out-of-sample.

Este artículo propone un predictor de conjunto para el aumento semanal del número de casos confirmados de COVID­19 en la ciudad de Nueva York a nivel de código postal. Dentro de un marco de promediación de modelo bayesiano, la línea de base es una regresión de Poisson para datos de recuento. El conjunto de covariables incluye términos autorregresivos, efectos espaciales y variables demográficas y socioeconómicas. Los resultados para la segunda ola de la pandemia de coronavirus muestran que estos regresores son más significativos para predecir el número de nuevos casos confirmados a medida que se desarrolla la pandemia. Tanto las previsiones puntuales como las de intervalo muestran una fuerte capacidad de predicción, tanto dentro como fuera de la muestra.

Prolonged avoidance training exacerbates OCD-like behaviors in a rodent model.

Obsessive-compulsive disorder (OCD) is characterized by compulsive behaviors that often resemble avoidance of perceived danger. OCD can be treated with exposure-with-response prevention (ERP) therapy in which patients are exposed to triggers but are encouraged to refrain from compulsions, to extinguish compulsive responses. The compulsions of OCD are strengthened by many repeated exposures to triggers, but little is known about the effects of extended repetition of avoidance behaviors on extinction. Here we assessed the extent to which overtraining of active avoidance affects subsequent extinction-with-response prevention (Ext-RP) as a rodent model of ERP, in which rats are extinguished to triggers, while the avoidance option is prevented. Male rats conditioned for 8d or 20d produced similar avoidance behavior to a tone paired with a shock, however, the 20d group showed a severe impairment of extinction during Ext-RP, as well as heightened anxiety. Furthermore, the majority of overtrained (20d) rats (75%) exhibited persistent avoidance following Ext-RP. In the 8d group, only a minority of rats (37%) exhibited persistent avoidance, and this was associated with elevated activity (c-Fos) in the prelimbic cortex and nucleus accumbens. In the 20d group, the minority of non-persistent rats (25%) showed elevated activity in the insular-orbital cortex and paraventricular thalamus. Lastly, extending the duration of Ext-RP prevented the deleterious effects of overtraining on extinction and avoidance. These rodent findings suggest that repeated expression of compulsion-like behaviors biases individuals toward persistent avoidance and alters avoidance circuits, thereby reducing the effectiveness of current extinction-based therapies.

The phosphatidic acid pathway enzyme PlsX plays both catalytic and channeling roles in bacterial phospholipid synthesis.

PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.

Membrane fluidity adjusts the insertion of the transacylase PlsX to regulate phospholipid biosynthesis in Gram-positive bacteria.

PlsX plays a central role in the coordination of fatty acid and phospholipid biosynthesis in Gram-positive bacteria. PlsX is a peripheral membrane acyltransferase that catalyzes the conversion of acyl-ACP to acyl-phosphate, which is in turn utilized by the polytopic membrane acyltransferase PlsY on the pathway of bacterial phospholipid biosynthesis. We have recently studied the interaction between PlsX and membrane phospholipids in vivo and in vitro, and observed that membrane association is necessary for the efficient transfer of acyl-phosphate to PlsY. However, understanding the molecular basis of such a channeling mechanism remains a major challenge. Here, we disentangle the binding and insertion events of the enzyme to the membrane, and the subsequent catalysis. We show that PlsX membrane binding is a process mostly mediated by phospholipid charge, whereas fatty acid saturation and membrane fluidity remarkably influence the membrane insertion step. Strikingly, the PlsXL254E mutant, whose biological functionality was severely compromised in vivo but remains catalytically active in vitro, was able to superficially bind to phospholipid vesicles, nevertheless, it loses the insertion capacity, strongly supporting the importance of membrane insertion in acyl-phosphate delivery. We propose a mechanism in which membrane fluidity governs the insertion of PlsX and thus regulates the biosynthesis of phospholipids in Gram-positive bacteria. This model may be operational in other peripheral membrane proteins with an unprecedented impact in drug discovery/development strategies.

Isothermal Titration Calorimetry to Determine Apparent Dissociation Constants (K d) and Stoichiometry of Interaction (n) of C-di-GMP Binding Proteins.

Isothermal titration calorimetry (ITC) is a commonly used biophysical technique that enables the quantitative characterization of intermolecular interactions in solution. Based on enthalpy changes (ΔH) upon titration of the binding partner (e.g., a small-molecule ligand such as c-di-GMP) to the molecule of interest (e.g., a receptor protein), the resulting binding isotherms provide information on the equilibrium association/dissociation constants (K a, K d) and stoichiometry of binding (n), as well as on changes in the Gibbs free energy (ΔG) and entropy (ΔS) along the interaction. Here we present ITC experiments used for the characterization of c-di-GMP binding proteins and discuss advantages and potential caveats in the interpretation of results.

Filaments and fingers: Novel structural aspects of the single septin from Chlamydomonas reinhardtii.

Septins are filament-forming GTP-binding proteins involved in many essential cellular events related to cytoskeletal dynamics and maintenance. Septins can self-assemble into heterocomplexes, which polymerize into highly organized, cell membrane-interacting filaments. The number of septin genes varies among organisms, and although their structure and function have been thoroughly studied in opisthokonts (including animals and fungi), no structural studies have been reported for other organisms. This makes the single septin from Chlamydomonas (CrSEPT) a particularly attractive model for investigating whether functional homopolymeric septin filaments also exist. CrSEPT was detected at the base of the flagella in Chlamydomonas, suggesting that CrSEPT is involved in the formation of a membrane-diffusion barrier. Using transmission electron microscopy, we observed that recombinant CrSEPT forms long filaments with dimensions comparable with those of the canonical structure described for opisthokonts. The GTP-binding domain of CrSEPT purified as a nucleotide-free monomer that hydrolyzes GTP and readily binds its analog guanosine 5'-3-O-(thio)triphosphate. We also found that upon nucleotide binding, CrSEPT formed dimers that were stabilized by an interface involving the ligand (G-interface). Across this interface, one monomer supplied a catalytic arginine to the opposing subunit, greatly accelerating the rate of GTP hydrolysis. This is the first report of an arginine finger observed in a septin and suggests that CrSEPT may act as its own GTP-activating protein. The finger is conserved in all algal septin sequences, suggesting a possible correlation between the ability to form homopolymeric filaments and the accelerated rate of hydrolysis that it provides.

Mechanistic insights into c-di-GMP-dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa.

Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ(54)-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ's AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP-complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs.