Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 16 de 16
Filtrar
Mais filtros










Base de dados
Tipo de estudo
Intervalo de ano de publicação
1.
Science ; 373(6556)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385369

RESUMO

Capturing the heterogeneous phenotypes of microbial populations at relevant spatiotemporal scales is highly challenging. Here, we present par-seqFISH (parallel sequential fluorescence in situ hybridization), a transcriptome-imaging approach that records gene expression and spatial context within microscale assemblies at a single-cell and molecule resolution. We applied this approach to the opportunistic pathogen Pseudomonas aeruginosa, analyzing about 600,000 individuals across dozens of conditions in planktonic and biofilm cultures. We identified numerous metabolic- and virulence-related transcriptional states that emerged dynamically during planktonic growth, as well as highly spatially resolved metabolic heterogeneity in sessile populations. Our data reveal that distinct physiological states can coexist within the same biofilm just several micrometers away, underscoring the importance of the microenvironment. Our results illustrate the complex dynamics of microbial populations and present a new way of studying them at high resolution.


Assuntos
Pseudomonas aeruginosa/genética , Transcriptoma , Biofilmes/crescimento & desenvolvimento , Proteínas de Fímbrias/genética , Flagelina/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Hibridização in Situ Fluorescente , Fenótipo , Plâncton/genética , Plâncton/crescimento & desenvolvimento , Plâncton/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Piocinas/biossíntese , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Célula Única , Análise Espaço-Temporal , Virulência/genética
2.
Elife ; 92020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32930660

RESUMO

Phenazines are natural bacterial antibiotics that can protect crops from disease. However, for most crops it is unknown which producers and specific phenazines are ecologically relevant, and whether phenazine biodegradation can counter their effects. To better understand their ecology, we developed and environmentally-validated a quantitative metagenomic approach to mine for phenazine biosynthesis and biodegradation genes, applying it to >800 soil and plant-associated shotgun-metagenomes. We discover novel producer-crop associations and demonstrate that phenazine biosynthesis is prevalent across habitats and preferentially enriched in rhizospheres, whereas biodegrading bacteria are rare. We validate an association between maize and Dyella japonica, a putative producer abundant in crop microbiomes. D. japonica upregulates phenazine biosynthesis during phosphate limitation and robustly colonizes maize seedling roots. This work provides a global picture of phenazines in natural environments and highlights plant-microbe associations of agricultural potential. Our metagenomic approach may be extended to other metabolites and functional traits in diverse ecosystems.


Assuntos
Produtos Agrícolas/microbiologia , Metagenômica , Microbiota , Fenazinas/metabolismo , Microbiologia do Solo , Poluentes do Solo/metabolismo , Solo/química , Agricultura , Biodegradação Ambiental , Gammaproteobacteria/fisiologia , Especificidade da Espécie , Regulação para Cima , Zea mays/microbiologia
3.
mBio ; 10(5)2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31594819

RESUMO

N-Acyl homoserine lactone (AHL) quorum sensing (QS) controls expression of over 200 genes in Pseudomonas aeruginosa. There are two AHL regulatory systems: the LasR-LasI circuit and the RhlR-RhlI system. We mapped transcription termination sites affected by AHL QS in P. aeruginosa, and in doing so we identified AHL-regulated small RNAs (sRNAs). Of interest, we noted that one particular sRNA was located within the rhlI locus. We found that rhlI, which encodes the enzyme that produces the AHL N-butanoyl-homoserine lactone (C4-HSL), is controlled by a 5' untranslated region (UTR)-derived sRNA we name RhlS. We also identified an antisense RNA encoded opposite the beginning of the rhlI open reading frame, which we name asRhlS. RhlS accumulates as wild-type cells enter stationary phase and is required for the production of normal levels of C4-HSL through activation of rhlI translation. RhlS also directly posttranscriptionally regulates at least one other unlinked gene, fpvA. The asRhlS appears to be expressed at maximal levels during logarithmic growth, and we suggest RhlS may act antagonistically to the asRhlS to regulate rhlI translation. The rhlI-encoded sRNAs represent a novel aspect of RNA-mediated tuning of P. aeruginosa QS.IMPORTANCE The opportunistic human pathogen Pseudomonas aeruginosa possesses multiple quorum sensing systems that regulate and coordinate production of virulence factors and adaptation to different environments. Despite extensive research, the regulatory elements that play a role in this complex network are still not fully understood. By using several RNA sequencing techniques, we were able to identify a small regulatory RNA we named RhlS. RhlS increases translation of RhlI, a key enzyme in the quorum sensing pathway, and represses the fpvA mRNA encoding one of the siderophore pyoverdine receptors. Our results highlight a new regulatory layer of P. aeruginosa quorum sensing and contribute to the growing understanding of the role regulatory RNAs play in bacterial physiology.


Assuntos
4-Butirolactona/análogos & derivados , Regiões 5' não Traduzidas , Regulação Bacteriana da Expressão Gênica , Ligases/metabolismo , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum , Pequeno RNA não Traduzido/metabolismo , Fatores de Transcrição/metabolismo , 4-Butirolactona/metabolismo , Proteínas de Bactérias/metabolismo , Ligases/genética , Pseudomonas aeruginosa/genética , Pequeno RNA não Traduzido/genética , Fatores de Transcrição/genética
4.
Pediatr Infect Dis J ; 38(3): 297-299, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29742642

RESUMO

A signal transducer and activator of transcription-1-deficient patient presented with prolonged fever, cachexia, anemia, hypoalbuminemia and finally relapsing debilitating mycobacterial osteomyelitis while receiving a previously effective antimycobacterial treatment. Progression despite rigorous workup and multiple antibiotics prompted shotgun metagenomics revealing adenovirus in liver samples. Brincidofovir led to a complete, sustained clinical recovery, including osteomyelitis, probably attributed to reversal of adenovirus-induced immune dysregulation.


Assuntos
Infecções por Adenoviridae/diagnóstico , Infecções por Adenoviridae/tratamento farmacológico , Antivirais/uso terapêutico , Citosina/análogos & derivados , Organofosfonatos/uso terapêutico , Fator de Transcrição STAT1/deficiência , Infecções por Adenoviridae/imunologia , Citosina/uso terapêutico , Humanos , Hospedeiro Imunocomprometido , Fígado/imunologia , Fígado/virologia , Masculino , Metagenômica , Osteomielite/tratamento farmacológico , Osteomielite/microbiologia , Resultado do Tratamento , Adulto Jovem
5.
Proc Natl Acad Sci U S A ; 115(52): 13359-13364, 2018 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-30545912

RESUMO

To overcome the action of antibiotics, bacteria have evolved a variety of different strategies, such as drug modification, target mutation, and efflux pumps. Recently, we performed a genome-wide analysis of Listeria monocytogenes gene expression after growth in the presence of antibiotics, identifying genes that are up-regulated upon antibiotic treatment. One of them, lmo0762, is a homolog of hflX, which encodes a heat shock protein that rescues stalled ribosomes by separating their two subunits. To our knowledge, ribosome splitting has never been described as an antibiotic resistance mechanism. We thus investigated the role of lmo0762 in antibiotic resistance. First, we demonstrated that lmo0762 is an antibiotic resistance gene that confers protection against lincomycin and erythromycin, and that we renamed hflXr (hflX resistance). We show that hflXr expression is regulated by a transcription attenuation mechanism relying on the presence of alternative RNA structures and a small ORF encoding a 14 amino acid peptide containing the RLR motif, characteristic of macrolide resistance genes. We also provide evidence that HflXr is involved in ribosome recycling in presence of antibiotics. Interestingly, L. monocytogenes possesses another copy of hflX, lmo1296, that is not involved in antibiotic resistance. Phylogenetic analysis shows several events of hflXr duplication in prokaryotes and widespread presence of hflXr in Firmicutes. Overall, this study reveals the Listeria hflXr as the founding member of a family of antibiotic resistance genes. The resistance conferred by this gene is probably of importance in the environment and within microbial communities.


Assuntos
Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Listeria monocytogenes/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/fisiologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Evolução Molecular , Proteínas de Ligação ao GTP/genética , Listeria monocytogenes/genética , Testes de Sensibilidade Microbiana , Filogenia , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo
6.
mBio ; 9(5)2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30254125

RESUMO

Prokaryotic genomes encode a plethora of small noncoding RNAs (ncRNAs) that fine-tune the expression of specific genes. The vast majority of known bacterial ncRNAs are encoded from within intergenic regions, where their expression is controlled by promoter and terminator elements, similarly to protein-coding genes. In addition, recent studies have shown that functional ncRNAs can also be derived from gene 3' untranslated regions (3'UTRs) via an alternative biogenesis pathway, in which the ncRNA segment is separated from the mRNA via RNase cleavage. Here, we report the detection of a large set of decay-generated noncoding RNAs (decRNAs), many of which are completely embedded within protein-coding mRNA regions rather than in the UTRs. We show that these decRNAs are "carved out" of the mRNA through the action of RNase E and that they are predicted to fold into highly stable RNA structures, similar to those of known ncRNAs. A subset of these decRNAs is predicted to interact with Hfq or ProQ or both, which act as ncRNA chaperones, and some decRNAs display evolutionarily conserved sequences and conserved expression patterns between different species. These results suggest that mRNA protein-coding regions may harbor a large set of potentially functional small RNAs.IMPORTANCE Bacteria and archaea utilize regulatory small noncoding RNAs (ncRNAs) to control the expression of specific genetic programs. These ncRNAs are almost exclusively encoded within intergenic regions and are independently transcribed. Here, we report on a large set ncRNAs that are "carved out" from within the protein-coding regions of Escherichia coli mRNAs by cellular RNases. These protected mRNA fragments fold into energetically stable RNA structures, reminiscent of those of intergenic regulatory ncRNAs. In addition, a subset of these ncRNAs coprecipitate with the major ncRNA chaperones Hfq and ProQ and display evolutionarily conserved sequences and conserved expression patterns between different bacterial species. Our data suggest that protein-coding genes can potentially act as a reservoir of regulatory ncRNAs.


Assuntos
Escherichia coli/genética , Fases de Leitura Aberta/genética , Pequeno RNA não Traduzido/genética , Transcriptoma , Sequência Conservada , DNA Intergênico , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Fator Proteico 1 do Hospedeiro/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
7.
PLoS Genet ; 14(4): e1007354, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29668692

RESUMO

Bacterial operons synchronize the expression of multiple genes by placing them under the control of a shared promoter. It was previously shown that polycistronic transcripts can undergo differential RNA decay, leaving some genes within the polycistron more stable than others, but the extent of regulation by differential mRNA decay or its evolutionary conservation remains unknown. Here, we find that a substantial fraction of E. coli genes display non-uniform mRNA stoichiometries despite being coded from the same operon. We further show that these altered operon stoichiometries are shaped post-transcriptionally by differential mRNA decay, which is regulated by RNA structures that protect specific regions in the transcript from degradation. These protective RNA structures are generally coded within the protein-coding regions of the regulated genes and are frequently evolutionarily conserved. Furthermore, we provide evidence that differences in ribosome densities across polycistronic transcript segments, together with the conserved structural RNA elements, play a major role in the differential decay process. Our results highlight a major role for differential mRNA decay in shaping bacterial transcriptomes.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Estabilidade de RNA/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequência Conservada , Endorribonucleases/metabolismo , Genes Bacterianos , Modelos Biológicos , Conformação de Ácido Nucleico , Óperon , Processamento Pós-Transcricional do RNA , RNA Bacteriano/química , RNA Mensageiro/química , Ribossomos/metabolismo , Transcriptoma
8.
Nucleic Acids Res ; 46(13): 6797-6805, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29669055

RESUMO

Transcription termination in bacteria can occur either via Rho-dependent or independent (intrinsic) mechanisms. Intrinsic terminators are composed of a stem-loop RNA structure followed by a uridine stretch and are known to terminate in a precise manner. In contrast, Rho-dependent terminators have more loosely defined characteristics and are thought to terminate in a diffuse manner. While transcripts ending in an intrinsic terminator are protected from 3'-5' exonuclease digestion due to the stem-loop structure of the terminator, it remains unclear what protects Rho-dependent transcripts from being degraded. In this study, we mapped the exact steady-state RNA 3' ends of hundreds of Escherichia coli genes terminated either by Rho-dependent or independent mechanisms. We found that transcripts generated from Rho-dependent termination have precise 3'-ends at steady state. These termini were localized immediately downstream of energetically stable stem-loop structures, which were not followed by uridine rich sequences. We provide evidence that these structures protect Rho-dependent transcripts from 3'-5' exonucleases such as PNPase and RNase II, and present data localizing the Rho-utilization (rut) sites immediately downstream of these protective structures. This study represents the first extensive in-vivo map of exact RNA 3'-ends of Rho-dependent transcripts in E. coli.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , RNA Mensageiro/química , Regiões Terminadoras Genéticas , Terminação da Transcrição Genética , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
9.
Curr Opin Microbiol ; 36: 111-117, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28414973

RESUMO

Antibiotic resistance genes are commonly regulated by sophisticated mechanisms that activate gene expression in response to antibiotic exposure. Growing evidence suggest that cis-acting non-coding RNAs play a major role in regulating the expression of many resistance genes, specifically those which counteract the effects of translation-inhibiting antibiotics. These ncRNAs reside in the 5'UTR of the regulated gene, and sense the presence of the antibiotics by recruiting translating ribosomes onto short upstream open reading frames (uORFs) embedded in the ncRNA. In the presence of translation-inhibiting antibiotics ribosomes arrest over the uORF, altering the RNA structure of the regulator and switching the expression of the resistance gene to 'ON'. The specificity of these riboregulators is tuned to sense-specific classes of antibiotics based on the length and composition of the respective uORF. Here we review recent work describing new types of antibiotic-sensing RNA-based regulators and elucidating the molecular mechanisms by which they function to control antibiotic resistance in bacteria.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Regulação da Expressão Gênica , RNA não Traduzido/genética , Regiões 5' não Traduzidas , Bactérias/genética , Humanos , Mutação , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Mensageiro/genética
10.
Nucleic Acids Res ; 45(2): 886-893, 2017 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-27574119

RESUMO

A common strategy for regulation of gene expression in bacteria is conditional transcription termination. This strategy is frequently employed by 5'UTR cis-acting RNA elements (riboregulators), including riboswitches and attenuators. Such riboregulators can assume two mutually exclusive RNA structures, one of which forms a transcriptional terminator and results in premature termination, and the other forms an antiterminator that allows read-through into the coding sequence to produce a full-length mRNA. We developed a machine-learning based approach, which, given a 5'UTR of a gene, predicts whether it can form the two alternative structures typical to riboregulators employing conditional termination. Using a large positive training set of riboregulators derived from 89 human microbiome bacteria, we show high specificity and sensitivity for our classifier. We further show that our approach allows the discovery of previously unidentified riboregulators, as exemplified by the detection of new LeuA leaders and T-boxes in Streptococci Finally, we developed PASIFIC (www.weizmann.ac.il/molgen/Sorek/PASIFIC/), an online web-server that, given a user-provided 5'UTR sequence, predicts whether this sequence can adopt two alternative structures conforming with the conditional termination paradigm. This webserver is expected to assist in the identification of new riboswitches and attenuators in the bacterial pan-genome.


Assuntos
Bactérias/genética , Simulação por Computador , Regulação Bacteriana da Expressão Gênica , Modelos Biológicos , Terminação da Transcrição Genética , Aprendizado de Máquina , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , Curva ROC
11.
Nat Microbiol ; 1(10): 16143, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27670118

RESUMO

Transcription termination sets the 3' end boundaries of RNAs and plays key roles in gene regulation. Although termination has been well studied in bacteria, the signals that mediate termination in archaea remain poorly understood. Here, we applied term-seq to comprehensively map RNA 3' termini, with single-base precision, in two phylogenetically distant archaea: Methanosarcina mazei and Sulfolobus acidocaldarius. Comparison of RNA 3' ends across hundreds of genes revealed the sequence composition of transcriptional terminators in each organism, highlighting both common and divergent characteristics between the different archaeal phyla. We find that, in contrast to bacteria, a considerable portion of archaeal genes are controlled by multiple consecutive terminators, generating several alternative 3' untranslated region isoforms for >30% of the genes. These alternative isoforms often present marked length differences, implying that archaea can employ regulation via alternative 3' untranslated regions, similar to eukaryotes. Although most of the terminators are intergenic, we discover numerous cases in which termination of one gene occurs within the coding region of a downstream gene, implying that leaky termination may tune inter-transcript stoichiometry in multi-gene operons. These results provide the first high-throughput maps of transcriptional terminators in archaea and point to an evolutionary path linking bacterial and eukaryal non-coding regulatory strategies.


Assuntos
Regiões 3' não Traduzidas , Archaea/genética , Regulação da Expressão Gênica em Archaea , Isoformas de RNA/genética , RNA Arqueal/genética , Terminação da Transcrição Genética , Proteínas Arqueais/genética , Perfilação da Expressão Gênica , Genes Arqueais , RNA não Traduzido
12.
Nucleic Acids Res ; 44(W1): W46-53, 2016 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-27154273

RESUMO

Whole-transcriptome sequencing studies from recent years revealed an unexpected complexity in transcriptomes of bacteria and archaea, including abundant non-coding RNAs, cis-antisense transcription and regulatory untranslated regions (UTRs). Understanding the functional relevance of the plethora of non-coding RNAs in a given organism is challenging, especially since some of these RNAs were attributed to 'transcriptional noise'. To allow the search for conserved transcriptomic elements we produced comparative transcriptome maps for multiple species across the microbial tree of life. These transcriptome maps are detailed in annotations, comparable by gene families, and BLAST-searchable by user provided sequences. Our transcriptome collection includes 18 model organisms spanning 10 phyla/subphyla of bacteria and archaea that were sequenced using standardized RNA-seq methods. The utility of the comparative approach, as implemented in our web server, is demonstrated by highlighting genes with exceptionally long 5'UTRs across species, which correspond to many known riboswitches and further suggest novel putative regulatory elements. Our study provides a standardized reference transcriptome to major clinically and environmentally important microbial phyla. The viewer is available at http://exploration.weizmann.ac.il/TCOL, setting a framework for comparative studies of the microbial non-coding genome.


Assuntos
Archaea/genética , Bactérias/genética , RNA Arqueal/genética , RNA Bacteriano/genética , RNA não Traduzido/genética , Transcriptoma , Interface Usuário-Computador , Regiões 5' não Traduzidas , Archaea/classificação , Bactérias/classificação , Mapeamento Cromossômico , Gráficos por Computador , Filogenia , Riboswitch , Análise de Sequência de RNA
13.
Science ; 352(6282): aad9822, 2016 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-27120414

RESUMO

Riboswitches and attenuators are cis-regulatory RNA elements, most of which control bacterial gene expression via metabolite-mediated, premature transcription termination. We developed an unbiased experimental approach for genome-wide discovery of such ribo-regulators in bacteria. We also devised an experimental platform that quantitatively measures the in vivo activity of all such regulators in parallel and enables rapid screening for ribo-regulators that respond to metabolites of choice. Using this approach, we detected numerous antibiotic-responsive ribo-regulators that control antibiotic resistance genes in pathogens and in the human microbiome. Studying one such regulator in Listeria monocytogenes revealed an attenuation mechanism mediated by antibiotic-stalled ribosomes. Our results expose broad roles for conditional termination in regulating antibiotic resistance and provide a tool for discovering riboswitches and attenuators that respond to previously unknown ligands.


Assuntos
Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica , Estudo de Associação Genômica Ampla/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Riboswitch/genética , Terminação da Transcrição Genética , Regiões 3' não Traduzidas/genética , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Enterococcus faecalis/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/genética , Genoma Bacteriano/genética , Humanos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Ribossomos/metabolismo , Análise de Sequência de RNA/métodos
14.
Science ; 345(6199): 940-3, 2014 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-25146292

RESUMO

Riboswitches are ligand-binding elements contained within the 5' untranslated regions of bacterial transcripts, which generally regulate expression of downstream open reading frames. Here, we show that in Listeria monocytogenes, a riboswitch that binds vitamin B12 controls expression of a noncoding regulatory RNA, Rli55. Rli55, in turn, controls expression of the eut genes, whose products enable ethanolamine utilization and require B12 as a cofactor. Defects in ethanolamine utilization, or in its regulation by Rli55, significantly attenuate Listeria virulence in mice. Rli55 functions by sequestering the two-component response regulator EutV by means of a EutV-binding site contained within the RNA. Thus, Rli55 is a riboswitch-regulated member of the small group of regulatory RNAs that function by sequestering a protein and reveals a distinctive mechanism of signal integration in bacterial gene regulation.


Assuntos
Etanolamina/metabolismo , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , RNA não Traduzido/metabolismo , Riboswitch , Vitamina B 12/metabolismo , Regiões 5' não Traduzidas , Animais , Listeria monocytogenes/metabolismo , Listeria monocytogenes/virologia , Camundongos , Camundongos Endogâmicos BALB C , Óperon , Elementos de Resposta
15.
Microbiology (Reading) ; 158(Pt 11): 2859-2869, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22977090

RESUMO

The Gram-negative bacterium Pseudomonas syringae pv. tomato (Pst) is the causal agent of speck disease in tomato. Pst pathogenicity depends on a type III secretion system that delivers effector proteins into host cells, where they promote disease by manipulating processes to the advantage of the pathogen. Previous studies identified seven Pst effectors that inhibit growth when expressed in yeast under normal growth conditions, suggesting that they interfere with cellular processes conserved in yeast and plants. We hypothesized that effectors also target conserved cellular processes that are required for yeast growth only under stress conditions. We therefore examined phenotypes induced by expression of Pst effectors in yeast grown in the presence of various stressors. Out of 29 effectors tested, five (HopX1, HopG1, HopT1-1, HopH1 and AvrPtoB) displayed growth inhibition phenotypes only in combination with stress conditions. Viability assays revealed that the HopX1 effector caused loss of cell viability under prolonged osmotic stress. Using transcription reporters, we found that HopX1 attenuated the activation of the high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway, which is responsible for yeast survival under osmotic stress, while other MAPK pathways were mildly affected by HopX1. Interestingly, HopX1-mediated phenotypes in yeast were dependent on the putative transglutaminase catalytic triad of the effector. This study enlarges the pool of phenotypes available for the functional analysis of Pst type III effectors in yeast, and exemplifies how analysis of phenotypes detected in yeast under stress conditions can lead to the identification of eukaryotic cellular processes affected by bacterial effectors.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Pseudomonas syringae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Expressão Gênica , Glicerol/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Concentração Osmolar , Doenças das Plantas/microbiologia , Pseudomonas syringae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
Mol Plant Microbe Interact ; 24(3): 305-14, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21062109

RESUMO

The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of spot disease in tomato and pepper. X. campestris pv. vesicatoria pathogenicity depends on a type III secretion system delivering effector proteins into the host cells. We hypothesized that some X. campestris pv. vesicatoria effectors target conserved eukaryotic cellular processes and examined phenotypes induced by their expression in yeast. Out of 21 effectors tested, 14 inhibited yeast growth in normal or stress conditions. Viability assay revealed that XopB and XopF2 attenuated cell proliferation, while AvrRxo1, XopX, and XopE1 were cytotoxic. Inspection of morphological features and DNA content of yeast cells indicated that cytotoxicity caused by XopX and AvrRxo1 was associated with cell-cycle arrest at G0/1. Interestingly, XopB, XopE1, XopF2, XopX, and AvrRxo1 that inhibited growth in yeast also caused phenotypes, such as chlorosis and cell death, when expressed in either host or nonhost plants. Finally, the ability of several effectors to cause phenotypes in yeast and plants was dependent on their putative catalytic residues or localization motifs. This study supports the use of yeast as a heterologous system for functional analysis of X. campestris pv. vesicatoria type III effectors, and sets the stage for identification of their eukaryotic molecular targets and modes of action.


Assuntos
Proteínas de Bactérias/metabolismo , Viabilidade Microbiana/genética , Xanthomonas vesicatoria/genética , Xanthomonas vesicatoria/patogenicidade , Leveduras/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Fase G1 , Expressão Gênica , Lycopersicon esculentum/microbiologia , Dados de Sequência Molecular , Fenótipo , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Fase de Repouso do Ciclo Celular , Tabaco/microbiologia , Xanthomonas vesicatoria/metabolismo , Leveduras/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...