Caracterização de um plano plasmídio conjugativo de mycobacterium avium

• Ano de defesa: 2016
• Autor(a) principal: Machado, Gabriel Esquitini [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Conjugativo pma100; Mycobacterium avium; Plasmídio; Origem de replicação; Conjugação genética; Micobactérias não tuberculosas
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=3579620; http://repositorio.unifesp.br/handle/11600/48297

Objective: Characterization of the pMA100 conjugative plasmid from Mycobacterium avium, identifying its replication origin and conjugation mechanism. Material and Methods: To identify and describe the main components of pMA100, assembly and annotation were carried out coupled with bioinformatics analysis and review of literature. Possible regions containing the origin of replication were identified, considering characteristics already described for replication origins of other mycobacterial plasmids, and cloned by the construction of E. coli-Mycobacterium shuttle plasmids. Six recombinant plasmids were constructed, three involving the rep1 gene and different upstream regions, and three involving the rep2 gene and different upstream regions. Rapidly growing mycobacteria (RGM) (M. smegmatis mc2155, M. abscessus ATCC 19977) and slow growing mycobacteria (SGM) (M. bovis BCG Moreau and Pasteur) were transformed with the six recombinant plasmidsconstructed. Additionally, a conjugative mechanism of pMA100 was proposed based on bioinformatics analyses and literature review of its components. Results: Bioinformatics analyses showed that the pMA100 plasmid belongs to a family of SGM plasmids which is characterized by the presence of three conserved regions, related to the conjugation mechanism of this plasmid: one secretion system type 4 (SST4) locus, one secretion system type 7 (SST7) locus and the exonuclease V gene. The existence of a functional replication origin in pMA100 was proven with two constructions involving the rep1 gene: rep1-2539 and rep1-1708. Construction rep1-2539 generated transformant colonies with M. smegmatis mc2155, M. bovis BCG Pasteur and Moreau, while construction rep1-1708 generated transformant colonies only with M. smegmatis mc2155. Conclusions: The pMA100 plasmid has a functional origin of replication in the upstream region of the rep1 gene, and this plasmid conjugation probably occurs by a novel mechanism involving components of SST4, SST7 and exonuclease V.