RESUMO
Type III secretion systems are a prolific virulence determinant among Gram-negative bacteria. They are used to paralyze the host cell, which enables bacterial pathogens to establish often fatal infections-unless an effective therapeutic intervention is available. However, as a result of a catastrophic rise in infectious bacteria resistant to conventional antibiotics, these bacteria are again a leading cause of worldwide mortality. Hence, this report describes a pDM4-based site-directed mutagenesis strategy that is assisting in our foremost objective to better understand the fundamental workings of the T3SS, using Yersinia as a model pathogenic bacterium. Examples are given that clearly document how pDM4-mediated site-directed mutagenesis has been used to establish clean point mutations and in-frame deletion mutations that have been instrumental in identifying and understanding the molecular interactions between components of the Yersinia type III secretion system.
Assuntos
Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Mutagênese Sítio-Dirigida , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Alelos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Conjugação Genética , Genoma Bacteriano , Infecções por Bactérias Gram-Negativas/microbiologia , Mutação , Fenótipo , Plasmídeos/genética , Reação em Cadeia da Polimerase , Ligação Proteica , Mapeamento de Interação de Proteínas , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Yersinia/genética , Yersinia/metabolismoRESUMO
Post-transcriptional C-to-U RNA editing occurs at specific sites in plastid and plant mitochondrial transcripts. Members of the Arabidopsis pentatricopeptide repeat (PPR) motif-containing protein family and RNA-editing factor Interacting Protein (RIP, also known as MORF) family have been characterized as essential components of the RNA editing apparatus. Recent studies reveal that several organelle-targeted RNA recognition motif (RRM)-containing proteins are involved in either plastid or mitochondrial RNA editing. ORRM1 (Organelle RRM protein 1) is essential for plastid editing, whereas ORRM2, ORRM3 and ORRM4 are involved in mitochondrial RNA editing. The RRM domain of ORRM1, ORRM3 and ORRM4 is required for editing activity, whereas the auxiliary RIP and Glycine-Rich (GR) domains mediate the ORRM proteins' interactions with other editing factors. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome.