EXOCHELIN GENES IN MYCOBACTERIUM-SMEGMATIS - IDENTIFICATION OF AN ABCTRANSPORTER AND 2 NONRIBOSOMAL PEPTIDE SYNTHETASE GENES

Many strains of mycobacteria produce two ferric chelating substances t hat are termed exochelin (an excreted product) and mycobactin (a cell- associated product). These agents may function as iron acquisition sid erophores. To examine the genetics of the iron acquisition system in m ycobacteria, ultraviolet (UV) and transposon (Tn611) mutagenesis techn iques were used to generate exochelin-deficient mutants of Mycobacteri um smegmatis strains ATCC 607 and LR222 respectively. Mutants were ide ntified on CAS siderophore detection agar plates. Comparisons of the a mounts of GAS-reactive material excreted by the possible mutant strain s with that of the wild-type strains verified that seven UV mutant str ains and two confirmed transposition mutant strains were deficient in exochelin production. Cell-associated mycobactin production in the mut ants appeared to be normal. From the two transposon mutants, the mutat ed gene regions were cloned and identified by colony hybridization wit h an IS6100 probe, and the DNA regions flanking the transposon inserti on sites were then used as probes to clone the wild-type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 k b PstI fragment and a 4.8 kb Pstl/Sacl subclone of this fragment compl emented one transposon mutant (LUN2) and one UV mutant (R92), A 10.1 k b Sad fragment restored exochelin production to the other transposon m utant (LUNI). The nucleotide sequence of the 15.3 kb DNA region that s panned the two transposon insertion sites overlapped the 5' region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orien tation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC, The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displa yed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non-ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non-ribosomal peptide synthetases and th at the ABC transporter ExiT is responsible for exochelin excretion.