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1.
BMC Genomics ; 20(1): 807, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694530

RESUMO

BACKGROUND: Xanthomonas citri pv. citri (Xcc) is a citrus canker causing Gram-negative bacteria. Currently, little is known about the biological and molecular responses of Xcc to low temperatures. RESULTS: Results depicted that low temperature significantly reduced growth and increased biofilm formation and unsaturated fatty acid (UFA) ratio in Xcc. At low temperature Xcc formed branching structured motility. Global transcriptome analysis revealed that low temperature modulates multiple signaling networks and essential cellular processes such as carbon, nitrogen and fatty acid metabolism in Xcc. Differential expression of genes associated with type IV pilus system and pathogenesis are important cellular adaptive responses of Xcc to cold stress. CONCLUSIONS: Study provides clear insights into biological characteristics and genome-wide transcriptional analysis based molecular mechanism of Xcc in response to low temperature.

2.
Proc Natl Acad Sci U S A ; 116(44): 22331-22340, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31604827

RESUMO

It is highly intriguing how bacterial pathogens can quickly shut down energy-costly infection machinery once successful infection is established. This study depicts that mutation of repressor SghR increases the expression of hydrolase SghA in Agrobacterium tumefaciens, which releases plant defense signal salicylic acid (SA) from its storage form SA ß-glucoside (SAG). Addition of SA substantially reduces gene expression of bacterial virulence. Bacterial vir genes and sghA are differentially transcribed at early and later infection stages, respectively. Plant metabolite sucrose is a signal ligand that inactivates SghR and consequently induces sghA expression. Disruption of sghA leads to increased vir expression in planta and enhances tumor formation whereas mutation of sghR decreases vir expression and tumor formation. These results depict a remarkable mechanism by which A. tumefaciens taps on the reserved pool of plant signal SA to reprogram its virulence upon establishment of infection.

3.
mSphere ; 4(3)2019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142621

RESUMO

Sporisorium scitamineum is the fungal pathogen causing severe sugarcane smut disease that leads to massive economic losses globally. S. scitamineum invades host cane by dikaryotic hyphae, formed after sexual mating of two haploid sporidia of opposite mating type. Therefore, mating/filamentation is critical for S. scitamineum pathogenicity, while its molecular mechanisms remain largely unknown. The AGC (cyclic AMP [cAMP]-dependent protein kinase 1 [protein kinase A {PKA}], cGMP-dependent protein kinase [PKG], and protein kinase C [PKC]) kinase family is a group of serine/threonine (Ser/Thr) protein kinases conserved among eukaryotic genomes, serving a variety of physiological functions, including cell growth, metabolism, differentiation, and cell death. In this study, we identified an AGC kinase, named SsAgc1 (for S. scitamineum Agc1), and characterized its function by reverse genetics. Our results showed that SsAgc1 is critical for S. scitamineum mating/filamentation and pathogenicity, and oxidative stress tolerance under some circumstances. Transcriptional profiling revealed that the SsAgc1 signaling pathway may control expression of the genes governing fungal mating/filamentation and tryptophan metabolism, especially for tryptophol production. We showed that tryptophan and tryptophol could at least partially restore ssagc1Δ mating/filamentation. Overall, our work revealed a signaling pathway mediated by AGC protein kinases to regulate fungal mating/filamentation, possibly through sensing and responding to tryptophol as signal molecules.IMPORTANCE The AGC signaling pathway represents a conserved distinct signaling pathway in regulation of fungal differentiation and virulence, while it has not been identified or characterized in the sugarcane smut fungus Sporisorium scitamineum In this study, we identified a PAS domain-containing AGC kinase, SsAgc1, in S. scitamineum Functional analysis revealed that SsAgc1 plays a regulatory role on the fungal dimorphic switch.

4.
Genes (Basel) ; 10(5)2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31064142

RESUMO

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most important bacterial diseases of citrus, impacting both plant growth and fruit quality. Identifying and elucidating the roles of genes associated with pathogenesis has aided our understanding of the molecular basis of citrus-bacteria interactions. However, the complex virulence mechanisms of X. citri subsp. citri are still not well understood. In this study, we characterized the role of PhoP in X. citri subsp. citri using a phoP deletion mutant, ΔphoP. Compared with wild-type strain XHG3, ΔphoP showed reduced motility, biofilm formation, as well as decreased production of cellulase, amylase, and polygalacturonase. In addition, the virulence of ΔphoP on citrus leaves was significantly decreased. To further understand the virulence mechanisms of X. citri subsp. citri, high-throughput RNA sequencing technology (RNA-Seq) was used to compare the transcriptomes of the wild-type and mutant strains. Analysis revealed 1017 differentially-expressed genes (DEGs), of which 614 were up-regulated and 403 were down-regulated in ΔphoP. Gene ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses suggested that the DEGs were enriched in flagellar assembly, two-component systems, histidine metabolism, bacterial chemotaxis, ABC transporters, and bacterial secretion systems. Our results showed that PhoP activates the expression of a large set of virulence genes, including 22 type III secretion system genes and 15 type III secretion system effector genes, as well as several genes involved in chemotaxis, and flagellar and histidine biosynthesis. Two-step reverse-transcription polymerase chain reaction analysis targeting 17 genes was used to validate the RNA-seq data, and confirmed that the expression of all 17 genes, except for that of virB1, decreased significantly. Our results suggest that PhoP interacts with a global signaling network to co-ordinate the expression of multiple virulence factors involved in modification and adaption to the host environment during infection.

5.
Environ Microbiol ; 21(3): 959-971, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30537399

RESUMO

The fungal pathogen Sporisorium scitamineum causes sugarcane smut disease. The formation and growth of dikaryotic hypha after sexual mating is critical for S. scitamineum pathogenicity, however regulation of S. scitimineum mating has not been studied in detail. We identified and characterized the core components of the conserved cAMP/PKA pathway in S. scitamineum by reverse genetics. Our results showed that cAMP/PKA signalling pathway is essential for proper mating and filamentation, and thus critical for S. scitamineum virulence. We further demonstrated that an elevated intracellular ROS (reactive oxygen species) level promotes S. scitamineum mating-filamentation, via transcriptional regulation of ROS catabolic enzymes, and is under regulation of the cAMP/PKA signalling pathway. Furthermore, we found that fungal cAMP/PKA signalling pathway is also involved in regulation of host ROS response. Overall, our work displayed a positive role of elevated intracellular ROS in fungal differentiation and virulence.

6.
Front Microbiol ; 9: 2555, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30416495

RESUMO

In the phytopathogenic fungus Sporisorium scitamineum, sexual mating between two compatible haploid cells and the subsequent formation of dikaryotic hyphae is essential for infection. This process was shown to be commonly regulated by a mitogen-activated protein kinase (MAPK) and a cAMP/PKA signaling pathway in the corn smut fungus Ustilago maydis but remains largely unknown in S. scitamineum. In this study, we identified a conserved putative MAP kinase Kpp2 in S. scitamineum and named it as SsKpp2. The sskpp2Δ mutant displayed significant reduction in mating/filamentation, which could be partially restored by addition of cAMP or tryptophol, a quorum-sensing molecule identified in budding yeast. Transcriptional profiling showed that genes governing S. scitamineum mating or tryptophol biosynthesis were significantly differentially regulated in the sskpp2Δ mutant compared to the WT, under mating condition. Our results demonstrate that the MAP kinase SsKpp2 is required for S. scitamineum mating/filamentation likely through regulating the conserved pheromone signal transduction pathway and tryptophol production.

7.
Mol Plant Microbe Interact ; : MPMI07180187R, 2018 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-30156480

RESUMO

The plant pathogen Xanthomonas campestris pv. campestris produces diffusible signal factor (DSF) quorum sensing (QS) signals to regulate its biological functions and virulence. Our previous study showed that X. campestris pv. campestris utilizes host plant metabolites to enhance the biosynthesis of DSF family signals. However, it is unclear how X. campestris pv. campestris benefits from the metabolic products of the host plant. In this study, we observed that the host plant metabolites not only boosted the production of the DSF family signals but also modulated the expression levels of DSF-regulated genes in X. campestris pv. campestris. Infection with X. campestris pv. campestris induced changes in the expression of many sugar transporter genes in Arabidopsis thaliana. Exogenous addition of sucrose or glucose, which are the major products of photosynthesis in plants, enhanced DSF signal production and X. campestris pv. campestris pathogenicity in the Arabidopsis model. In addition, several sucrose hydrolase-encoding genes in X. campestris pv. campestris and sucrose invertase-encoding genes in the host plant were notably upregulated during the infection process. These enzymes hydrolyzed sucrose to glucose and fructose, and in trans expression of one of these enzymes, CINV1 of A. thaliana or XC_0805 of X. campestris pv. campestris, enhanced DSF signal biosynthesis in X. campestris pv. campestris in the presence of sucrose. Taken together, our findings demonstrate that X. campestris pv. campestris applies multiple strategies to utilize host plant sugars to enhance QS and pathogenicity.

8.
BMC Genomics ; 17: 354, 2016 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-27185248

RESUMO

BACKGROUND: Sporisorium scitamineum causes the sugarcane smut disease, one of the most serious constraints to global sugarcane production. S. scitamineum possesses a sexual mating system composed of two mating-type loci, a and b locus. We previously identified and deleted the b locus in S. scitamineum, and found that the resultant SsΔMAT-1b mutant was defective in mating and pathogenicity. RESULTS: To further understand the function of b-mating locus, we carried out transcriptome analysis by comparing the transcripts of the mutant strain SsΔMAT-1b, from which the SsbE1 and SsbW1 homeodomain transcription factors have previously been deleted, with those from the wild-type MAT-1 strain. Also the transcripts from SsΔMAT-1b X MAT-2 were compared with those from wild-type MAT-1 X MAT-2 mating. A total of 209 genes were up-regulated (p < 0.05) in the SsΔMAT-1b mutant, compared to the wild-type MAT-1 strain, while 148 genes down-regulated (p < 0.05). In the mixture, 120 genes were up-regulated (p < 0.05) in SsΔMAT-1b X MAT-2, which failed to mate, compared to the wild-type MAT-1 X MAT-2 mating, and 271 genes down-regulated (p < 0.05). By comparing the up- and down-regulated genes in these two sets, it was found that 15 up-regulated and 37 down-regulated genes were common in non-mating haploid and mating mixture, which indeed could be genes regulated by b-locus. Furthermore, GO and KEGG enrichment analysis suggested that carbon metabolism pathway and stress response mediated by Hog1 MAPK signaling pathway were altered in the non-mating sets. CONCLUSIONS: Experimental validation results indicate that the bE/bW heterodimeric transcriptional factor, encoded by the b-locus, could regulate S. scitamineum sexual mating and/or filamentous growth via modulating glucose metabolism and Hog1-mediating oxidative response.


Assuntos
Basidiomycota/fisiologia , Meio Ambiente , Perfilação da Expressão Gênica , Reprodução Assexuada/genética , Transcriptoma , Metabolismo dos Carboidratos/genética , Biologia Computacional/métodos , Bases de Dados de Ácidos Nucleicos , Regulação Fúngica da Expressão Gênica , Genes Fúngicos Tipo Acasalamento , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Anotação de Sequência Molecular
9.
Mol Plant Pathol ; 17(9): 1398-1408, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-26814706

RESUMO

Dickeya zeae is a causal agent of rice root rot disease. The pathogen is known to produce a range of virulence factors, including phytotoxic zeamines and extracellular enzymes, but the mechanisms of virulence regulation remain vague. In this study, we identified a SlyA/MarR family transcription factor SlyA in D. zeae strain EC1. Disruption of slyA significantly decreased zeamine production, enhanced swimming and swarming motility, reduced biofilm formation and significantly decreased pathogenicity on rice. Quantitative polymerase chain reaction (qPCR) analysis confirmed the role of SlyA in transcriptional modulation of a range of genes associated with bacterial virulence. In trans expression of slyA in expI mutants recovered the phenotypes of motility and biofilm formation, suggesting that SlyA is downstream of the acylhomoserine lactone-mediated quorum sensing pathway. Taken together, the findings from this study unveil a key transcriptional regulatory factor involved in the modulation of virulence factor production and overall pathogenicity of D. zeae EC1.


Assuntos
Proteínas de Bactérias/metabolismo , Enterobacteriaceae/patogenicidade , Oryza/microbiologia , Toxinas Biológicas/metabolismo , Biofilmes , Parede Celular/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Genes de Plantas , Genoma Bacteriano , Germinação , Macrolídeos/metabolismo , Movimento , Mutação/genética , Poliaminas/metabolismo , Sementes/microbiologia , Transcrição Genética , Virulência
10.
Fungal Genet Biol ; 86: 1-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26563415

RESUMO

Sporisorium scitamineum is the causal agent of sugarcane smut, which is one of the most serious constraints to global sugarcane production. S. scitamineum and Ustilago maydis are two closely related smut fungi, that are predicted to harbor similar sexual mating processes/system. To elucidate the molecular basis of sexual mating in S. scitamineum, we identified and deleted the ortholog of mating-specific U. maydis locus b, in S. scitamineum. The resultant b-deletion mutant was defective in mating and pathogenicity in S. scitamineum. Furthermore, a functional b locus heterodimer could trigger filamentous growth without mating in S. scitamineum, and functionally replace the b locus in U. maydis in terms of triggering aerial filament production and forming solopathogenic strains, which do not require sexual mating prior to pathogenicity on the host plants.


Assuntos
Genes Fúngicos Tipo Acasalamento , Saccharum/microbiologia , Ustilaginales/genética , Ustilaginales/patogenicidade , Sequência de Aminoácidos , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Genética Reversa , Ustilaginales/crescimento & desenvolvimento , Ustilago/genética , Ustilago/patogenicidade , Virulência
11.
Sci Rep ; 5: 8784, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25740758

RESUMO

Cyhalothrin is a common environmental pollutant which poses increased risks to non-target organisms including human beings. This study reported for the first time a newly isolated strain, Bacillus thuringiensis ZS-19 completely degraded cyhalothrin in minimal medium within 72 h. The bacterium transformed cyhalothrin by cleavage of both the ester linkage and diaryl bond to yield six intermediate products. Moreover, a novel degradation pathway of cyhalothrin in strain ZS-19 was proposed on the basis of the identified metabolites. In addition to degradation of cyhalothrin, this strain was found to be capable of degrading 3-phenoxybenzoic acid, a common metabolite of pyrethroids. Furthermore, strain ZS-19 participated in efficient degradation of a wide range of pyrethroids including cyhalothrin, fenpropathrinn, deltamethrin, beta-cypermethrin, cyfluthrin and bifenthrin. Taken together, our results provide insights into the mechanism of cyhalothrin degradation and also highlight the promising potentials of B.thuringiensis ZS-19 in bioremediation of pyrethroid-contaminated environment. This is the first report of (i) degradation of cyhalothrin and other pyrethroids by B.thuringiensis, (ii) identification of 3-phenoxyphenyl acetonitrile and N-(2-isoproxy-phenyl)-4-phenoxy-benzamide as the metabolites in the degradation pathway of pyrethroids, and (iii) a pathway of degradation of cyhalothrin by cleavage of both the ester linkage and diaryl bond in a microorganism.


Assuntos
Bacillus thuringiensis/metabolismo , Biodegradação Ambiental , Fungicidas Industriais/metabolismo , Redes e Vias Metabólicas , Nitrilos/metabolismo , Piretrinas/metabolismo , Bacillus thuringiensis/classificação , Bacillus thuringiensis/genética , Bacillus thuringiensis/isolamento & purificação , Benzoatos/metabolismo , Cinética , Metaboloma , Metabolômica/métodos , Filogenia , RNA Ribossômico 16S/genética
12.
Cell Res ; 25(3): 351-69, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25698579

RESUMO

The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.


Assuntos
Apoptose/genética , Senescência Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/genética , Proteína Supressora de Tumor p53/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Linhagem Celular , DNA/genética , DNA Ligase Dependente de ATP , DNA Ligases/biossíntese , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Humanos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Isoformas de Proteínas/genética , Interferência de RNA , RNA Interferente Pequeno , Rad51 Recombinase/biossíntese , Peixe-Zebra , Proteínas de Peixe-Zebra/biossíntese
13.
J Agric Food Chem ; 62(10): 2147-57, 2014 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-24576059

RESUMO

The widely used insecticide fenpropathrin in agriculture has become a public concern because of its heavy environmental contamination and toxic effects on mammals, yet little is known about the kinetic and metabolic behaviors of this pesticide. This study reports the degradation kinetics and metabolic pathway of fenpropathrin in Bacillus sp. DG-02, previously isolated from the pyrethroid-manufacturing wastewater treatment system. Up to 93.3% of 50 mg L(-1) fenpropathrin was degraded by Bacillus sp. DG-02 within 72 h, and the degradation rate parameters qmax, Ks, and Ki were determined to be 0.05 h(-1), 9.0 mg L(-1), and 694.8 mg L(-1), respectively. Analysis of the degradation products by gas chromatography-mass spectrometry led to identification of seven metabolites of fenpropathrin, which suggest that fenpropathrin could be degraded first by cleavage of its carboxylester linkage and diaryl bond, followed by degradation of the aromatic ring and subsequent metabolism. In addition to degradation of fenpropathrin, this strain was also found to be capable of degrading a wide range of synthetic pyrethroids including deltamethrin, λ-cyhalothrin, ß-cypermethrin, ß-cyfluthrin, bifenthrin, and permethrin, which are also widely used insecticides with environmental contamination problems with the degradation process following the first-order kinetic model. Bioaugmentation of fenpropathrin-contaminated soils with strain DG-02 significantly enhanced the disappearance rate of fenpropathrin, and its half-life was sharply reduced in the soils. Taken together, these results depict the biodegradation mechanisms of fenpropathrin and also highlight the promising potentials of Bacillus sp. DG-02 in bioremediation of pyrethroid-contaminated soils.


Assuntos
Bacillus/metabolismo , Inseticidas/metabolismo , Piretrinas/metabolismo , Poluentes do Solo/metabolismo , Bacillus/crescimento & desenvolvimento , Biodegradação Ambiental , Cromatografia Gasosa-Espectrometria de Massas , Cinética
14.
J Genet Genomics ; 41(2): 53-62, 2014 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-24576456

RESUMO

Zebrafish Δ113p53, an N-terminal truncated p53 isoform, is a p53-target gene that antagonises p53-mediated apoptotic activity. Interestingly, Δ113p53 does not act on p53 in a dominant-negative manner, but rather interferes with the p53 function by differentially modulating p53-target gene expression to protect cells from apoptosis. Previous studies showed that over-expressed Δ113p53 and p53 proteins formed a complex. However, it is not known whether endogenous p53 and Δ113p53 proteins also interact with each other, and if this interaction is required for Δ113p53 to inhibit the apoptotic activity of full-length p53. In this study, we used two available zebrafish p53 antibodies to address these questions. One, Zfp53-N, only recognises full-length p53, whereas the other, Zfp53-A7C10, detects both full-length p53 and Δ113p53. Using Zfp53-N for immunoprecipitation and Zfp53-A7C10 for detection, we demonstrated that endogenous Δ113p53 and full-length p53 induced by a DNA-damaging drug formed a complex in vivo. Furthermore, of the six Δ113p53 mutants we generated with different point mutations in the oligomerisation domain, two failed to interact with p53 and lost the ability to modulate p53-target gene expression and inhibit p53-induced cell apoptosis. However, those Δ113p53 mutants that could interact with p53 retained the ability to antagonise the apoptotic activity of p53. Therefore, our data demonstrated that protein-protein interaction between Δ113p53 and p53 is essential for the anti-apoptotic function of Δ113p53. In addition, the two Δ113p53 mutants that failed to interact with p53 are also useful for the study of the mechanisms of other functions of Δ113p53.


Assuntos
Apoptose , Mutação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Camptotecina/farmacologia , Dano ao DNA , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteína Supressora de Tumor p53/química , Proteínas de Peixe-Zebra/química
15.
PLoS One ; 8(9): e75450, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098697

RESUMO

The widely used insecticide beta-cypermethrin has become a public concern because of its environmental contamination and toxic effects on mammals. In this study, a novel beta-cypermethrin degrading enzyme designated as CMO was purified to apparent homogeneity from a Streptomyces sp. isolate capable of utilizing beta-cypermethrin as a growth substrate. The native enzyme showed a monomeric structure with a molecular mass of 41 kDa and pI of 5.4. The enzyme exhibited the maximal activity at pH 7.5 and 30°C. It was fairly stable in the pH range from 6.5-8.5 and at temperatures below 10°C. The enzyme activity was significantly stimulated by Fe(2+), but strongly inhibited by Ag(+), Al(3+), and Cu(2+). The enzyme catalyzed the degradation of beta-cypermethrin to form five products via hydroxylation and diaryl cleavage. A novel beta-cypermethrin detoxification pathway was proposed based on analysis of these products. The purified enzyme was identified as a monooxygenase by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis (MALDI-TOF-MS) and N-terminal protein sequencing. Given that all the characterized pyrethroid-degrading enzymes are the members of hydrolase family, CMO represents the first pyrethroid-degrading monooxygenase identified from environmental microorganisms. Taken together, our findings depict a novel pyrethroid degradation mechanism and indicate that the purified enzyme may be a promising candidate for detoxification of beta-cypermethrin and environmental protection.


Assuntos
Poluição Ambiental/prevenção & controle , Inseticidas/metabolismo , Oxigenases de Função Mista/química , Piretrinas/metabolismo , Streptomyces/enzimologia , Sequência de Bases , Concentração de Íons de Hidrogênio , Metais Pesados/farmacologia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/isolamento & purificação , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Peso Molecular , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Temperatura Ambiente
16.
Mol Plant Microbe Interact ; 26(11): 1294-301, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23883359

RESUMO

Dickeya zeae is the causal agent of rice foot rot and maize stalk rot diseases, which could cause severe economic losses. The pathogen is known to produce two phytotoxins known as zeamine and zeamine II which are also potent antibiotics against both gram-positive and gram-negative bacteria pathogens. Zeamine II is a long-chain aminated polyketide and zeamine shares the same polyketide structure as zeamine II, with an extra valine derivative moiety conjugated to the primary amino group of zeamine II. In this study, we have identified a gene designated as zmsK encoding a putative nonribosomal peptide synthase (NRPS) by screening of the transposon mutants defective in zeamine production. Different from most known NRPS enzymes, which are commonly multidomain proteins, ZmsK contains only a condensation domain. High-performance liquid chromatography and mass spectrometry analyses showed that the ZmsK deletion mutant produced only zeamine II but not zeamine, suggesting that ZmsK catalyzes the amide bond formation by using zeamine II as a substrate to generate zeamine. We also present evidence that a partially conserved catalytic motif within the condensation domain is critical for zeamine production. Furthermore, we show that deletion of zmsK substantially decreased the total antimicrobial activity and virulence of D. zeae. Our findings provide a new insight into the biosynthesis pathway of zeamines and the virulence mechanisms of the bacterial pathogen D. zeae.


Assuntos
Anti-Infecciosos/metabolismo , Enterobacteriaceae/enzimologia , Macrolídeos/metabolismo , Oryza/microbiologia , Peptídeo Sintases/genética , Poliaminas/metabolismo , Sequência de Aminoácidos , Anti-Infecciosos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão , Enterobacteriaceae/genética , Enterobacteriaceae/patogenicidade , Germinação , Macrolídeos/química , Espectrometria de Massas , Dados de Sequência Molecular , Oryza/fisiologia , Peptídeo Sintases/metabolismo , Fenótipo , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Brotos de Planta/microbiologia , Brotos de Planta/fisiologia , Poliaminas/química , Estrutura Terciária de Proteína , Sementes/microbiologia , Sementes/fisiologia , Alinhamento de Sequência , Deleção de Sequência , Virulência
17.
Nat Chem Biol ; 9(5): 339-43, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23542643

RESUMO

Pseudomonas aeruginosa uses a hierarchical quorum sensing (QS) network consisting of las, pqs and rhl regulatory elements to coordinate the expression of bacterial virulence genes. However, clinical isolates frequently contain loss-of-function mutations in the central las system. This motivated us to search for a mechanism that may functionally substitute las. Here we report identification of a new QS signal, IQS. Disruption of IQS biosynthesis paralyzes the pqs and rhl QS systems and attenuates bacterial virulence. Production of IQS is tightly controlled by las under normal culture conditions but is also activated by phosphate limitation, a common stressor that bacteria encounter during infections. Thus, these results have established an integrated QS system that connects the central las system and phosphate-stress response mechanism to the downstream pqs and rhl regulatory systems. Our discovery highlights the complexity of QS signaling systems and extends the gamut of QS and stress-response mechanisms.


Assuntos
Proteínas de Bactérias/metabolismo , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum , Transdução de Sinais , Estresse Fisiológico , Proteínas de Bactérias/genética , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/genética
18.
Bioresour Technol ; 132: 16-23, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23395753

RESUMO

Brevibacterium aureum DG-12, a new bacterial strain isolated from active sludge, was able to degrade and utilize cyfluthrin as a growth substrate in the mineral medium. Response surface methodology using central composite rotatable design of cultural conditions was successfully employed for optimization resulting in 88.6% degradation of cyfluthrin (50mgL(-1)) within 5days. The bacterium degraded cyfluthrin by cleavage of both the carboxylester linkage and diaryl bond to form 2,2,3,3-tetramethyl-cyclopropanemethanol, 4-fluoro-3-phenexy-benzoic acid, 3,5-dimethoxy phenol, and phenol, and subsequently transformed these compounds with a maximum specific degradation rate, half-saturation constant and inhibition constant of 1.0384day(-1), 20.4967mgL(-1), and 141.9013mgL(-1), respectively. A novel degradation pathway for cyfluthrin was proposed based on analysis of these metabolites. In addition, this strain was found capable of degrading a wide range of synthetic pyrethroid insecticides. Our results suggest that B. aureum DG-12 may be an ideal microorganism for bioremediation of the pyrethroid-contaminated environments.


Assuntos
Brevibacterium/metabolismo , Poluentes Ambientais/metabolismo , Inseticidas/metabolismo , Nitrilos/metabolismo , Piretrinas/metabolismo , Biodegradação Ambiental , Esgotos/microbiologia
19.
Mol Microbiol ; 87(1): 80-93, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23113660

RESUMO

The diffusible factor synthase XanB2, originally identified in Xanthomonas campestris pv. campestris (Xcc), is highly conserved across a wide range of bacterial species, but its substrate and catalytic mechanism have not yet been investigated. Here, we show that XanB2 is a unique bifunctional chorismatase that hydrolyses chorismate, the end-product of the shikimate pathway, to produce 3-hydroxybenzoic acid (3-HBA) and 4-HBA. 3-HBA and 4-HBA are respectively associated with the yellow pigment xanthomonadin biosynthesis and antioxidant activity in Xcc. We further demonstrate that XanB2 is a structurally novel enzyme with three putative domains. It catalyses 3-HBA and 4-HBA biosynthesis via a unique mechanism with the C-terminal YjgF-like domain conferring activity for 3-HBA biosynthesis and the N-terminal FGFG motif-containing domain responsible for 4-HBA biosynthesis. Furthermore, we show that Xcc produces coenzyme Q8 (CoQ8) via a new biosynthetic pathway independent of the key chorismate-pyruvate lyase UbiC. XanB2 is the alternative source of 4-HBA for CoQ8 biosynthesis. The similar CoQ8 biosynthetic pathway, xanthomonadin biosynthetic gene cluster and XanB2 homologues are well conserved in the bacterial species within Xanthomonas, Xylella, Xylophilus, Pseudoxanthomonas, Rhodanobacter, Frateuria, Herminiimonas and Variovorax, suggesting that XanB2 may be a conserved metabolic link between the shikimate pathway, ubiquinone and xanthomonadin biosynthetic pathways in diverse bacteria.


Assuntos
Anisóis/metabolismo , Carbono-Oxigênio Liases/metabolismo , Ácido Corísmico/metabolismo , Ácido Chiquímico/metabolismo , Ubiquinona/metabolismo , Xanthomonas campestris/enzimologia , Vias Biossintéticas/genética , Deleção de Genes , Hidroxibenzoatos/metabolismo , Xanthomonas campestris/genética , Xanthomonas campestris/metabolismo
20.
J Genet Genomics ; 39(9): 489-502, 2012 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-23021549

RESUMO

The use of reporter systems to analyze DNA double-strand break (DSB) repairs, based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce I, is usually carried out with cell lines. In this study, we developed three visual-plus quantitative assay systems for homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA) DSB repair pathways at the organismal level in zebrafish embryos. To initiate DNA DSB repair, we used two I-Sce I recognition sites in opposite orientation rather than the usual single site. The NHEJ, HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions, and the repair of DNA lesion caused by I-Sce I could be tracked by EGFP expression in the embryos. Apart from monitoring the intensity of green fluorescence, the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction (qPCR). Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos. Furthermore, while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52, respectively, NHEJ could only be impaired by the knockdown of ligaseIV (lig4) when the NHEJ construct was cut by I-Sce I in vivo. More interestingly, blocking NHEJ with lig4-MO increased the frequency of HR, but decreased the frequency of SSA. Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal, and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/análise , Recombinação Homóloga/genética , Animais , Células CHO , Linhagem Celular , Cricetinae , Reparo do DNA por Junção de Extremidades , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Ligases/genética , DNA Ligases/metabolismo , DNA de Cadeia Simples/genética , Proteínas de Fluorescência Verde , Peixe-Zebra/embriologia , Peixe-Zebra/genética
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