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Search for conserved sites in Mycobacterium tuberculosis DNA gyrase

D Zygala-Pytlos(1,2) A Minias(1) A Brzostek(1) J Dziadek(1)

1:Laboratory of Genetics and Physiology of Mycobacterium, Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland; 2:The Bio-Med-Chem Doctoral School of the University of Lodz and Lodz Institutes of the Polish Academy of Sciences, University of Lodz, Lodz, Poland

DNA gyrase is an enzyme necessary for the proper functioning of Mycobacterium tuberculosis (Mtb). It is the target of action of quinolones, a group of drugs used in the treatment of tuberculosis. Unfortunately, emerging mutations in the genes encoding subunits of this protein contribute to drug resistance and ineffectiveness of these compounds. The aim of the study is to search for conserved sites in DNA gyrase. These sites could serve as binding sites for new antitubercular compounds, showing greater effectiveness due to the lack of possibility of mutations at the binding site and hence restraining the development of drug resistance. On the basis of bioinformatic analyses, we selected the codons of the gyrA and gyrB genes under strong purifying selection. We chose amino acid substitutions in codons that did not cause deleterious changes in the protein based on the PredictSNP tool prediction. We genetically modified Mtb introducing selected point mutations into the genome. We selected 30 codons for testing. We obtained 18 mutants with point mutations, while in 12 cases, it was not possible to obtain a strain with a mutation in the selected codon. Conservative sites will be confirmed with a second attempt to mutate the site. We found codons that may be highly conserved in Mtb gyrase. These results require further verification. We also found that the majority of sites estimated to be under strong purifying selection by mathematical models are not hyper-conserved. The observable lack of substitutions at these sites in bacterial population remains to be explained.

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