RESUMO
We describe a gene expression system for use in mammalian cells that yields reproducible, inducible gene expression that can be modulated within the physiological range. A synthetic promoter library was generated from which representatives were selected that gave weak, intermediate-strength or strong promoter activity. Each promoter resulted in a tight expression range when used to drive single-copy reporter genes integrated at the same genome location in stable cell lines, in contrast to the broad range of expression typical of transiently transfected cells. To test this new expression system in neurodegenerative disease models, we used each promoter type to generate cell lines carrying single-copy genes encoding polyglutamine-containing proteins. Expression over a period of up to three months resulted in a proportion of cells developing juxtanuclear aggresomes whose rate of formation, penetrance, and morphology were expression-level dependent. At the highest expression levels, fibrillar aggregates deposit close to the nuclear envelope, indicating that cell proteostasis is overwhelmed by misfolded protein species. We also observed expression-level dependent, abnormal nuclear morphology in cells containing aggresomes, with up to â¼80% of cells affected. This system constitutes a valuable tool in gene regulation at different levels and allows the quantitative assessment of gene expression effects when developing disease models or investigating cell function through the introduction of gene constructs.
Assuntos
Regulação da Expressão Gênica/genética , Peptídeos/genética , Peptídeos/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas/genética , Proteínas/metabolismo , Sequência de Bases , Linhagem Celular , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Peptídeos/química , Agregados Proteicos/genética , Proteínas/químicaRESUMO
Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a ß-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either ß-galactosidase or ß-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.
RESUMO
Inhibition of ß-amyloid (Aß) aggregation is an attractive therapeutic and preventive strategy for the discovery of disease-modifying agents in Alzheimer's disease (AD). Phomopsis occulta is a new, salt-tolerant fungus isolated from mangrove Pongamia pinnata (L.) Pierre. We report here the inhibitory effects of secondary metabolites from Ph. occulta on the aggregation of Aß42. It was found that mycelia extracts (MEs) from Ph. occulta cultured with 0, 2, and 3 M NaCl exhibited inhibitory activity in an E. coli model of Aß aggregation. A water-soluble fraction, ME0-W-F1, composed of mainly small peptides, was able to reduce aggregation of an Aß42-EGFP fusion protein and an early onset familial mutation Aß42E22G-mCherry fusion protein in transfected HEK293 cells. ME0-W-F1 also antagonized the cytotoxicity of Aß42 in the neural cell line SH-SY5Y in dose-dependent manner. Moreover, SDS-PAGE and FT-IR analysis confirmed an inhibitory effect of ME0-W-F1 on the aggregation of Aß42 in vitro. ME0-W-F1 blocked the conformational transition of Aß42 from α-helix/random coil to ß-sheet, and thereby inhibited formation of Aß42 tetramers and high molecular weight oligomers. ME0-W-F1 and other water-soluble secondary metabolites from Ph. occulta therefore represent new candidate natural products against aggregation of Aß42, and illustrate the potential of salt tolerant fungi from mangrove as resources for the treatment of AD and other diseases.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Produtos Biológicos/farmacologia , Fungos/química , Fragmentos de Peptídeos/metabolismo , Peptídeos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Linhagem Celular , Células HEK293 , Humanos , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Peptídeos/química , Peptídeos/isolamento & purificação , Estrutura Secundária de Proteína/efeitos dos fármacosRESUMO
Serratia sp. strain ATCC 39006 is a Gram-negative bacterium and a member of the Enterobacteriaceae that produces various bioactive secondary metabolites, including the tripyrrole red pigment prodigiosin and the ß-lactam antibiotic 1-carbapenen-2-em-3-carboxylic acid (a carbapenem). This strain is the only member of the Enterobacteriaceae known to naturally produce gas vesicles, as flotation organelles. Here we present the genome sequence of this strain, which has served as a model for analysis of the biosynthesis and regulation of antibiotic production.
RESUMO
Gas vesicles are hollow intracellular proteinaceous organelles produced by aquatic Eubacteria and Archaea, including cyanobacteria and halobacteria. Gas vesicles increase buoyancy and allow taxis toward air-liquid interfaces, enabling subsequent niche colonization. Here we report a unique example of gas vesicle-mediated flotation in an enterobacterium; Serratia sp. strain ATCC39006. This strain is a member of the Enterobacteriaceae previously studied for its production of prodigiosin and carbapenem antibiotics. Genes required for gas vesicle synthesis mapped to a 16.6-kb gene cluster encoding three distinct homologs of the main structural protein, GvpA. Heterologous expression of this locus in Escherichia coli induced copious vesicle production and efficient cell buoyancy. Gas vesicle morphogenesis in Serratia enabled formation of a pellicle-like layer of highly vacuolated cells, which was dependent on oxygen limitation and the expression of ntrB/C and cheY-like regulatory genes within the gas-vesicle gene cluster. Gas vesicle biogenesis was strictly controlled by intercellular chemical signaling, through an N-acyl homoserine lactone, indicating that in this system the quorum-sensing molecule acts as a morphogen initiating organelle development. Flagella-based motility and gas vesicle morphogenesis were also oppositely regulated by the small RNA-binding protein, RsmA, suggesting environmental adaptation through physiological control of the choice between motility and flotation as alternative taxis modes. We propose that gas vesicle biogenesis in this strain represents a distinct mechanism of mobility, regulated by oxygen availability, nutritional status, the RsmA global regulatory system, and the quorum-sensing morphogen.
Assuntos
Proteínas de Bactérias/metabolismo , Vesículas Citoplasmáticas/metabolismo , Proteínas/metabolismo , Percepção de Quorum/fisiologia , Serratia/metabolismo , Proteínas de Bactérias/genética , Vesículas Citoplasmáticas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Genes Bacterianos/fisiologia , Família Multigênica/fisiologia , Proteínas/genética , Serratia/genéticaAssuntos
Continuidade da Assistência ao Paciente/organização & administração , Cuidados Paliativos/organização & administração , Atenção Primária à Saúde/organização & administração , Atenção à Saúde/organização & administração , Humanos , Medicina Estatal/organização & administração , Reino Unido , Adulto JovemRESUMO
Pectobacterium carotovorum SCRI193 is a phytopathogenic Gram-negative bacterium. In this study, we have identified a novel cryptic pigment biosynthetic locus in P. carotovorum SCRI193 which we have called the Pectobacterium orange pigment (pop) cluster. The pop cluster is flanked by two tRNA genes and contains genes that encode non-ribosomal peptide synthases and polyketide synthase and produces a negatively charged polar orange pigment. Orange pigment production is activated when an adjacent transcriptional activator sharing sequence similarity with the Erwinia virulence regulator (Evr) is overexpressed. Evr was shown to positively activate its own transcription and that of the pigment biosynthetic genes and an unlinked locus encoding a phenomycin homologue. In addition, the expression of Evr and orange pigment production was shown to be regulated by N-(3-oxohexanoyl)-HSL (OHHL) quorum sensing and have a virulence phenotype in potato. Finally, by comparative genomics and Southern blotting we demonstrate that this pigment biosynthetic cluster is present in multiple P. carotovorum spp., Pectobacterium brasiliensis 1692 and a truncated version of the cluster is present in Pectobacterium atrosepticum. The conserved nature of this cluster in P. carotovorum and P. brasiliensis suggests that the pop cluster has an important function in these broad-host-range soft rotting bacteria, which is no longer required in the narrow-host-range P. atrosepticum SCRI1043.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Pectobacterium carotovorum/fisiologia , Peptídeos/metabolismo , Pigmentos Biológicos/biossíntese , Percepção de Quorum , Fatores de Transcrição/metabolismo , Vias Biossintéticas/genética , Southern Blotting , DNA Bacteriano/química , DNA Bacteriano/genética , Ordem dos Genes , Genes Bacterianos , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Família Multigênica , Pectobacterium carotovorum/genética , Doenças das Plantas/microbiologia , Análise de Sequência de DNA , Solanum tuberosum/microbiologia , VirulênciaRESUMO
Analogues of prodigiosin, a tripyrrolic pigment produced by Serratia species with potent immunosuppressive and anticancer activities, have been produced by feeding synthetic analogues of the normal precursor MBC to mutants of Serratia sp. ATCC 39006 or to engineered strains of Escherichia coli; in this way it has been shown that the prodigiosin synthesising enzyme, PigC, has a relaxed substrate-specificity.
Assuntos
Enzimas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Prodigiosina/síntese química , Prodigiosina/metabolismo , Enzimas/genética , Glicosilfosfatidilinositóis/classificação , Estrutura Molecular , Mutação/genética , Prodigiosina/análogos & derivados , Prodigiosina/química , Serratia/enzimologia , Serratia/genética , Especificidade por SubstratoRESUMO
Bacterial prodiginines are a family of red-pigmented, tripyrrolic compounds that display numerous biological activities, including antibacterial, antifungal, antiprotozoal, antimalarial, immunosuppressive and anticancer properties. Recently, significant progress has been made in understanding the biosynthesis and regulation of bacterial prodiginines. An understanding of the biosynthesis of prodiginines will allow engineering of bacterial strains capable of synthesizing novel prodiginines through rational design and mutasynthesis experiments. Bacterial prodiginines and synthetic derivatives are effective proapoptotic agents with multiple cellular targets, and they are active against numerous cancer cell lines, including multidrug-resistant cells, with little or no toxicity towards normal cell lines. A synthetic derivative, GX15-070 (Obatoclax), developed through structure-activity relationship studies of the pyrrolic ring A of GX15, is in multiple Phase I and II clinical trials in both single and dual-agent studies to treat different types of cancer. Therefore, prodiginines have real therapeutic potential in the clinic.
Assuntos
Antineoplásicos/farmacologia , Bactérias/química , Proliferação de Células/efeitos dos fármacos , Imunossupressores/farmacologia , Prodigiosina/análogos & derivados , Linhagem Celular Tumoral , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Prodigiosina/farmacologiaRESUMO
The red-pigmented prodiginines are bioactive secondary metabolites produced by both Gram-negative and Gram-positive bacteria. Recently, these tripyrrole molecules have received renewed attention owing to reported immunosuppressive and anticancer properties. The enzymes involved in the biosynthetic pathways for the production of two of these molecules, prodigiosin and undecylprodigiosin, are now known. However, the biochemistry of some of the reactions is still poorly understood. The physiology and regulation of prodiginine production in Serratia and Streptomyces are now well understood, although the biological role of these pigments in the producer organisms remains unclear. However, research into the biology of pigment production will stimulate interest in the bioengineering of strains to synthesize useful prodiginine derivatives.