RESUMO
Chromosomally acquired streptomycin resistance is frequently due to mutations in the gene encoding the ribosomal protein S12, rpsL. The presence of several rRNA operons (rrn) and a single rpsL gene in most bacterial genomes prohibits the isolation of streptomycin-resistant mutants in which resistance is mediated by mutations in the 16S rRNA gene (rrs). Three strains were constructed in this investigation: Mycobacterium smegmatis rrnB, M. smegmatis rpsL(3+), and M. smegmatis rrnB rpsL(3+). M. smegmatis rrnB carries a single functional rrn operon, i.e., rrnA (comprised of 16S, 23S, and 5S rRNA genes) and a single rpsL+ gene; M. smegmatis rpsL(3+) is characterized by the presence of two rrn operons (rrnA and rrnB) and three rpsL+ genes; and M. smegmatis rrnB rpsL(3+) carries a single functional rrn operon (rrnA) and three rpsL+ genes. By genetically altering the number of rpsL and rrs alleles in the bacterial genome, mutations in rrs conferring streptomycin resistance could be selected, as revealed by analysis of streptomycin-resistant derivatives of M. smegmatis rrnB rpsL(3+). Besides mutations well known to confer streptomycin resistance, novel streptomycin resistance conferring mutations were isolated. Most of the mutations were found to map to a functional pseudoknot structure within the 530 loop region of the 16S rRNA. One of the mutations observed, i.e., 524G-->C, severely distorts the interaction between nucleotides 524G and 507C, a Watson-Crick interaction which has been thought to be essential for ribosome function. The use of the single rRNA allelic M. smegmatis strain should help to elucidate the principles of ribosome-drug interactions.
Assuntos
Farmacorresistência Bacteriana/genética , Mycobacterium smegmatis/genética , RNA Ribossômico 16S/genética , Estreptomicina/farmacologia , Alelos , Antibacterianos/farmacologia , DNA Bacteriano/análise , Mutação , Mycobacterium smegmatis/efeitos dos fármacos , Conformação de Ácido Nucleico , RNA Ribossômico 16S/químicaRESUMO
Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxifies reactive oxygen species, and DNA repair systems, which repair damage resulting from oxidative stress. To (i) determine the relative importance of the DNA repair system when oxidative stress is encountered by the Mycobacterium tuberculosis complex during infection of the host and to (ii) provide improved mycobacterial hosts as live carriers to express foreign antigens, the recA locus was inactivated by allelic exchange in Mycobacterium bovis BCG. The recA mutants are sensitive to DNA-damaging agents and show increased susceptibility to metronidazole, the first lead compound active against the dormant M. tuberculosis complex. Surprisingly, the recA genotype does not affect the in vitro dormancy response, nor does the defect in the DNA repair system lead to attenuation as determined in a mouse infection model. The recA mutants will be a valuable tool for further development of BCG as an antigen delivery system to express foreign antigens and as a source of a genetically stable vaccine against tuberculosis.
Assuntos
Alquilantes/farmacologia , Deleção de Genes , Mycobacterium bovis , Recombinases Rec A/genética , Tuberculose/microbiologia , Animais , Antibacterianos/farmacologia , Western Blotting , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Resistência Microbiana a Medicamentos , Metronidazol/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/genética , Mycobacterium bovis/fisiologia , Mycobacterium bovis/efeitos da radiação , Recombinases Rec A/metabolismo , Proteínas Ribossômicas/genética , Transformação Bacteriana , Raios Ultravioleta , VirulênciaRESUMO
Integrative vectors expressing foreign genes are used as tools for the development of recombinant vaccines in mycobacteria since it is assumed that these vectors are stably maintained even without antibiotic selection. We here demonstrate that integration-proficient vectors are lost from the mycobacterial genome in high frequency. Loss of integrated vectors occurred in recA+ and in recA-strains, indicating a RecA-independent mechanism. Loss of the integrated vector was prevented when integrase gene function was carried on a separate plasmid that is unable to replicate in mycobacteria, indicating that excision is a function of integrase. By providing attP in cis and integrase function in trans, vectors integrating at the attB site are stably maintained, even when carrying genes that deleteriously affect the host.
