BackgroundBacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition.Methods and Principal FindingsHere we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity.ConclusionsWe have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.

Microorganisms produce siderophores to facilitate iron uptake and even though this trait has been extensively studied, there is growing evidence suggesting that siderophores may have other physiological roles aside from iron acquisition. In support of this notion, we previously linked the archetypal siderophore enterobactin with oxidative stress alleviation. To further characterize this association, we studied the sensitivity of Escherichia coli strains lacking different components of the enterobactin system to the classical oxidative stressors hydrogen peroxide and paraquat. We observed that strains impaired in enterobactin production, uptake and hydrolysis were more susceptible to the oxidative damage caused by both compounds than the wild-type strain. In addition, meanwhile iron supplementation had little impact on the sensitivity, the reducing agent ascorbic acid alleviated the oxidative stress and therefore significantly decreased the sensitivity to the stressors. This indicated that the enterobactin-mediated protection is independent of its ability to scavenge iron. Furthermore, enterobactin supplementation conferred resistance to the entE mutant but did not have any protective effect on the fepG and fes mutants. Thus, we inferred that only after enterobactin is hydrolysed by Fes in the cell cytoplasm and iron is released, the free hydroxyl groups are available for radical stabilization. This hypothesis was validated testing the ability of enterobactin to scavenge radicals in vitro. Given the strong connection between enterobactin and oxidative stress, we studied the transcription of the entE gene and the concomitant production of the siderophore in response to such kind of stress. Interestingly, we observed that meanwhile iron represses the expression and production of the siderophore, hydrogen peroxide and paraquat favour these events even if iron is present. Our results support the involvement of enterobactin as part of the oxidative stress response and highlight the existence of a novel regulation mechanism for enterobactin biosynthesis.

Microcin J25 (MccJ25) uptake by Escherichia coli requires the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbD, ExbB, and SbmA. MccJ25 appears to have two intracellular targets: (i) RNA polymerase (RNAP), which has been described in E. coli and Salmonella enterica serovars, and (ii) the respiratory chain, reported only in S. enterica serovars. In the current study, it is shown that the observed difference between the actions of microcin on the respiratory chain in E. coli and S. enterica is due to the relatively low microcin uptake via the chromosomally encoded FhuA. Higher expression by a plasmid-encoded FhuA allowed greater uptake of MccJ25 by E. coli strains and the consequent inhibition of oxygen consumption. The two mechanisms, inhibition of RNAP and oxygen consumption, are independent of each other. Further analysis revealed for the first time that MccJ25 stimulates the production of reactive oxygen species (O 2˙؊ ) in bacterial cells, which could be the main reason for the damage produced on the membrane respiratory chain.MccJ25 is active on gram-negative bacteria related to the producer strain, including some pathogenic strains (43,44,55). Four plasmid genes, mcjABCD, are involved in MccJ25 production: mcjA, mcjB, and mcjC code for an MccJ25 precursor and two processing enzymes required for the in vivo synthesis of the mature peptide, respectively, and mcjD encodes the McjD immunity protein (53). McjD, a homologous ABC exporter family protein, participates in MccJ25 secretion (52). Thus, immunity is mediated by active efflux of the peptide, keeping its intracellular concentration below a critical level (53). Recently, it has been demonstrated that YojI, a chromosomal protein with ABC-type exporter homology (36), is also able to export MccJ25 from the cells (14). TolC, an E. coli outer membrane protein, is necessary for MccJ25 secretion mediated by either McjD or YojI (11,14). On the other hand, the uptake of MccJ25 by E. coli is dependent on the outer membrane receptor FhuA (15, 45) and the four inner membrane proteins TonB, ExbD, ExbB, and SbmA, the first three of which constitute the Ton complex (38), while the last one acts as a transporter (46).Convincing evidence showing that RNA polymerase (RNAP) is the target for MccJ25 action in E. coli was previously provided by our laboratory. The peptide inhibits the enzyme activity by obstructing the secondary channel and consequently preventing access of the substrates to its active sites (1,12,34,57). Later, it was demonstrated that MccJ25 can bind and penetrate into the phospholipid monolayer and disrupt the electric potential of liposomes composed of phospholipids from gram-negative bacteria (5, 40). These results encouraged the study of the effect of MccJ25 on the bacterial membrane. MccJ25 was found to disrupt the membrane potential inhibiting oxygen consumption in Salmonella enterica serovar Newport (41) and S. enterica serovar Typhimurium transformed with a plasmid carrying fhuA from E. coli (55), suggesting the presence of a se...