Characterization of MbaS, an iron responsive sigma factor of Burkholderia pseudomallei

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Hauglund, Melissa J.
Oregon Health & Science University
Gram-negative bacteria; Melioidosis; Siderophores; Bacterial proteins; Protein engineering; Burkholderia pseudomallei; Sigma Factor; Transcription Initiation Site; Bacterial Proteins; Amino Acid Substitution
thesis / dissertation description
Burkholderia pseudomallei, a Gram-negative saprophyte endemic to Southeast Asia and Northern Australia, is the causative agent of melioidosis. Although numerous studies were initiated to identify and characterize virulence mechanisms utilized by this human pathogen, characterization of the iron acquisition systems is still in its infancy. Presently, only the malleobactin siderophore system of B. pseudomallei has been described. The work presented herein is a genetic characterization of the iron responsive sigma factor MbaS. We generated a two-plasmid system for screening the functionality of MbaS when expressed in E. coli and a second single plasmid system for the identification of functional promoter elements and translation initiation. We demonstrated that an ATG codon located 90 nucleotides upstream of the annotated start codon is required for expression from the mbaS promoter and that this upstream promoter region, which contains a putative Fur binding site, is responsive to iron.