cAMP Receptor Research Articles

Metabolomics reveals that the cAMP receptor protein regulates nitrogen and peptidoglycan synthesis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis requires extensive sensing and response to environment for its successful survival and pathogenesis, and signalling by cyclic adenosine 3′,5′-monophosphate (cAMP) is an important mechanism. cAMP regulates expression of target genes via interaction with downstream proteins, one of which is cAMP receptor protein (CRP), a global transcriptional regulator. Previous genomic works had identified regulon of CRP and investigated transcriptional changes in crp deletion mutant, however a link to downstream metabolomic events were lacking, which would help better understand roles of CRP. This work aims at investigating changes at metabolome level in M. tuberculosis crp deletion mutant combining untargeted LC-MS analysis and 13C isotope tracing analysis. The results were compared with previously published RNA sequencing data. We identified increasing abundances of metabolites related to nitrogen metabolism including ornithine, citrulline and glutamate derivatives, while 13C isotope labelling analysis further showed changes in turnover of these metabolites and amino acids, suggesting regulatory roles of CRP in nitrogen metabolism. Upregulation of diaminopimelic acid and its related genes also suggested role of CRP in regulation of peptidoglycan synthesis. This study provides insights on metabolomic aspects of cAMP-CRP regulatory pathway in M. tuberculosis and links to previously published transcriptomic data drawing a more complete map.

Structural Energy Landscapes and Plasticity of the Microstates of Apo Escherichia coli cAMP Receptor Protein.

The theory for allostery has evolved to a modern energy landscape ensemble theory, the major feature of which is the existence of multiple microstates in equilibrium. The properties of microstates are not well defined due to their transient nature. Characterization of apo protein microstates is important because the specific complex of the ligand-bound microstate defines the biological function. The information needed to link biological function and structure is a quantitative correlation of the energy landscapes between the apo and holo protein states. We employed the Escherichia coli cAMP receptor protein (CRP) system to test the features embedded in the ensemble theory because multiple crystalline apo and holo structures are available. Small angle X-ray scattering data eliminated one of the three apo states but not the other two. We defined the underlying energy landscape differences among the apo microstates by employing the computation algorithm COREX/BEST. The same connectivity patterns among residues in apo CRP are retained upon binding of cAMP. The microstates of apo CRP differ from one another by minor structural perturbations, resulting in changes in the energy landscapes of the various domains of CRP. Using the differences in energy landscapes among these apo states, we computed the cAMP binding energetics that were compared with solution biophysical results. Only one of the three apo microstates yielded data consistent with the solution data. The relative magnitude of changes in energy landscapes embedded in various apo microstates apparently defines the ultimate outcome of the cooperativity of binding.

The Cyclic AMP Receptor Protein Regulates Quorum Sensing and Global Gene Expression in Yersinia pestis during Planktonic Growth and Growth in Biofilms.

Cyclic AMP (cAMP) receptor protein (Crp) is an important transcriptional regulator of Yersinia pestis Expression of crp increases during pneumonic plague as the pathogen depletes glucose and forms large biofilms within lungs. To better understand control of Y. pestis Crp, we determined a 1.8-Å crystal structure of the protein-cAMP complex. We found that compared to Escherichia coli Crp, C helix amino acid substitutions in Y. pestis Crp did not impact the cAMP dependency of Crp to bind DNA promoters. To investigate Y. pestis Crp-regulated genes during plague pneumonia, we performed RNA sequencing on both wild-type and Δcrp mutant bacteria growing in planktonic and biofilm states in minimal media with glucose or glycerol. Y. pestis Crp was found to dramatically alter expression of hundreds of genes in a manner dependent upon carbon source and growth state. Gel shift assays confirmed direct regulation of the malT and ptsG promoters, and Crp was then linked to Y. pestis growth on maltose as a sole carbon source. Iron regulation genes ybtA and fyuA were found to be indirectly regulated by Crp. A new connection between carbon source and quorum sensing was revealed as Crp was found to regulate production of acyl-homoserine lactones (AHLs) through direct and indirect regulation of genes for AHL synthetases and receptors. AHLs were subsequently identified in the lungs of Y. pestis-infected mice when crp expression was highest in Y. pestis biofilms. Thus, in addition to the well-studied pla gene, other Crp-regulated genes likely have important functions during plague infection.IMPORTANCE Bacterial pathogens have evolved extensive signaling pathways to translate environmental signals into changes in gene expression. While Crp has long been appreciated for its role in regulating metabolism of carbon sources in many bacterial species, transcriptional profiling has revealed that this protein regulates many other aspects of bacterial physiology. The plague pathogen Y. pestis requires this global regulator to survive in blood, skin, and lungs. During disease progression, this organism adapts to changes within these niches. In addition to regulating genes for metabolism of nonglucose sugars, we found that Crp regulates genes for virulence, metal acquisition, and quorum sensing by direct or indirect mechanisms. Thus, this single transcriptional regulator, which responds to changes in available carbon sources, can regulate multiple critical behaviors for causing disease.

Fumarate dependent protein composition under aerobic and anaerobic growth conditions in Escherichia coli

In the absence of sugars, C4-dicarboxylates (C4DC) like fumarate represent important substrates for growth of Escherichia coli. Aerobically, C4DCs are oxidized to CO2 whereas anaerobically, C4DCs are used for fumarate respiration. In order to determine the impact of fumarate under aerobic and anaerobic conditions, proteomes of E. coli W3110 grown aerobically or anaerobically with fumarate and/or the non-C4DC substrate glycerol were comparatively profiled by nanoLC-MS/MS. Membrane enrichment allowed sensitive detection of membrane proteins. A total of 1657 proteins of which 646 and 374 were assigned to the cytosol or membrane, respectively, were covered. Presence of fumarate triggered changes (≥ 2fold) to the levels of 211 and 76 proteins under aerobic and anaerobic growth, respectively. The fumarate induced changes included proteins encoded by genes regulated by the C4DC two-component system DcuS-DcuR (DctA, DcuB, FumB, FrdABC proteins) catalyzing uptake and initial catabolic steps. Many of the proteins displaying altered levels are not part of the DcuS-DcuR regulon, including proteins of citric acid cycle and associated pathways (aerobic), proteins involved in motility and chemotaxis (anaerobic), and oxidative stress. Their genes are mostly preceded by cAMP receptor protein (CRP) sites, some by DcuR-like sites. Testing of selected genes confirmed regulation by CRP and DcuS-DcuR. SignificanceGlobal protein profiling of the soluble and the membrane fraction provides a comprehensive view on the protein pattern of E. coli grown aerobically and anaerobically with or without fumarate. The results disclose during aerobic growth besides the known impact of the C4-dicarboxylates (C4DC) on carbon utilization and citric acid cycle major adaptations in amino acid metabolism. In contrast, protein alterations in the presence of fumarate under anaerobic conditions point to enhanced motility and chemotaxis. Only proteins (transporters, initial metabolic steps) feeding external C4DCs to the central pathways were regulated by the C4DC two-component system DcuS-DcuR, whereas other protein levels were controlled in an indirect manner by CRP triggered catabolite control and other mechanisms. Consequently, metabolic and transcriptional regulation by C4DCs is apparently effected by a network of the DcuS-DcuR system with important contribution by catabolite control.

An average optimal control approach to the set stabilization problem for boolean control networks

AbstractThis paper addresses the set stabilization problem for deterministic Boolean control networks (BCNs). An optimal control approach is investigated to solve the problems by using the semi‐tensor product of matrices, where a policy iteration algorithm for the set stabilization problem is deduced. Finally, the intervention problem of a cAMP receptor protein is addressed in the framework of the set stabilization problem. The problem is solved to validate the effectiveness of the proposed policy iteration approach for a practical application.

Roles of CytR, an anti-activator of cyclic-AMP receptor protein (CRP) on flagellar expression and virulence in uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infection (UTI), a common bacterial infectious disease. This bacterium invades the urinary tract cells, where it aggregates, and subsequently forms multicellular colonies termed intracellular bacterial communities (IBCs). The motility of the bacteria plays a key role in the mechanism of virulence in the host bladder. Here, we show that CytR is a modulator of bacterial internalization and aggregation within the bladder epithelial cells sustained by CRP in UPEC. Mutational analyses and gel-shift assays indicated that CytR represses the expression of flhD, thereby encoding a master regulator for flagellar expression that is responsible for bacterial motility when CRP is present, whereas CRP is an activator of flhD expression. Thus, elevated flagellar expression was involved in promoted virulence in the cytR mutant. These combined observations suggest another regulatory layer of flagellar expression and the role of CytR in UPEC virulence.

Differential modulation of energy landscapes of cyclic AMP receptor protein (CRP) as a regulatory mechanism for class II CRP-dependent promoters

The Escherichia coli cAMP receptor protein, CRP, is a homodimeric global transcription activator that employs multiple mechanisms to modulate the expression of hundreds of genes. These mechanisms require different interfacial interactions among CRP, RNA, and DNA of varying sequences. The involvement of such a multiplicity of interfaces requires a tight control to ensure the desired phenotype. CRP-dependent promoters can be grouped into three classes. For decades scientists in the field have been puzzled over the differences in mechanisms between class I and II promoters. Using a new crystal structure, IR spectroscopy, and computational analysis, we defined the energy landscapes of WT and 14 mutated CRPs to determine how a homodimeric protein can distinguish nonpalindromic DNA sequences and facilitate communication between residues located in three different activation regions (AR) in CRP that are ∼30 Å apart. We showed that each mutation imparts differential effects on stability among the subunits and domains in CRP. Consequently, the energetic landscapes of subunits and domains are different, and CRP is asymmetric. Hence, the same mutation can exert different effects on ARs in class I or II promoters. The effect of a mutation is transmitted through a network by long-distance communication not necessarily relying on physical contacts between adjacent residues. The mechanism is simply the sum of the consequences of modulating the synchrony of dynamic motions of residues at a distance, leading to differential effects on ARs in different subunits. The computational analysis is applicable to any system and potentially with predictive capability.

Serotype-shifting gene rfbT is a direct transcriptional target of cAMP receptor protein (CRP) in V. cholerae O1

Ogawa and Inaba are two main serotypes of O1 V. cholerae and alternate among cholera epidemics. The rfbT gene encodes a methyltransferase and is required for Ogawa serotype. The Inaba serotype is the consequence of genetic alterations in rfbT gene which results in loss-of-function enzyme product. However, the expression and regulation of rfbT has not been understood yet. Here we demonstrated that a global regulator, cAMP receptor protein (CRP), positively regulates rfbT transcription through a non-canonical CRP binding site (CBS) in its promoter region. This finding is supported by the analyses of rfbT mRNA abundance, rfbT–lacZ fusions and electrophoretic mobility shift assay (EMSA). The analyses of rfbT mRNA level in wild type (WT), Δcrp, and lower or higher level of cAMP backgrounds revealed that CRP is required for rfbT expression in response to intracellular cAMP level. Subsequent rfbT–lacZ fusions and EMSA collectively displayed that cAMP-CRP complex transcriptionally activates rfbT through directly binding to CBS in rfbT promoter region. Consistently, serological microagglutination test showed that crp deletion resulted in at least 4-fold decrease in titer of Ogawa serum compared to its WT. These results expanded our knowledge of understanding the genetic determinants and probable regulatory mechanism of V. cholerae O1 serotype shift between Ogawa and Inaba.

Quorum Sensing and Metabolic State of the Host Control Lysogeny-Lysis Switch of Bacteriophage T1.

Bacterial viruses, or bacteriophages, are highly abundant in the biosphere and have a major impact on microbial populations. Many examples of phage interactions with their hosts, including establishment of dormant lysogenic and active lytic states, have been characterized at the level of the individual cell. However, much less is known about the dependence of these interactions on host metabolism and signal exchange within bacterial communities. In this report, we describe a lysogenic state of the enterobacterial phage T1, previously known as a classical lytic phage, and characterize the underlying regulatory circuitry. We show that the transition from lysogeny to lysis depends on bacterial population density, perceived via interspecies autoinducer 2. Lysis is further controlled by the metabolic state of the cell, mediated by the cyclic-3',5'-AMP (cAMP) receptor protein (CRP) of the host. We hypothesize that such combinations of cell density and metabolic sensing may be common in phage-host interactions.IMPORTANCE The dynamics of microbial communities are heavily shaped by bacterium-bacteriophage interactions. But despite the apparent importance of bacteriophages, our understanding of the mechanisms controlling phage dynamics in bacterial populations, and particularly of the differences between the decisions that are made in the dormant lysogenic and active lytic states, remains limited. In this report, we show that enterobacterial phage T1, previously described as a lytic phage, is able to undergo lysogeny. We further demonstrate that the lysogeny-to-lysis decision occurs in response to changes in the density of the bacterial population, mediated by interspecies quorum-sensing signal AI-2, and in the metabolic state of the cell, mediated by cAMP receptor protein. We hypothesize that this strategy enables the phage to maximize its chances of self-amplification and spreading in bacterial population upon induction of the lytic cycle and that it might be common in phage-host interactions.

CRP Interacts Specifically With Sxy to Activate Transcription in Escherichia coli.

Horizontal gene transfer through natural competence is an important driving force of bacterial evolution and antibiotic resistance development. In several Gram-negative pathogens natural competence is regulated by the concerted action of cAMP receptor protein (CRP) and the transcriptional co-regulator Sxy through a subset of CRP-binding sites (CRP-S sites) at genes encoding competence factors. Despite the wealth of knowledge on CRP’s structure and function it is not known how CRP and Sxy act together to activate transcription. In order to get an insight into the regulatory mechanism by which these two proteins activate gene expression, we performed a series of mutational analyses on CRP and Sxy. We found that CRP contains a previously uncharacterized region necessary for Sxy dependent induction of CRP-S sites, here named “Sxy Interacting Region” (SIR) encompassing residues Q194 and L196. Lost promoter induction in SIR mutants could be restored in the presence of specific complementary Sxy mutants, presenting evidence for a direct interaction of CRP and Sxy proteins in transcriptional activation. Moreover, we identified constitutive mutants of Sxy causing higher levels of CRP-S site promoter activation than wild-type Sxy. Both suppressor and constitutive mutations are located within the same area of Sxy.

The Crk adapter protein is essential for Drosophila embryogenesis, where it regulates multiple actin-dependent morphogenic events.

Small Src homology domain 2 (SH2) and 3 (SH3) adapter proteins regulate cell fate and behavior by mediating interactions between cell surface receptors and downstream signaling effectors in many signal transduction pathways. The CT10 regulator of kinase (Crk) family has tissue-specific roles in phagocytosis, cell migration, and neuronal development and mediates oncogenic signaling in pathways like that of Abelson kinase. However, redundancy among the two mammalian family members and the position of the Drosophila gene on the fourth chromosome precluded assessment of Crk’s full role in embryogenesis. We circumvented these limitations with short hairpin RNA and CRISPR technology to assess Crk’s function in Drosophila morphogenesis. We found that Crk is essential beginning in the first few hours of development, where it ensures accurate mitosis by regulating orchestrated dynamics of the actin cytoskeleton to keep mitotic spindles in syncytial embryos from colliding. In this role, it positively regulates cortical localization of the actin-related protein 2/3 complex (Arp2/3), its regulator suppressor of cAMP receptor (SCAR), and filamentous actin to actin caps and pseudocleavage furrows. Crk loss leads to the loss of nuclei and formation of multinucleate cells. We also found roles for Crk in embryonic wound healing and in axon patterning in the nervous system, where it localizes to the axons and midline glia. Thus, Crk regulates diverse events in embryogenesis that require orchestrated cytoskeletal dynamics.

EtcABC, a Putative EII Complex, Regulates Type 3 Fimbriae via CRP-cAMP Signaling in Klebsiella pneumoniae.

Biofilm formation by Klebsiella pneumoniae on indwelling medical devices increases the risk of infection. Both type 1 and type 3 fimbriae are important factors in biofilm formation by K. pneumoniae. We found that a putative enzyme II (EII) complex of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), etcA (EIIA)-etcB (EIIB)-etcC (EIIC), regulated biofilm and type 3 fimbriae formation by K. pneumoniae STU1. In this study, the regulatory mechanism of etcABC in K. pneumoniae type 3 fimbriae formation was investigated. We found via quantitative RT-PCR that overexpression of etcABC enhanced the transcription level of the mrk operon, which is involved in type 3 fimbriae synthesis, and reduced the transcription level of the fim operon, which is involved in type 1 fimbriae synthesis. To gain further insight into the role of etcABC in type 3 fimbriae synthesis, we analyzed the region upstream of the mrk operon and found the potential cyclic 3′5′-adenosine monophosphate (cAMP) receptor protein (CRP) binding site. After crp was deleted in K. pneumoniae STU1 and two clinical isolates, these three crp mutant strains could not express MrkA, the major subunit of the fimbrial shaft, indicating that CRP positively regulated type 3 fimbriae synthesis. Moreover, a crp mutant overexpressing etcABC could not express MrkA, indicating that the regulation of type 3 fimbriae by etcABC was dependent on CRP. In addition, deletion of cyaA, which encodes the adenylyl cyclase that synthesizes cAMP, and deletion of crr, which encodes the glucose-specific EIIA, led to a reduction in lac operon regulation and therefore bacterial lactose uptake in K. pneumoniae. Exogenous cAMP but not etcABC overexpression compensated for the role of cyaA in bacterial lactose uptake. However, either etcABC overexpression or exogenous cAMP compensated for the role of crr in bacterial lac operon regulation that would eventually restore lactose uptake. We also found via ELISA and the luxCDABE reporter system that overexpression of etcABC increased intracellular cAMP levels and the transcription level of crp, respectively, in K. pneumoniae. In conclusion, overexpression of etcABC positively regulated cAMP production and cAMP-CRP activity to activate the mrk operon, resulting in increased type 3 fimbriae synthesis in K. pneumoniae.

Overexpression of the adenylate cyclase gene cyaC facilitates current generation by Shewanella oneidensis in bioelectrochemical systems

Electrochemically active bacteria (EAB) are capable of electrochemical interactions with electrodes via extracellular electron transfer (EET) pathways and serve as essential components in bioelectrochemical systems. Previous studies have suggested that EAB, such as Shewanella oneidensis MR-1, use cyclic AMP (cAMP) receptor proteins to coordinately regulate the expression of catabolic and EET-related genes, prompting us to hypothesize that the intracellular cAMP concentration is an important factor determining the electrochemical activities of EAB. The present study constructed an MR-1 mutant, cyaC-OE, that overexpressed cyaC, a gene encoding a membrane-bound class III adenylate cyclase, and examined its electrochemical and transcriptomic characteristics. We show that the intracellular cAMP concentration in cyaC-OE is more than five times that in wild-type MR-1, and that cya-OE generates approximately two-fold higher current in BES than the wild-type strain. In addition, the expression of genes involved in EET and anaerobic carbon catabolism is up-regulated in cya-OE compared to that in the wild-type strain. These results suggest that increasing the intracellular cAMP level is a promising approach for constructing EAB with high catabolic and electrochemical activities.

Cyclic AMP induction of Dictyostelium prespore gene expression requires autophagy

Dictyostelium discoideum amoebas display colonial multicellularity where starving amoebas aggregate to form migrating slugs and fruiting bodies consisting of spores and three supporting cell types. To resolve the cell signalling mechanism that control sporulation, we use insertional mutagenesis of amoebas transformed with fusion constructs of spore genes and red fluorescent protein. We identified the defective gene in a mutant lacking spore gene expression as the autophagy gene Atg7. Directed knock-out of atg7 and of autophagy genes like atg5 and atg9 yielded a similar phenotype, with lack of viable spores and excessive differentiation of stalk cells. The atg7-, atg5- and atg9- cells were specifically defective in cAMP induction of prespore genes, but showed enhanced cAMP stimulation of prestalk genes at the same developmental stage. The lack of prespore gene induction in the autophagy mutants was not due to deleterious effects of loss of autophagy on known components of the cAMP pathway, such as cAMP receptors and their cAMP-induced phosphorylation and internalization, PKA and the transcription factors SpaA and GbfA, or to lack of NH3 production by proteolysis, which was previously suggested to stimulate the spore pathway. Our continued mutagenesis approach is the most likely to yield the intriguing link between autophagy and prespore gene induction.

A novel reporter system for cyclic AMP mediated gene expression in mammalian cells based on synthetic transgene expression system

Cyclic AMP (cAMP) is an important second messenger that mediates various biological functions in both prokaryotes and eukaryotes. Due to the ever increasing significance in studying the function and modulation of cAMP-based signaling, it is important to develop a protein-based biosensor that reports the cAMP mediated gene expression. Based on a synthetic transgene approach, an artificial mammalian transactivator was developed by fusing a transcriptional regulatory element cAMP receptor protein (CRP) of Escherichia coli to the VP16 transactivation domain of Herpes simplex virus in a mammalian expression vector (pLA1) that activates CRP specific operator site present in a chimeric promoter (OCRP- PhCMVmin- Luciferase) in a concentration dependent manner in mammalian cells. Our results reveal that the engineered transactivator report the gene expression mediated by cAMP directly in mammalian cells and this cAMP reporter system works irrespective of Protein kinase A (PKA) - cyclic AMP response element binding protein (CREB) - cyclic AMP response element (CRE) signaling since the luciferase activity mediated by synthetic gene construct is seen even in the presence of PKA inhibitor H-89 (derived from H-8 (N-[2-(methylamino) ethyl]-5-isoquinoline-sulfonamide). Furthermore this synthetic transcription factor plays a significant role in reporting and mediating cAMP signaling in tumorigenic cells which possess an aberrant cAMP signaling due to PKA and CREB mutations.

Insights into the pharmacology of GPER/GPR30 and its involvement in gallstone formation

The prevalence of gallstone disease is a major public health issue globally, and due to complex etiology of gallstones, there are a limited number of pharmaceuticals available. While the G protein‐coupled estrogen receptor (GPR30/GPER) has been shown to induce gallstone formation, little is known about its chemical biology and pharmacology. Building upon previous computational modeling of GPER, a novel series of antagonists were designed and synthesized. These novel compounds were assessed for their therapeutic value via calcium mobilization, cyclic AMP, and estrogen receptor a and β binding assays. From this series of compounds, one compound, SG‐1, exhibited superior antagonism and selectivity for GPER. The potential therapeutic value of SG‐1 was further validated in an in vivo mouse model of gallstone formation. Data revealed that SG‐1 treatment results in a dose‐dependent reduction in gallstone prevalence in estrogen‐treated ovariectomized mice.Support or Funding InformationSupported by startup funds from Saint Louis University (C.A.) and this work was supported in part by research grants DK73917 and DK101793 (to D.W.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Kinetic changes in Ga cycling can increase cAMP accumulation while decreasing G protein‐coupled receptor kinase‐mediated receptor desensitization

A G protein‐coupled receptor interacts with the GDP‐bound Gαβγ trimer, G protein‐coupled receptor kinases (GRKs), and in part arrestins through the cytosolic surface of the transmembrane helixes, suggesting that there is either competition for binding to the same surface or an orchestrated interchange between binding partners allowing for proper interactions and signaling. Experimentally induced competition for the intracellular Gαβγ trimer binding site results in a reduction in GRK and Gα function. Here we present a Gαs chimera, called sis, containing most of the helical domain of Gαi1. Initially, sis was designed to study GRK4γ interaction with Gαs and GRK4γ‐mediated receptor desensitization. However, sis prevented various GRK‐mediated reductions in cAMP accumulation. Therefore, we tested the hypothesis that sis disrupts normal orchestration of the Gαβγ trimer and GRK/arrestin function, resulting in an apparent Gα‐bias. To exclude any background effects of endogenous Gαs, we used GnasE2−/E2−‐MEFs re‐expressing Gαs (control) or sis and measured cAMP accumulation, receptor‐Gs kinetics, GRK function, and receptor internalization. The sis helical domain did not significantly affect the GEF activity of endogenously expressed β2‐AR or Gαβγ trimer formation; however, GRK function and cAMP production were altered. GRK2, 4γ, 5, and 6 failed to attenuate isoproterenol‐mediated cAMP accumulation in sis‐expressing cells, but all GRKs attenuated cAMP accumulation in Gs‐expressing cells. Expectedly, the rate of receptor endocytosis was reduced in sis‐expressing cells. Additionally, the production of cAMP in the Gs and sis cells was not equal. With phosphodiesterases inhibited, isoproterenol generated approximately 2.5‐fold more cAMP in sis cells compared to controls; moreover, in the same conditions forskolin was 50% less effective in sis cells. The original classification of adenylyl cyclase indicates that the effects of Gαs and forskolin are synergistic leading to greater cAMP production than either component alone. Therefore, our cAMP data suggests that interaction between sis and adenylyl cyclase is compromised, and sis likely cycles back to the receptor rapidly blocking GRK association and allowing itself to be reactivated. Supporting this conclusion, the rate of Gαs‐mediated cAMP accumulation was marginally faster than sis‐mediated cAMP accumulation (0.404 ± 0.127 s−1 vs. 0.178 ± 0.022 s−1) and the rate of cAMP degradation was greatly attenuated, resulting in a much larger area under the curve (2.4‐fold). These data mirror the previous end‐point cAMP measurements and further support the conclusion that sis allows the β2‐AR to remain active for a longer duration, resulting in greater cAMP accumulation.Support or Funding InformationWesternU MSPS program funds and NIH grants DK073911 (MB), DK079307 (EKJ) and HL109002 (EKJ) supported this work.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NaCl triggers the CRP-dependent increase of cAMP in Mycobacterium tuberculosis

The second messenger 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) has been shown to be involved in the regulation of many biological processes ranging from carbon catabolite repression in bacteria to cell signalling in eukaryotes. In mycobacteria, the role of cAMP and the mechanisms utilized by the bacterium to adapt to and resist immune and pharmacological sterilization remain poorly understood. Among the stresses encountered by bacteria, ionic and non-ionic osmotic stresses are among the best studied. However, in mycobacteria, the link between ionic osmotic stress, particularly sodium chloride, and cAMP has been relatively unexplored. Using a targeted metabolic analysis combined with stable isotope tracing, we show that the pathogenic Mycobacterium tuberculosis but not the opportunistic pathogen Mycobacterium marinum nor the non-pathogenic Mycobacterium smegmatis responds to NaCl stress via an increase in intracellular cAMP levels. We further showed that this increase in cAMP is dependent on the cAMP receptor protein and in part on the threonine/serine kinase PnkD, which has previously been associated with the NaCl stress response in mycobacteria.

Transcriptional regulation of fatty acid cis-trans isomerization in the solvent-tolerant soil bacterium, Pseudomonas putida F1.

One key to the success of Pseudomonas spp. is their ability to reside in hostile environments. Pseudomonas spp. possess a cis-trans isomerase (Cti) an enzyme that converts the cis-unsaturated fatty acids (FAs) of the membrane lipids to their trans-isomers to rigidify the membrane and thereby resist stresses. Whereas the posttranslational Cti regulation has been previously reported, transcriptional cti regulation remains to be studied in more details. Here, we have studied cti transcriptional regulation in the solvent-tolerant strain Pseudomonas putida F1. Two cti transcriptional start sites (cti-279 and cti-77) were identified with cti-279 transcript being dominant. Expression of cti was found to increase with temperature increase, addition of the organic solvent, octanol and in the stationary growth phase. We found that cti expression was repressed by the cyclic-AMP receptor protein (Crp) and repression required the cyclic-AMP ligand of Crp. Production of trans-unsaturated FAs was found to decrease after 24 h of growth. Although this decrease was accompanied by an increase in cyclopropane FA content, this was not at the expense of trans-unsaturated FAs demonstrating the absence of competition between Cti and Cfa in FA modification.

Plasticity of Promoter-Core Sequences Allows Bacteria to Compensate for the Loss of a Key Global Regulatory Gene

Transcription regulatory networks (TRNs) are of central importance for both short-term phenotypic adaptation in response to environmental fluctuations and long-term evolutionary adaptation, with global regulatory genes often being targets of natural selection in laboratory experiments. Here, we combined evolution experiments, whole-genome resequencing, and molecular genetics to investigate the driving forces, genetic constraints, and molecular mechanisms that dictate how bacteria can cope with a drastic perturbation of their TRNs. The crp gene, encoding a major global regulator in Escherichia coli, was deleted in four different genetic backgrounds, all derived from the Long-Term Evolution Experiment (LTEE) but with different TRN architectures. We confirmed that crp deletion had a more deleterious effect on growth rate in the LTEE-adapted genotypes; and we showed that the ptsG gene, which encodes the major glucose-PTS transporter, gained CRP (cyclic AMP receptor protein) dependence over time in the LTEE. We then further evolved the four crp-deleted genotypes in glucose minimal medium, and we found that they all quickly recovered from their growth defects by increasing glucose uptake. We showed that this recovery was specific to the selective environment and consistently relied on mutations in the cis-regulatory region of ptsG, regardless of the initial genotype. These mutations affected the interplay of transcription factors acting at the promoters, changed the intrinsic properties of the existing promoters, or produced new transcription initiation sites. Therefore, the plasticity of even a single promoter region can compensate by three different mechanisms for the loss of a key regulatory hub in the E. coli TRN.