RESUMO
Stigmatella aurantiaca displays a complex developmental life cycle in response to starvation conditions that results in the formation of tree-like fruiting bodies capable of producing spores. The phage Mx8, first isolated from the close relative Myxococcus xanthus, is unable to infect S. aurantiaca cells and integrate into the genome. However, plasmids containing Mx8 fragments encoding the integrase and attP are able to integrate at the attB locus in the S. aurantiaca genome by site-specific recombination. After recombination between attP and attB, the S. aurantiaca cells were incapable of building normal fruiting bodies but formed clumps and fungus-like structures characteristic of intermediate stages of development displayed by the wild type. We identified two tRNA genes, trnD and trnV, encoding tRNA(Asp) and tRNA(Val), respectively, composing an operon at the attB locus of S. aurantiaca. Integration of attP-containing plasmids resulted in the incorporation of the t(Mx8) terminator sequence, in addition to a short sequence of Mx8 DNA downstream of trnD. The integrant was unable to process the trnD transcript at the normal 3' processing site and displayed a lower level of expression of the trnVD operon. In addition, several developmentally regulated proteins were no longer produced in mutants following insertion at the attB locus. We hypothesize that the integration of the t(Mx8) terminator sequence results in reduced levels of mature tRNA(Asp) and tRNA(Val) and that altered protein production during development is thereby responsible for the observed phenotype. The trnVD locus thus defines a new developmental checkpoint for Stigmatella aurantiaca.
Assuntos
Sítios de Ligação Microbiológicos/genética , Regulação Bacteriana da Expressão Gênica , Óperon/genética , RNA de Transferência de Ácido Aspártico/genética , RNA de Transferência de Valina/genética , Stigmatella aurantiaca/fisiologia , Proteínas de Bactérias/metabolismo , Bacteriófagos/enzimologia , Sequência de Bases , Genes Bacterianos , Teste de Complementação Genética , Integrases/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Óperon/fisiologia , Plasmídeos/genética , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Valina/metabolismo , Alinhamento de Sequência , Esporos Bacterianos/crescimento & desenvolvimento , Stigmatella aurantiaca/genética , Proteínas Virais/genéticaRESUMO
Despite the considerable progress molecular genetics of filamentous fungi has made during the past decade, there is still an urgent need for efficiently working selectable markers for fungal transformation. Using Pichia pastoris as a host, we describe the development of a new dominant selectable marker of prokaryotic origin. This system, termed sor(R), is based upon the resistance of the bacterial enzyme acetyl-CoA carboxylase (ACCase) to the macrocyclic polyketide soraphen A, a potent inhibitor of fungal ACCase produced by the myxobacterium Sorangium cellulosum. In this study, we firstly demonstrate that the integration of a single sor(R) cassette into the P. pastoris genome confers resistance to elevated concentrations of soraphen A. Furthermore, it has been shown that the versatility of this marker can be considerably increased by splitting the sor(R) cassette, especially when successive transformations are performed on the same strain. As pronounced sensitivity to soraphen A is the rule among filamentous fungi, we expect the sor(R) marker to be a widely applicable tool for fungal transformation.
Assuntos
Antibacterianos/farmacologia , Marcadores Genéticos/genética , Macrolídeos/farmacologia , Pichia/efeitos dos fármacos , Pichia/genética , Transformação Genética/genética , Acetil-CoA Carboxilase/genética , Southern Blotting , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Fúngico/química , DNA Fúngico/genética , Reação em Cadeia da PolimeraseRESUMO
Agrobacterium tumefaciens was used to stably transform the entomopathogenic deuteromycete Beauveria bassiana to hygromycin B resistance by integration of the hph gene of Escherichia coli into the fungal genome. The transformation protocol was optimized to generate a library of insertion mutants of Beauveria. Transformation frequencies around 10(-4) and suppression of background growth were achieved. Over 90% of the AIM mutants investigated contained single-copy T-DNA integrations at different chromosomal locations. Integrated T-DNAs were re-isolated from ten transformants by a marker rescue approach. When the sequences flanking these T-DNAs were compared with the corresponding locations of the wild-type genome, truncations of T-DNA borders were found to be common, while none of the sites of integration had suffered deletion or rearrangement. Thus, AIM can be considered a promising tool for insertional mutagenesis studies of entomopathogenic filamentous fungi.