Caracterização de um plano plasmídio conjugativo de mycobacterium avium

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.Objetivo: Caracterização do plasmídio conjugativo pMA100 de Mycobacterium avium, identificando sua origem de replicação e mecanismo de conjugação. Material e Métodos: A montagem e anotação de pMA100 foram realizadas aliadas a uma análise bioinformática e revisão da literatura para identificar e descrever os principais componentes desse plasmidio. Possíveis regiões contendo a origem de replicação de pMA100 foram identificadas, considerando as características de origens de replicação já descritas para outros plasmídios de micobactérias, e clonadas mediante a construção de plasmídios shuttle E. coli-Mycobacterium. Seis plasmídios recombinantes foram construídos, três envolvendo o gene rep1 e diferentes regiões a montante deste gene, e outros três envolvendo o gene rep2 e diferentes regiões a montante deste gene. Essas construções foram utilizadas na transformação de micobactérias de crescimento rápido (MCR) (M. smegmatis mc2155, M. abscessus ATCC 19977) e micobatérias de crescimento lento (MCL) (M. bovis BCG Moreau e Pasteur). Adicionalmente, um mecanismo de conjugação foi proposto com base na análise bioinformática de pMA100 e revisão da literatura sobre seus componentes. Resultados: A análise bioinformática revelou que o plasmídio pMA100 pertence a um grupo de plasmídios de MCL que se caracteriza pela presença de três regiões conservadas, relacionadas ao mecanismo de conjugação deste plasmídio: um locus do sistema de secreção tipo 4 (SST4), um locus do sistema de secreção tipo 7 (SST7) e um gene de exonuclease V. A existência de uma origem de replicação de pMA100 funcional em micobactérias foi comprovada em duas construções envolvendo o gene rep1: rep1-2539 e rep1-1708. A construção rep1-2539 gerou colônias transformantes em M. smegmatis mc2155, M. bovis BCG Moreau e Pasteur, enquanto que a construção rep1-1708 gerou colônias transformantes somente em M. smegmatis mc2155. Conclusões: O plasmídio pMA100 possui uma origem de replicação funcional na região a montante do gene rep1, e o processo de conjugação deste plasmídio possivelmente ocorre por um mecanismo inédito, envolvendo componentes do SST4, SST7 e a proteína exonuclease V.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Characterization of mycobacteria and mycobacteriophages isolated from compost at the São Paulo Zoo Park Foundation in Brazil and creation of the new mycobacteriophage Cluster U

Abstract Background A large collection of sequenced mycobacteriophages capable of infecting a single host strain of Mycobacterium smegmatis shows considerable genomic diversity with dozens of distinctive types (clusters) and extensive variation within those sharing evident nucleotide sequence similarity. Here we profiled the mycobacterial components of a large composting system at the São Paulo zoo. Results We isolated and sequenced eight mycobacteriophages using Mycobacterium smegmatis mc2155 as a host. None of these eight phages infected any of mycobacterial strains isolated from the same materials. The phage isolates span considerable genomic diversity, including two phages (Barriga, Nhonho) related to Subcluster A1 phages, two Cluster B phages (Pops, Subcluster B1; Godines, Subcluster B2), three Subcluster F1 phages (Florinda, Girafales, and Quico), and Madruga, a relative of phage Patience with which it constitutes the new Cluster U. Interestingly, the two Subcluster A1 phages and the three Subcluster F1 phages have genomic relationships indicating relatively recent evolution within a geographically isolated niche in the composting system. Conclusions We predict that composting systems such as those used to obtain these mycobacteriophages will be a rich source for the isolation of additional phages that will expand our view of bacteriophage diversity and evolution