RESUMO
Promoters of genes encoding superoxide dismutase (sodA) and peptide methionine sulfoxide reductase (msrA) from Cory-nebacterium glutamicum were cloned and sequenced. Promoter region analysis of sodA-msrA was unable to identify putative sites of fixed eventual regulators except for possible sites of fixed OxyR and integra-tion host factor. A study of the regulation of these genes was performed using the lacZ gene of Escherichia coli as a reporter placed under the control of sequences downstream of sodA and msrA. In silico analysis was used to identify regulators in the genome of C. glutamicum, which revealed the absence of homologs of soxRS and arcA and the presence of inactive oxyR and putative candidates of the homologs of ahpC, ohrR, integration host factor, furA, IdeR, diphtheria toxin repressor, and mntR.
Assuntos
Corynebacterium glutamicum/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Metionina Sulfóxido Redutases/genética , Estresse Oxidativo/fisiologia , Superóxido Dismutase/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Corynebacterium glutamicum/metabolismo , Corynebacterium glutamicum/efeitos da radiação , Metionina Sulfóxido Redutases/biossíntese , Estresse Oxidativo/genética , Regiões Promotoras Genéticas , Estresse Fisiológico , Superóxido Dismutase/biossínteseRESUMO
In an attempt to clone the ORF of the nptII gene of Escherichia coli K12 (ATCC 10798), two degenerate primers were designed based on the nptII sequence of its Tn5 transposon. The nptII ORF was placed under the control of the E. coli hybrid trc promoter, in the pKK388-1 vector, transformed into E. coli DH5α ΔrecA (recombinant, deficient strain). Transferred cells were tested for ampicillin, tetracycline, kanamycin, neomycin, geneticin, paromomycin, penicillin, and UV resistance. The neomycin phosphotransferase gene of E. coli was cloned successfully and conferred kanamycin, neomycin, geneticin, and paromomycin resistance to recombinant DH5α; this did not inhibit insertion of additional antibiotic resistance against ampicillin and tetracycline, meaning the trc promoter can express two different genes carried by two different plasmids harbored in the same cell. This resistance conferral process could be considered as an emulation of horizontal gene transfer occurring in nature and would be a useful tool for understanding mechanisms of evolution of multidrug-resistant strains.