Genomics characterization of the endosymbiont bacterium of the tripanosomatídeo Crithidia deanei and of its DNA topoisomerases
The present work is focused in the endosymbiont type II topoisomerases, due to their essential roles not only in the replication and transcription processes but also at the recombination and chromosome segregation processes. There are two Type II topoisomerases in bacteria: DNA gyrase and DNA Topoisomerase IV. DNA gyrase is the only topoisomerase capable of introducing negative supercoiling in the DNA, while topoisomerase IV is mainly involved in the decatenation of the newly replicated chromosomes during the chromosome segregation. For this study a genomic library from the endosymbiont was generated in pUC18 and 7.500 clones were initially sequenced, with a coverage range of 1.2 Mb. The obtained sequences were analyzed through the BLAST Algorithm. The analysis showed that the endosymbiont genome has a high homology with the genome of bacteria belonging to the Bordetella genus but diverges at their A+T content. The endosymbiont has a 70% A+T content while the Bordetella genus has only a 32% A+T content in their genomes. This strategy allowed us to identify the coding sequences for DNA gyrase subunit A (gyrA) and subunit B (gyrB). We also identified the gene that encodes the topoisomerase III (Topo III), but we were not able to find the genes for topo IV and topo I (topoisomerase I), which altogether with DNA gyrase and topo III compose the whole topoisomerase repertoire found in other bacteria. Based on gyrA and gyrB gene sequences primers were designed for the targets amplification by PCR. The genes gyrA and gyrB were expressed in E. coli. The recombinant proteins were purified for polyclonal antiserum production. Western blot assay identified specific polypeptides with corresponding molecular weights to those predicted form the gene sequences of gyrA and gyrB. Immunofluorescence analysis showed that the antibodies raised against DNA gyrase subunits recognize specific antigens allover the endosymbiont cytoplasm. The gyrA e gyrB genes were also expressed in a baculovirus expression system to obtain soluble recombinant protein. The two subunits were purified in metal affinity matrix (Ni-NTA). This work also allowed us to identify some endosymbiont genes which their products are involved in biosynthetic pathways that provide many metabolites necessary for the host cell, such as that involved in the ornithine metabolism, which has already been identified through biochemical studies.
Advisor:Stenio Perdigão Fragoso
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Crithidia DNA topoisomerases tipo II deanei Genome Topoisomerases Type
Date of Publication:10/03/2006