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Allosteric transcription factors undergo binding events at inducer binding sites as well as at distinct DNA binding domains, and it is difficult to disentangle the structural and functional consequences of these two classes of interactions. We compare the ability of two statistical mechanical models-the Monod-Wyman-Changeux (MWC) and the Koshland-Némethy-Filmer (KNF) models of protein conformational change-to characterize the multi-step activation mechanism of the broadly acting cyclic-AMP receptor protein (CRP). We first consider the allosteric transition resulting from cyclic-AMP binding to CRP, then analyze how CRP binds to its operator, and finally investigate the ability of CRP to activate gene expression. We use these models to examine a beautiful recent experiment that created a single-chain version of the CRP homodimer, creating six mutants using all possible combinations of the wild type, D53H, and S62F subunits. We demonstrate that the MWC model can explain the behavior of all six mutants using a small, self-consistent set of parameters whose complexity scales with the number of subunits, providing a significant benefit over previous models. In comparison, the KNF model not only leads to a poorer characterization of the available data but also fails to generate parameter values in line with the available structural knowledge of CRP. In addition, we discuss how the conceptual framework developed here for CRP enables us to not merely analyze data retrospectively, but has the predictive power to determine how combinations of mutations will interact, how double mutants will behave, and how each construct would regulate gene expression.

The transcription mechanism of genetic information from DNA to RNA is efficiently controlled by regulatory proteins, such as catabolite activator protein (CAP), and their ligands. When cyclic AMP (cAMP) binds to CAP, the complex forms a dimer and binds specifically to DNA to activate the transcription mechanism. On the other hand, when cyclic GMP (cGMP) binds to CAP, the complex has no marked effect on the mechanism. In our previous study, based on molecular dynamics (MD) and ab initio fragment molecular orbital (FMO) methods, we elucidated which residues of CAP are important for the specific interactions between CAP and DNA in the CAP-monomer+DNA + cAMP complex. However, this monomer model for CAP cannot describe real interactions between the CAP-dimer and DNA because CAPs form a dimer before binding to DNA. Accordingly, here, we investigated stable structures and their electronic states for the CAP-dimer+DNA complex with cAMP or cGMP ligand, to clarify the influence of ligand-binding on the interactions between CAP-dimer and DNA. The MD simulations elucidated that the DNA-binding domains of CAP-dimer behave differently depending on the ligand bound to the CAP-dimer. In addition, FMO calculations revealed that the binding energy between CAP-dimer and DNA for the CAP-dimer+DNA + cAMP complex is larger than that for the CAP-dimer+DNA + cGMP complex, being consistent with experiments. It was also highlighted that the Arg185 and Lys188 residues of CAP-dimer are important for the binding between CAP-dimer and DNA. These results provide useful information for proposing new compounds that efficiently control the transcription mechanism.

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.

Glutathione (GSH) is the most abundant antioxidant in all living organisms. Previously, we have shown that a deletion mutant in the glutathione synthetase gene (ΔgshB) decreases the expression of type III secretion system (T3SS) genes of Pseudomonas aeruginosa. However, the mechanism remains elusive. In this study, a comprehensive transcriptomic analysis of the GSH-deficient mutant ΔgshAΔgshB was used to elucidate the role of GSH in the pathogenesis of P. aeruginosa. The data show that the expression of genes in T3SS, type VI secretion system (T6SS) and some regulatory genes were impaired. ΔgshAΔgshB was attenuated in a mouse model of acute pneumonia, swimming and swarming motilities, and biofilm formation. Under T3SS inducing conditions, GSH enhanced the expression of T3SS in both wild-type PAO1 and ΔgshAΔgshB, but not in Δvfr. Genetic complementation of Δvfr restored the ability of GSH to induce the expression of T3SS genes. Site-directed mutagenesis based substitution of cysteine residues with alanine in Vfr protein abolished the induction of T3SS genes by GSH, confirming that GSH regulates T3SS genes through Vfr. Exposure to H2O2 decreased free thiol content on Vfr, indicating that the protein was sensitive to redox modification. Importantly, GSH restored the oxidized Vfr to reduced state. Collectively, these results suggest that GSH serves as an intracellular redox signal sensed by Vfr to upregulate T3SS expression in P. aeruginosa. Our work provides new insights into the role of GSH in P. aeruginosa pathogenesis. Published version

Many organisms produce endogenous hydrogen sulfide (H

Cyclic di-AMP (c-di-AMP) is a second messenger involved in diverse metabolic processes, including osmolyte uptake, cell wall homeostasis, and antibiotic and heat resistance. In Lactococcus lactis, a lactic acid bacterium which is used in the dairy industry and as a cell factory in biotechnological processes, the only reported interaction partners of c-di-AMP are the pyruvate carboxylase and BusR, the transcription regulator of the busAB operon for glycine betaine uptake. However, recent studies uncovered a major role of c-di-AMP in the control of potassium homeostasis, and potassium is the signal that triggers c-di-AMP synthesis. In this study, we have identified KupA and KupB, which belong to the Kup/HAK/KT family, as novel c-di-AMP binding proteins. Both proteins are high-affinity potassium transporters, and their transport activities are inhibited by binding of c-di-AMP. Thus, in addition to the well-studied Ktr/Trk potassium channels, KupA and KupB represent a second class of potassium transporters that are subject to inhibition by c-di-AMP. IMPORTANCE Potassium is an essential ion in every living cell. Even though potassium is the most abundant cation in cells, its accumulation can be toxic. Therefore, the level of potassium has to be tightly controlled. In many Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in the control of potassium homeostasis by binding to potassium transporters and regulatory proteins and RNA molecules. In the lactic acid bacterium Lactococcus lactis, none of these conserved c-di-AMP-responsive molecules are present. In this study, we demonstrate that the KupA and KupB proteins of L. lactis IL1403 are high-affinity potassium transporters and that their transport activity is inhibited by the second messenger c-di-AMP.

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.

The metal-reducing gammaproteobacterium Shewanella oneidensis reduces iodate (IO(3)(−)) as an anaerobic terminal electron acceptor. Microbial IO(3)(−) electron transport pathways are postulated to terminate with nitrate (NO(3)(−)) reductase, which reduces IO(3)(−) as an alternative electron acceptor. Recent studies with S. oneidensis, however, have demonstrated that NO(3)(−) reductase is not involved in IO(3)(−) reduction. The main objective of the present study was to determine the metal reduction and protein secretion genes required for IO(3)(−) reduction by Shewanella oneidensis with lactate, formate, or H(2) as the electron donor. With all electron donors, the type I and type V protein secretion mutants retained wild-type IO(3)(−) reduction activity, while the type II protein secretion mutant lacking the outer membrane secretin GspD was impaired in IO(3)(−) reduction. Deletion mutants lacking the cyclic AMP receptor protein (CRP), cytochrome maturation permease CcmB, and inner membrane-tethered c-type cytochrome CymA were impaired in IO(3)(−) reduction with all electron donors, while deletion mutants lacking c-type cytochrome MtrA and outer membrane β-barrel protein MtrB of the outer membrane MtrAB module were impaired in IO(3)(−) reduction with only lactate as an electron donor. With all electron donors, mutants lacking the c-type cytochromes OmcA and MtrC of the metal-reducing extracellular electron conduit MtrCAB retained wild-type IO(3)(−) reduction activity. These findings indicate that IO(3)(−) reduction by S. oneidensis involves electron donor-dependent metal reduction and protein secretion pathway components, including the outer membrane MtrAB module and type II protein secretion of an unidentified IO(3)(−) reductase to the S. oneidensis outer membrane. IMPORTANCE Microbial iodate (IO(3)(−)) reduction is a major component in the biogeochemical cycling of iodine and the bioremediation of iodine-contaminated environments; however, the molecular mechanism of microbial IO(3)(−) reduction is poorly understood. Results of the present study indicate that outer membrane (type II) protein secretion and metal reduction genes encoding the outer membrane MtrAB module of the extracellular electron conduit MtrCAB are required for IO(3)(−) reduction by S. oneidensis. On the other hand, the metal-reducing c-type cytochrome MtrC of the extracellular electron conduit is not required for IO(3)(−) reduction by S. oneidensis. These findings indicate that the IO(3)(−) electron transport pathway terminates with an as yet unidentified IO(3)(−) reductase that associates with the outer membrane MtrAB module to deliver electrons extracellularly to IO(3)(−).

The human pathogen Pseudomonas aeruginosa can cause both acute infections and chronic biofilm-based infections. Expression of acute virulence factors is positively regulated by cAMP, whereas biofilm formation is positively regulated by c-di-GMP. We provide evidence that increased levels of cAMP, caused by either a lack of degradation or increased production, inhibit P. aeruginosa biofilm formation. cAMP-mediated inhibition of P. aeruginosa biofilm formation required Vfr, and involved a reduction of the level of c-di-GMP, as well as reduced production of biofilm matrix components. A mutant screen and characterization of defined knockout mutants suggested that a subset of c-di-GMP-degrading phosphodiesterases is involved in cAMP-Vfr-mediated biofilm inhibition in P. aeruginosa.

Increasing cell apoptosis is one of the major causes of intervertebral disc degeneration (IDD). β-ecdysterone has been demonstrated to protect PC12 cells against neurotoxicity. A previous study revealed that β‑ecdysterone may be involved in the regulation of autophagy in osteoblasts. Therefore, we hypothesized that β‑ecdysterone may possess therapeutic effects on IDD via autophagy stimulation. The effect of β‑ecdysterone on IDD was explored by in vitro experiments. The results demonstrated that β‑ecdysterone attenuated the apoptosis induced by tert‑butyl hydroperoxide via promoting autophagy in nucleus pulposus cells. Beclin‑1, an indispensable protein for the stimulation of autophagy, is upregulated and stabilized by β‑ecdysterone in a dose‑ and time‑dependent manner in nucleus pulposus cells. Inhibition of autophagy with 3‑methyladenine partially abrogated the protective function of β‑ecdysterone against apoptosis of nucleus pulposus cells, indicating that autophagy participated in the protective effect of β‑ecdysterone on IDD. Additionally, β‑ecdysterone promoted the expression of anabolic genes while inhibiting the expression of catabolic genes in nucleus pulposus cells. Collectively, the present study demonstrated that β‑ecdysterone may protect nucleus pulposus cells against apoptosis by autophagy stimulation and ameliorate disc degeneration, which indicates that β‑ecdysterone may be a potential therapeutic agent for IDD.

Metformin is the first-line therapy for treating type 2 diabetes and a promising anti-aging drug. We set out to address the fundamental question of how gut microbes and nutrition, key regulators of host physiology, affect the effects of metformin. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we developed a high-throughput four-way screen to define the underlying host-microbe-drug-nutrient interactions. We show that microbes integrate cues from metformin and the diet through the phosphotransferase signaling pathway that converges on the transcriptional regulator Crp. A detailed experimental characterization of metformin effects downstream of Crp in combination with metabolic modeling of the microbiota in metformin-treated type 2 diabetic patients predicts the production of microbial agmatine, a regulator of metformin effects on host lipid metabolism and lifespan. Our high-throughput screening platform paves the way for identifying exploitable drug-nutrient-microbiome interactions to improve host health and longevity through targeted microbiome therapies. VIDEO ABSTRACT. ispartof: Cell vol:178 issue:6 pages:1299-1312 ispartof: location:United States status: published

The Escherichia coli cyclic AMP receptor protein (CRP or catabolite activator protein, CAP) provides a textbook example of bacterial transcriptional regulation and is one of the best studied transcription factors in biology. For almost five decades a large number of mutants, evolved in vivo or engineered in vitro, have shed light on the molecular structure and mechanism of CRP. Here, we review previous work, providing an overview of studies describing the isolation of CRP mutants. Furthermore, we present new data on deep sequencing of different bacterial populations that have evolved under selective pressure that strongly favors mutations in the crp locus. Our new approach identifies more than 100 new CRP mutations and paves the way for a deeper understanding of this fascinating bacterial master regulator. Keywords: cAMP receptor protein, Carbon catabolite repression, Global transcriptional regulation, Experimental evolution

We present a rare comparison of structures of the same protein but generated by different potentials. We used four popular water potentials (SPC, TIP3P, TIP4P, TIP5P) in conjunction with the equally popular ff99SB. However, the ff12SB protein potential was used with TI3P only. Simulations (60 ns) were run on the catabolite activator protein (CAP), which is a textbook case of allosteric interaction. Overall, all potentials generated largely similar structures but failed to reproduce a crucial structural feature determined by NMR experiment. This example shows the need to develop next-generation potentials. Graphical abstract Catabolite activator protein.

ADP-glucose is the precursor of glycogen biosynthesis in bacteria, and a compound abundant in the starchy plant organs ingested by many mammals. Here we show that the enteric species Escherichia coli is capable of scavenging exogenous ADP-glucose for use as a glycosyl donor in glycogen biosynthesis and feed the adenine nucleotide pool. To unravel the molecular mechanisms involved in this process, we screened the E. coli single-gene deletion mutants of the Keio collection for glycogen content in ADP-glucose-containing culture medium. In comparison to wild-type (WT) cells, individual ∆nupC and ∆nupG mutants lacking the cAMP/CRP responsive inner-membrane nucleoside transporters NupC and NupG displayed reduced glycogen contents and slow ADP-glucose incorporation. In concordance, ∆cya and ∆crp mutants accumulated low levels of glycogen and slowly incorporated ADP-glucose. Two-thirds of the glycogen-excess mutants identified during screening lacked functions that underlie envelope biogenesis and integrity, including the RpoE specific RseA anti-sigma factor. These mutants exhibited higher ADP-glucose uptake than WT cells. The incorporation of either ∆crp, ∆nupG or ∆nupC null alleles sharply reduced the ADP-glucose incorporation and glycogen content initially witnessed in ∆rseA cells. Overall, the data showed that E. coli incorporates extracellular ADP-glucose through a cAMP/CRP-regulated process involving the NupC and NupG nucleoside transporters that is facilitated under envelope stress conditions., This work was partially supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) (grant numbers BIO2013-49125-C2-1-P and BIO2016-78747-P). A.M.V. is a Career Researcher of the Consejo Nacional de Investigaciones Cientificas y Técnicas de Argentina (CONICET) and Professor of Micribiology at the National University of Rosario (U.N.R., Argentina). A.M.V. expresses his gratitude to the Ministerio de Educación y Cultura, the Consejo Superior de Investigaciones Científicas, and the Public University of Navarra for financial support.

Shewanella oneidensis MR-1 is a facultative anaerobe that respires using a variety of electron acceptors. Although this organism is incapable of fermentative growth in the absence of electron acceptors, its genome encodes LdhA (a putative fermentative NADH-dependent d-lactate dehydrogenase [d-LDH]) and Dld (a respiratory quinone-dependent d-LDH). However, the physiological roles of LdhA in MR-1 are unclear. Here, we examined the activity, transcriptional regulation, and traits of deletion mutants to gain insight into the roles of LdhA in the anaerobic growth of MR-1. Analyses of d-LDH activity in MR-1 and the ldhA deletion mutant confirmed that LdhA functions as an NADH-dependent d-LDH that catalyzes the reduction of pyruvate to d-lactate. In vivo and in vitro assays revealed that ldhA expression was positively regulated by the cyclic-AMP receptor protein, a global transcription factor that regulates anaerobic respiratory pathways in MR-1, suggesting that LdhA functions in coordination with anaerobic respiration. Notably, we found that a deletion mutant of all four NADH dehydrogenases (NDHs) in MR-1 (ΔNDH mutant) retained the ability to grow on N-acetylglucosamine under fumarate-respiring conditions, while an additional deletion of ldhA or dld deprived the ΔNDH mutant of this growth ability. These results indicate that LdhA-Dld serves as a bypass of NDH in electron transfer from NADH to quinones. Our findings suggest that the LdhA-Dld system manages intracellular redox balance by utilizing d-lactate as a temporal electron sink under electron acceptor-limited conditions. IMPORTANCE NADH-dependent LDHs are conserved among diverse organisms and contribute to NAD(+) regeneration in lactic acid fermentation. However, this type of LDH is also present in nonfermentative bacteria, including members of the genus Shewanella, while their physiological roles in these bacteria remain unknown. Here, we show that LdhA (an NADH-dependent d-LDH) works in concert with Dld (a quinone-dependent d-LDH) to transfer electrons from NADH to quinones during sugar catabolism in S. oneidensis MR-1. Our results indicate that d-lactate acts as an intracellular electron mediator to transfer electrons from NADH to membrane quinones. In addition, d-lactate serves as a temporal electron sink when respiratory electron acceptors are not available. Our study suggests novel physiological roles for d-LDHs in providing nonfermentative bacteria with catabolic flexibility under electron acceptor-limited conditions.

Binding to mucin is the initial step for enteropathogens to establish pathogenesis. An open reading frame, gbpA, of Vibrio vulnificus was identified and characterized in this study. Compared with wild type, the gbpA mutant was impaired in binding to mucin-agar and the mucin-secreting HT29-methotrexate cells, and the impaired mucin binding was restored by the purified GbpA provided exogenously. The gbpA mutant had attenuated virulence and ability of intestinal colonization in a mouse model, indicating that GbpA is a mucin-binding protein and essential for pathogenesis of V. vulnificus. The gbpA transcription was growth phase-dependent, reaching a maximum during the exponential phase. The Fe-S cluster regulator (IscR) and the cyclic AMP receptor protein (CRP) coactivated, whereas SmcR, a LuxR homologue, repressed gbpA. The cellular levels of IscR, CRP, and SmcR were not significantly affected by one another, indicating that the regulator proteins function cooperatively to regulate gbpA rather than sequentially in a regulatory cascade. The regulatory proteins directly bind upstream of the gbpA promoter PgbpA. DNase I protection assays, together with the deletion analyses of PgbpA, demonstrated that IscR binds to two specific sequences centered at -164.5 and -106, and CRP and SmcR bind specifically to the sequences centered at -68 and -45, respectively. Furthermore, gbpA was induced by exposure to H2O2, and the induction appeared to be mediated by elevated intracellular levels of IscR. Consequently, the combined results indicated that IscR, CRP, and SmcR cooperate for precise regulation of gbpA during the V. vulnificus pathogenesis.

The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence determinant and a potential target for antivirulence drugs. One candidate target is ExsA, a member of the AraC family of DNA-binding proteins required for expression of the T3SS. A previous study identified small molecules based on an N -hydroxybenzimidazole scaffold that inhibit the DNA-binding activity of several AraC proteins, including ExsA. In this study, we further characterized a panel of N -hydroxybenzimidazoles. The half-maximal inhibitory concentrations (IC 50 s) for the tested N -hydroxybenzimidazoles ranged from 8 to 45 μM in DNA-binding assays. Each of the N -hydroxybenzimidazoles protected mammalian cells from T3SS-dependent cytotoxicity, and protection correlated with reduced T3SS gene expression in a coculture infection model. Binding studies with the purified ExsA DNA-binding domain (i.e., lacking the amino-terminal self-association domain) confirmed that the activity of N -hydroxybenzimidazoles results from interactions with the DNA-binding domain. The interaction is specific, as an unrelated DNA-binding protein (Vfr) was unaffected by N -hydroxybenzimidazoles. ExsA homologs that control T3SS gene expression in Yersinia pestis , Aeromonas hydrophila , and Vibrio parahaemolyticus were also sensitive to N -hydroxybenzimidazoles. Although ExsA and Y. pestis LcrF share 79% sequence identity in the DNA-binding domain, differential sensitivities to several of the N -hydroxybenzimidazoles were observed. Site-directed mutagenesis based on in silico docking of inhibitors to the DNA-binding domain, and on amino acid differences between ExsA and LcrF, resulted in the identification of several substitutions that altered the sensitivity of ExsA to N -hydroxybenzimidazoles. Development of second-generation compounds targeted to the same binding pocket could lead to drugs with improved pharmacological properties.

Bacterial persisters represent a small number of slow-growing antibiotic-tolerant cells among populations of rapidly growing cells, and are the main cause of frequent recurrent infections. MqsR-MqsA, the toxin-antitoxin (TA) pair, is the most frequently induced TA system associated with antibiotic persistence in Escherichia coli. In this study, we show that the cyclic AMP receptor protein (CRP) indirectly upregulates mqsRA transcription. We also show that CRP plays an important role in antibiotic persistence, which seems to be partially mediated through MqsRA. Overall, this study highlights the role of CRP as an important regulator of antibiotic persistence in E. coli.

The CRISPR/Cas system protects bacteria against bacteriophage and plasmids through a sophisticated mechanism where cas operon plays a crucial role consisting of cse1 and cas3. However, comprehensive studies on the regulation of cas3 operon of the Type I-E CRISPR/Cas system are scarce. Herein, we investigated the regulation of cas3 in Escherichia coli. The mutation in gcvP or crp reduced the CRISPR/Cas system interference ability and increased bacterial susceptibility to phage, when the casA operon of the CRISPR/Cas system was activated. The silence of the glycine cleavage system (GCS) encoded by gcvTHP operon reduced cas3 expression. Adding N5, N10-methylene tetrahydrofolate (N5, N10-mTHF), which is the product of GCS-catalyzed glycine, was able to activate cas3 expression. In addition, a cAMP receptor protein (CRP) encoded by crp activated cas3 expression via binding to the cas3 promoter in response to cAMP concentration. Since N5, N10-mTHF provides one-carbon unit for purine, we assumed GCS regulates cas3 through associating with CRP. It was evident that the mutation of gcvP failed to further reduce the cas3 expression with the crp deletion. These results illustrated a novel regulatory pathway which GCS and CRP co-regulate cas3 of the CRISPR/Cas system and contribute to the defence against invasive genetic elements, where CRP is indispensable for GCS regulation of cas3 expression.

BPI-inducible protein A (BipA) is a conserved ribosome-associated GTPase in bacteria that is structurally similar to other GTPases associated with protein translation, including IF2, EF-Tu, and EF-G. Its binding site on the ribosome appears to overlap those of these translational GTPases. Mutations in the bipA gene cause a variety of phenotypes, including cold and antibiotics sensitivities and decreased pathogenicity, implying that BipA may participate in diverse cellular processes by regulating translation. According to recent studies, a bipA-deletion strain of Escherichia coli displays a ribosome assembly defect at low temperature, suggesting that BipA might be involved in ribosome assembly. To further investigate BipA’s role in ribosome biogenesis, here, we compared and analyzed the ribosomal protein compositions of MG1655 WT and bipA-deletion strains at 20 °C. Aberrant 50S ribosomal subunits (i.e. 44S particles) accumulated in the bipA-deletion strain at 20 °C, and the ribosomal protein L6 was absent in these 44S particles. Furthermore, bipA expression was significantly stimulated at 20 °C, suggesting that it encodes a cold shock–inducible GTPase. Moreover, the transcriptional regulator cAMP receptor protein (CRP) positively promoted bipA expression only at 20 °C. Importantly, GFP and α-glucosidase refolding assays revealed that BipA has chaperone activity. Our findings indicate that BipA is a cold shock–inducible GTPase that participates in 50S ribosomal subunit assembly by incorporating the L6 ribosomal protein into the 44S particle during the assembly.

Survival of bacteria in ever-changing habitats with fluctuating nutrient supplies requires rapid adaptation of their metabolic capabilities. To this end, carbohydrate metabolism is governed by complex regulatory networks including posttranscriptional mechanisms that involve small regulatory RNAs (sRNAs) and RNA-binding proteins. sRNAs limit the response to substrate availability and set the threshold or time required for induction and repression of carbohydrate utilization systems. Carbon catabolite repression (CCR) also involves sRNAs. In Enterobacteriaceae , sRNA Spot 42 cooperates with the transcriptional regulator cyclic AMP (cAMP)-receptor protein (CRP) to repress secondary carbohydrate utilization genes when a preferred sugar is consumed. In pseudomonads, CCR operates entirely at the posttranscriptional level, involving RNA-binding protein Hfq and decoy sRNA CrcZ. Moreover, sRNAs coordinate fluxes through central carbohydrate metabolic pathways with carbohydrate availability. In Gram-negative bacteria, the interplay between RNA-binding protein CsrA and its cognate sRNAs regulates glycolysis and gluconeogenesis in response to signals derived from metabolism. Spot 42 and cAMP-CRP jointly downregulate tricarboxylic acid cycle activity when glycolytic carbon sources are ample. In addition, bacteria use sRNAs to reprogram carbohydrate metabolism in response to anaerobiosis and iron limitation. Finally, sRNAs also provide homeostasis of essential anabolic pathways, as exemplified by the hexosamine pathway providing cell envelope precursors. In this review, we discuss the manifold roles of bacterial sRNAs in regulation of carbon source uptake and utilization, substrate prioritization, and metabolism.

Allosteric transcription factors undergo binding events both at their inducer binding sites as well as at distinct DNA binding domains, and it is often difficult to disentangle the structural and functional consequences of these two classes of interactions. In this work, we compare the ability of two statistical mechanical models – the Monod-Wyman-Changeux (MWC) and the Koshland-Némethy-Filmer (KNF) models of protein conformational change – to characterize the multi-step activation mechanism of the broadly acting cyclic-AMP receptor protein (CRP). We first consider the allosteric transition resulting from cyclic-AMP binding to CRP, then analyze how CRP binds to its operator, and finally investigate the ability of CRP to activate gene expression. In light of these models, we examine data from a beautiful recent experiment that created a single-chain version of the CRP homodimer, thereby enabling each subunit to be mutated separately. Using this construct, six mutants were created using all possible combinations of the wild type subunit, a D53H mutant subunit, and an S62F mutant subunit. We demonstrate that both the MWC and KNF models can explain the behavior of all six mutants using a small, self-consistent set of parameters. In comparing the results, we find that the MWC model slightly outperforms the KNF model in the quality of its fits, but more importantly the parameters inferred by the MWC model are more in line with structural knowledge of CRP. In addition, we discuss how the conceptual framework developed here for CRP enables us to not merely analyze data retrospectively, but has the predictive power to determine how combinations of mutations will interact, how double mutants will behave, and how each construct would regulate gene expression.

Klebsiella pneumoniae is an opportunistic pathogen, and its hypervirulent variants cause serious invasive community-acquired infections. A genomic view of K. pneumoniae NTUH-2044 for the carbohydrate phosphotransferase system (PTS) found a putative fructose PTS, namely, the Frw PTS gene cluster. The deletion mutant and the complemented mutant of frwC (KP1_1992), which encodes the putative fructose-specific enzyme IIC, were constructed, and the phenotypes were characterized. This transmembrane PTS protein is responsible for fructose utilization. frwC deletion can enhance biofilm formation and capsular polysaccharide (CPS) biosynthesis but decreases the growth rate and lethality in mice. frwC expression was repressed in the cyclic AMP receptor protein (CRP) mutant. Electrophoretic mobility shift assay showed that CRP can directly bind to the promoter of frwC These results indicated that frwC expression is controlled by CRP directly and that such regulation contributes to bacterial growth, CPS synthesis, and the virulence of the Δcrp strain. The findings help elucidate fructose metabolism and the CRP regulatory mechanism in K. pneumoniae.

Introduction: Frailty can adversely affect the outcomes of allogeneic hematopoietic stem cell transplantation (alloHSCT) but is difficult to measure in busy transplant clinics. The limited published studies have used dedicated trained persons and comprehensive geriatric assessment (GA) tools, which are time consuming (Muffly LS, Haematologica 2014; Holmes HM, J Geriatr Oncol 2014; Rodrigues M, J Geriatr Oncol 2019). The difficulty in application of GA tools by transplant clinicians, residents and nurses in their clinics has resulted in low adoption rates in routine practice. At our center we adopted selected tests for frailty and function which could be conducted during pre-transplant consultation in a busy clinic, without extra waiting time for patients, and using existing staff. The Timed up and Go test (TUGT) was adopted as it could be done in any closed clinic room, without need for a corridor. Thus it was considered safer than a gait speed test and was even applicable to patients in "isolation". We aim to share a preliminary analysis of the applicability and correlation between our selected frailty assessment with transplant outcomes and complications. Methods: Patients referred for transplant underwent the following assessments conducted by different providers. All ages were included. Relevant tests and source of data are as follows: Frailty and function by clinician evaluating (a) Clinical Frailty scale (CFS) with 9 points based on clinical judgement (Rockwood 2005) (b) Lawton's Instrumental activities of daily living (IADL). Objective physical performance by nursing BMT coordinator using (a) TUGT and (b) Grip strength using hydraulic "Jamar" hand dynamometer conducted in clinic room at time of documentation. Self assessment by patient completing (a) Self-rated health (SRH) question and (b) a question on falls. Blood tests (a) CRP (b) Albumin. The present study is a single center prospective observational study. Patients who did not proceed to transplant were excluded. Ninety-six consecutive adult allo-HSCT patients were eligible for the present analysis, updated on July 2019. The parameters were individually correlated with overall survival (OS), non-relapse mortality (NRM), cumulative incidence (cum.Inc) of acute GVHD, median time of transplant hospitalization and readmissions. Multivariate analysis was not performed in this pilot study due to limited number of patients and low frequency of adverse events. Results: Baseline characteristics and main post-transplant information are noted in Table 1. Median follow up of cohort was 5 months. Table 2 shows the main outcomes (with normal values). For the entire cohort the median OS at 6 months was 73.9% (range 61.7-82.8), NRM at day+100 was 8.7% (range 2.6-14.7), Cum.Inc of Acute GVHD 41.1% (range 30.1-52.1), Cum.Inc gr II-IV acute GVHD was 25.7% (range 15.6-35.9). Relapse occurred in 8 cases (8.3%) and deaths in 23 (23.9%). A TUGT of more than 10 seconds and raised CRP predicted poor OS (p10 seconds, SRH question Conclusions: Our pilot study shows that with selected brief tools, frailty and functionality can be assessed as part of routine clinical practice in allogeneic-stem cell transplantation in all age groups without extra waiting time for patients or additional human resources. TUGT is a useful prognostic tool which can be conducted in a clinic room and correlates with OS, NRM, and duration of hospitalization. Larger number of patients and longer follow-up will help to evaluate the different assessment modalities as prognostic tools in allo-HSCT and their wider applicability. Disclosures Michelis: CSL Behring: Other: Financial Support. Mattsson:Gilead: Honoraria; Celgene: Honoraria; Therakos: Honoraria.

Yersinia pestis is a dangerous bacterial pathogen that can cause plague. Both RovA and cyclic AMP receptor protein (cAMP-CRP) are required for regulating biofilm- and virulence-related genes in Y. pestis. In this study, the transcriptional regulation between RovA and cAMP-CRP were analyzed by using primer extension, quantitative RT-PCR, LacZ fusion, and electrophoretic mobility shift assay. The results indicated that RovA repressed crp transcription in an indirect manner, while that RovA had no regulatory action on cyaA at the transcriptional level. In addition, cAMP-CRP did not regulate the transcription of rovA. Taken together with our previous results, complex regulatory interactions of RovA, cAMP-CRP, and PhoP/PhoQ in Y. pestis were revealed, which would promote us gain deeper understanding about coordinative modulation of biofilm- and virulence-related regulator genes.

The bacterium Vibrio cholerae is native to aquatic environments and can switch lifestyles to cause disease in humans. Lifestyle switching requires modulation of genetic systems for quorum sensing, intestinal colonization, and toxin production. Much of this regulation occurs at the level of gene expression and is controlled by transcription factors. In this work, we have mapped the binding of cAMP receptor protein (CRP) and RNA polymerase across the V. cholerae genome. We show that CRP is an integral component of the regulatory network that controls lifestyle switching. Focusing on a locus necessary for toxin transport, we demonstrate CRP-dependent regulation of gene expression in response to host colonization. Examination of further CRP-targeted genes reveals that this behavior is commonplace. Hence, CRP is a key regulator of many V. cholerae genes in response to lifestyle changes., IMPORTANCE Cholera is an infectious disease that is caused by the bacterium Vibrio cholerae. Best known for causing disease in humans, the bacterium is most commonly found in aquatic ecosystems. Hence, humans acquire cholera following ingestion of food or water contaminated with V. cholerae. Transition between an aquatic environment and a human host triggers a lifestyle switch that involves reprogramming of V. cholerae gene expression patterns. This process is controlled by a network of transcription factors. In this paper, we show that the cAMP receptor protein (CRP) is a key regulator of V. cholerae gene expression in response to lifestyle changes.

The D NA d amage r esponse ddrI gene encodes a transcription regulator belonging to the cAMP receptor protein (CRP) family. Cells devoid of the DdrI protein exhibit a pleiotropic phenotype, including growth defects and sensitivity to DNA-damaging agents and to oxidative stress. Here, we show that the absence of the DdrI protein also confers sensitivity to heat shock treatment, and several genes involved in heat shock response were shown to be upregulated in a DdrI-dependent manner. Interestingly, expression of the Escherichia coli CRP partially compensates for the absence of the DdrI protein. Microscopic observations of Δ ddrI mutant cells revealed an increased proportion of two-tetrad and anucleated cells in the population compared to the wild-type strain, indicating that DdrI is crucial for the completion of cell division and/or chromosome segregation. We show that DdrI is also involved in the megaplasmid MP1 stability and in efficient plasmid transformation by facilitating the maintenance of the incoming plasmid in the cell. The in silico prediction of putative DdrI binding sites in the D. radiodurans genome suggests that hundreds of genes, belonging to several functional groups, may be regulated by DdrI. In addition, the DdrI protein absolutely requires cAMP for in vitro binding to specific target sequences, and it acts as a dimer. All these data underline the major role of DdrI in D. radiodurans physiology under normal and stress conditions by regulating, both directly and indirectly, a cohort of genes involved in various cellular processes, including central metabolism and specific responses to diverse harmful environments. IMPORTANCE Deinococcus radiodurans has been extensively studied to elucidate the molecular mechanisms responsible for its exceptional ability to withstand lethal effects of various DNA-damaging agents. A complex network, including efficient DNA repair, protein protection against oxidation, and diverse metabolic pathways, plays a crucial role for its radioresistance. The regulatory networks orchestrating these various pathways are still missing. Our data provide new insights into the crucial contribution of the transcription factor DdrI for the D. radiodurans ability to withstand harmful conditions, including UV radiation, mitomycin C treatment, heat shock, and oxidative stress. Finally, we highlight that DdrI is also required for accurate cell division, for maintenance of plasmid replicons, and for central metabolism processes responsible for the overall cell physiology.

The two-partner secretion system ExlBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [ K eq ] of ≈2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ , resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers. IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.

Feed-forward loops (FFLs) represent an important and basic network motif to understand specific biological functions. Cyclic-AMP (cAMP) receptor protein (CRP), a transcription factor (TF), mediates catabolite repression and regulates more than 400 genes in response to changes in intracellular concentrations of cAMP in Escherichia coli. CRP participates in some FFLs, such as araBAD and araFGH operons and adapts to fluctuating environmental nutrients, thereby enhancing the survivability of E. coli. Although computational simulations have been conducted to explore the potential functionality of FFLs, a comprehensive study on the functions of all structural types on the basis of in vivo data is lacking. Moreover, the regulatory role of CRP-mediated FFLs (CRP-FFLs) remains obscure. We identified 393 CRP-FFLs in E. coli using EcoCyc and RegulonDB. Dose&ndash, response genomic microarray of E. coli revealed dynamic gene expression of each target gene of CRP-FFLs in response to a range of cAMP dosages. All eight types of FFLs were present in CRP regulon with various expression patterns of each CRP-FFL, which were further divided into five functional groups. The microarray and reported regulatory relationships identified 202 CRP-FFLs that were directly regulated by CRP in these eight types of FFLs. Interestingly, 34% (147/432) of genes were directly regulated by CRP and CRP-regulated TFs, which indicates that these CRP-regulated genes were also regulated by other CRP-regulated TFs responding to environmental signals through CRP-FFLs. Furthermore, we applied gene ontology annotation to reveal the biological functions of CRP-FFLs.

Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3’UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3’UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3’UTR as a hub for post-transcriptional control of Salmonella invasion gene expression., Author summary Salmonella infection is one of the major causes of foodborne illness worldwide. During infection, Salmonella expresses a set of virulence genes encoded in discrete regions of the genome. The expression of these genes is tightly regulated, being specific for different stages of the Salmonella infection process. While many regulatory mechanisms that lead to the activation of infection-related gene expression have been described, little is known about silencing mechanisms under conditions when expression is not needed and may rather represent a burden than a benefit for the bacterial fitness. Here, we report a condition-specific silencing mechanism of bacterial virulence. That is, the global transcriptional regulator CRP-cAMP represses, indirectly through a post-transcriptional mechanism, the expression of the major Salmonella virulence regulator HilD. In bacteria, post-transcriptional regulation has so far been mainly focused on 5’ untranslated regions (5’UTR). Remarkably, here we describe a molecular mechanism targeting the 3’untranslated region (3’UTR) of the mRNA of the major regulator of Salmonella virulence by a small non-coding RNA under the transcriptional control of the global regulator CRP-cAMP. Our data highlight the importance of 3’UTR in the regulation of gene expression in bacteria.

Despite the identification of many genes and pathways involved in the persistence phenomenon of bacteria, the mechanisms of persistence are not well understood. Here, using Escherichia coli as a model, we identified polynucleotide phosphorylase (PNPase) as a key regulator in persister formation. We successfully constructed pnp knockout mutant strain and its complemented strain, and exposed the pnp knockout mutant and complemented strain to antibiotics and stress conditions. The results showed that, compared with the wild-type W3110, the pnp knockout strain had defect in persistence to antibiotics and stress conditions, and the persistence to antibiotics and stresses was restored upon complementation. RNA-Seq was performed to identify the transcriptome profile in the pnp knockout strain compared with wild-type strain W3110, and the data revealed that 242 (166 up-regulated, and 76 down-regulated) genes were differentially expressed in the pnp knockout mutant strain. KEGG pathway analysis of the up-regulated genes showed that they were mostly mapped to metabolism and virulence pathways, most of which are positively regulated by the global regulator cyclic AMP receptor protein (CRP). Similarly, the transcription level of the crp gene in the pnp-deletion strain increased 3.22-fold in the early stationary phase. We further explored the indicators of cellular metabolism of the pnp-deletion strain, the persistence phenotype of the pnp and crp double-deletion mutant, and the transcriptional activity of crp gene. Our results indicate that PNPase controls cellular metabolism by negatively regulating the crp operon at the post-transcriptional level by targeting the 5’- Untranslated Region (UTR) of the crp transcript. This study offers new insight about the persister mechanisms and provides new targets for development of new drugs against persisters for more effective treatment of persistent bacterial infections.

Mutations in the gene encoding the protein kinase A (PKA) catalytic subunit α have been found to be responsible for cortisol-producing adenomas (CPAs). In this study, we identified by whole-exome sequencing the somatic mutation p.S54L in the PRKACB gene, encoding the catalytic subunit β (Cβ) of PKA, in a CPA from a patient with severe Cushing syndrome. Bioluminescence resonance energy transfer and surface plasmon resonance assays revealed that the mutation hampers formation of type I holoenzymes and that these holoenzymes were highly sensitive to cAMP. PKA activity, measured both in cell lysates and with recombinant proteins, based on phosphorylation of a synthetic substrate, was higher under basal conditions for the mutant enzyme compared with the WT, while maximal activity was lower. These data suggest that at baseline the PRKACB p.S54L mutant drove the adenoma cells to higher cAMP signaling activity, probably contributing to their autonomous growth. Although the role of PRKACB in tumorigenesis has been suggested, we demonstrated for the first time to our knowledge that a PRKACB mutation can lead to an adrenal tumor. Moreover, this observation describes another mechanism of PKA pathway activation in CPAs and highlights the particular role of residue Ser54 for the function of PKA.

Septicemia-causing Vibrio vulnificus produces at least three exoproteases, VvpE, VvpS, and VvpM, all of which participate in interactions with human cells. Expression of VvpE and VvpS is induced in the stationary phase by multiple transcription factors, including sigma factor S, SmcR, and the cAMP-cAMP receptor protein (cAMP-CRP) complex. Distinct roles of VvpM, such as induction of apoptosis, lead us to hypothesize VvpM expression is different from that of the other exoproteases. Its transcription, which was found to be independent of sigma S, is induced at the early exponential phase and then becomes negligible upon entry into the stationary phase. SmcR and CRP were studied regarding the control of vvpM expression. Transcription of vvpM was repressed by SmcR and cAMP-CRP complex individually, which specifically bound to the regions −2 to +20 and +6 to +27, respectively, relative to the vvpM transcription initiation site. Derepression of vvpM gene expression was 10- to 40-fold greater in an smcR crp double mutant than in single-gene mutants. Therefore, these results show that the expression of V. vulnificus exoproteases is differentially regulated, and in this way, distinct proteases can engage in specific interactions with a host. IMPORTANCE An opportunistic human pathogen, Vibrio vulnificus produces multiple extracellular proteases that are involved in diverse interactions with a host. The total exoproteolytic activity is detected mainly in the supernatants of the high-cell-density cultures. However, some proteolytic activity derived from a metalloprotease, VvpM, was present in the supernatants of the low-cell-density cultures sampled at the early growth period. In this study, we present the regulatory mechanism for VvpM expression via repression by at least two transcription factors. This type of transcriptional regulation is the exact opposite of those for expression of the other V. vulnificus exoproteases. Differential regulation of each exoprotease's production then facilitates the pathogen's participation in the distinct interactions with a host.

A variety of bacteria, including Escherichia coli, are known to enter the viable but non-culturable (VBNC) state under various stress conditions. During this state, cells lose colony-forming activities on conventional agar plates while retaining signs of viability. Diverse environmental stresses including starvation induce the VBNC state. However, little is known about the genetic mechanism inducing this state. Here, we aimed to reveal the genetic determinants of the VBNC state of E. coli. We hypothesized that the VBNC state is a process wherein specific gene products important for colony formation are depleted during the extended period of stress conditions. If so, higher expression of these genes would maintain colony-forming activities, thereby restraining cells from entering the VBNC state. From an E. coli plasmid-encoded ORF library, we identified genes that were responsible for maintaining high colony-forming activities after exposure to starvation condition. Among these, cpdA encoding cAMP phosphodiesterase exhibited higher performance in the maintenance of colony-forming activities. As cpdA overexpression decreases intracellular cAMP, cAMP or its complex with cAMP-receptor protein (CRP) may negatively regulate colony-forming activities under stress conditions. We confirmed this using deletion mutants lacking adenylate cyclase or CRP. These mutants fully maintained colony-forming activities even after a long period of starvation, while wild-type cells lost most of this activity. Thus, we concluded that the lack of cAMP-CRP effectively retains high colony-forming activities, indicating that cAMP-CRP acts as a positive regulator necessary for the induction of the VBNC state in E. coli.

Haemophilus parasuis causes Glässer's disease and pneumonia, incurring serious economic losses in the porcine industry. In this study, natural competence was investigated in H. parasuis. We found competence genes in H. parasuis homologous to ones in Haemophilus influenzae and a high consensus battery of Sxy-dependent cyclic AMP (cAMP) receptor protein (CRP-S) regulons using bioinformatics. High rates of natural competence were found from the onset of stationary-phase growth condition to mid-stationary phase (OD600 from 0.29 to 1.735); this rapidly dropped off as cells reached mid-stationary phase (OD600 from 1.735 to 1.625). As a whole, bacteria cultured in liquid media were observed to have lower competence levels than those grown on solid media plates. We also revealed that natural transformation in this species is stable after 200 passages and is largely dependent on DNA concentration. Transformation competition experiments showed that heterogeneous DNA cannot outcompete intraspecific natural transformation, suggesting an endogenous uptake sequence or other molecular markers may be important in differentiating heterogeneous DNA. We performed qRT-PCR targeting multiple putative competence genes in an effort to compare bacteria pre-cultured in TSB++ vs. TSA++ and SC1401 vs. SH0165 to determine expression profiles of the homologs of competence-genes in H. influenzae. Taken together, this study is the first to investigate natural transformation in H. parasuis based on a highly naturally transformable strain SC1401.

Cyclic AMP receptor protein (CRP), a global regulator in Escherichia coli, regulates more than 180 genes via two roles: activation and repression. Few methods are available for predicting the regulatory roles from the binding sites of transcription factors. This work proposes an accurate method PredCRP to derive an optimised model (named PredCRP-model) and a set of four interpretable rules (named PredCRP-ruleset) for predicting and analysing the regulatory roles of CRP from sequences of CRP-binding sites. A dataset consisting of 169 CRP-binding sites with regulatory roles strongly supported by evidence was compiled. The PredCRP-model, using 12 informative features of CRP-binding sites, and cooperating with a support vector machine achieved a training and test accuracy of 0.98 and 0.93, respectively. PredCRP-ruleset has two activation rules and two repression rules derived using the 12 features and the decision tree method C4.5. This work further screened and identified 23 previously unobserved regulatory interactions in Escherichia coli. Using quantitative PCR for validation, PredCRP-model and PredCRP-ruleset achieved a test accuracy of 0.96 (=22/23) and 0.91 (=21/23), respectively. The proposed method is suitable for designing predictors for regulatory roles of all global regulators in Escherichia coli. PredCRP can be accessed at https://github.com/NctuICLab/PredCRP.

ZnO quantum dots (QDs) are very well known for their antimicrobial activity against several bacteria, however, we still do not know any protein targets of ZnO QDs. In order to determine possible protein target, interaction of ZnO QDs was studied with CRP (Cyclic AMP Receptor Protein), a global transcription regulator protein. Binding between ZnO QDs and E. coli CRP was mainly studied by isothermal titration calorimetry (ITC), structural changes of protein were monitored by fluorescence and circular dichroism spectroscopy, and in-vitro transcription assay was used to asses CRP activity. Result shows that both electrostatic and hydrophobic interactions are involved in CRP-ZnO binding. Different spectroscopic investigation revealed that ZnO binding to CRP leads to extensive unfolding and destabilization, which ultimately leads to protein aggregation. It was also observed that in presence of ZnO dimerization ability of CRP was sharply reduced. In-vitro transcription assay also shows that CRP activity gets severely compromised on ZnO binding. All our data suggests that ZnO QD binding to CRP and consequent structural and functional changes most probably plays a crucial role in ZnO QD induced antimicrobial action.

Endothelial Per-Arnt-Sim domain protein-1/hypoxia-inducible factor-2α (EPAS-1/ HIF-2α) is a catabolic transcription factor that regulates osteoarthritis (OA)-related cartilage destruction. Here, we examined whether microRNA-365 (miR-365) affects interleukin (IL)-1β-induced expression of catabolic factors in chondrocytes via regulation of HIF-2α. MiR-365 levels were significantly decreased in human OA cartilage relative to normal cartilage. Overexpression of miR-365 significantly suppressed IL-1β-induced expression of HIF-2α in human articular chondrocytes. Pharmacological inhibition of various IL-1β-associated signaling pathways revealed mitogen-activated protein kinase and nuclear factor-κB as the primary pathways driving IL-1β-mediated decreases in miR-365 and subsequent increase in HIF-2α expression. Using a luciferase reporter assay encoding the 3′ untranslated region (UTR) of human HIF-2α mRNA, we showed that overexpression of miR-365 significantly suppressed IL-1β-induced up-regulation of HIF-2α. AGO2 RNA-immunoprecipitation (IP) assay demonstrated that miR-365 and HIF-2α mRNA were enriched in the AGO2-IP fraction in miR-365-transfected primary chondrocytes compared to miR-con-transfected cells, indicating that HIF-2α is a target of miR-365. Furthermore, miR-365 overexpression significantly suppressed IL-1β-induced expression of catabolic factors, including cyclooxygenase-2 and matrix metalloproteinase-1, -3 and -13, in chondrocytes. In pellet culture of primary chondrocytes miR-365 prevented IL-1β-stimulated extracellular matrix loss and matrix metalloproteinase-13 expression. MiR-365 regulates IL-1β-stimulated catabolic effects in human chondrocytes by modulating HIF-2α expression.

Understanding the evolution of a new metabolic capability in full mechanistic detail is challenging, as causative mutations may be masked by non-essential "hitchhiking" mutations accumulated during the evolutionary trajectory. We have previously used adaptive laboratory evolution of a rationally engineered ancestor to generate an Escherichia coli strain able to utilize CO2 fixation for sugar synthesis. Here, we reveal the genetic basis underlying this metabolic transition. Five mutations are sufficient to enable robust growth when a non-native Calvin–Benson–Bassham cycle provides all the sugar-derived metabolic building blocks. These mutations are found either in enzymes that affect the efflux of intermediates from the autocatalytic CO2 fixation cycle toward biomass (prs, serA, and pgi), or in key regulators of carbon metabolism (crp and ppsR). Using suppressor analysis, we show that a decrease in catalytic capacity is a common feature of all mutations found in enzymes. These findings highlight the enzymatic constraints that are essential to the metabolic stability of autocatalytic cycles and are relevant to future efforts in constructing non-native carbon fixation pathways., An E. coli strain able to use CO2 fixation for sugar synthesis was previously generated by experimental evolution of an engineered strain. Here, Herz et al. show that specific mutations in five genes, encoding carbon metabolism enzymes or key regulators, are sufficient to enable robust growth of the strain.

Fluoroquinolone antibiotics are prescribed for the treatment of Salmonella enterica infections, but resistance to this family of antibiotics is growing. Here we report that loss of the global regulatory protein cyclic AMP (cAMP) receptor protein (CRP) or its allosteric effector, cAMP, reduces susceptibility to fluoroquinolones. A Δ crp mutation was synergistic with the primary fluoroquinolone resistance allele gyrA83 , thus able to contribute to clinically relevant resistance. Decreased susceptibility to fluoroquinolones could be partly explained by decreased expression of the outer membrane porin genes ompA and ompF with a concomitant increase in the expression of the ciprofloxacin resistance efflux pump gene acrB in Δ crp cells. Expression of gyrAB , which encode the DNA supercoiling enzyme GyrAB, which is blocked by fluoroquinolones, and expression of topA , which encodes the dominant supercoiling-relaxing enzyme topoisomerase I, were unchanged in Δ crp cells. Yet Δ crp cells maintained a more relaxed state of DNA supercoiling, correlating with an observed increase in topoisomerase IV ( parCE ) expression. Surprisingly, the Δ crp mutation had the unanticipated effect of enhancing fitness in the presence of fluoroquinolone antibiotics, which can be explained by the observation that exposure of Δ crp cells to ciprofloxacin had the counterintuitive effect of restoring wild-type levels of DNA supercoiling. Consistent with this, Δ crp cells did not become elongated or induce the SOS response when challenged with ciprofloxacin. These findings implicate the combined action of multiple drug resistance mechanisms in Δ crp cells: reduced permeability and elevated efflux of fluoroquinolones coupled with a relaxed DNA supercoiling state that buffers cells against GyrAB inhibition by fluoroquinolones.

Many proteobacteria modulate a suite of catabolic genes using the second messenger cyclic 3', 5'-AMP (cAMP) and the cAMP receptor protein (CRP). Together, the cAMP-CRP complex regulates target promoters, usually by activating transcription. In the canonical model, the phosphotransferase system (PTS), and in particular the EIIA(Glc) component for glucose uptake, provides a mechanistic link that modulates cAMP levels depending on glucose availability, resulting in more cAMP and activation of alternative catabolic pathways when glucose is unavailable. Within the Vibrionaceae, cAMP-CRP appears to play the classical role in modulating metabolic pathways; however, it also controls functions involved in natural competence, bioluminescence, pheromone signaling, and colonization of animal hosts. For this group of marine bacteria, chitin is an ecologically relevant resource, and chitin's monomeric sugar N-acetylglucosamine (NAG) supports robust growth while also triggering regulatory responses. Recent studies with Vibrio fischeri indicate that NAG and glucose uptake share EIIA(Glc), yet the responses of cAMP-CRP to these two carbon sources are starkly different. Moreover, control of cAMP levels appears to be more dominantly controlled by export and degradation. Perhaps more surprisingly, although CRP may require cAMP, its activity can be controlled in response to glucose by a mechanism independent of cAMP levels. Future studies in this area promise to shed new light on the role of cAMP and CRP.

Bacteria possess signal transduction pathways capable of sensing and responding to a wide variety of signals. The Cpx envelope stress response, composed of the sensor histidine kinase CpxA and the response regulator CpxR, senses and mediates adaptation to insults to the bacterial envelope. The Cpx response has been implicated in the regulation of a number of envelope-localized virulence determinants across bacterial species. Here, we show that activation of the Cpx pathway in Vibrio cholerae El Tor strain C6706 leads to a decrease in expression of the major virulence factors in this organism, cholera toxin (CT) and the toxin-coregulated pilus (TCP). Our results indicate that this occurs through the repression of production of the ToxT regulator and an additional upstream transcription factor, TcpP. The effect of the Cpx response on CT and TCP expression is mostly abrogated in a cyclic AMP receptor protein (CRP) mutant, although expression of the crp gene is unaltered. Since TcpP production is controlled by CRP, our data suggest a model whereby the Cpx response affects CRP function, which leads to diminished TcpP, ToxT, CT, and TCP production.

Bacterial pathogens adapt to changing environments within their hosts, and the signaling molecule adenosine 3', 5'-cyclic monophosphate (cAMP) facilitates this process. In this study, we characterized in vivo DNA binding and gene regulation by the cAMP-responsive protein CRP in M. bovis BCG as a model for tuberculosis (TB)-complex bacteria. Chromatin immunoprecipitation followed by deep-sequencing (ChIP-seq) showed that CRP associates with ∼900 DNA binding regions, most of which occur within genes. The most highly enriched binding region was upstream of a putative copper transporter gene (ctpB), and crp-deleted bacteria showed increased sensitivity to copper toxicity. Detailed mutational analysis of four CRP binding sites upstream of the virulence-associated Rv0249c-Rv0247c succinate dehydrogenase genes demonstrated that CRP directly regulates Rv0249c-Rv0247c expression from two promoters, one of which requires sequences intragenic to Rv0250c for maximum expression. The high percentage of intragenic CRP binding sites and our demonstration that these intragenic DNA sequences significantly contribute to biologically relevant gene expression greatly expand the genome space that must be considered for gene regulatory analyses in mycobacteria. These findings also have practical implications for an important bacterial pathogen in which identification of mutations that affect expression of drug target-related genes is widely used for rapid drug resistance screening.

Peptidoglycan degradative enzymes have important roles at many stages during the bacterial life cycle, and it is critical that these enzymes be stringently regulated to avoid compromising the integrity of the cell wall. How this regulation is exerted is of considerable interest: promoter-based control and protein-protein interactions are known to be employed; however, other regulatory mechanisms are almost certainly involved. In the actinobacteria, a class of muralytic enzymes - the 'resuscitation-promoting factors' (Rpfs) - orchestrates the resuscitation of dormant cells. In this study, we have taken a holistic approach to exploring the mechanisms governing RpfA function using the model bacterium Streptomyces coelicolor and have uncovered unprecedented multilevel regulation that is coordinated by three second messengers. Our studies show that RpfA is subject to transcriptional control by the cyclic AMP receptor protein, riboswitch-mediated transcription attenuation in response to cyclic di-AMP, and growth stage-dependent proteolysis in response to ppGpp accumulation. Furthermore, our results suggest that these control mechanisms are likely applicable to cell wall lytic enzymes in other bacteria.