Genetic and structural analysis of the Legionella pneumophila siderophore, legiobactin.

摘要

Under iron stress, Legionella pneumophila, the etiologic agent of Legionnaires. Disease, secretes legiobactin, a hydrophilic, nonclassical siderophore that is reactive in the chrome azurol S (CAS) assay. My work has led to the identification of two genes involved in the expression of legiobactin. The two genes, designated as lbtAB, are in an operon that appears to have a Fur-regulated promoter. The protein product of lbtA has significant homology to hydroxamate synthetases and FrgA, a previously described iron-regulated protein important for L. pneumophila growth in macrophages. lbtB encodes a protein homologous to members of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtA were defective for legiobactin, consistently producing 40-70% less CAS-reactivity in low iron chemically defined medium (CDM) than wildtype. Unlike wildtype, lbtA mutant CDM culture supernatants did not support growth of iron-limited wildtype bacteria in 2'2'-dipyridyl-containing buffered charcoal yeast extract (BCYE) agar and a ferrous iron transport mutant (feoB) on BCYE agar without added iron. Mutants lacking lbtB similarly secreted 40-70% less CAS reactivity than wildtype, presumably due to their siderophore export defect. Interestingly, while lbtB mutant supernatants did not support growth of feoB, the lbtB mutants could promote growth when plated on top of the feoB indicator strain, likely due to legiobactin release upon cell lysis. Complementation of the lbt mutants restored siderophore expression as measured by the CAS assay and bioassays. The lbtA mutant, albeit defective for siderophore production, replicated as wildtype did in macrophages and amoebae. However, since it is possible that legiobactin is a redundant iron uptake determinant, the siderophore may still be important for L. pneumophila iron acquisition in the host or the environment. Legiobactin was purified and demonstrated to be the growth-promoting element in CAS-reactive supernatants. Legiobactin was shown to bind iron and it likely does this through its supposed carbonyl oxygens. Although the presence of an amide bond formed by LbtA has yet to be demonstrated, it is probable that this type of bond is utilized in the formation of legiobactin. Legiobactin may be similar in structure to other carboxylate type siderophores such as staphyloferrins A and B, rhizoferrin or rhizobactin.