Analysis of genes involved in siderophore-mediated iron uptake in mycobacteria

Abstract

This work was aimed at identifying genes involved in iron sequestration by mycobacteria and focused on the pathway of iron uptake by the mycobacterial siderophores. On blue chrome azural S (CAS) indicator plates, the secretion of exochelin generates a yellow halo around mycobacteria colonies. Six thousand chemically mutagenized colonies of Mycobacterium smegmatis {dollar}\rm mc\sp2155{dollar} were screened and nineteen mutants were identified that lost the ability to produce halos on CAS plates. Thirteen of these mutants were complemented by a single cosmid. Sequence analysis of this cosmid revealed nine open reading frames (ORFs); many were homologous to transporter proteins.;In previous work, the fxbA gene was identified and proven to be involved in exochelin biosynthesis. The fxbA, fxbB, and fxbC genes are in the same region of M. smegmatis chromosomal DNA. Primer extension analysis has been used to map the transcription start site of the exochelin biosynthesis gene fxbA. A translational fusion of fxbA with {dollar}\beta{dollar}-galactosidase was used to demonstrate iron regulation of the gene. In the fxbA promoter region, two sequences that have homology to the Gram-positive DtxR iron box were identified.;In the process of screening exochelin deficient mutants in M. smegmatis, we identified two mutants which failed to grow on CAS and made larger halos on CAS plates. Complementation analysis using a Mycobacterium bovis BCG cosmid library showed that these mutants are biotin auxotrophs and have mutations on the bioA and bioF gene.;Transposon mutagenesis was performed on Mycobacterium tuberculosis and siderophore deficient mutants were screened on CAS medium. A library of 8,000 M. tuberculosis transposon mutants was generated. Southern analysis of chromosomal DNA showed that the transposon insertions are random. All of the transposon mutants were screened on CAS plates for iron uptake defective (halo loss) mutants and more than 50 colonies showed altered halo phenotypes. The chromosomal DNA adjacent to the transposon insertions was sequenced. Sequence analysis demonstrated that two mutants, {dollar}\rm mc\sp23014{dollar} and {dollar}\rm mc\sp23019,{dollar} have the transposon inserted in separate regions of a large gene that has homology to polyketide biosynthesis genes. The remaining mutants have the transposon inserted in M. tuberculosis homologues of thiosulfate sulfur transferase, imidazole glycerol phosphate dehydratase, and alcohol dehydrogenase respectively. (Abstract shortened by UMI.).