Functional characterization of siderophore synthesis in Mycobacterium smegmatis: Identification of new mycobactin synthesis genes

Siderophores are involved in many high affinity iron uptake systems and are virulence or growth factors for many bacteria. Most mycobacteria produce cell associated and excreted forms of the siderophore mycobactin. Two mycobactin mutants were generated through insertional mutagenesis (Zhu, 1999). The mutated gene regions were characterized in this study. One mutant contained an insertion in a gene homologous to mbtE, a known mycobactin synthesis gene in the mbt gene cluster of M. tuberculosis described by Quadri (1998). These cells displayed no mycobactin when analyzed spectrophotometrically or by high performance thin layer chromatography (HPTLC).; The second mycobactin mutant contained an insertion in a gene homologous to fadD33 of M. tuberculosis, a fatty acyl-CoA synthetase gene located between an acyl carrier protein gene and the fatty acyl-CoA dehydrogenase gene, fadE14. Mycobactin preparations from this mutant exhibited an absorbance spectrum similar to previously published mycobactin spectra, however, Rf values less than those for mycobactin were observed on HPTLC. Nuclear magnetic resonance studies demonstrated the absence of a double bond in the alkyl side chain of the mutant mycobactin product. A novel iron chelator containing a para-hydroxy aryl was present in both mutant and wild type extracts. However, because less mycobactin is made by the mutant strain, this form of iron chelator was present in greater amounts in this preparation. Electron paramagnetic resonance analysis confirmed coordinating and relaxation differences between the mutant and wild type mycobactins. Minimal inhibitory concentrations of the iron chelator ethylenediamine-di( o-hydroxyphenylacetic acid (EDDA)), were the same for each mycobactin mutant strain and were much less than that of the wild type strain, Mycobacterium smegmatis, LR222.; The major excreted siderophore from M. smegmatis is exochelin. Exochelin/mycobactin double mutants were constructed by transposon mutagenesis and homologous recombination. Exochelin negative strains were identified on chrome azurol S agar. Growth of double mutant strains was less than that of wild type strains, exochelin mutants and mycobactin mutants in iron restricted conditions. Growth of these strains in iron restricted conditions suggests that mutations in these siderophore systems are not lethal for M. smegmatis and that secondary methods of iron acquisition are capable of supporting growth.