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Burkholderia is an important bacterial genus containing species of ecological, biotechnological and pathogenic interest which are widely distributed in the natural environment. This study describes the development of molecular methods to identify Burkholderia and also examines the genetic basis for their tolerance to halogenated pollutants. The recA gene is a useful target for speciation of B. cepacia complex bacteria. Novel PCR primers were evaluated by testing their specificity against a panel of Burkholderia species. Nucleotide sequencing and phylogenetic analysis of 869 bp of the recA gene differentiated between putative and known Burkholderia species, including the B. cepacia complex. A genus-specific recA PCR yielding 385 bp was also able to identify all Burkholderia examined. Phylogenetic analysis of 188 novel recA genes clarified the taxonomic position of several important Burkholderia strains, revealing the presence of four novel B. cepacia complex recA lineages. Although the recA phylogeny could not differentiate B. cepacia complex strains recovered from clinical infection versus the natural environment, it facilitated the identification of clonal strain types of from both niches. A rec/4-based cultivation-independent approach was used to examine Burkholderia diversity associated with the maize rhizosphere and cord forming fungi. Robust sequence datasets were created from the 869 bp recA fragments and screened to identify recA phylotypes matching B. cepacia complex species previously cultivated from the maize samples. Burkholderia related to known species as well as novel phylogenies were identified from the maize and fungal samples in both cases the phylogenetic resolution of recA was sufficient to distinguish all individual species. Transposon mutagenesis of B. vietnamiensis G4 was used to elucidate the genetic basis of resistance to halogenated phenol derivatives. Mutations resulting in increased susceptibility to 2,4-dichlorophenol included genes encoding enolase, toluene tolerance, regulators and a lipoprotein. Gene complementation restored the resistant phenotype of two mutants, a GTP-binding protein and a methyltransferase gene. |
