cAMP Receptor Research Articles

The interplay between metabolic stochasticity and cAMP-CRP regulation in single E. coli cells

The inherent stochasticity of metabolism raises a critical question for understanding homeostasis: are cellular processes regulated in response to internal fluctuations? Here, we show that, in E. coli cells under constant external conditions, catabolic enzyme expression continuously responds to metabolic fluctuations. The underlying regulatory feedback is enabled by the cyclic AMP (cAMP) and cAMP receptor protein (CRP) system, which controls catabolic enzyme expression based on metabolite concentrations. Using single-cell microscopy, genetic constructs in which this feedback is disabled, and mathematical modeling, we show how fluctuations circulate through the metabolic and genetic network at sub-cell-cycle timescales. Modeling identifies four noise propagation modes, including one specific to CRP regulation. Together, these modes correctly predict noise circulation at perturbed cAMP levels. The cAMP-CRP system may thus have evolved to control internal metabolic fluctuations in addition to external growth conditions. We conjecture that second messengers may more broadly function to achieve cellular homeostasis.

A simple mechanism for integration of quorum sensing and cAMP signalling in Vibrio cholerae

Many bacteria use quorum sensing to control changes in lifestyle. The process is regulated by microbially derived ‘autoinducer’ signalling molecules, that accumulate in the local environment. Individual cells sense autoinducer abundance, to infer population density, and alter their behaviour accordingly. In Vibrio cholerae, quorum-sensing signals are transduced by phosphorelay to the transcription factor LuxO. Unphosphorylated LuxO permits expression of HapR, which alters global gene expression patterns. In this work, we have mapped the genome-wide distribution of LuxO and HapR in V. cholerae. Whilst LuxO has a small regulon, HapR targets 32 loci. Many HapR targets coincide with sites for the cAMP receptor protein (CRP) that regulates the transcriptional response to carbon starvation. This overlap, also evident in other Vibrio species, results from similarities in the DNA sequence bound by each factor. At shared sites, HapR and CRP simultaneously contact the double helix and binding is stabilised by direct interaction of the two factors. Importantly, this involves a CRP surface that usually contacts RNA polymerase to stimulate transcription. As a result, HapR can block transcription activation by CRP. Thus, by interacting at shared sites, HapR and CRP integrate information from quorum sensing and cAMP signalling to control gene expression. This likely allows V. cholerae to regulate subsets of genes during the transition between aquatic environments and the human host.

A simple mechanism for integration of quorum sensing and cAMP signalling in Vibrio cholerae

Many bacteria use quorum sensing to control changes in lifestyle. The process is regulated by microbially derived ‘autoinducer’ signalling molecules, that accumulate in the local environment. Individual cells sense autoinducer abundance, to infer population density, and alter their behaviour accordingly. In Vibrio cholerae, quorum-sensing signals are transduced by phosphorelay to the transcription factor LuxO. Unphosphorylated LuxO permits expression of HapR, which alters global gene expression patterns. In this work, we have mapped the genome-wide distribution of LuxO and HapR in V. cholerae. Whilst LuxO has a small regulon, HapR targets 32 loci. Many HapR targets coincide with sites for the cAMP receptor protein (CRP) that regulates the transcriptional response to carbon starvation. This overlap, also evident in other Vibrio species, results from similarities in the DNA sequence bound by each factor. At shared sites, HapR and CRP simultaneously contact the double helix and binding is stabilised by direct interaction of the two factors. Importantly, this involves a CRP surface that usually contacts RNA polymerase to stimulate transcription. As a result, HapR can block transcription activation by CRP. Thus, by interacting at shared sites, HapR and CRP integrate information from quorum sensing and cAMP signalling to control gene expression. This likely allows V. cholerae to regulate subsets of genes during the transition between aquatic environments and the human host.

Pro-inflammatory action of formoterol in human bronchial epithelia

Despite the wide usage of β2-adrenoceptor agonists in asthma treatment, they do have side effects such as aggravating inflammation. We previously reported that isoprenaline induced Cl- secretion and IL-6 release via cAMP-dependent pathways in human bronchial epithelia, but the mechanisms underlying the inflammation-aggravation effects of β2-adrenoceptor agonists remain pooly understood. In this study, we investigated formoterol, a more specific β2-adrenoceptor agonist, -mediated signaling pathways involved in the production of IL-6 and IL-8 in 16HBE14o- human bronchial epithelia. The effects of formoterol were detected in the presence of PKA, exchange protein directly activated by cAMP (EPAC), cystic fibrosis transmembrane conductance regulator (CFTR), extracellular signal-regulated protein kinase (ERK)1/2 and Src inhibitors. The involvement of β-arrestin2 was determined using siRNA knockdown. Our results indicate that formoterol can induce IL-6 and IL-8 secretion in concentration-dependent manner. The PKA-specific inhibitor, H89, partially inhibited IL-6 release, but not IL-8. Another intracellular cAMP receptor, EPAC, was not involved in either IL-6 or IL-8 release. PD98059 and U0126, two ERK1/2 inhibitors, blocked IL-8 while attenuated IL-6 secretion induced by formoterol. Furthermore, formoterol-induced IL-6 and IL-8 release was attenuated by Src inhibitors, namely dasatinib and PP1, and CFTRinh172, a CFTR inhibitor. In addition, knockdown of β-arrestin2 by siRNA only suppressed IL-8 release when a high concentration of formoterol (1 μM) was used. Taken together, our results suggest that formoterol stimulates IL-6 and IL-8 release which involves PKA/Src/ERK1/2 and/or β-arrestin2 signaling pathways.

Designing glucose utilization "highway" for recombinant biosynthesis

cAMP receptor protein (CRP) is known as a global regulatory factor mainly mediating carbon source catabolism. Herein, we successfully engineered CRP to develop microbial chassis cells with improved recombinant biosynthetic capability in minimal medium with glucose as single carbon source. The obtained best-performing cAMP-independent CRPmu9 mutant conferred both faster cell growth and a 133-fold improvement in expression level of lac promoter in presence of 2% glucose, compared with strain under regulation of CRPwild-type. Promoters free from “glucose repression” are advantageous for recombinant expression, as glucose is a frequently used inexpensive carbon source in high-cell-density fermentations. Transcriptome analysis demonstrated that the CRP mutant globally rewired cell metabolism, displaying elevated tricarboxylic acid cycle activity; reduced acetate formation; increased nucleotide biosynthesis; and improved ATP synthesis, tolerance, and stress-resistance activity. Metabolites analysis confirmed the enhancement of glucose utilization with the upregulation of glycolysis and glyoxylate-tricarboxylic acid cycle. As expected, an elevated biosynthetic capability was demonstrated with vanillin, naringenin and caffeic acid biosynthesis in strains regulated by CRPmu9. This study has expanded the significance of CRP optimization into glucose utilization and recombinant biosynthesis, beyond the conventionally designated carbon source utilization other than glucose. The Escherichiacoli cell regulated by CRPmu9 can be potentially used as a beneficial chassis for recombinant biosynthesis.

Therapeutic effects on the development of heart failure with preserved ejection fraction by the sodium-glucose cotransporter 2 inhibitor dapagliflozin in type 2 diabetes

BackgroundHeart failure with preserved ejection fraction (HFpEF) is a common disease with high morbidity and lacks effective treatment. We investigated the protective effects of the long-term application of the sodium-glucose cotransporter 2 inhibitor (SGLT2i) dapagliflozin on diabetes-associated HFpEF in a rat model. Serum proteomics and metabolomics analysis were also conducted in type 2 diabetic patients with HFpEF treated with dapagliflozin.MethodsMale Zucker diabetic fatty (ZDF) rats were used as a model of diabetic cardiomyopathy. From weeks 16 to 28, animals were given a vehicle or dapagliflozin (1 mg/kg) once daily. Primary blood biochemistry indices, echocardiography, histopathology, and cardiac hemodynamics were determined during the study period. The key markers of myocardial fibrosis, nitro-oxidative stress, inflammation, apoptosis, autophagy, and AMPK/mTOR signaling were examined. Additionally, healthy controls and individuals with type 2 diabetes were enrolled and 16 serum samples from 4 groups were randomly selected. Serum proteome and metabolome changes after dapagliflozin treatment were analyzed in diabetic individuals with HFpEF.ResultsDapagliflozin effectively prevented the development of HFpEF in rats with diabetes by mitigating nitro-oxidative stress, pro-inflammatory cytokines, myocardial hypertrophy, and fibrosis, reducing apoptosis, and restoring autophagy through AMPK activating and mTOR pathway repressing. Proteomics and metabolomics revealed that cholesterol and high-density lipoprotein particle metabolism, nicotinate and nicotinamide metabolism, arginine biosynthesis, and cAMP and peroxisome proliferator-activated receptor (PPAR) signaling are the major disturbed pathways in HFpEF patients treated with dapagliflozin.ConclusionLong-term treatment with dapagliflozin significantly prevented the development of HFpEF in diabetic rats. Dapagliflozin could be a promising therapeutic strategy in managing HFpEF individuals with type 2 diabetes.

Unleashing the Influence of cAMP Receptor Protein: The Master Switch of Bacteriocin Export in Pectobacterium carotovorum subsp. carotovorum.

Pectobacterium carotovorum subsp. carotovorum (Pcc) is a Gram-negative phytopathogenic bacterium that produces carocin, a low-molecular-weight bacteriocin that can kill related strains in response to factors in the environment such as UV exposure or nutritional deficiency. The function of the catabolite activator protein (CAP), also known as the cyclic AMP receptor protein (CRP), as a regulator of carocin synthesis was examined. The crp gene was knocked out as part of the investigation, and the outcomes were assessed both in vivo and in vitro. Analysis of the DNA sequence upstream of the translation initiation site of carocin S3 revealed two putative binding sites for CRP that were confirmed using a biotinylated probe pull-down experiment. This study revealed that the deletion of crp inhibited genes involved in extracellular bacteriocin export via the flagellar type III secretion system and impacted the production of many low-molecular-weight bacteriocins. The biotinylated probe pull-down test demonstrated that when UV induction was missing, CRP preferentially attached to one of the two CAP sites while binding to both when UV induction was present. In conclusion, our research aimed to simulate the signal transduction system that controls the expression of the carocin gene in response to UV induction.

Δ9-Tetrahydrocannabinol Treatment Modifies Insulin Secretion in Pancreatic Islets from Prediabetic Mice Under Hypercaloric Diet.

Background: The endocannabinoid system over-activation is associated with type-2 diabetes mellitus onset, involving physiological, metabolic, and genetic alterations in pancreatic islets. The use of Δ9-Tetrahydrocannabinol (THC) as treatment is still controversial since its effects and mechanisms on insulin secretion are unclear. The aim of this study was to evaluate the effects of THC treatment in pancreatic islets from prediabetic mice. Methods: Prediabetes was induced in mice by hypercaloric diet, and then treated with THC for 3 weeks. Blood glucose and body weight were determined, after behavior tests. Histological changes were evaluated in whole pancreas; in isolated islets we analyzed the effect of THC exposure in glucose-stimulated insulin secretion (GSIS), gene expression, intracellular cyclic adenosine monophosphate (cAMP), and cytosolic calcium changes. Results: THC treatment in prediabetic mice enhanced anxiety and antidepressive behavior without changes in food ingestion, decreased oral-glucose tolerance test, plasma insulin and weight, with small alterations on pancreatic histology. In isolated islets from healthy mice THC increased GSIS, cAMP, and CB1 receptor (CB1r) expression, meanwhile calcium release was diminished. Small changes were observed in islets from prediabetic mice. Conclusions: THC treatment improves some clinical parameters in prediabetic mice, however, in isolated islets, modifies GSIS, intracellular calcium and gene expression, suggesting specific effects related to diabetes evolution.

A Direct Comparison of Targeted Muscle Reinnervation and Regenerative Peripheral Nerve Interfaces to Prevent Neuroma Pain.

Targeted muscle reinnervation (TMR) and regenerative peripheral nerve interface (RPNI) surgeries manage neuroma pain; however, there remains considerable discord regarding the best treatment strategy. We provide a direct comparison of TMR and RPNI surgery using a rodent model for the treatment of neuroma pain. The tibial nerve of 36 Fischer rats was transected and secured to the dermis to promote neuroma formation. Pain was assessed using mechanical stimulation at the neuroma site (direct pain) and von Frey analysis at the footpad (to assess tactile allodynia from collateral innervation). Once painful neuromas were detected 6 weeks later, animals were randomized to experimental groups: (a) TMR to the motor branch to biceps femoris, (b) RPNI with an extensor digitorum longus graft, (c) neuroma excision, and (d) neuroma in situ. The TMR/RPNIs were harvested to confirm muscle reinnervation, and the sensory ganglia and nerves were harvested to assess markers of regeneration, pain, and inflammation. Ten weeks post-TMR/RPNI surgery, animals had decreased pain scores compared with controls ( P < .001) and they both demonstrated neuromuscular junction reinnervation. Compared with neuroma controls, immunohistochemistry showed that sensory neuronal cell bodies of TMR and RPNI showed a decrease in regeneration markers phosphorylated cyclic AMP receptor binding protein and activation transcription factor 3 and pain markers transient receptor potential vanilloid 1 and neuropeptide Y ( P < .05). The nerve and dorsal root ganglion maintained elevated Iba-1 expression in all cohorts. RPNI and TMR improved pain scores after neuroma resection suggesting both may be clinically feasible techniques for improving outcomes for patients with nerve injuries or those undergoing amputation.

Quantitative model for genome-wide cyclic AMP receptor protein binding site identification and characteristic analysis

Cyclic AMP receptor proteins (CRPs) are important transcription regulators in many species. The prediction of CRP-binding sites was mainly based on position-weighted matrixes (PWMs). Traditional prediction methods only considered known binding motifs, and their ability to discover inflexible binding patterns was limited. Thus, a novel CRP-binding site prediction model called CRPBSFinder was developed in this research, which combined the hidden Markov model, knowledge-based PWMs and structure-based binding affinity matrixes. We trained this model using validated CRP-binding data from Escherichia coli and evaluated it with computational and experimental methods. The result shows that the model not only can provide higher prediction performance than a classic method but also quantitatively indicates the binding affinity of transcription factor binding sites by prediction scores. The prediction result included not only the most knowns regulated genes but also 1089 novel CRP-regulated genes. The major regulatory roles of CRPs were divided into four classes: carbohydrate metabolism, organic acid metabolism, nitrogen compound metabolism and cellular transport. Several novel functions were also discovered, including heterocycle metabolic and response to stimulus. Based on the functional similarity of homologous CRPs, we applied the model to 35 other species. The prediction tool and the prediction results are online and are available at: https://awi.cuhk.edu.cn/∼CRPBSFinder.

Impact of the cAMP-cAMP Receptor Protein Regulatory Complex on Lipopolysaccharide Modifications and Polymyxin B Resistance in Escherichia coli.

Gram-negative bacteria have a unique cell surface that can be modified to maintain bacterial fitness in diverse environments. A well-defined example is the modification of the lipid A component of lipopolysaccharide (LPS), which promotes resistance to polymyxin antibiotics and antimicrobial peptides. In many organisms, such modifications include the addition of the amine-containing constituents 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN). Addition of pEtN is catalyzed by EptA, which uses phosphatidylethanolamine (PE) as its substrate donor, resulting in production of diacylglycerol (DAG). DAG is then quickly recycled into glycerophospholipid (GPL) synthesis by the DAG kinase A (DgkA) to produce phosphatidic acid, the major GPL precursor. Previously, we hypothesized that loss of DgkA recycling would be detrimental to the cell when LPS is heavily modified. Instead, we found that DAG accumulation inhibits EptA activity, preventing further degradation of PE, the predominant GPL of the cell. However, DAG inhibition of pEtN addition results in complete loss of polymyxin resistance. Here, we selected for suppressors to find a mechanism of resistance independent of DAG recycling or pEtN modification. Disrupting the gene encoding the adenylate cyclase, cyaA, fully restored antibiotic resistance without restoring DAG recycling or pEtN modification. Supporting this, disruptions of genes that reduce CyaA-derived cAMP formation (e.g., ptsI) or disruption of the cAMP receptor protein, Crp, also restored resistance. We found that loss of the cAMP-CRP regulatory complex was necessary for suppression and that resistance arises from a substantial increase in l-Ara4N-modified LPS, bypassing the need for pEtN modification. IMPORTANCE Gram-negative bacteria can alter the structure of their LPS to promote resistance to cationic antimicrobial peptides, including polymyxin antibiotics. Polymyxins are considered last-resort antibiotics for treatment against multidrug-resistant Gram-negative organisms. Here, we explore how changes in general metabolism and carbon catabolite repression pathways can alter LPS structure and influence polymyxin resistance.

The cAMP receptor protein (CRP) enhances the competitive nature of Salmonella Typhimurium

The cAMP receptor protein (CRP) is a global regulatory protein. We evaluated the role of CRP in starvation physiology in Salmonella Typhimurium. The Δcrpmutant survived 10days of starvation. However, in a co-culture with the wild type in nutrient-rich medium, Δcrpdied within 48h. Similar co-culture results were observed with Escherichia coli and Staphylococcus aureus. Our study showed that the Δcrpmutant was not killed by toxins and the Type IV secretion system of the WT. The possibility of viable but non-culturable cells (VBNC) was also ruled out. However, when the overall metabolism of the co-culture was slowed down (anaerobic condition, inhibition by antibiotics and low temperature) that improved the survival of Δcrpin co-culture. But one more significant observation was that the Δcrpmutant survived in nutrient-free co-culture conditions. These two observations suggest that CRP protein is essential for efficient nutrient assimilation in a competitive environment. The cells without CRP protein are unable to evaluate the energy balance within the cell, and the cell spends energy to absorb nutrients. But the wild type cell absorbs nutrients at a faster rate than Δcrpmutant. This leads to a situation wherein the Δcrpis spending energy to absorb the nutrients but is unable to compete with the wild type. This futile metabolism leads to death. Hence, this study shows that CRP is a metabolism modulator in a complex nutrient environment. This study also highlights the need for innovative growth conditions to understand the unique function of a gene.

Structural Basis of Dual Specificity of Sinorhizobium meliloti Clr, a cAMP and cGMP Receptor Protein.

In bacteria, the most prevalent receptor proteins of 3',5'-cyclic AMP (cAMP) and 3',5'-cyclic GMP (cGMP) are found among transcription factors of the Crp-Fnr superfamily. The prototypic Escherichia coli catabolite activator protein (CAP) represents the main Crp cluster of this superfamily and is known to bind cAMP and cGMP but to mediate transcription activation only in its cAMP-bound state. In contrast, both cyclic nucleotides mediate transcription activation by Sinorhizobium meliloti Clr, mapping to cluster G of Crp-like proteins. We present crystal structures of Clr-cAMP and Clr-cGMP bound to the core motif of the palindromic Clr DNA binding site (CBS). We show that both cyclic nucleotides shift ternary Clr-cNMP-CBS-DNA complexes (where cNMP is cyclic nucleotide monophosphate) to almost identical active conformations, unlike the situation known for the E. coli CAP-cNMP complex. Isothermal titration calorimetry measured similar affinities of cAMP and cGMP binding to Clr in the presence of CBS core motif DNA (equilibrium dissociation constant for cNMP (KDcNMP], ~7 to 11 μM). However, different affinities were determined in the absence of this DNA (KDcGMP, ~24 μM; KDcAMP, ~6 μM). Sequencing of Clr-coimmunoprecipitated DNA as well as electrophoretic mobility shift and promoter-probe assays expanded the list of experimentally proven Clr-regulated promoters and CBS. This comprehensive set of CBS features conserved nucleobases that are consistent with the sequence readout through interactions of Clr amino acid residues with these nucleobases, as revealed by the Clr-cNMP-CBS-DNA crystal structures. IMPORTANCE Cyclic 3',5'-AMP (cAMP) and cyclic 3',5'-GMP (cGMP) are both long known as important nucleotide secondary messengers in eukaryotes. This is also the case for cAMP in prokaryotes, whereas a signaling role for cGMP in this domain of life has been recognized only recently. Catabolite repressor proteins (CRPs) are the most ubiquitous bacterial cAMP receptor proteins. Escherichia coli CAP, the prototypic transcription regulator of the main Crp cluster, binds both cyclic mononucleotides, but only the CAP-cAMP complex promotes transcription activation. In contrast, Crp cluster G proteins studied so far are activated by cGMP or by both cAMP and cGMP. Here, we report a structural analysis of the cAMP- and cGMP-activatable cluster G member Clr from Sinorhizobium meliloti, how binding of cAMP and cGMP shifts Clr to its active conformation, and the structural basis of its DNA binding site specificity.

Optimization of FK-506 production in Streptomyces tsukubaensis by modulation of Crp-mediated regulation

FK-506 is a potent immunosuppressive macrocyclic polyketide with growing pharmaceutical interest, produced by Streptomyces tsukubaensis. However, due to low levels synthesized by the wild-type strain, biotechnological production of FK-506 is rather limited. Optimization strategies to enhance the productivity of S. tsukubaensis by means of genetic engineering have been established. In this work primarily global regulatory aspects with respect to the FK-506 biosynthesis have been investigated with the focus on the global Crp (cAMP receptor protein) regulator. In expression analyses and protein-DNA interaction studies, the role of Crp during FK-506 biosynthesis was elucidated. Overexpression of Crp resulted in two-fold enhancement of FK-506 production in S. tsukubaensis under laboratory conditions. Further optimizations using fermentors proved that the strategy described in this study can be transferred to industrial scale, presenting a new approach for biotechnological FK-506 production.Key Points• The role of the global Crp (cAMP receptor protein) regulator for FK-506 biosynthesis in S. tsukubaensis was demonstrated• Crp overexpression in S. tsukubaensis was applied as an optimization strategy to enhance FK-506 and FK-520 production resulting in two-fold yield increase

Proximity Protein Labeling In Dictyostelium With Engineered Ascorbic Acid Peroxidase 2.

To fully understand any cellular process, we not only need to identify the proteins implicated, but also how the protein network is structurally and spatially organized and changes over time. However, the dynamic nature of many protein interactions involved in cellular signaling pathways continues to be the bottleneck in mapping and studying protein networks. Fortunately, a recently developed proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells allows the identification of weak and/or transient protein interactions with spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium, using the cAMP receptor cAR1 as example. Coupled to the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium's proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium.

Ampicillin-controlled glucose metabolism manipulates the transition from tolerance to resistance in bacteria

The mechanism(s) of how bacteria acquire tolerance and then resistance to antibiotics remains poorly understood. Here, we show that glucose abundance decreases progressively as ampicillin-sensitive strains acquire resistance to ampicillin. The mechanism involves that ampicillin initiates this event via targeting pts promoter and pyruvate dehydrogenase (PDH) to promote glucose transport and inhibit glycolysis, respectively. Thus, glucose fluxes into pentose phosphate pathway to generate reactive oxygen species (ROS) causing genetic mutations. Meanwhile, PDH activity is gradually restored due to the competitive binding of accumulated pyruvate and ampicillin, which lowers glucose level, and activates cyclic adenosine monophosphate (cAMP)/cAMP receptor protein (CRP) complex. cAMP/CRP negatively regulates glucose transport and ROS but enhances DNA repair, leading to ampicillin resistance. Glucose and Mn2+ delay the acquisition, providing an effective approach to control the resistance. The same effect is also determined in the intracellular pathogen Edwardsiella tarda. Thus, glucose metabolism represents a promising target to stop/delay the transition of tolerance to resistance.

CAMP Activation of the cAMP Receptor Protein, a Model Bacterial Transcription Factor.

The active and inactive structures of the Escherichia coli cAMP receptor protein (CRP), a model bacterial transcription factor, are compared to generate a paradigm in the cAMP-induced activation of CRP. The resulting paradigm is shown to be consistent with numerous biochemical studies of CRP and CRP*, a group of CRP mutants displaying cAMP-free activity. The cAMP affinity of CRP is dictated by two factors: (i) the effectiveness of the cAMP pocket and (ii) the protein equilibrium of apo-CRP. How these two factors interplay in determining the cAMP affinity and cAMP specificity of CRP and CRP* mutants are discussed. Both the current understanding and knowledge gaps of CRP-DNA interactions are also described. This review ends with a list of several important CRP issues that need to be addressed in the future.

Mycobacterial Regulatory Systems Involved in the Regulation of Gene Expression Under Respiration-Inhibitory Conditions.

Mycobacterium tuberculosis is the causative agent of tuberculosis. M. tuberculosis can survive in a dormant state within the granuloma, avoiding the host-mounting immune attack. M. tuberculosis bacilli in this state show increased tolerance to antibiotics and stress conditions, and thus the transition of M. tuberculosis to the nonreplicating dormant state acts as an obstacle to tuberculosis treatment. M. tuberculosis in the granuloma encounters hostile environments such as hypoxia, nitric oxide, reactive oxygen species, low pH, and nutrient deprivation, etc., which are expected to inhibit respiration of M. tuberculosis. To adapt to and survive in respiration-inhibitory conditions, it is required for M. tuberculosis to reprogram its metabolism and physiology. In order to get clues to the mechanism underlying the entry of M. tuberculosis to the dormant state, it is important to understand the mycobacterial regulatory systems that are involved in the regulation of gene expression in response to respiration inhibition. In this review, we briefly summarize the information regarding the regulatory systems implicated in upregulation of gene expression in mycobacteria exposed to respiration-inhibitory conditions. The regulatory systems covered in this review encompass the DosSR (DevSR) two-component system, SigF partner switching system, MprBA-SigE-SigB signaling pathway, cAMP receptor protein, and stringent response.

Phylogeny-wide analysis of G-protein coupled receptors in social amoebas and implications for the evolution of multicellularity.

G-protein coupled receptors (GPCRs) are seven-transmembrane proteins and constitute the largest group of receptors within eukaryotes. The presence of a large set of GPCRs in the unicellular Amoebozoa was surprising and is indicative of the largely undiscovered environmental sensing capabilities in this group. Evolutionary transitions from unicellular to multicellular lifestyles, like we see in social amoebas, have occurred several times independently in the Amoebozoa, and GPCRs may have been co-opted for new functions in cell-cell communication. Methods We have analysed a set of GPCRs from fully sequenced Amoebozoan genomes by Bayesian inference, compared their phylogenetic distribution and domain composition, and analysed their temporal and spatial expression patterns in five species of dictyostelids. Results We found evidence that most GPCRs are conserved deeply in the Amoebozoa and are probably performing roles in general cell functions and complex environmental sensing. All families of GPCRs (apart from the family 4 fungal pheromone receptors) are present in dictyostelids with family 5 being the largest and family 2 the one with the fewest members. For the first time, we identify the presence of family 1 rhodopsin-like GPCRs in dictyostelids. Some GPCRs have been amplified in the dictyostelids and in specific lineages thereof and through changes in expression patterns may have been repurposed for signalling in multicellular development. Discussion Our phylogenetic analysis suggests that GPCR families 1, 2 and 6 already diverged early in the Amoebozoa, whereas families 3 and 5 expanded later within the dictyostelids. The family 6 cAMP receptors that have experimentally supported roles in multicellular development in dictyostelids ( carA-carD; tasA/B) originated at the root of all dictyostelids and only have weakly associated homologs in Physarum polycephalum. Our analysis identified candidate GPCRs which have evolved in the dictyostelids and could have been co-opted for multicellular development.

Genetically Engineering Escherichia coli to Produce Xylitol from Corncob Hydrolysate without Lime Detoxification.

Before fermentation with hemicellulosic hydrolysate as a substrate, it is generally necessary to detoxify the toxic substances that are harmful to microorganism growth. Cyclic AMP receptor protein (CRP) is a global regulator, and mutation of its key sites may have an important impact on E. coli virulence tolerance. Using corncob hydrolysate without ion-exchange or lime detoxification as the substrate, shake flask fermentation experiments showed that CRP mutant IS5-dG (I112L, T127G, A144T) produced 18.4 g/L of xylitol within 34 h, and the OD600 was 9.7 at 24 h; these values were 41.5% and 21.3% higher than those of the starting strain, IS5-d, respectively. This mutant produced 82 g/L of xylitol from corncob hydrolysate without ion-exchange or lime detoxification during fed-batch fermentation in a 15-L bioreactor, with a productivity of 1.04 g/L/h; these values were 173% and 174% higher than the starting strain, respectively. To our knowledge, this is the highest xylitol concentration and productivity produced by microbial fermentation using completely non-detoxified hemicellulosic hydrolysate as the substrate to date. This study also showed that alkali neutralization, high temperature sterilization, and fermentation of the hydrolysate had important effects on the xylose loss rate and xylitol production.