RESUMO
Using a transposon mutagenesis approach, we have identified a mutant of Burkholderia pseudomallei that is auxotrophic for branched chain amino acids. The transposon was shown to have interrupted the ilvI gene encoding the large subunit of the acetolactate synthase enzyme. Compared to the wild type, this mutant was significantly attenuated in a murine model of disease. Mice inoculated intraperitoneally with the auxotrophic mutant, 35 days prior to challenge, were protected against a challenge dose of 6,000 median lethal doses of wild-type B. pseudomallei.
Assuntos
Aminoácidos de Cadeia Ramificada/biossíntese , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Melioidose/etiologia , Animais , Burkholderia pseudomallei/imunologia , Burkholderia pseudomallei/patogenicidade , Feminino , Genótipo , Melioidose/imunologia , Melioidose/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Mutação , FenótipoAssuntos
Proteínas de Bactérias/genética , Francisella tularensis/genética , Genoma Bacteriano , Análise de Sequência de DNA , Aminoácidos/biossíntese , Proteínas de Bactérias/metabolismo , Biologia Computacional , DNA Bacteriano/genética , Biblioteca Gênica , Genes Bacterianos , Humanos , Fases de Leitura Aberta , Purinas/biossínteseRESUMO
Francisella tularensis is the etiological agent of tularemia, a serious disease in several Northern hemisphere countries. The organism has fastidious growth requirements and is very poorly understood at the genetic and molecular levels. Given the lack of data on this organism, we undertook the sample sequencing of its genome. A random library of DNA fragments from a highly virulent strain (Schu 4) of F. tularensis was constructed and the nucleotide sequences of 13,904 cloned fragments were determined and assembled into 353 contigs. A total of 1.83 Mb of nucleotide sequence was obtained that had a G+C content of 33.2%. Genes located on plasmids pOM1 and pNFL10, which had been previously isolated from low virulence strains of F. tularensis, were absent but all of the other known F. tularensis genes were represented in the assembled data. F. tularensis Schu4 was able to grow in the absence of aromatic amino acids and orthologues of genes which could encode enzymes in the shikimate pathway in other bacteria were identified in the assembled data. Genes that could encode all of the enzymes in the purine biosynthetic and most of the en- zymes in the purine salvage pathways were also identified. This data will be used to develop defined rationally attenuated mutants of F. tularensis, which could be used as replacements for the existing genetically undefined live vaccine strain.
