Marc Folcher

Allosteric control of cyclic di-GMP signaling

Cyclic di-guanosine monophosphate is a bacterial second messenger that has been implicated in biofilm formation, antibiotic resistance, and persistence of pathogenic bacteria in their animal host. Although the enzymes responsible for the regulation of cellular levels of c-di-GMP, diguanylate cyclases (DGC) and phosphodiesterases, have been identified recently, little information is available on the molecular mechanisms involved in controlling the activity of these key enzymes or on the specific interactions of c-di-GMP with effector proteins. By using a combination of genetic, biochemical, and modeling techniques we demonstrate that an allosteric binding site for c-di-GMP (I-site) is responsible for non-competitive product inhibition of DGCs. The I-site was mapped in both multi- and single domain DGC proteins and is fully contained within the GGDEF domain itself. In vivo selection experiments and kinetic analysis of the evolved I-site mutants led to the definition of an RXXD motif as the core c-di-GMP binding site. Based on these results and based on the observation that the I-site is conserved in a majority of known and potential DGC proteins, we propose that product inhibition of DGCs is of fundamental importance for c-di-GMP signaling and cellular homeostasis. The definition of the I-site binding pocket provides an entry point into unraveling the molecular mechanisms of ligand-protein interactions involved in c-di-GMP signaling and makes DGCs a valuable target for drug design to develop new strategies against biofilm-related diseases.

Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression

Second messengers control a wide range of important cellular functions in eukaryotes and prokaryotes. Here we show that cyclic di-GMP, a global bacterial second messenger, promotes cell cycle progression in Caulobacter crescentus by mediating the specific degradation of the replication initiation inhibitor CtrA. During the G1-to-S-phase transition, both CtrA and its cognate protease ClpXP dynamically localize to the old cell pole, where CtrA is rapidly degraded. Sequestration of CtrA to the cell pole depends on PopA, a newly identified cyclic di-GMP effector protein. PopA itself localizes to the cell pole and directs CtrA to this subcellular site via the direct interaction with a mediator protein, RcdA. We present evidence that c-di-GMP regulates CtrA degradation during the cell cycle by controlling the dynamic sequestration of the PopA recruitment factor to the cell pole. Furthermore, we show that cell cycle timing of CtrA degradation relies on converging pathways responsible for substrate and protease localization to the old cell pole. This is the first report that links cyclic di-GMP to protein dynamics and cell cycle control in bacteria.

DgrA is a member of a new family of cyclic diguanosine monophosphate receptors and controls flagellar motor function in Caulobacter crescentus.

Bacteria are able to switch between two mutually exclusive lifestyles, motile single cells and sedentary multicellular communities that colonize surfaces. These behavioral changes contribute to an increased fitness in structured environments and are controlled by the ubiquitous bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP). In response to changing environments, fluctuating levels of c-di-GMP inversely regulate cell motility and cell surface adhesins. Although the synthesis and breakdown of c-di-GMP has been studied in detail, little is known about the downstream effector mechanisms. Using affinity chromatography, we have isolated several c-di-GMP-binding proteins from Caulobacter crescentus. One of these proteins, DgrA, is a PilZ homolog involved in mediating c-di-GMP-dependent control of C. crescentus cell motility. Biochemical and structural analysis of DgrA and homologs from C. crescentus, Salmonella typhimurium, and Pseudomonas aeruginosa demonstrated that this protein family represents a class of specific diguanylate receptors and suggested a general mechanism for c-di-GMP binding and signal transduction. Increased concentrations of c-di-GMP or DgrA blocked motility in C. crescentus by interfering with motor function rather than flagellar assembly. We present preliminary evidence implicating the flagellar motor protein FliL in DgrA-dependent cell motility control.

Pharmaceutically controlled designer circuit for the treatment of the metabolic syndrome

Synthetic biology has significantly advanced the design of genetic devices that can reprogram cellular activities and provide novel treatment strategies for future gene- and cell-based therapies. However, many metabolic disorders are functionally linked while developing distinct diseases that are difficult to treat using a classic one-drug-one-disease intervention scheme. For example, hypertension, hyperglycemia, obesity, and dyslipidemia are interdependent pathologies that are collectively known as the metabolic syndrome, the prime epidemic of the 21st century. We have designed a unique therapeutic strategy in which the clinically licensed antihypertensive drug guanabenz (Wytensin) activates a synthetic signal cascade that stimulates the secretion of metabolically active peptides GLP-1 and leptin. Therefore, the signal transduction of a chimeric trace-amine–associated receptor 1 (cTAAR1) was functionally rewired via cAMP and cAMP-dependent phosphokinase A (PKA)-mediated activation of the cAMP-response element binding protein (CREB1) to transcription of synthetic promoters containing CREB1-specific cAMP response elements. Based on this designer signaling cascade, it was possible to use guanabenz to dose-dependently control expression of GLP-1-FcmIgG-Leptin, a bifunctional therapeutic peptide hormone that combines the glucagon-like peptide 1 (GLP-1) and leptin via an IgG-Fc linker. In mice developing symptoms of the metabolic syndrome, this three-in-one treatment strategy was able to simultaneously attenuate hypertension and hyperglycemia as well as obesity and dyslipidemia. Using a clinically licensed drug to coordinate expression of therapeutic transgenes combines drug- and gene-based therapies for coordinated treatment of functionally related metabolic disorders.

Role of Acid Metabolism in Streptomyces coelicolor Morphological Differentiation and Antibiotic Biosynthesis

Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in Streptomyces coelicolor. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent K(m) values of CitA for acetyl coenzyme A (acetyl-CoA) (32 microM) and oxaloacetate (17 microM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD(+), NADH, ATP, ADP, isocitrate, or alpha-ketoglutarate). The corresponding gene (citA) was cloned and sequenced, allowing construction of a citA mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that citA encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the citA mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains, citA and bldJ mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that citA defines a new stage of the Streptomyces developmental cascade.

Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant

Synthetic devices for traceless remote control of gene expression may provide new treatment opportunities in future gene- and cell-based therapies. Here we report the design of a synthetic mind-controlled gene switch that enables human brain activities and mental states to wirelessly programme the transgene expression in human cells. An electroencephalography (EEG)-based brain–computer interface (BCI) processing mental state-specific brain waves programs an inductively linked wireless-powered optogenetic implant containing designer cells engineered for near-infrared (NIR) light-adjustable expression of the human glycoprotein SEAP (secreted alkaline phosphatase). The synthetic optogenetic signalling pathway interfacing the BCI with target gene expression consists of an engineered NIR light-activated bacterial diguanylate cyclase (DGCL) producing the orthogonal second messenger cyclic diguanosine monophosphate (c-di-GMP), which triggers the stimulator of interferon genes (STING)-dependent induction of synthetic interferon-β promoters. Humans generating different mental states (biofeedback control, concentration, meditation) can differentially control SEAP production of the designer cells in culture and of subcutaneous wireless-powered optogenetic implants in mice.

Roles of Aconitase in Growth, Metabolism, and Morphological Differentiation of Streptomyces coelicolor

The studies of aconitase presented here, along with those of citrate synthase (P. H. Viollier, W. Minas, G. E. Dale, M. Folcher, and C. J. Thompson, J. Bacteriol. 183:3184-3192, 2001), were undertaken to investigate the role of the tricarboxylic acid (TCA) cycle in Streptomyces coelicolor development. A single aconitase activity (AcoA) was detected in protein extracts of cultures during column purification. The deduced amino acid sequence of the cloned acoA gene constituted the N-terminal sequence of semipurified AcoA and was homologous to bacterial A-type aconitases and bifunctional eukaryotic aconitases (iron regulatory proteins). The fact that an acoA disruption mutant (BZ4) did not grow on minimal glucose media in the absence of glutamate confirmed that this gene encoded the primary vegetative aconitase catalyzing flux through the TCA cycle. On glucose-based complete medium, BZ4 had defects in growth, antibiotic biosynthesis, and aerial hypha formation, partially due to medium acidification and accumulation of citrate. The inhibitory effects of acids and citrate on BZ4 were partly suppressed by buffer or by introducing a citrate synthase mutation. However, the fact that growth of an acoA citA mutant remained impaired, even on a nonacidogenic carbon source, suggested alternative functions of AcoA. Immunoblots revealed that AcoA was present primarily during substrate mycelial growth on solid medium. Transcription of acoA was limited to the early growth phase in liquid cultures from a start site mapped in vitro and in vivo.

Molecular characterization and transcriptional analysis of a multidrug resistance gene cloned from the pristinamycin‐producing organism, Streptomyces pristinaespiralis

A multidrug resistance gene (mdr) has been cloned from Streptomyces pristinaespiralis, a producer of two antibiotics having synergistic activities together known as pristinamycin. This gene, ptr, provides resistance not only to two structurally dissimilar compounds (pristinamycin I, PI; pristinamycin II, PII) and the natural pristinamycin mixture but also to rifampicin. Mutagenesis and subcloning of ptr localized it to a 2 kb region which was sequenced and analysed. It contained an open reading frame of 1506 bp which encoded a putative membrane protein with 14 hydrophobic domains, and showed sequence similarity to a superfamily of bacterial proteins that employ transmembrane electrochemical gradients to catalyse active efflux of various antibiotics and toxic compounds. Ptr was most similar to a subfamily which included other mdr genes and antibiotic transport genes associated with antibiotic biosynthetic gene clusters in actinomycetes. In vitro coupled transcription‐translation experiments were used to identify the ptr gene product. Analysis of the upstream region did not reveal a divergently transcribed repressor gene, as is the case for several related resistance determinants involved in antibiotic transport, suggesting that ptr is regulated by a different mechanism. Transcriptional analyses of this gene, carried out in both S. pristinaespiralis and Streptomyces lividans, indicated the same transcriptional start point and predicted −10 and −35 hexamers which were somewhat similar to Streptomyces vegetative‐type promoters.

A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells

Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid, we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3, Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells, whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine.

A Synthetic Erectile Optogenetic Stimulator Enabling Blue‐Light‐Inducible Penile Erection

Precise spatiotemporal control of physiological processes by optogenetic devices inspired by synthetic biology may provide novel treatment opportunities for gene- and cell-based therapies. An erectile optogenetic stimulator (EROS), a synthetic designer guanylate cyclase producing a blue-light-inducible surge of the second messenger cyclic guanosine monophosphate (cGMP) in mammalian cells, enabled blue-light-dependent penile erection associated with occasional ejaculation after illumination of EROS-transfected corpus cavernosum in male rats. Photostimulated short-circuiting of complex psychological, neural, vascular, and endocrine factors to stimulate penile erection in the absence of sexual arousal may foster novel advances in the treatment of erectile dysfunction.