Differential ligand-selective control of opposing enzymatic activities within a bifunctional c-di-GMP enzyme.

Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a second messenger that modulates bacterial cellular processes, including biofilm formation. While proteins containing both c-di-GMP synthesizing (GGDEF) and c-di-GMP hydrolyzing (EAL) domains are widely predicted in bacterial genomes, it is poorly understood how domains with opposing enzymatic activity are regulated within a single polypeptide. Herein, we report the characterization of a globin-coupled sensor protein (GCS) from Paenibacillus dendritiformis (DcpG) with bifunctional c-di-GMP enzymatic activity. DcpG contains a regulatory sensor globin domain linked to diguanylate cyclase (GGDEF) and phosphodiesterase (EAL) domains that are differentially regulated by gas binding to the heme; GGDEF domain activity is activated by the Fe(II)-NO state of the globin domain, while EAL domain activity is activated by the Fe(II)-O2 state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation of P. dendritiformis in response to gaseous environment, with nitric oxide conditions leading to the greatest amount of biofilm formation. The ability of DcpG to differentially control GGDEF and EAL domain activity in response to ligand binding is likely due to the unusual properties of the globin domain, including rapid ligand dissociation rates and high midpoint potentials. Using structural information from small-angle X-ray scattering and negative stain electron microscopy studies, we developed a structural model of DcpG, providing information about the regulatory mechanism. These studies provide information about full-length GCS protein architecture and insight into the mechanism by which a single regulatory domain can selectively control output domains with opposing enzymatic activities.

BusR senses bipartite DNA binding motifs by a unique molecular ruler architecture.

The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with proteins such as potassium channels, the second messenger also specifically binds to transcription factors, thereby altering the processes in the cell on the transcriptional level. We here describe the structural and biochemical characterization of BusR from the human pathogen Streptococcus agalactiae. BusR is a member of a yet structurally uncharacterized subfamily of the GntR family of transcription factors that downregulates transcription of the genes for the BusA (OpuA) glycine-betaine transporter upon c-di-AMP binding. We report crystal structures of full-length BusR, its apo and c-di-AMP bound effector domain, as well as cryo-EM structures of BusR bound to its operator DNA. Our structural data, supported by biochemical and biophysical data, reveal that BusR utilizes a unique domain assembly with a tetrameric coiled-coil in between the binding platforms, serving as a molecular ruler to specifically recognize a 22 bp separated bipartite binding motif. Binding of c-di-AMP to BusR induces a shift in equilibrium from an inactivated towards an activated state that allows BusR to bind the target DNA, leading to transcriptional repression.

Mapping genetic changes in the cAMP-signaling cascade in human atria.

AIM: To obtain a quantitative expression profile of the main genes involved in the cAMP-signaling cascade in human control atria and in different cardiac pathologies. METHODS AND RESULTS: Expression of 48 target genes playing a relevant role in the cAMP-signaling cascade was assessed by RT-qPCR. 113 samples were obtained from right atrial appendages (RAA) of patients in sinus rhythm (SR) with or without atrium dilation, paroxysmal atrial fibrillation (AF), persistent AF or heart failure (HF); and left atrial appendages (LAA) from patients in SR or with AF. Our results show that right and left atrial appendages in donor hearts or from SR patients have similar expression values except for AC7 and PDE2A. Despite the enormous chamber-dependent variability in the gene-expression changes between pathologies, several distinguishable patterns could be identified. PDE8A, PI3Kγ and EPAC2 were upregulated in AF. Different phosphodiesterase (PDE) families showed specific pathology-dependent changes. CONCLUSION: By comparing mRNA-expression patterns of the cAMP-signaling cascade related genes in right and left atrial appendages of human hearts and across different pathologies, we show that 1) gene expression is not significantly affected by cardioplegic solution content, 2) it is appropriate to use SR atrial samples as controls, and 3) many genes in the cAMP-signaling cascade are affected in AF and HF but only few of them appear to be chamber (right or left) specific. TOPIC: Genetic changes in human diseased atria. TRANSLATIONAL PERSPECTIVE: The cyclic AMP signaling pathway is important for atrial function. However, expression patterns of the genes involved in the atria of healthy and diseased hearts are still unclear. We give here a general overview of how different pathologies affect the expression of key genes in the cAMP signaling pathway in human right and left atria appendages. Our study may help identifying new genes of interest as potential therapeutic targets or clinical biomarkers for these pathologies and could serve as a guide in future gene therapy studies.

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis.

In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.

Reciprocal growth control by competitive binding of nucleotide second messengers to a metabolic switch in Caulobacter crescentus.

Bacteria use small signalling molecules such as (p)ppGpp or c-di-GMP to tune their physiology in response to environmental changes. It remains unclear whether these regulatory networks operate independently or whether they interact to optimize bacterial growth and survival. We report that (p)ppGpp and c-di-GMP reciprocally regulate the growth of Caulobacter crescentus by converging on a single small-molecule-binding protein, SmbA. While c-di-GMP binding inhibits SmbA, (p)ppGpp competes for the same binding site to sustain SmbA activity. We demonstrate that (p)ppGpp specifically promotes Caulobacter growth on glucose, whereas c-di-GMP inhibits glucose consumption. We find that SmbA contributes to this metabolic switch and promotes growth on glucose by quenching the associated redox stress. The identification of an effector protein that acts as a central regulatory hub for two global second messengers opens up future studies on specific crosstalk between small-molecule-based regulatory networks.

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.

DNA damage and growth hormone hypersecretion in pituitary somatotroph adenomas.

Drivers of sporadic benign pituitary adenoma growth are largely unknown. Whole-exome sequencing of 159 prospectively resected pituitary adenomas showed that somatic copy number alteration (SCNA) rather than mutation is a hallmark of hormone-secreting adenomas and that SCNAs correlate with adenoma phenotype. Using single-gene SCNA pathway analysis, we observed that both cAMP and Fanconi anemia DNA damage repair pathways were affected by SCNAs in growth hormone-secreting (GH-secreting) somatotroph adenomas. As somatotroph differentiation and GH secretion are dependent on cAMP activation and we previously showed DNA damage, aneuploidy, and senescence in somatotroph adenomas, we studied links between cAMP signaling and DNA damage. Stimulation of cAMP in C57BL/6 mouse primary pituitary cultures using forskolin or a long-acting GH-releasing hormone (GHRH) analog increased GH production and DNA damage measured by H2AX phosphorylation and a comet assay. Octreotide, a somatostatin receptor ligand that targets somatotroph adenoma GH secretion in patients with acromegaly, inhibited cAMP and GH and reversed DNA damage induction. In vivo long-acting GHRH treatment also induced pituitary DNA damage in mice. We conclude that cAMP, which induces somatotroph proliferation and GH secretion, may concomitantly induce DNA damage, potentially linking hormone hypersecretion to SCNA and genome instability. These results elucidating somatotroph adenoma pathophysiology identify pathways for targeted treatment.

Cyclic di-GMP cyclase SSFG_02181 from Streptomyces ghanaensis ATCC14672 regulates antibiotic biosynthesis and morphological differentiation in streptomycetes.

Streptomycetes are filamentous bacteria famous for their ability to produce a vast majority of clinically important secondary metabolites. Both complex morphogenesis and onset of antibiotic biosynthesis are tightly linked in streptomycetes and require series of specific signals for initiation. Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, one of the well-known bacterial second messengers, has been recently shown to govern morphogenesis and natural product synthesis in Streptomyces by altering the activity of the pleiotropic regulator BldD. Here we report a role of the heme-binding diguanylate cyclase SSFG_02181 from Streptomyces ghanaensis in the regulation of the peptidoglycan glycosyltransferase inhibitor moenomycin A biosynthesis. Deletion of ssfg_02181 reduced the moenomycin A accumulation and led to a precocious sporulation, while the overexpression of the gene blocked sporogenesis and remarkably improved antibiotic titer. We also demonstrate that BldD negatively controls the expression of ssfg_02181, which stems from direct binding of BldD to the ssfg_02181 promoter. Notably, the heterologous expression of ssfg_02181 in model Streptomyces spp. arrested morphological progression at aerial mycelium level and strongly altered the production of secondary metabolites. Altogether, our work underscores the significance of c-di-GMP-mediated signaling in natural product biosynthesis and pointed to extensively applicable approach to increase antibiotic production levels in streptomycetes.

cGMP Signaling in Cardiovascular Diseases: Linking Genotype and Phenotype.

Cyclic guanosine 3',5'-monophosphate (cGMP) is the key second messenger molecule in nitric oxide signaling. Its rapid generation and fate, but also its role in mediating acute cellular functions has been extensively studied. In the past years, genetic studies suggested an important role for cGMP in affecting the risk of chronic cardiovascular diseases, for example, coronary artery disease and myocardial infarction. Here, we review the role of cGMP in atherosclerosis and other cardiovascular diseases and discuss recent genetic findings and identified mechanisms. Finally, we highlight open questions and promising research topics.

The cyanobacterial phytochrome 2 regulates the expression of motility-related genes through the second messenger cyclic di-GMP.

The cyanobacterial phytochrome Cph2 is a light-dependent diguanylate cyclase of the cyanobacterium Synechocystis 6803. Under blue light, Cph2-dependent increase in the cellular c-di-GMP concentration leads to inhibition of surface motility and enhanced flocculation of cells in liquid culture. However, the targets of second messenger signalling in this cyanobacterium and its mechanism of action remained unclear. Here, we determined the cellular concentrations of cAMP and c-di-GMP in wild-type and Δcph2 cells after exposure to blue and green light. Inactivation of cph2 completely abolished the blue-light dependent increase in c-di-GMP content. Therefore, a microarray analysis with blue-light grown wild-type and Δcph2 mutant cells was used to identify c-di-GMP dependent alterations in transcript accumulation. The increase in the c-di-GMP content alters expression of genes encoding putative cell appendages, minor pilins and components of chemotaxis systems. The mRNA encoding the minor pilins pilA5-pilA6 was negatively affected by high c-di-GMP content under blue light, whereas the minor pilin encoding operon pilA9-slr2019 accumulates under these conditions, suggesting opposing functions of the respective gene sets. Artificial overproduction of c-di-GMP leads to similar changes in minor pilin gene expression and supports previous findings that c-di-GMP is important for flocculation via the function of minor pilins. Mutational and gene expression analysis further suggest that SyCRP2, a CRP-like transcription factor, is involved in regulation of minor pilin and putative chaperone usher pili gene expression.

Secondary nucleotide messenger c-di-GMP exerts a global control on natural product biosynthesis in streptomycetes.

Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, is a ubiquitous second messenger controlling diverse cellular processes in bacteria. In streptomycetes, c-di-GMP plays a crucial role in a complex morphological differentiation by modulating an activity of the pleiotropic regulator BldD. Here we report that c-di-GMP plays a key role in regulating secondary metabolite production in streptomycetes by altering the expression levels of bldD. Deletion of cdgB encoding a diguanylate cyclase in Streptomycesghanaensis reduced c-di-GMP levels and the production of the peptidoglycan glycosyltransferase inhibitor moenomycin A. In contrast to the cdgB mutant, inactivation of rmdB, encoding a phosphodiesterase for the c-di-GMP hydrolysis, positively correlated with the c-di-GMP and moenomycin A accumulation. Deletion of bldD adversely affected the synthesis of secondary metabolites in S. ghanaensis, including the production of moenomycin A. The bldD-deficient phenotype is partly mediated by an increase in expression of the pleiotropic regulatory gene wblA. Genetic and biochemical analyses demonstrate that a complex of c-di-GMP and BldD effectively represses transcription of wblA, thus preventing sporogenesis and sustaining antibiotic synthesis. These results show that manipulation of the expression of genes controlling c-di-GMP pool has the potential to improve antibiotic production as well as activate the expression of silent gene clusters.

Cardioprotective GLP-1 metabolite prevents ischemic cardiac injury by inhibiting mitochondrial trifunctional protein-α.

Mechanisms mediating the cardioprotective actions of glucagon-like peptide 1 (GLP-1) were unknown. Here, we show in both ex vivo and in vivo models of ischemic injury that treatment with GLP-1(28-36), a neutral endopeptidase-generated (NEP-generated) metabolite of GLP-1, was as cardioprotective as GLP-1 and was abolished by scrambling its amino acid sequence. GLP-1(28-36) enters human coronary artery endothelial cells (caECs) through macropinocytosis and acts directly on mouse and human coronary artery smooth muscle cells (caSMCs) and caECs, resulting in soluble adenylyl cyclase Adcy10-dependent (sAC-dependent) increases in cAMP, activation of protein kinase A, and cytoprotection from oxidative injury. GLP-1(28-36) modulates sAC by increasing intracellular ATP levels, with accompanying cAMP accumulation lost in sAC-/- cells. We identify mitochondrial trifunctional protein-α (MTPα) as a binding partner of GLP-1(28-36) and demonstrate that the ability of GLP-1(28-36) to shift substrate utilization from oxygen-consuming fatty acid metabolism toward oxygen-sparing glycolysis and glucose oxidation and to increase cAMP levels is dependent on MTPα. NEP inhibition with sacubitril blunted the ability of GLP-1 to increase cAMP levels in coronary vascular cells in vitro. GLP-1(28-36) is a small peptide that targets novel molecular (MTPα and sAC) and cellular (caSMC and caEC) mechanisms in myocardial ischemic injury.

Glucocorticoids rapidly activate cAMP production via Gαs to initiate non-genomic signaling that contributes to one-third of their canonical genomic effects.

Glucocorticoids are widely used for the suppression of inflammation, but evidence is growing that they can have rapid, non-genomic actions that have been unappreciated. Diverse cell signaling effects have been reported for glucocorticoids, leading us to hypothesize that glucocorticoids alone can swiftly increase the 3',5'-cyclic adenosine monophosphate (cAMP) production. We found that prednisone, fluticasone, budesonide, and progesterone each increased cAMP levels within 3 minutes without phosphodiesterase inhibitors by measuring real-time cAMP dynamics using the cAMP difference detector in situ assay in a variety of immortalized cell lines and primary human airway smooth muscle (HASM) cells. A membrane- impermeable glucocorticoid showed similarly rapid stimulation of cAMP, implying that responses are initiated at the cell surface. siRNA knockdown of Gαs virtually eliminated glucocorticoid-stimulated cAMP responses, suggesting that these drugs activate the cAMP production via a G protein-coupled receptor. Estradiol had small effects on cAMP levels but G protein estrogen receptor antagonists had little effect on responses to any of the glucocorticoids tested. The genomic and non-genomic actions of budesonide were analyzed by RNA-Seq analysis of 24 hours treated HASM, with and without knockdown of Gαs . A 140-gene budesonide signature was identified, of which 48 genes represent a non-genomic signature that requires Gαs signaling. Collectively, this non-genomic cAMP signaling modality contributes to one-third of the gene expression changes induced by glucocorticoid treatment and shifts the view of how this important class of drugs exerts its effects.

The Vc2 Cyclic di-GMP-Dependent Riboswitch of Vibrio cholerae Regulates Expression of an Upstream Putative Small RNA by Controlling RNA Stability.

Cyclic di-GMP (c-di-GMP) is a bacterial second messenger molecule that is important in the biology of Vibrio cholerae, but the molecular mechanisms by which this molecule regulates downstream phenotypes have not been fully characterized. We have previously shown that the Vc2 c-di-GMP-binding riboswitch, encoded upstream of the gene tfoY, functions as an off switch in response to c-di-GMP. However, the mechanism by which c-di-GMP controls expression of tfoY has not been fully elucidated. During our studies of this mechanism, we determined that c-di-GMP binding to Vc2 also controls the abundance and stability of upstream noncoding RNAs with 3' ends located immediately downstream of the Vc2 riboswitch. Our results suggest these putative small RNAs (sRNAs) are not generated by transcriptional termination but rather by preventing degradation of the upstream untranslated RNA when c-di-GMP is bound to Vc2.IMPORTANCE Riboswitches are typically RNA elements located in the 5' untranslated region of mRNAs. They are highly structured and specifically recognize and respond to a given chemical cue to alter transcription termination or translation initiation. In this work, we report a novel mechanism of riboswitch-mediated gene regulation in Vibrio cholerae whereby a 3' riboswitch, named Vc2, controls the stability of upstream untranslated RNA upon binding to its cognate ligand, the second messenger cyclic di-GMP, leading to the accumulation of previously undescribed putative sRNAs. We further demonstrate that binding of the ligand to the riboswitch prevents RNA degradation. As binding of riboswitches to their ligands often produces compactly structured RNA, we hypothesize this mechanism of gene regulation is widespread.

Signal Transduction of Streptococci by Cyclic Dinucleotide Second Messengers.

Since the discovery of cyclic dimeric guanosine 3',5'-monophosphate (c-di-GMP) in 1987, the role of cyclic dinucleotides in signal pathways has been extensively studied. Many receptors and effectors of cyclic dinucleotides have been identified which play important roles in cellular processes. Example of such effectors include cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP)-binding proteins and endoplasmic reticulum membrane adaptor. Accumulating evidence indicate that cyclic dinucleotides act as second messengers that not only regulate the bacterial physiological processes but also affect host immune responses during infections. Streptococci species, which produce cyclic dinucleotides, are responsible for many human diseases. Numerous studies suggest that the cyclic dinucleotides are vital in signal transduction pathways as second messengers and influence the progression of infectious diseases. Here, we provide an overview of the molecular principles of cyclic dinucleotides synthesis and degradation and discuss recent progress on streptococcal signal transduction pathways by cyclic dinucleotide second messengers and their role in regulating host immune reaction. This review will provide a better understanding of the molecular mechanisms of streptococcal cyclic dinucleotide second messengers thereby revealing novel targets for preventing infections.

microRNA-142-mediated repression of phosphodiesterase 3B critically regulates peripheral immune tolerance.

Tregs play a fundamental role in immune tolerance via control of self-reactive effector T cells (Teffs). This function is dependent on maintenance of a high intracellular cAMP concentration. A number of microRNAs are implicated in the maintenance of Tregs. In this study, we demonstrate that peripheral immune tolerance is critically dependent on posttranscriptional repression of the cAMP-hydrolyzing enzyme phosphodiesterase-3b (Pde3b) by microRNA-142-5p (miR-142-5p). In this manner, miR-142-5p acts as an immunometabolic regulator of intracellular cAMP, controlling Treg suppressive function. Mir142 was associated with a super enhancer bound by the Treg lineage-determining transcription factor forkhead box P3 (FOXP3), and Treg-specific deletion of miR-142 in mice (TregΔ142) resulted in spontaneous, lethal, multisystem autoimmunity, despite preserved numbers of phenotypically normal Tregs. Pharmacological inhibition and genetic ablation of PDE3B prevented autoimmune disease and reversed the impaired suppressive function of Tregs in TregΔ142 animals. These findings reveal a critical molecular switch, specifying Treg function through the modulation of a highly conserved, cell-intrinsic metabolic pathway. Modulation of this pathway has direct relevance to the pathogenesis and treatment of autoimmunity and cancer.

Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM.

c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.

Protein kinase D1 deletion in adipocytes enhances energy dissipation and protects against adiposity.

Nutrient overload in combination with decreased energy dissipation promotes obesity and diabetes. Obesity results in a hormonal imbalance, which among others activates G protein-coupled receptors utilizing diacylglycerol (DAG) as secondary messenger. Protein kinase D1 (PKD1) is a DAG effector, which integrates multiple nutritional and hormonal inputs, but its physiological role in adipocytes is unknown. Here, we show that PKD1 promotes lipogenesis and suppresses mitochondrial fragmentation, biogenesis, respiration, and energy dissipation in an AMP-activated protein kinase (AMPK)-dependent manner. Moreover, mice lacking PKD1 in adipocytes are resistant to diet-induced obesity due to elevated energy expenditure. Beiging of adipocytes promotes energy expenditure and counteracts obesity. Consistently, deletion of PKD1 promotes expression of the ß3-adrenergic receptor (ADRB3) in a CCAAT/enhancer binding protein (C/EBP)-α- and δ-dependent manner, which leads to the elevated expression of beige markers in adipocytes and subcutaneous adipose tissue. Finally, deletion of PKD1 in adipocytes improves insulin sensitivity and ameliorates liver steatosis. Thus, depletion of PKD1 in adipocytes increases energy dissipation by several complementary mechanisms and might represent an attractive strategy to treat obesity and its related complications.

Seven-transmembrane receptor protein RgsP and cell wall-binding protein RgsM promote unipolar growth in Rhizobiales.

Members of the Rhizobiales (class of α-proteobacteria) display zonal peptidoglycan cell wall growth at one cell pole, contrasting with the dispersed mode of cell wall growth along the sidewalls of many other rod-shaped bacteria. Here we show that the seven-transmembrane receptor (7TMR) protein RgsP (SMc00074), together with the putative membrane-anchored peptidoglycan metallopeptidase RgsM (SMc02432), have key roles in unipolar peptidoglycan formation during growth and at mid-cell during cell division in Sinorhizobium meliloti. RgsP is composed of a periplasmic globular 7TMR-DISMED2 domain, a membrane-spanning region, and cytoplasmic PAS, GGDEF and EAL domains. The EAL domain confers phosphodiesterase activity towards the second messenger cyclic di-GMP, a key regulatory player in the transition between bacterial lifestyles. RgsP and RgsM localize to sites of zonal cell wall synthesis at the new cell pole and cell divison site, suggesting a role in cell wall biogenesis. The two proteins are essential for cell wall biogenesis and cell growth. Cells depleted of RgsP or RgsM had an altered muropeptide composition and RgsM binds to peptidoglycan. RgsP and RgsM orthologs are functional when interchanged between α-rhizobial species pointing to a conserved mechanism for cell wall biogenesis/remodeling within the Rhizobiales. Overall, our findings suggest that RgsP and RgsM contribute to the regulation of unipolar cell wall biogenesis in α-rhizobia.

HpoR, a novel c-di-GMP effective transcription factor, links the second messenger's regulatory function to the mycobacterial antioxidant defense.

Cyclic di-GMP (c-di-GMP) is a global signaling molecule that widely modulates diverse cellular processes. However, whether or not the c-di-GMP signal participates in regulation of bacterial antioxidant defense is unclear, and the involved regulators remain to be explored. In this study, we characterized HpoR as a novel c-di-GMP effective transcription factor and found a link between the c-di-GMP signal and the antioxidant regulation in Mycobacterium smegmatis. H2O2 stress induces c-di-GMP accumulation in M. smegmatis. High level of c-di-GMP triggers expression of a redox gene cluster, designated as hpoR operon, which is required for the mycobacterial H2O2 resistance. HpoR acts as an inhibitor of the hpoR operon and recognizes a 12-bp motif sequence within the upstream regulatory region of the operon. c-di-GMP specifically binds with HpoR at a ratio of 1:1. Low concentrations of c-di-GMP stimulate the DNA-binding activity of HpoR, whereas high concentrations of the signal molecule inhibit the activity. Strikingly, high level of c-di-GMP de-represses the intracellular association of HpoR with the regulatory region of the hpoR operon in M. smegmatis and enhances the mycobacterial H2O2 resistance. Therefore, we report a novel c-di-GMP effective regulator in mycobacteria, which extends the second messenger's function to bacterial antioxidant defense.