Mutational Analysis of a Role for Salicylic Acid in Iron Metabolism of Mycobacterium smegmatis

Abstract

The role of salicylic acid in iron metabolism was examined in two wild-type strains (mc 2 155 and NCIMB 8548) and three mutant strains (mc 2 1292 [lacking exochelin], SM3 [lacking iron-dependent repressor protein IdeR] and S99 [a salicylate-requiring auxotroph derived in this study]) of Mycobacterium smegmatis. Synthesis of salicylate in SM3 was derepressed even in the presence of iron, as was synthesis of the siderophores exochelin, mycobactin, and carboxymycobactin. S99 was dependent on salicylate for growth and failed to grow with the three ferrisiderophores, suggesting that salicylate fulfills an additional function(s) other than being a precursor of mycobactin and carboxymycobactin. Salicylic acid at 100 g/ml repressed the formation of a 29-kDa cell envelope protein (putative exochelin receptor protein) in S99 grown both iron deficiently and iron sufficiently. In contrast, synthesis of this protein was affected only under iron-limited conditions in the parent strain, mc 2 155, and remained unaltered in SM3, suggesting an interaction between the IdeR protein and salicylate. Thus, salicylate may also function as a signal molecule for recognition of cellular iron status. Growth of all strains and mutants with p-aminosalicylate (PAS) at 100 g/ml increased salicylate accumulation between three-and eightfold under both iron-limited and iron-sufficient growth conditions and decreased mycobactin accumulation by 40 to 80% but increased carboxymycobactin accumulation by 50 to 55%. Thus, although PAS inhibited salicylate conversion to mycobactin, presumptively by blocking salicylate AMP kinase, PAS also interferes with the additional functions of salicylate, as its effect was heightened in S99 when the salicylate concentration was minimal.Mycobacterium smegmatis, a saprophytic mycobacterium, secretes two extracellular siderophores (exochelin and carboxymycobactin) and has an intracellular siderophore termed mycobactin (28, 30). De Voss et al. (8) demonstrated the importance of the siderophores mycobactin and carboxymycobactin in M. tuberculosis by showing that a mutant incapable of producing these two siderophores failed to grow within macrophages. Carboxymycobactin is structurally related to mycobactin but is probably not derived from it (2). While carboxymycobactin is the sole extracellular siderophore of pathogenic mycobacteria (30), it is only a minor one in saprophytic mycobacteria, which use exochelin as the major siderophore for iron acquisition (28). Salicylic acid is produced extracellularly by most mycobacteria in greatly increased quantities (up to 17 g/ml) when grown under iron-deficient conditions (28, 33) as opposed to iron-sufficient conditions. Although salicylate is a precursor of mycobactin (31), mycobactin could not spare the requirement for salicylate in a salicylate-requiring auxotrophic mutant of M. smegmatis (32), suggesting that salicylate fulfills a second but unknown role in this bacterium.Salicylate and its derivatives are important as analgesics, antipyretics, anticoagulants, and anti-infl...

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