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1.
Nature ; 582(7811): 246-252, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32499648

RESUMEN

A wealth of specialized neuroendocrine command systems intercalated within the hypothalamus control the most fundamental physiological needs in vertebrates1,2. Nevertheless, we lack a developmental blueprint that integrates the molecular determinants of neuronal and glial diversity along temporal and spatial scales of hypothalamus development3. Here we combine single-cell RNA sequencing of 51,199 mouse cells of ectodermal origin, gene regulatory network (GRN) screens in conjunction with genome-wide association study-based disease phenotyping, and genetic lineage reconstruction to show that nine glial and thirty-three neuronal subtypes are generated by mid-gestation under the control of distinct GRNs. Combinatorial molecular codes that arise from neurotransmitters, neuropeptides and transcription factors are minimally required to decode the taxonomical hierarchy of hypothalamic neurons. The differentiation of γ-aminobutyric acid (GABA) and dopamine neurons, but not glutamate neurons, relies on quasi-stable intermediate states, with a pool of GABA progenitors giving rise to dopamine cells4. We found an unexpected abundance of chemotropic proliferation and guidance cues that are commonly implicated in dorsal (cortical) patterning5 in the hypothalamus. In particular, loss of SLIT-ROBO signalling impaired both the production and positioning of periventricular dopamine neurons. Overall, we identify molecular principles that shape the developmental architecture of the hypothalamus and show how neuronal heterogeneity is transformed into a multimodal neural unit to provide virtually infinite adaptive potential throughout life.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Hipotálamo/citología , Hipotálamo/embriología , Morfogénesis , Animales , Diferenciación Celular , Linaje de la Célula , Dopamina/metabolismo , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Ectodermo/citología , Ectodermo/metabolismo , Femenino , Neuronas GABAérgicas/citología , Neuronas GABAérgicas/metabolismo , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo , Ácido Glutámico/metabolismo , Hipotálamo/metabolismo , Masculino , Ratones , Morfogénesis/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Neuropéptidos/metabolismo , Neurotransmisores/metabolismo , Receptores Inmunológicos/metabolismo , Regulón/genética , Transducción de Señal , Factores de Transcripción/metabolismo , Ácido gamma-Aminobutírico/metabolismo
2.
Nat Commun ; 11(1): 2936, 2020 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-32522993

RESUMEN

Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes, many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR-mediated translational control induces the expression of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one-carbon metabolism. Concomitantly, the cells become resistant to the folate-based antimetabolites Methotrexate and Pemetrexed, establishing a direct link between UPR-driven changes to gene expression and resistance to pharmacological treatment.


Asunto(s)
Antimetabolitos/farmacología , Ácido Fólico/farmacología , Regulón/genética , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genética , Animales , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Humanos , Metotrexato/farmacología , Pemetrexed/farmacología , Proteoma/efectos de los fármacos , Proteoma/genética , Regulón/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Transcriptoma/genética
3.
Nat Commun ; 11(1): 2427, 2020 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-32415193

RESUMEN

Electronic information can be transmitted to cells directly from microelectronics via electrode-activated redox mediators. These transmissions are decoded by redox-responsive promoters which enable user-specified control over biological function. Here, we build on this redox communication modality by establishing an electronic eCRISPR conduit of information exchange. This system acts as a biological signal processor, amplifying signal reception and filtering biological noise. We electronically amplify bacterial quorum sensing (QS) signaling by activating LasI, the autoinducer-1 synthase. Similarly, we filter out unintended noise by inhibiting the native SoxRS-mediated oxidative stress response regulon. We then construct an eCRISPR based redox conduit in both E. coli and Salmonella enterica. Finally, we display eCRISPR based information processing that allows transmission of spatiotemporal redox commands which are then decoded by gelatin-encapsulated E. coli. We anticipate that redox communication channels will enable biohybrid microelectronic devices that could transform our abilities to electronically interpret and control biological function.


Asunto(s)
Sistemas CRISPR-Cas , Ingeniería Genética/métodos , Oxidación-Reducción , Electroquímica , Electrodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ferricianuros/química , Regulación Bacteriana de la Expresión Génica , Estrés Oxidativo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Piocianina/química , Percepción de Quorum , Regulón , Salmonella enterica/metabolismo , Espectrometría de Fluorescencia
4.
Nucleic Acids Res ; 48(9): 4811-4826, 2020 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-32282918

RESUMEN

The phosphorylation pattern of Pol2 CTD Y1S2P3T4S5P6S7 repeats comprises an informational code coordinating transcription and RNA processing. cis-trans isomerization of CTD prolines expands the scope of the code in ways that are not well understood. Here we address this issue via analysis of fission yeast peptidyl-prolyl isomerase Pin1. A pin1Δ allele that does not affect growth per se is lethal in the absence of cleavage-polyadenylation factor (CPF) subunits Ppn1 and Swd22 and elicits growth defects absent CPF subunits Ctf1 and Dis2 and termination factor Rhn1. Whereas CTD S2A, T4A, and S7A mutants thrive in combination with pin1Δ, a Y1F mutant does not, nor do CTD mutants in which half the Pro3 or Pro6 residues are replaced by alanine. Phosphate-acquisition genes pho1, pho84 and tgp1 are repressed by upstream lncRNAs and are sensitive to changes in lncRNA 3' processing/termination. pin1Δ hyper-represses PHO gene expression and erases the de-repressive effect of CTD-S7A. Transcriptional profiling delineated sets of 56 and 22 protein-coding genes that are down-regulated and up-regulated in pin1Δ cells, respectively, 77% and 100% of which are downregulated/upregulated when the cis-proline-dependent Ssu72 CTD phosphatase is inactivated. Our results implicate Pin1 as a positive effector of 3' processing/termination that acts via Ssu72.


Asunto(s)
Regulación Fúngica de la Expresión Génica , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Fosfoproteínas Fosfatasas/genética , Procesamiento de Término de ARN 3' , Proteínas de Schizosaccharomyces pombe/genética , Terminación de la Transcripción Genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Eliminación de Gen , Peptidilprolil Isomerasa de Interacción con NIMA/química , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Proteínas Nucleares/genética , Fosfatos/metabolismo , Fosforilación , Dominios Proteicos/genética , Pirofosfatasas/genética , ARN Polimerasa II/genética , RNA-Seq , Regulón , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Serina/metabolismo , Treonina/metabolismo
5.
Arch Microbiol ; 202(6): 1359-1368, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32157346

RESUMEN

The epidemic diarrheal disease cholera is caused by the Gram-negative bacterium Vibrio cholerae. V. cholerae virulence factors include the toxin-coregulated pilus (TCP) and cholera toxin, which are major factors responsible for host colonization and production of diarrhea. Expression of cholera toxin and TCP genes is controlled by the ToxR regulon. The ToxR regulon includes the transcriptional activators ToxR, TcpP, and ToxT. ToxT directly initiates transcription of the cholera toxin and TCP genes. TcpP and ToxR are necessary for expression of toxT. TcpP and ToxR activity requires TcpH and ToxS, respectively. Additionally, ToxR is able to directly initiate transcription of the cholera toxin genes independent of TcpP and ToxT. TCP is required early in infection to colonize the small intestine, then cholera toxin is expressed later in infection to produce diarrhea. We tested whether stringent response, the low nutrient stress response, was involved in regulation of virulence genes. Using an infant mouse model, we found that V. cholerae strains with deletions of the stringent response genes were unable to colonize the small intestine. We further tested these stringent response-null mutants and found that stringent response was necessary for TCP expression, although effects on cholera toxin expression were not significant. We then tested whether stringent response regulation of TCP occurred through the ToxR regulon. We found that stringent response induced toxT and tcpPH expression, while repressing toxRS. This differential regulation of ToxR and TcpP may explain the differential expression of TCP and cholera toxin in vivo.


Asunto(s)
Proteínas Bacterianas/genética , Cólera/microbiología , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica/genética , Regulón/genética , Factores de Transcripción/genética , Vibrio cholerae/genética , Vibrio cholerae/patogenicidad , Factores de Virulencia/genética , Animales , Eliminación de Gen , Ratones
6.
PLoS Pathog ; 16(3): e1008332, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32130269

RESUMEN

Pathogens of the Streptococcus genus inhabit many different environmental niches during the course of an infection in a human host and the bacteria must adjust their metabolism according to available nutrients. Despite their lack of the citric-acid cycle, some streptococci proliferate in niches devoid of a readily available carbohydrate source. Instead they rely on carbohydrate scavenging for energy acquisition, which are obtained from the host. Here we discover a two-component system (TCS07) of Streptococcus pneumoniae that responds to glycoconjugated structures on proteins present on the host cells. Using next-generation RNA sequencing we find that the uncharacterized TCS07 regulon encodes proteins important for host-glycan processing and transporters of the released glycans, as well as intracellular carbohydrate catabolizing enzymes. We find that a functional TCS07 allele is required for growth on the glycoconjugated model protein fetuin. Consistently, we see a TCS07-dependent activation of the glycan degradation pathway. Thus, we pinpoint the molecular constituents responsible for sensing host derived glycans and link this to the induction of the proteins necessary for glycan degradation. Furthermore, we connect the TCS07 regulon to virulence in a mouse model, thereby establishing that host-derived glycan-metabolism is important for infection in vivo. Finally, a comparative phylogenomic analysis of strains from the Streptococcus genus reveal that TCS07 and most of its regulon is specifically conserved in species that utilize host-glycans for growth.


Asunto(s)
Proteínas Bacterianas/metabolismo , Infecciones Neumocócicas/metabolismo , Polisacáridos/metabolismo , Streptococcus pneumoniae/metabolismo , Animales , Proteínas Bacterianas/genética , Genoma Bacteriano , Interacciones Huésped-Patógeno , Humanos , Ratones , Infecciones Neumocócicas/microbiología , Regulón , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pneumoniae/patogenicidad , Virulencia
7.
PLoS One ; 15(2): e0226232, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32109244

RESUMEN

Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungal pathogen Sclerotinia sclerotiorum. PA23 produces several inhibitory compounds that are under control of a complex regulatory network. Included in this cascade is the PhzRI quorum sensing (QS) system, which plays an essential role in PA23 biocontrol, as well as CsaRI and AurRI, which have not yet been characterized in PA23. The focus of the current study was to employ RNA sequencing to explore the spectrum of PA23 genes under QS control. In this work, we investigated genes under the control of the main QS transcriptional regulator, PhzR, as well as those differentially expressed in an AHL-deficient strain, PA23-6863, which constitutively expresses an AiiA lactonase, rendering the strain QS defective. Transcriptomic profiling revealed 545 differentially expressed genes (365 downregulated; 180 upregulated) in the phzR mutant and 534 genes (382 downregulated; 152 upregulated) in the AHL-deficient PA23-6863. In both strains, decreased expression of phenazine, pyrrolnitrin, and exoprotease biosynthetic genes was observed. We have previously reported that QS activates expression of these genes and their encoded products. In addition, elevated siderophore and decreased chitinase gene expression was observed in the QS-deficient stains, which was confirmed by phenotypic analysis. Inspection of the promoter regions revealed the presence of "phz-box" sequences in only 58 of the 807 differentially expressed genes, suggesting that much of the QS regulon is indirectly regulated. Consistent with this notion, 41 transcriptional regulators displayed altered expression in one or both of the QS-deficient strains. Collectively, our findings indicate that QS governs expression of approximately 13% of the PA23 genome affecting diverse functions ranging from secondary metabolite production to general metabolism.


Asunto(s)
Control Biológico de Vectores , Pseudomonas chlororaphis/genética , Percepción de Quorum/genética , Regulón/genética , Proteínas Bacterianas/genética , Hidrolasas de Éster Carboxílico/deficiencia , Movimiento Celular/genética , Quitinasas/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Mutantes , RNA-Seq , Sideróforos/genética , Transactivadores/genética , Transcriptoma
8.
Nucleic Acids Res ; 48(7): 3567-3590, 2020 04 17.
Artículo en Inglés | MEDLINE | ID: mdl-32086516

RESUMEN

To sustain iron homeostasis, microorganisms have evolved fine-tuned mechanisms for uptake, storage and detoxification of the essential metal iron. In the human pathogen Aspergillus fumigatus, the fungal-specific bZIP-type transcription factor HapX coordinates adaption to both iron starvation and iron excess and is thereby crucial for virulence. Previous studies indicated that a HapX homodimer interacts with the CCAAT-binding complex (CBC) to cooperatively bind bipartite DNA motifs; however, the mode of HapX-DNA recognition had not been resolved. Here, combination of in vivo (genetics and ChIP-seq), in vitro (surface plasmon resonance) and phylogenetic analyses identified an astonishing plasticity of CBC:HapX:DNA interaction. DNA motifs recognized by the CBC:HapX protein complex comprise a bipartite DNA binding site 5'-CSAATN12RWT-3' and an additional 5'-TKAN-3' motif positioned 11-23 bp downstream of the CCAAT motif, i.e. occasionally overlapping the 3'-end of the bipartite binding site. Phylogenetic comparison taking advantage of 20 resolved Aspergillus species genomes revealed that DNA recognition by the CBC:HapX complex shows promoter-specific cross-species conservation rather than regulon-specific conservation. Moreover, we show that CBC:HapX interaction is absolutely required for all known functions of HapX. The plasticity of the CBC:HapX:DNA interaction permits fine tuning of CBC:HapX binding specificities that could support adaptation of pathogens to their host niches.


Asunto(s)
Aspergillus fumigatus/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factor de Unión a CCAAT/metabolismo , Proteínas Fúngicas/metabolismo , Hierro/metabolismo , Regiones Promotoras Genéticas , Secuencia Rica en At , Aspergillus fumigatus/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Sitios de Unión , ADN de Hongos/química , ADN de Hongos/metabolismo , Evolución Molecular , Proteínas Fúngicas/química , Mutación , Motivos de Nucleótidos , Unión Proteica , Dominios Proteicos , Regulón , Sideróforos/metabolismo , Resonancia por Plasmón de Superficie , Factores de Transcripción/química , Factores de Transcripción/metabolismo
9.
Science ; 367(6473): 96-100, 2020 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-31896718

RESUMEN

Changes in both the coding sequence of transcriptional regulators and in the cis-regulatory sequences recognized by these regulators have been implicated in the evolution of transcriptional circuits. However, little is known about how they evolved in concert. We describe an evolutionary pathway in fungi where a new transcriptional circuit (a-specific gene repression by the homeodomain protein Matα2) evolved by coding changes in this ancient regulator, followed millions of years later by cis-regulatory sequence changes in the genes of its future regulon. By analyzing a group of species that has acquired the coding changes but not the cis-regulatory sites, we show that the coding changes became necessary for the regulator's deeply conserved function, thereby poising the regulator to jump-start formation of the new circuit.


Asunto(s)
Evolución Molecular , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Redes Reguladoras de Genes , Proteínas de Homeodominio/genética , Levaduras/genética , Código Genético , Regulón , Transcripción Genética
10.
Nat Commun ; 11(1): 557, 2020 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-31992699

RESUMEN

Hydrogen sulfide (H2S) is involved in numerous pathophysiological processes and shares overlapping functions with CO and •NO. However, the importance of host-derived H2S in microbial pathogenesis is unknown. Here we show that Mtb-infected mice deficient in the H2S-producing enzyme cystathionine ß-synthase (CBS) survive longer with reduced organ burden, and that pharmacological inhibition of CBS reduces Mtb bacillary load in mice. High-resolution respirometry, transcriptomics and mass spectrometry establish that H2S stimulates Mtb respiration and bioenergetics predominantly via cytochrome bd oxidase, and that H2S reverses •NO-mediated inhibition of Mtb respiration. Further, exposure of Mtb to H2S regulates genes involved in sulfur and copper metabolism and the Dos regulon. Our results indicate that Mtb exploits host-derived H2S to promote growth and disease, and suggest that host-directed therapies targeting H2S production may be potentially useful for the management of tuberculosis and other microbial infections.


Asunto(s)
Sulfuro de Hidrógeno/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Animales , Cobre/metabolismo , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Citocinas/sangre , Modelos Animales de Enfermedad , Complejo IV de Transporte de Electrones/metabolismo , Metabolismo Energético , Femenino , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Homeostasis , Pulmón/patología , Macrófagos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium tuberculosis/genética , Células RAW 264.7 , Regulón , Azufre/metabolismo , Transcriptoma , Tuberculosis
11.
Proc Natl Acad Sci U S A ; 117(3): 1689-1699, 2020 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-31915298

RESUMEN

Streptococcus pneumoniae is an opportunistic human pathogen that utilizes the competence regulon, a quorum-sensing circuitry, to acquire antibiotic resistance genes and initiate its attack on the human host. Interception of the competence regulon can therefore be utilized to study S. pneumoniae cell-cell communication and behavioral changes, as well as attenuate S. pneumoniae infectivity. Herein we report the design and synthesis of cyclic dominant negative competence-stimulating peptide (dnCSP) analogs capable of intercepting the competence regulon in both S. pneumoniae specificity groups with activities at the low nanomolar range. Structural analysis of lead analogs provided important insights as to the molecular mechanism that drives CSP receptor binding and revealed that the pan-group cyclic CSPs exhibit a chimeric hydrophobic patch conformation that resembles the hydrophobic patches required for both ComD1 and ComD2 binding. Moreover, the lead cyclic dnCSP, CSP1-E1A-cyc(Dap6E10), was found to possess superior pharmacological properties, including improved resistance to enzymatic degradation, while remaining nontoxic. Lastly, CSP1-E1A-cyc(Dap6E10) was capable of attenuating mouse mortality during acute pneumonia caused by both group 1 and group 2 S. pneumoniae strains. This cyclic pan-group dnCSP is therefore a promising drug lead scaffold against S. pneumoniae infections that could be administered individually or utilized in combination therapy to augment the effects of current antimicrobial agents.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Percepción de Quorum/efectos de los fármacos , Streptococcus pneumoniae/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Infecciones Neumocócicas/tratamiento farmacológico , Unión Proteica , Regulón/efectos de los fármacos
12.
Plant Mol Biol ; 102(4-5): 447-462, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31898148

RESUMEN

KEY MESSAGE: ZjICE2 works as a positive regulator in abiotic stress responses and ZjICE2 is a valuable genetic resource to improve abiotic stress tolerance in the molecular breeding program of Zoysia japonica. The basic helix-loop-helix (bHLH) family transcription factors (TFs) play an important role in response to biotic or abiotic stresses in plants. However, the functions of bHLH TFs in Zoysia japonica, one of the warm-season turfgrasses, remain poorly understood. Here, we identified ZjICE2 from Z. japonica, a novel MYC-type bHLH transcription factor that was closely related to ICE homologs in the phylogenetic tree, and its expression was regulated by various abiotic stresses. Transient expression of ZjICE2-GFP in onion epidermal cells revealed that ZjICE2 was a nuclear-localized protein. Also, ZjICE2 bound the MYC cis-element in the promoter of dehydration responsive element binding 1 of Z. japonica (ZjDREB1) using yeast one-hybrid assay. A phenotypic analysis showed that overexpression of the ZjICE2 in Arabidopsis enhanced tolerance to cold, drought, and salt stresses. The transgenic Arabidopsis and Z. japonica accumulated more transcripts of cold-responsive DREB/CBFs and their downstream genes than the wild type (WT) after cold treatment. Furthermore, the transgenic plants exhibited an enhanced Reactive oxygen species (ROS) scavenging ability, which resulted in an efficient maintenance of oxidant-antioxidant homeostasis. In addition, overexpression of the ZjICE2 in Z. japonica displayed intensive cold tolerance with increases in chlorophyll contents and photosynthetic efficiency. Our study suggests that ZjICE2 works as a positive regulator in abiotic stress responses and the ICE-DREB/CBFs response pathway involved in cold stress tolerance is also conserved in Z. japonica. These results provide a valuable genetic resource for the molecular breeding program especially for warm-season grasses as well as other leaf crop plants.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/fisiología , Poaceae/fisiología , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Frío , Respuesta al Choque por Frío , Sequías , Filogenia , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/fisiología , Poaceae/genética , Regulón , Tolerancia a la Sal , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Activación Transcripcional
13.
FASEB J ; 34(1): 1038-1051, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31914596

RESUMEN

Over recent years several examples of randomly switching methyltransferases, associated with Type III restriction-modification (R-M) systems, have been described in pathogenic bacteria. In every case examined, changes in simple DNA sequence repeats result in variable methyltransferase expression and result in global changes in gene expression, and differentiation of the bacterial cell into distinct phenotypes. These epigenetic regulatory systems are called phasevarions, phase-variable regulons, and are widespread in bacteria, with 17.4% of Type III R-M system containing simple DNA sequence repeats. A distinct, recombination-driven random switching system has also been described in Streptococci in Type I R-M systems that also regulate gene expression. Here, we interrogate the most extensive and well-curated database of R-M systems, REBASE, by searching for all possible simple DNA sequence repeats in the hsdRMS genes that encode Type I R-M systems. We report that 7.9% of hsdS, 2% of hsdM, and of 4.3% of hsdR genes contain simple sequence repeats that are capable of mediating phase variation. Phase variation of both hsdM and hsdS genes will lead to differential methyltransferase expression or specificity, and thereby the potential to control phasevarions. These data suggest that in addition to well characterized phasevarions controlled by Type III mod genes, and the previously described Streptococcal Type I R-M systems that switch via recombination, approximately 10% of all Type I R-M systems surveyed herein have independently evolved the ability to randomly switch expression via simple DNA sequence repeats.


Asunto(s)
Epigénesis Genética , Repeticiones de Microsatélite , Regulón , Proteínas Bacterianas/genética , Biología Computacional , ADN/análisis , Metilación de ADN , Metilasas de Modificación del ADN/metabolismo , Enzimas de Restricción-Modificación del ADN/genética , Enzimas de Restricción-Modificación del ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo I/genética , Fusobacterium nucleatum , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Mannheimia haemolytica , Metiltransferasas/metabolismo , Fenotipo , Pseudomonas aeruginosa , Salmonella enterica
15.
Artículo en Inglés | MEDLINE | ID: mdl-31759173

RESUMEN

In many γ-proteobacteria, FadR is recognized as a global transcriptional regulator: in addition to being the most prominent regulator for FA biosynthesis and degradation, the protein also mediates expression of many genes in diverse biological processes. In Shewanella oneidensis, a bacterium renowned for its respiratory versatility, FadR directly controls only a few genes. However, the FadR loss substantially increases BCFA contents and impairs growth. In this study, we showed that FadR is required to activate a number of important FA biosynthesis genes, including fabA, fabB, and fabH1. Although most of these genes are controlled by FadR in a direct manner, they are not critically responsible for the phenotypes resulting from the FadR depletion. Subsequent investigations identified BKD encoded by the bkd operon as the critical factor for enhanced BCFA production. In the absence of FadR, the bkd operon is derepressed, resulting in elevated conversion of 3MOP to 3-methylbutanoyl-CoA, one of the direct substrates for BCFA synthesis. We further showed that the growth defect of the fadR mutant is due to BCAA shortage, a scenario also attributable to excessive BKD: 3MOP, the common substrate for both BCFA and BCAA, is disproportionately used for BCFA synthesis, leading to reduced production of BCAA. Collectively, our findings reveal that the S. oneidensis FadR regulon is surely larger than previously proposed and a new mechanism by which FadR impacts bacterial physiology.


Asunto(s)
3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/metabolismo , Proteínas Bacterianas/metabolismo , Ácidos Grasos/biosíntesis , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas Represoras/metabolismo , Shewanella/fisiología , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/genética , Aminoácidos de Cadena Ramificada/biosíntesis , Proteínas Bacterianas/genética , Vías Biosintéticas/genética , Isoleucina/metabolismo , Mutación , Operón/genética , Regulón/fisiología , Proteínas Represoras/genética
16.
BMC Genomics ; 20(1): 942, 2019 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-31810444

RESUMEN

BACKGROUND: Mycobacterium smegmatis is a saprophytic bacterium frequently used as a genetic surrogate to study pathogenic Mycobacterium tuberculosis. The PrrAB two-component genetic regulatory system is essential in M. tuberculosis and represents an attractive therapeutic target. In this study, transcriptomic analysis (RNA-seq) of an M. smegmatis ΔprrAB mutant was used to define the PrrAB regulon and provide insights into the essential nature of PrrAB in M. tuberculosis. RESULTS: RNA-seq differential expression analysis of M. smegmatis wild-type (WT), ΔprrAB mutant, and complementation strains revealed that during in vitro exponential growth, PrrAB regulates 167 genes (q < 0.05), 57% of which are induced in the WT background. Gene ontology and cluster of orthologous groups analyses showed that PrrAB regulates genes participating in ion homeostasis, redox balance, metabolism, and energy production. PrrAB induced transcription of dosR (devR), a response regulator gene that promotes latent infection in M. tuberculosis and 21 of the 25 M. smegmatis DosRS regulon homologues. Compared to the WT and complementation strains, the ΔprrAB mutant exhibited an exaggerated delayed growth phenotype upon exposure to potassium cyanide and respiratory inhibition. Gene expression profiling correlated with these growth deficiency results, revealing that PrrAB induces transcription of the high-affinity cytochrome bd oxidase genes under both aerobic and hypoxic conditions. ATP synthesis was ~ 64% lower in the ΔprrAB mutant relative to the WT strain, further demonstrating that PrrAB regulates energy production. CONCLUSIONS: The M. smegmatis PrrAB two-component system regulates respiratory and oxidative phosphorylation pathways, potentially to provide tolerance against the dynamic environmental conditions experienced in its natural ecological niche. PrrAB positively regulates ATP levels during exponential growth, presumably through transcriptional activation of both terminal respiratory branches (cytochrome c bc1-aa3 and cytochrome bd oxidases), despite transcriptional repression of ATP synthase genes. Additionally, PrrAB positively regulates expression of the dormancy-associated dosR response regulator genes in an oxygen-independent manner, which may serve to fine-tune sensory perception of environmental stimuli associated with metabolic repression.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Mutación , Mycobacterium smegmatis/fisiología , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Prueba de Complementación Genética , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Cianuro de Potasio/farmacología , Regulón , Análisis de Secuencia de ARN/métodos
17.
PLoS Genet ; 15(10): e1008263, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31626625

RESUMEN

In Bacillus subtilis, the extracytoplasmic function σ factor σM regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. The anti-σ factors YhdL and YhdK form a complex that restricts the basal activity of σM, and the absence of YhdL leads to runaway expression of the σM regulon and cell death. Here, we report that this lethality can be suppressed by gain-of-function mutations in yidC1 (spoIIIJ), which encodes the major YidC membrane protein insertase in B. subtilis. B. subtilis PY79 YidC1 (SpoIIIJ) contains a single amino acid substitution in a functionally important hydrophilic groove (Q140K), and this allele suppresses the lethality of high σM. Analysis of a library of YidC1 variants reveals that increased charge (+2 or +3) in the hydrophilic groove can compensate for high expression of the σM regulon. Derepression of the σM regulon induces secretion stress, oxidative stress and DNA damage responses, all of which can be alleviated by the YidC1Q140K substitution. We further show that the fitness defect caused by high σM activity is exacerbated in the absence of the SecDF protein translocase or σM-dependent induction of the Spx oxidative stress regulon. Conversely, cell growth is improved by mutation of specific σM-dependent promoters controlling operons encoding integral membrane proteins. Collectively, these results reveal how the σM regulon has evolved to up-regulate membrane-localized complexes involved in cell wall synthesis, and to simultaneously counter the resulting stresses imposed by regulon induction.


Asunto(s)
Bacillus subtilis/genética , Integrasas/genética , Proteínas de Transporte de Membrana/genética , Factor sigma/genética , Bacillus subtilis/crecimiento & desarrollo , Membrana Celular/genética , Pared Celular/genética , Daño del ADN/genética , Regulación Bacteriana de la Expresión Génica , Mutación/genética , Operón/genética , Regiones Promotoras Genéticas , Regulón/genética
18.
Nat Commun ; 10(1): 4596, 2019 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-31601799

RESUMEN

Many of the regulatory features governing erythrocyte specification, maturation, and associated disorders remain enigmatic. To identify new regulators of erythropoiesis, we utilize a functional genomic screen for genes affecting expression of the erythroid marker CD235a/GYPA. Among validating hits are genes coding for the N6-methyladenosine (m6A) mRNA methyltransferase (MTase) complex, including, METTL14, METTL3, and WTAP. We demonstrate that m6A MTase activity promotes erythroid gene expression programs through selective translation of ~300 m6A marked mRNAs, including those coding for SETD histone methyltransferases, ribosomal components, and polyA RNA binding proteins. Remarkably, loss of m6A marks results in dramatic loss of H3K4me3 marks across key erythroid-specific KLF1 transcriptional targets (e.g., Heme biosynthesis genes). Further, each m6A MTase subunit and a subset of their mRNAs targets are required for human erythroid specification in primary bone-marrow derived progenitors. Thus, m6A mRNA marks promote the translation of a network of genes required for human erythropoiesis.


Asunto(s)
Adenosina/análogos & derivados , Eritropoyesis/genética , Biosíntesis de Proteínas , Adenosina/genética , Antígenos CD34/genética , Antígenos CD34/metabolismo , Células de la Médula Ósea/fisiología , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Regulación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Leucemia Eritroblástica Aguda/genética , Metiltransferasas/genética , Regiones Promotoras Genéticas , Factores de Empalme de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulón
19.
BMC Bioinformatics ; 20(1): 466, 2019 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-31500560

RESUMEN

BACKGROUND: Although many of the genic features in Mycobacterium abscessus have been fully validated, a comprehensive understanding of the regulatory elements remains lacking. Moreover, there is little understanding of how the organism regulates its transcriptomic profile, enabling cells to survive in hostile environments. Here, to computationally infer the gene regulatory network for Mycobacterium abscessus we propose a novel statistical computational modelling approach: BayesIan gene regulatory Networks inferreD via gene coExpression and compaRative genomics (BINDER). In tandem with derived experimental coexpression data, the property of genomic conservation is exploited to probabilistically infer a gene regulatory network in Mycobacterium abscessus.Inference on regulatory interactions is conducted by combining 'primary' and 'auxiliary' data strata. The data forming the primary and auxiliary strata are derived from RNA-seq experiments and sequence information in the primary organism Mycobacterium abscessus as well as ChIP-seq data extracted from a related proxy organism Mycobacterium tuberculosis. The primary and auxiliary data are combined in a hierarchical Bayesian framework, informing the apposite bivariate likelihood function and prior distributions respectively. The inferred relationships provide insight to regulon groupings in Mycobacterium abscessus. RESULTS: We implement BINDER on data relating to a collection of 167,280 regulator-target pairs resulting in the identification of 54 regulator-target pairs, across 5 transcription factors, for which there is strong probability of regulatory interaction. CONCLUSIONS: The inferred regulatory interactions provide insight to, and a valuable resource for further studies of, transcriptional control in Mycobacterium abscessus, and in the family of Mycobacteriaceae more generally. Further, the developed BINDER framework has broad applicability, useable in settings where computational inference of a gene regulatory network requires integration of data sources derived from both the primary organism of interest and from related proxy organisms.


Asunto(s)
Biología Computacional/métodos , Redes Reguladoras de Genes , Mycobacterium abscessus/genética , Programas Informáticos , Área Bajo la Curva , Bacterias/genética , Simulación por Computador , Regulación Bacteriana de la Expresión Génica , Curva ROC , Regulón/genética
20.
Int J Med Microbiol ; 309(8): 151353, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31521502

RESUMEN

Tuberculosis (TB) is the deadly infectious disease challenging the public health globally and its impact is further aggravated by co-infection with HIV and the emergence of drug resistant strains of Mycobacterium tuberculosis. In this study, we attempted to characterise the Rv2004c encoded protein, a member of DosR regulon, for its role in drug resistance. In silico docking analysis revealed that Rv2004c binds with streptomycin (SM). Phosphotransferase assay demonstrated that Rv2004c possibly mediates SM resistance through the aminoglycoside phosphotransferase activity. Further, E. coli expressing Rv2004c conferred resistance to 100µM of SM in liquid broth cultures indicating a mild aminoglycoside phosphotransferase activity of Rv2004c. Moreover, we investigated the role of MSMEG_3942 (an orthologous gene of Rv2004c) encoded protein in intracellular survival, its effect on in-vitro growth and its expression in different stress conditions by over expressing it in Mycobacterium smegmatis (M. smegmatis). MSMEG_3942 overexpressing recombinant M. smegmatis strains grew faster in acidic medium and also showed higher bacillary counts in infected macrophages when compared to M. smegmatis transformed with vector alone. Our results are likely to contribute to the better understanding of the involvement of Rv2004c in partial drug resistance, intracellular survival and adaptation of bacilli to stress conditions.


Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Macrófagos/microbiología , Mycobacterium smegmatis/efectos de los fármacos , Proteínas Quinasas/genética , Estreptomicina/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Humanos , Kanamicina Quinasa/metabolismo , Simulación del Acoplamiento Molecular , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Unión Proteica , Regulón , Células THP-1
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