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1.
World J Microbiol Biotechnol ; 35(8): 127, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375931

RESUMO

Aeromonas hydrophila is a Gram-negative bacterium that causes serious infections in aquaculture and exhibits significant multidrug resistance. The LysR-type transcriptional regulator (LTTR) family proteins are a well-known group of transcriptional regulators involved in diverse physiological functions. However, the role of LTTRs in the regulation of bacterial resistance to antibiotics is still largely unknown. In this study, to further investigate the role of four putative LTTR family proteins (A0KIU1, A0KJ82, A0KPK0, and A0KQ63) in antibiotic resistance in A. hydrophila, their genes were cloned and overexpressed in engineered Escherichia coli. After the optimization of experimental conditions including incubation time, temperature, and IPTG concentration, these proteins were successfully purified, and their specific antibodies against mice were obtained. Using western blot analysis, we found that these LTTR family proteins were downregulated in A. hydrophila following antibiotic treatment, indicating that they may be involved in the regulation of antibiotic resistance. Additionally, minimum inhibitory concentration (MIC) assays of chloramphenicol (CM), chlortetracycline (CTC), ciprofloxacin (CF), furazolidone (FZ), and balofloxacin (BF) in E. coli showed that overexpression of these LTTRs led to increased sensitivity to several antibiotics. To further validate their functional role in antibiotic resistance, we demonstrated that bacteria with loss of A0KQ63 (ΔAHA_3980) exhibited multi-drug resistance properties. Our results indicate that these LTTR family proteins may play an important role in the antibiotic resistance of A. hydrophila, and the that underlying mechanisms controlling antibiotic resistance should be further investigated.


Assuntos
Aeromonas hydrophila/efeitos dos fármacos , Aeromonas hydrophila/genética , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Fatores de Transcrição/metabolismo , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Western Blotting , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Expressão Gênica , Perfilação da Expressão Gênica , Genes Bacterianos , Camundongos , Testes de Sensibilidade Microbiana , Fatores de Transcrição/análise , Fatores de Transcrição/genética
2.
Microbiol Res ; 226: 48-54, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284944

RESUMO

The Burkholderia pseudomallei complex consists of six phylogenetically related Gram-negative bacterial species that include environmental saprophytes and mammalian pathogens. These microbes possess multiple type VI secretion systems (T6SS) that provide a fitness advantage in diverse niches by translocating effector molecules into prokaryotic and eukaryotic cells in a contact-dependent manner. Several recent studies have elucidated the regulation and function of T6SS-2, a novel contact-independent member of the T6SS family. Expression of the T6SS-2 gene cluster is repressed by OxyR, Zur and TctR and is activated by GvmR and reactive oxygen species (ROS). The last two genes of the T6SS-2 gene cluster encode a zincophore (TseZ) and a manganeseophore (TseM) that are exported into the extracellular milieu in a contact-independent fashion when microbes encounter oxidative stress. TseZ and TseM bind Zn2+ and Mn2+, respectively, and deliver them to bacteria where they provide protection against the lethal effects of ROS. The TonB-dependent transporters that interact with TseZ and TseM, and actively transport Zn2+ and Mn2+ across the outer membrane, have also been identified. Finally, T6SS-2 provides a contact-independent growth advantage in nutrient limited environments and is critical for virulence in Galleria mellonella larvae, but is dispensable for virulence in rodent models of infection.


Assuntos
Proteínas de Bactérias/genética , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Manganês/metabolismo , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Zinco/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Burkholderia pseudomallei/classificação , Regulação Bacteriana da Expressão Gênica , Genes Reguladores/genética , Homeostase , Larva , Proteínas de Membrana Transportadoras/genética , Metiltransferases , Família Multigênica , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Virulência/genética
3.
Gene ; 719: 144007, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31357024

RESUMO

Congenital bilateral absence of vas deferens (CBAVD), a frequent cause of obstructive azoospermia and male infertility in Chinese, is mainly due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This study aim to explore the promoter region of CFTR gene in CBAVD patients and study the mutations by functional analysis, and to discuss the significance of mutation testing in this area. We performed screening analysis on 65 CBAVD patients and 50 controls to detect mutations in the CFTR gene, and studied the functions of promoter mutations using reporter gene constructs, transient transfection techniques and subsequent assessment of transcriptional activity and expression levels. Mutations c.-195C>A and c.-34C>T in the promoter region of the CFTR gene were detected in 4 of our Chinese CBAVD patients, one of which was novel (c.-195C>A) and located in the conservative area, as well as the binding site of SP1 transcription factor through the prediction of bioinformatics analysis. By reverse transcription qPCR assay and luciferase assay, we validated it as a functional disease-causing variant that down-regulates the CFTR gene expression, and this effect was related to the amount of transcription factors. This study was the first to explore the promoter region of the CFTR gene in Chinese, and we believe that mutations in this region are associated with Chinese CBAVD patients. We also suggest a systematic strategy for genotyping Chinese CBAVD couples, which should help in developing reproductive counseling.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Doenças Urogenitais Masculinas/genética , Mutação , Regiões Promotoras Genéticas , Ducto Deferente/anormalidades , Adulto , Linhagem Celular , China , Regulação para Baixo , Genes Reguladores , Aconselhamento Genético , Humanos , Masculino , Reprodução , Adulto Jovem
4.
Appl Microbiol Biotechnol ; 103(12): 4917-4929, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31073877

RESUMO

Undesirable flavor caused by excessive higher alcohols restrains the development of the wheat beer industry. To clarify the regulation mechanism of the metabolism of higher alcohols in wheat beer brewing by the top-fermenting yeast Saccharomyces cerevisiae S17, the effect of temperature on the fermentation performance and transcriptional levels of relevant genes was investigated. The strain S17 produced 297.85 mg/L of higher alcohols at 20 °C, and the production did not increase at 25 °C, reaching about 297.43 mg/L. Metabolite analysis and transcriptome sequencing showed that the metabolic pathways of branched-chain amino acids, pyruvate, phenylalanine, and proline were the decisive factors that affected the formation of higher alcohols. Fourteen most promising genes were selected to evaluate the effects of single-gene deletions on the synthesis of higher alcohols. The total production of higher alcohols by the mutants Δtir1 and Δgap1 was reduced by 23.5 and 19.66% compared with the parent strain S17, respectively. The results confirmed that TIR1 and GAP1 are crucial regulatory genes in the metabolism of higher alcohols in the top-fermenting yeast. This study provides valuable knowledge on the metabolic pathways of higher alcohols and new strategies for reducing the amounts of higher alcohols in wheat beer.


Assuntos
Álcoois/metabolismo , Cerveja/microbiologia , Fermentação , Genes Reguladores , Saccharomyces cerevisiae/genética , Temperatura Ambiente , Reatores Biológicos , Aromatizantes , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Redes e Vias Metabólicas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Paladar
5.
Microb Pathog ; 132: 201-207, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31077753

RESUMO

Subclinical necrotic enteritis (SNE) broadly occurs in boilers, which reduces the growth performance by causing serious economic and social problems. The following study was conducted to better understand the molecular mechanism of the SNE on liver inflammation and to examine the innovative prevention of Lactobacillus johnsonii BS15 upon SNE. The research was based on the regulatory molecular mechanism of Lactobacillus johnsonii BS15, and its effect on liver inflammatory pathways in the broiler with SNE infection. Day old one hundred and eighty (Cobb 500) broiler chickens were distributed into 3 groups (control, SNE and BS15 group) and reared for 28 days. RNA sequencing was used for the analysis of gene expression extracted from liver samples. Gene expression was detected with the help of quantitative real-time PCR (qRT-PCR). RNA-Seq analysis revealed altered expressions of genes involved in liver inflammatory pathway. A total number of 385 genes were found as differentially expressed (DEGs) in the liver samples that belonged to SNE group as compared with the control liver samples (p < 0.05). Out of those 385 genes, 117 were down-regulated and 268 were up-regulated. The DEGs related to liver inflammation between control group and SNE group or SNE and BS15 groups, included cluster of differentiation 80 (CD80), Interleukin 1 beta (IL1B), Phosphoinositide 3- Kinase regulatory subunit 5 (PIK3R5), Toll-like receptor 4 (TLR4), Toll-like receptor 2 A (TLR2A), and proto-oncogene protein (FOS). The RNA-Seq analysis provided DEGs expression and this result was validated by qRT-PCR. Results confirmed that these genes are essential in the regulation of liver inflammation in the SNE infected chickens. Findings of current research indicated that the hepatic inflammation could be induced by SNE in broilers. Simultaneously, effects of SNE infection on liver could be subsided by improved TLRs signaling pathway with the naturally present prophylactic strategy as BS15.


Assuntos
Enterite/metabolismo , Perfilação da Expressão Gênica/métodos , Inflamação/genética , Lactobacillus johnsonii/fisiologia , Fígado/metabolismo , Probióticos/farmacologia , Animais , Antígeno B7-1 , Galinhas , Clostridium perfringens , Regulação para Baixo , Enterite/prevenção & controle , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reguladores , Inflamação/tratamento farmacológico , Interleucina-1beta , Fígado/efeitos dos fármacos , Fígado/patologia , Doenças das Aves Domésticas/prevenção & controle , Análise de Sequência de RNA , Transdução de Sinais , Transcriptoma , Regulação para Cima
6.
Nat Commun ; 10(1): 2236, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110181

RESUMO

Genome-wide association studies (GWAS) have identified more than 50,000 unique associations with common human traits. While this represents a substantial step forward, establishing the biology underlying these associations has proven extremely difficult. Even determining which cell types and which particular gene(s) are relevant continues to be a challenge. Here, we conduct a cell-specific pathway analysis of the latest GWAS in multiple sclerosis (MS), which had analyzed a total of 47,351 cases and 68,284 healthy controls and found more than 200 non-MHC genome-wide associations. Our analysis identifies pan immune cell as well as cell-specific susceptibility genes in T cells, B cells and monocytes. Finally, genotype-level data from 2,370 patients and 412 controls is used to compute intra-individual and cell-specific susceptibility pathways that offer a biological interpretation of the individual genetic risk to MS. This approach could be adopted in any other complex trait for which genome-wide data is available.


Assuntos
Regulação da Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Esclerose Múltipla/genética , Biologia de Sistemas/métodos , Genes Reguladores/genética , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único
7.
PLoS Genet ; 15(4): e1008068, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30969965

RESUMO

The roles of histone demethylation in the regulation of plant flowering, disease resistance, rhythmical response, and seed germination have been elucidated recently; however, how histone demethylation affects leaf senescence remains largely unclear. In this study, we exploited yeast one-hybrid (Y1H) to screen for the upstream regulators of NONYELLOWING1 (NYE1), and identified RELATIVE OF EARLY FLOWERING6 (REF6), a histone H3 lysine 27 tri-methylation (H3K27me3) demethylase, as a putative binding protein of NYE1 promoter. By in vivo and in vitro analyses, we demonstrated that REF6 directly binds to the motif CTCGYTY in NYE1/2 promoters through its zinc finger domain and positively regulates their expression. Loss-of-function of REF6 delayed chlorophyll (Chl) degradation, whereas overexpression of REF6 accelerated Chl degradation. Subsequently, we revealed that REF6 positively regulates the general senescence process by directly up-regulating ETHYLENE INSENSITIVE 2 (EIN2), ORESARA1 (ORE1), NAC-LIKE, ACTIVATED BY AP3/PI (NAP), PYRUVATE ORTHOPHOSPHATE DIKINASE (PPDK), PHYTOALEXIN DEFICIENT 4 (PAD4), LIPOXYGENASE 1 (LOX1), NAC DOMAIN CONTAINING PROTEIN 3 (AtNAC3), and NAC TRANSCRIPTION FACTOR-LIKE 9 (NTL9), the key regulatory and functional genes predominantly involved in the regulation of developmental leaf senescence. Importantly, loss-of-function of REF6 increased H3K27me3 levels at all the target Senescence associated genes (SAGs). We therefore conclusively demonstrate that H3K27me3 methylation represents an epigenetic mechanism prohibiting the premature transcriptional activation of key developmentally up-regulated senescence regulatory as well as functional genes in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Arabidopsis/crescimento & desenvolvimento , Sítios de Ligação/genética , Clorofila/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genes Reguladores , Modelos Genéticos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas
8.
Appl Microbiol Biotechnol ; 103(10): 4089-4102, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30937499

RESUMO

Teicoplanin is a frontline glycopeptide antibiotic produced by Actinoplanes teichomyceticus. It is used to treat complicated cases of infection, including pediatric ones, caused by Gram-positive pathogens. There is a steady interest in elucidating the genetic mechanisms determining teicoplanin production, as they would help overproduce known teicoplanins and discover novel glycopeptides. Herein, we investigate the transcriptional organization of the tei biosynthetic gene cluster and the roles of the cluster-situated regulatory genes in controlling teicoplanin production and self-resistance in A. teichomyceticus. We demonstrate that the tei cluster is organized into nine polygenic and nine monogenic transcriptional units. Most of tei biosynthetic genes are subjected to StrR-like Tei15* control, which, in turn, appears to be regulated by LuxR-type Tei16*. Expression of the genes conferring teicoplanin self-resistance in A. teichomyceticus is not co-regulated with antibiotic production. The gene tei31*, coding for a putative DNA binding protein, is not expressed under teicoplanin producing conditions and is dispensable for antibiotic production. Finally, phylogenesis reconstruction of the glycopeptide cluster-encoded regulators reveals two main clades of StrR-like regulators. Tei15* and close orthologues form one of these clades; the second clade is composed by orthologues of Bbr and Dbv4, governing the biosynthesis of balhimycin and teicoplanin-like A40926, respectively. In addition, the LuxR-type Tei16* appears unrelated to the LuxR-like Dbv3, which is controlling A40926 biosynthesis. Our results shed new light on teicoplanin biosynthesis regulation and on the evolution of novel and old glycopeptide biosynthetic gene clusters.


Assuntos
Antibacterianos/biossíntese , Vias Biossintéticas/genética , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Teicoplanina/biossíntese , Farmacorresistência Bacteriana , Perfilação da Expressão Gênica , Ordem dos Genes , Óperon
9.
Appl Microbiol Biotechnol ; 103(11): 4511-4523, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30982107

RESUMO

XYR1 is the key transcription activator for cellulase gene expression in the model filamentous fungus Trichoderma reesei, which is widely applied in the industry due to its excellent capability of secreting a large quantity of cellulases. Despite the essential role of XYR1, the regulation of its expression in T. reesei cellulolytic response is poorly understood. In this study, we identified a transcription factor RXE1 exhibiting strong binding activity to the xyr1 promoter using yeast one-hybrid screen. RXE1 homologs exist in quite a few filamentous fungi but none of them have been assessed regarding their functional involvement in plant cell wall degradation. Knockdown of rxe1 in T. reesei using a copper-mediated RNAi system not only abrogated conidiation, but also remarkably compromised xyr1 and cellulase gene expression. The defective cellulase but not conidia production in the rxe1-knockdown strain was fully rescued by the constitutive expression of XYR1. Our study thus identified a novel transcriptional regulator controlling xyr1 and cellulase gene expression, which will contribute to elaborating the intricate network of cellulase gene regulation in T. reesei.


Assuntos
Celulase/biossíntese , Regulação Fúngica da Expressão Gênica , Genes Reguladores , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Trichoderma/genética , Trichoderma/metabolismo , Celulase/genética , DNA Fúngico/metabolismo , Técnicas de Silenciamento de Genes , Testes Genéticos , Regiões Promotoras Genéticas , Ligação Proteica
10.
Toxicon ; 161: 50-56, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30849453

RESUMO

Sterigmatocystin (STC) is structurally close to the mycotoxin aflatoxin B1 as it shares its biosynthetic pathway with aflatoxins. The purpose of the present study was to investigate the short-term (24 h) effects of STC contaminated diet at different doses (1 mg, 2 mg and 4 mg STC kg-1 feed) in one year old common carp juveniles. Liver samples were taken in 8-h intervals. The markers of the lipid peroxidation showed moderate changes after the application of sterigmatocystin-contaminated diet, significant elevations were only observed in the lowest applied dose group of sterigmatocystin after 16 h of exposure. Reduced glutathione content showed higher levels than control group after 16 h of exposure as effect of low dose of sterigmatocystin. Glutathione peroxidase (GPX4) activity was lower than control in the group treated with 2 mg STC kg-1 feed after 24 h of exposure. Gene expression measurements of keap1, nrf2, gpx4a, gpx4b and gss genes revealed a dual response. Down-regulation or near control values were observed 8 h after exposure which was followed by an induction 16 and 24 h after exposure. In case of gsr, gene expression values returned to control levels by the 24th hour. In summary, these results suggest that lower doses of STC caused oxidative stress earlier than higher doses, which efficiently activated the Keap1-Nrf2 pathway, while higher doses revealed long-drawn activation of this pathway.


Assuntos
Carpas/genética , Carpas/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Esterigmatocistina/farmacologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reguladores/efeitos dos fármacos , Glutationa Peroxidase/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , RNA/genética , RNA/metabolismo
11.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 33(3): 349-355, 2019 Mar 15.
Artigo em Chinês | MEDLINE | ID: mdl-30874395

RESUMO

Objective: To investigate the effect of accessory gene regulator C (agr C) specific binding peptides (named N1) on the biofilm formation of Staphylococcus epidermidis on the surface of polyvinyl chloride (PVC) materials in vitro. Methods: Firstly, the two strains (ATCC35984, ATCC12228) were cultured with N1 at concentrations of 100, 200, 400, 800, and 1 600 µg/mL, respectively. The control group was cultured with agrC specific binding unrelated peptides (named N0) at the same concentrations and the absorbance ( A) value was measured after 24 hours to determine the optimal bacteriostatic concentration of N1. The two strains were cultured with N1 and N0 of the optimal concentration, respectively. The A values were measured at 6, 12, 18, 24, 30, and 48 hours to observe the effect of N1 on the biofilm formation ability of Staphylococcus epidermidis. On this basis, the surface structure of the biofilm on the surface of PVC material was observed by scanning electron microscopy after 6, 12, 18, 24, and 30 hours of incubation with PVC material sheet. The thickness of the biofilm was observed by laser confocal microscopy after 6, 12, 18, and 24 hours of incubation with ATCC35984 strain. Results: The optimal bacteriostatic concentration of N1 was 800 µg/mL. ATCC 12228 strain did not form obvious biofilm after being cultured with N1 and N0. When ATCC35984 strain was cultured with N1 and N0 for 12 hours, the difference in biofilm formation ability between groups N1 and N0 was statistically significant ( P<0.05), but there was no significant difference at 6, 18, 24, 30, and 48 hours ( P>0.05). Scanning electron microscopy examination showed that mature biofilm structure was observed in ATCC35984 strain and was not observed in ATCC12228 strain. Laser confocal microscopy observation showed that the number of bacteria in the group N1 was significantly lower than that in the group N0 at 12 hours, and the most of bacteria were dead bacteria. There was no significant difference in the number of bacteria at 6, 18, and 24 hours, and the most of them were live bacteria. The biofilm thickness of group N1 was significantly lower than that of group N0 at 12 and 18 hours ( P<0.05). Conclusion: The intensity of N1 inhibiting the formation of Staphylococcus epidermidis biofilm is dose-dependent. During the aggregation period, N1 can inhibit the biofilm formation by hindering the bacterial growth and aggregation. The inhibition effect on mature biofilm is not obvious.


Assuntos
Biofilmes , Peptídeos , Cloreto de Polivinila , Staphylococcus epidermidis , Genes Bacterianos , Genes Reguladores , Microscopia Eletrônica de Varredura , Peptídeos/farmacologia , Staphylococcus epidermidis/crescimento & desenvolvimento
12.
J Ind Microbiol Biotechnol ; 46(5): 649-655, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30798437

RESUMO

Pseudonocardia autotrophica was previously identified to produce a toxicity-reduced and solubility-improved disaccharide-containing anti-fungal compound belonging to the tetraene-family, Nystatin-like Pseudonocardia Polyene A1 (NPP A1). Subsequently NPP B1, a novel derivative harboring a heptaene core structure, was produced by a pathway-engineered Pseudonocardia strain through inactivation of the specific enoly reductase gene domain in the NPP biosynthetic gene cluster. Although in vitro and in vivo efficacy and toxicity studies indicate that NPP B1 is a promising lead antifungal compound, further improvement is required to increase the extremely low production yield in the pathway-engineered strain. To overcome this challenge, we performed the N-methyl-N'-nitro-N-nitrosoguanidine (NTG) iterative random mutagenesis, followed by zone-of-inhibition agar plug assay. After three rounds of the mutagenesis-and-screening protocol, the production yield of NPP B1 increased to 6.25 mg/L, which is more than an eightfold increase compared to the parental strain. The qRT-PCR analysis revealed that transcripts of the NPP B1 biosynthetic genes were increased in the mutant strain. Interestingly, an endogenous 125-kb plasmid was found to be eliminated through this mutagenesis. To further improve the NPP B1 production yield, the 32-kb NPP-specific regulatory gene cluster was cloned and overexpressed in the mutant strain. The chromosomal integration of the extra copy of the six NPP-specific regulatory genes led to an additional increase of NPP B1 yield to 31.6 mg/L, which is the highest production level of NPP B1 ever achieved by P. autotrophica strains. These results suggest that a synergistic combination of both the traditional and genetic strain improvement approaches is a very efficient strategy to stimulate the production of an extremely low-level metabolite (such as NPP B1) in a pathway-engineered rare actinomycetes strain.


Assuntos
Actinobacteria/metabolismo , Nistatina/biossíntese , Polienos/metabolismo , Actinobacteria/genética , Actinomycetales/genética , Antifúngicos/química , Dissacarídeos/metabolismo , Genes Reguladores , Microbiologia Industrial , Família Multigênica , Mutagênese , Plasmídeos/metabolismo , Engenharia de Proteínas , Açúcares
13.
PLoS Genet ; 15(2): e1007784, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30759082

RESUMO

Endogenous small RNAs (sRNAs) and Argonaute proteins are ubiquitous regulators of gene expression in germline and somatic tissues. sRNA-Argonaute complexes are often expressed in gametes and are consequently inherited by the next generation upon fertilization. In Caenorhabditis elegans, 26G-RNAs are primary endogenous sRNAs that trigger the expression of downstream secondary sRNAs. Two subpopulations of 26G-RNAs exist, each of which displaying strongly compartmentalized expression: one is expressed in the spermatogenic gonad and associates with the Argonautes ALG-3/4; plus another expressed in oocytes and in embryos, which associates with the Argonaute ERGO-1. The determinants and dynamics of gene silencing elicited by 26G-RNAs are largely unknown. Here, we provide diverse new insights into these endogenous sRNA pathways of C. elegans. Using genetics and deep sequencing, we dissect a maternal effect of the ERGO-1 branch of the 26G-RNA pathway. We find that maternal primary sRNAs can trigger the production of zygotic secondary sRNAs that are able to silence targets, even in the absence of zygotic primary triggers. Thus, the interaction of maternal and zygotic sRNA populations, assures target gene silencing throughout animal development. Furthermore, we explore other facets of 26G-RNA biology related to the ALG-3/4 branch. We find that sRNA abundance, sRNA pattern of origin and the 3' UTR length of target transcripts are predictors of the regulatory outcome by the Argonautes ALG-3/4. Lastly, we provide evidence suggesting that ALG-3 and ALG-4 regulate their own mRNAs in a negative feedback loop. Altogether, we provide several new regulatory insights on the dynamics, target regulation and self-regulation of the endogenous RNAi pathways of C. elegans.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Reguladores/genética , Interferência de RNA/fisiologia , Zigoto/fisiologia , Regiões 3' não Traduzidas/genética , Animais , Proteínas Argonauta/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Inativação Gênica/fisiologia , Células Germinativas/fisiologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética
14.
Database (Oxford) ; 20192019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30806704

RESUMO

Super-enhancers (SEs) are enriched with a cluster of mediator binding sites, which are major contributors to cell-type-specific gene expression. Currently, a large quantity of long non-coding RNAs has been found to be transcribed from or to interact with SEs, which constitute super-enhancer associated long non-coding RNAs (SE-lncRNAs). These SE-lncRNAs play essential roles in transcriptional regulation through controlling SEs activity to regulate a broad range of physiological and pathological processes, especially tumorigenesis. However, the pathological functions of SE-lncRNAs in tumorigenesis are still obscure. In this paper, we characterized 5056 SE-lncRNAs and their associated genes by analysing 102 SE data sets. Then, we analysed their expression profiles and prognostic information derived from 19 cancer types to identify cancer-related SE-lncRNAs and to explore their potential functions. In total, 436 significantly differentially expressed SE-lncRNAs and 2035 SE-lncRNAs with high prognostic values were identified. Additionally, 3935 significant correlations between SE-lncRNAs and their regulatory genes were further validated by calculating their correlation coefficients in each cancer type. Finally, the SELER database incorporating the aforementioned data was provided for users to explore their physiological and pathological functions to comprehensively understand the blocks of living systems.


Assuntos
Bases de Dados Genéticas , Elementos Facilitadores Genéticos , Neoplasias/genética , RNA Longo não Codificante/genética , Transcrição Genética , Regulação Neoplásica da Expressão Gênica , Genes Reguladores , Humanos
15.
Int J Med Microbiol ; 309(2): 85-96, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30606691

RESUMO

The two-component system response regulator ArlR and the global regulator MgrA in Staphylococcus aureus participated in numerous biological processes including biofilm formation inhibition. Previous studies have shown that these two regulators could function as a regulatory cascade. Rbf is a positive regulator of biofilm formation enhancing the production of PIA (polysaccharide intercellular adhesin). Here we have demonstrated that both ArlR and MgrA can directly bind to the promoter of rbf and repress its expression. ArlR and MgrA can also directly bind to the promoter of ica operon and enhance the expression of icaA and PIA production, revealing that the ArlR-MgrA regulatory cascade controls PIA-dependent biofilm formation. In addition, we have found that Rbf can directly bind to the aur promoter and repress the expression of aur, which encodes a protease initiating a protease cascade to inhibit protein-mediated biofilm formation. Moreover, our data indicate that the ArlR-MgrA regulatory cascade can promote the expression of aur by directly binding to its promoter and inhibit protein-mediated biofilm formation. These findings shed light on the molecular mechanisms of both PIA-dependent and protein-mediated biofilm formation modulated by the ArlR-MgrA regulatory cascade and the new role of Rbf in protein-mediated biofilm formation, and broaden our understanding of the biofilm formation regulation in S. aureus.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Polissacarídeos Bacterianos/metabolismo , Staphylococcus aureus/crescimento & desenvolvimento , DNA Bacteriano/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Staphylococcus aureus/genética
16.
J Med Microbiol ; 68(2): 263-278, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30628877

RESUMO

PURPOSE: Burkholderia pseudomallei, the tier 1 agent of melioidosis, is a saprophytic microbe that causes endemic infections in tropical regions such as South-East Asia and Northern Australia. It is globally distributed, challenging to diagnose and treat, infectious by several routes including inhalation, and has potential for adversarial use. B. pseudomallei strain MSHR5848 produces two colony variants, smooth (S) and rough (R), which exhibit a divergent range of morphological, biochemical and metabolic phenotypes, and differ in macrophage and animal infectivity. We aimed to characterize two major phenotypic differences, analyse gene expression and study the regulatory basis of the variation. METHODOLOGY: Phenotypic expression was characterized by DNA and RNA sequencing, microscopy, and differential bacteriology. Regulatory genes were identified by cloning and bioinformatics.Results/Key findings. Whereas S produced larger quantities of extracellular DNA, R was upregulated in the production of a unique chromosome 1-encoded Siphoviridae-like bacteriophage, φMSHR5848. Exploratory transcriptional analyses revealed significant differences in variant expression of genes encoding siderophores, pili assembly, type VI secretion system cluster 4 (T6SS-4) proteins, several exopolysaccharides and secondary metabolites. A single 3 base duplication in S was the only difference that separated the variants genetically. It occurred upstream of a cluster of bacteriophage-associated genes on chromosome 2 that were upregulated in S. The first two genes were involved in regulating expression of the multiple phenotypes distinguishing S and R. CONCLUSION: Bacteriophage-associated proteins have a major role in the phenotypic expression of MSHR5848. The goals are to determine the regulatory basis of this phenotypic variation and its role in pathogenesis and environmental persistence of B. pseudomallei.


Assuntos
Bacteriófagos/genética , Burkholderia pseudomallei/genética , Melioidose/microbiologia , Bacteriófagos/isolamento & purificação , Bacteriófagos/ultraestrutura , Burkholderia pseudomallei/classificação , Burkholderia pseudomallei/virologia , Clonagem Molecular , Biologia Computacional , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Viral/análise , DNA Viral/química , DNA Viral/isolamento & purificação , Duplicação Gênica/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Humanos , Microscopia Eletrônica , Família Multigênica , Myoviridae/genética , Myoviridae/isolamento & purificação , Myoviridae/ultraestrutura , Fenótipo , RNA Bacteriano/análise , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificação , Análise de Sequência de DNA , Análise de Sequência de RNA
17.
Medicina (Kaunas) ; 55(1)2019 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-30658502

RESUMO

Colorectal cancer (CRC) is the second most common cause of cancer-related death in the world, but early diagnosis ameliorates the survival of CRC. This report aimed to identify molecular biomarker signatures in CRC. We analyzed two microarray datasets (GSE35279 and GSE21815) from the Gene Expression Omnibus (GEO) to identify mutual differentially expressed genes (DEGs). We integrated DEGs with protein⁻protein interaction and transcriptional/post-transcriptional regulatory networks to identify reporter signaling and regulatory molecules; utilized functional overrepresentation and pathway enrichment analyses to elucidate their roles in biological processes and molecular pathways; performed survival analyses to evaluate their prognostic performance; and applied drug repositioning analyses through Connectivity Map (CMap) and geneXpharma tools to hypothesize possible drug candidates targeting reporter molecules. A total of 727 upregulated and 99 downregulated DEGs were detected. The PI3K/Akt signaling, Wnt signaling, extracellular matrix (ECM) interaction, and cell cycle were identified as significantly enriched pathways. Ten hub proteins (ADNP, CCND1, CD44, CDK4, CEBPB, CENPA, CENPH, CENPN, MYC, and RFC2), 10 transcription factors (ETS1, ESR1, GATA1, GATA2, GATA3, AR, YBX1, FOXP3, E2F4, and PRDM14) and two microRNAs (miRNAs) (miR-193b-3p and miR-615-3p) were detected as reporter molecules. The survival analyses through Kaplan⁻Meier curves indicated remarkable performance of reporter molecules in the estimation of survival probability in CRC patients. In addition, several drug candidates including anti-neoplastic and immunomodulating agents were repositioned. This study presents biomarker signatures at protein and RNA levels with prognostic capability in CRC. We think that the molecular signatures and candidate drugs presented in this study might be useful in future studies indenting the development of accurate diagnostic and/or prognostic biomarker screens and efficient therapeutic strategies in CRC.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/tratamento farmacológico , Proteína Semelhante a ELAV 2/genética , Genes Reguladores/genética , Genes Reporter/genética , MicroRNAs/genética , Terapia de Alvo Molecular , Fatores de Transcrição/genética , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Bases de Dados Genéticas , Diagnóstico Precoce , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores Imunológicos/uso terapêutico , Estimativa de Kaplan-Meier , Prognóstico , Transdução de Sinais , Análise de Sobrevida , Biologia de Sistemas/métodos
18.
Mem. Inst. Oswaldo Cruz ; 114: e190105, 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1012671

RESUMO

BACKGROUND Healthcare-associated infections caused by bacteria such as Pseudomonas aeruginosa are a major public health problem worldwide. Gene regulatory networks (GRN) computationally represent interactions among regulatory genes and their targets. They are an important approach to help understand bacterial behaviour and to provide novel ways of overcoming scientific challenges, including the identification of potential therapeutic targets and the development of new drugs. OBJECTIVES The goal of this study was to reconstruct the multidrug-resistant (MDR) P. aeruginosa GRN and to analyse its topological properties. METHODS The methodology used in this study was based on gene orthology inference using the reciprocal best hit method. We used the genome of P. aeruginosa CCBH4851 as the basis of the reconstruction process. This MDR strain is representative of the sequence type 277, which was involved in an endemic outbreak in Brazil. FINDINGS We obtained a network with a larger number of regulatory genes, target genes and interactions as compared to the previously reported network. Topological analysis results are in accordance with the complex network representation of biological processes. MAIN CONCLUSIONS The properties of the network were consistent with the biological features of P. aeruginosa. To the best of our knowledge, the P. aeruginosa GRN presented here is the most complete version available to date.


Assuntos
Humanos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Infecções por Pseudomonas/imunologia , Genes Reguladores/imunologia , Brasil/epidemiologia , Genes MDR/genética
19.
PLoS One ; 13(12): e0209611, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30589856

RESUMO

Placental dysfunction is implicated in many pregnancy complications, including preeclampsia and preterm birth (PTB). While both these syndromes are influenced by environmental risk factors, they also have a substantial genetic component that is not well understood. Precisely controlled gene expression during development is crucial to proper placental function and often mediated through gene regulatory enhancers. However, we lack accurate maps of placental enhancer activity due to the challenges of assaying the placenta and the difficulty of comprehensively identifying enhancers. To address the gap in our knowledge of gene regulatory elements in the placenta, we used a two-step machine learning pipeline to synthesize existing functional genomics studies, transcription factor (TF) binding patterns, and evolutionary information to predict placental enhancers. The trained classifiers accurately distinguish enhancers from the genomic background and placental enhancers from enhancers active in other tissues. Genomic features collected from tissues and cell lines involved in pregnancy are the most predictive of placental regulatory activity. Applying the classifiers genome-wide enabled us to create a map of 33,010 predicted placental enhancers, including 4,562 high-confidence enhancer predictions. The genome-wide placental enhancers are significantly enriched nearby genes associated with placental development and birth disorders and for SNPs associated with gestational age. These genome-wide predicted placental enhancers provide candidate regions for further testing in vitro, will assist in guiding future studies of genetic associations with pregnancy phenotypes, and aid interpretation of potential mechanisms of action for variants found through genetic studies.


Assuntos
Elementos Facilitadores Genéticos , Genes Reguladores , Estudo de Associação Genômica Ampla , Genômica , Placenta/metabolismo , Mapeamento Cromossômico , Biologia Computacional/métodos , Feminino , Genômica/métodos , Humanos , Aprendizado de Máquina , Anotação de Sequência Molecular , Gravidez , Curva ROC
20.
MBio ; 9(6)2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30563896

RESUMO

Human infection with Cryptococcus causes up to a quarter of a million AIDS-related deaths annually and is the most common cause of nonviral meningitis in the United States. As an opportunistic fungal pathogen, Cryptococcus neoformans is distinguished by its ability to adapt to diverse host environments, including plants, amoebae, and mammals. In the present study, comparative transcriptomics of the fungus within human cerebrospinal fluid identified expression profiles representative of low-nutrient adaptive responses. Transcriptomics of fungal isolates from a cohort of HIV/AIDS patients identified high expression levels of an alternative carbon nutrient transporter gene, STL1, to be associated with poor early fungicidal activity, an important clinical prognostic marker. Mouse modeling and pathway analysis demonstrated a role for STL1 in mammalian pathogenesis and revealed that STL1 expression is regulated by a novel multigene regulatory mechanism involving the CAC2 subunit of the chromatin assembly complex 1, CAF-1. In this pathway, the global regulator of virulence gene VAD1 was found to transcriptionally regulate a cryptococcal homolog of a cytosolic protein, Ecm15, in turn required for nuclear transport of the Cac2 protein. Derepression of STL1 by the CAC2-containing CAF-1 complex was mediated by Cac2 and modulated binding and suppression of the STL1 enhancer element. Derepression of STL1 resulted in enhanced survival and growth of the fungus in the presence of low-nutrient, alternative carbon sources, facilitating virulence in mice. This study underscores the utility of ex vivo expression profiling of fungal clinical isolates and provides fundamental genetic understanding of saprophyte adaption to the human host.IMPORTANCE Cryptococcus is a fungal pathogen that kills an estimated quarter of a million individuals yearly and is the most common cause of nonviral meningitis in the United States. The fungus is carried in about 10% of the adult population and, after reactivation, causes disease in a wide variety of immunosuppressed individuals, including the HIV infected and patients receiving transplant conditioning, cancer therapy, or corticosteroid therapy for autoimmune diseases. The fungus is widely carried in the soil but can also cause infections in plants and mammals. However, the mechanisms for this widespread ability to infect a variety of hosts are poorly understood. The present study identified adaptation to low nutrients as a key property that allows the fungus to inhabit these diverse environments. Further studies identified a nutrient transporter gene, STL1, to be upregulated under low nutrients and to be associated with early fungicidal activity, a marker of poor clinical outcome in a cohort of HIV/AIDS patients. Understanding molecular mechanisms involved in adaptation to the human host may help to design better methods of control and treatment of widely dispersed fungal pathogens such as Cryptococcus.


Assuntos
Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Redes e Vias Metabólicas/genética , Serina-Treonina Quinases TOR/genética , Fatores de Virulência/genética , Adulto , Animais , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Genes Reguladores , Infecções por HIV/microbiologia , Interações Hospedeiro-Patógeno/genética , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Meningite Criptocócica/líquido cefalorraquidiano , Camundongos , Camundongos Endogâmicos CBA , Pessoa de Meia-Idade , Ensaios Clínicos Controlados Aleatórios como Assunto , Virulência , Adulto Jovem
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