RESUMO
The development of natural competence by bacteria in situ is considered one of the main factors limiting transformation-mediated gene exchanges in the environment. Ralstonia solanacearum is a plant pathogen that is also a naturally transformable bacterium that can develop the competence state during infection of its host. We have attempted to determine whether this bacterium could become the recipient of plant genes. We initially demonstrated that plant DNA was released close to the infecting bacteria. We constructed and tested various combinations of transgenic plants and recipient bacteria to show that the effectiveness of such transfers was directly related to the ratio of the complexity of the plant genome to the number of copies of the transgene.
Assuntos
Betaproteobacteria/genética , Técnicas de Transferência de Genes , Genoma de Planta , Plantas Geneticamente Modificadas/genética , Transgenes/genética , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologiaRESUMO
We have explored the potential of using the apramycin resistance gene as a marker in mycobacterial gene transfer studies. Shuttle plasmids available for both electroporation and conjugation studies have been constructed, and we have successfully validated the use of the apramycin resistance gene as a component of cloning vectors for Mycobacterium smegmatis, M. bovis BCG, and M. tuberculosis.
Assuntos
Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/genética , Mycobacterium/genética , Nebramicina/análogos & derivados , Conjugação Genética , Eletroporação , Técnicas de Transferência de Genes , Engenharia Genética , Vetores Genéticos , Mycobacterium/efeitos dos fármacos , Nebramicina/farmacologia , Transformação GenéticaRESUMO
Repeated attempts at isolating the Frankia endophyte of Coriaria spp. have not yielded infective microbial cultures that could fulfil Koch's postulates. In order to circumvent the critical isolation step, nodule endophytes of Coriaria were characterized directly by means of specific amplification of nodule DNA (PCR) followed by sequencing of part of the 16S rDNA gene. Three closely related sequences were obtained from nodules originating from France, Mexico and New Zealand, containing unique sequences different from all other Frankia strains characterized so far. The sequences obtained were closest (with 5 or 6 substitutions) to those of Frankia alni and those of Casuarina-infective Frankia strains, respectively. Two nucleotides unique to the Coriaria endophyte sequences were used to define specific primers, resulting in a hybridization test that could discriminate between Frankia DNAs originating from Coriaria nodules and those recovered from all cultured Frankia strains tested. The endophytes of Coriaria thus appear to form a distinct Frankia lineage.
Assuntos
Actinomycetales/genética , Actinomycetales/isolamento & purificação , DNA Fúngico/genética , Plantas/microbiologia , Actinomycetales/classificação , Sequência de Bases , Primers do DNA/genética , DNA Fúngico/isolamento & purificação , DNA Ribossômico/genética , Ecossistema , Variação Genética , Dados de Sequência Molecular , Filogenia , Plantas/genética , SimbioseRESUMO
In order to develop a rapid and specific detection test for bacteria in soil, we improved a method based on the polymerase chain reaction (PCR). Each step of the protocol, including direct lysis of cells, DNA purification, and PCR amplification, was optimized. To increase the efficiency of lysis, a step particularly critical for some microorganisms which resist classical techniques, we used small soil samples (100 mg) and various lytic treatments, including sonication, microwave heating, and thermal shocks. Purification of nucleic acids was achieved by passage through up to three Elutip d columns. Finally, PCR amplifications were optimized via biphasic protocols using booster conditions, lower denaturation temperatures, and addition of formamide. Two microorganisms were used as models: Agrobacterium tumefaciens, which is naturally absent from the soil used and was inoculated to calibrate the validity of the protocol, and Frankia spp., an actinomycete indigenous to the soil used. Specific primers were characterized either in the plasmid-borne vir genes for A. tumefaciens or in the variable regions of the 16S ribosomal gene for Frankia spp. Specific detection of the inoculated A. tumefaciens strain was routinely obtained when inocula ranged from 10(7) to 10(3) cells. Moreover, the strong correlation we observed between the size of the inocula and the results of the PCR reactions permitted assessment of the validity of the protocol in enumerating the number of microbial cells present in a soil sample. This allowed us to estimate the indigenous population of Frankia spp. at 0.2 x 10(5) genomes (i.e., amplifiable target sequences) per g of soil.
Assuntos
Bactérias/isolamento & purificação , DNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase , Microbiologia do Solo , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/crescimento & desenvolvimento , Agrobacterium tumefaciens/isolamento & purificação , Bactérias/química , Bacteriólise , Sequência de Bases , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Streptomyces/química , Streptomyces/crescimento & desenvolvimento , Streptomyces/isolamento & purificaçãoRESUMO
The nature of the LD50 test and its central place in the conceptual development of pharmacology is reviewed. The results of the test and possible uses are outlined. Some guidelines are set out for the development of alternatives to LD50 tests. Some circumstances are suggested in which it seems likely that some form of the test will continue to be required. It is concluded that the problem of replacing the LD50 test with apparently more elegant methods is part of the larger and more fundamental problem of bringing science to the study and regulation of toxicology.
