A small RNA promotes siderophore production through transcriptional and metabolic remodeling.
ABSTRACT: Siderophores are essential factors for iron (Fe) acquisition in bacteria during colonization and infection of eukaryotic hosts, which restrain iron access through iron-binding protein, such as lactoferrin and transferrin. The synthesis of siderophores by Escherichia coli is considered to be fully regulated at the transcriptional level by the Fe-responsive transcriptional repressor Fur. Here we characterized two different pathways that promote the production of the siderophore enterobactin via the action of the small RNA RyhB. First, RyhB is required for normal expression of an important enterobactin biosynthesis polycistron, entCEBAH. Second, RyhB directly represses the translation of cysE, which encodes a serine acetyltransferase that uses serine as a substrate for cysteine biosynthesis. Reduction of CysE activity by RyhB allows serine to be used as building blocks for enterobactin synthesis through the nonribosomal peptide synthesis pathway. Thus, RyhB plays an essential role in siderophore production and may modulate bacterial virulence through optimization of siderophore production.
Project description:<h4>Background</h4>Iron is essential for bacterial survival. Bacterial siderophores are small molecules with unmatched capacity to scavenge iron from proteins and the extracellular milieu, where it mostly occurs as insoluble Fe<sup>3+</sup>. Siderophores chelate Fe<sup>3+</sup> for uptake into the cell, where it is reduced to soluble Fe<sup>2+</sup>. Siderophores are key molecules in low soluble iron conditions. The ability of bacteria to synthesize proprietary siderophores may have increased bacterial evolutionary fitness; one way that bacteria diversify siderophore structure is by incorporating different polyamine backbones while maintaining the catechol moieties.<h4>Results</h4>We report that Serratia plymuthica V4 produces a variety of siderophores, which we term the siderome, and which are assembled by the concerted action of enzymes encoded in two independent gene clusters. Besides assembling serratiochelin A and B with diaminopropane, S. plymuthica utilizes putrescine and the same set of enzymes to assemble photobactin, a siderophore found in the bacterium Photorhabdus luminescens. The enzymes encoded by one of the gene clusters can independently assemble enterobactin. A third, independent operon is responsible for biosynthesis of the hydroxamate siderophore aerobactin, initially described in Enterobacter aerogenes. Mutant strains not synthesizing polyamine-siderophores significantly increased enterobactin production levels, though lack of enterobactin did not impact the production of serratiochelins. Knocking out SchF0, an enzyme involved in the assembly of enterobactin alone, significantly reduced bacterial fitness.<h4>Conclusions</h4>This study shows the natural occurrence of serratiochelins, photobactin, enterobactin, and aerobactin in a single bacterial species and illuminates the interplay between siderophore biosynthetic pathways and polyamine production, indicating routes of molecular diversification. Given its natural yields of diaminopropane (97.75??mol/g DW) and putrescine (30.83??mol/g DW), S. plymuthica can be exploited for the industrial production of these compounds.
Project description:<h4>Background</h4>Bacteria 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.<h4>Methods and principal findings</h4>Here 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.<h4>Conclusions</h4>We 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.
Project description:Siderophores, small iron-binding molecules secreted by many microbial species, capture environmental iron for transport back into the cell. Vibrio cholerae synthesizes and uses the catechol siderophore vibriobactin and also uses siderophores secreted by other species, including enterobactin produced by Escherichia coli. E. coli secretes both canonical cyclic enterobactin and linear enterobactin derivatives likely derived from its cleavage by the enterobactin esterase Fes. We show here that V. cholerae does not use cyclic enterobactin but instead uses its linear derivatives. V. cholerae lacked both a receptor for efficient transport of cyclic enterobactin and enterobactin esterase to promote removal of iron from the ferrisiderophore complex. To further characterize the transport of catechol siderophores, we show that the linear enterobactin derivatives were transported into V. cholerae by either of the catechol siderophore receptors IrgA and VctA, which also transported the synthetic siderophore MECAM [1,3,5-N,N',N?-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene]. Vibriobactin is transported via the additional catechol siderophore receptor ViuA, while the Vibrio fluvialis siderophore fluvibactin was transported by all three catechol receptors. ViuB, a putative V. cholerae siderophore-interacting protein (SIP), functionally substituted for the E. coli ferric reductase YqjH, which promotes the release of iron from the siderophore in the bacterial cytoplasm. In V. cholerae, ViuB was required for the use of vibriobactin but was not required for the use of MECAM, fluvibactin, ferrichrome, or the linear derivatives of enterobactin. This suggests the presence of another protein in V. cholerae capable of promoting the release of iron from these siderophores.Vibrio cholerae is a major human pathogen and also serves as a model for the Vibrionaceae, which include other serious human and fish pathogens. The ability of these species to persist and acquire essential nutrients, including iron, in the environment is epidemiologically important but not well understood. In this work, we characterize the ability of V. cholerae to acquire iron by using siderophores produced by other organisms. We resolve confusion in the literature regarding its ability to use the Escherichia coli siderophore enterobactin and identify the receptor and TonB system used for the transport of several siderophores. The use of some siderophores did not require the ferric reductase ViuB, suggesting that an uncharacterized ferric reductase is present in V. cholerae.
Project description:In Escherichia coli, the small regulatory noncoding RNA (sRNA) RyhB and the global ferric uptake regulator (Fur) mediate iron acquisition and storage control. Iron is both essential and potentially toxic for most living organisms, making the precise maintenance of iron homeostasis necessary for survival. While the roles of these regulators in iron homeostasis have been well studied in a nonpathogenic E. coli strain, their impact on the production of virulence-associated factors is still unknown for a pathogenic E. coli strain. We thus investigated the roles of RyhB and Fur in iron homeostasis and virulence of the uropathogenic E. coli (UPEC) strain CFT073. In a murine model of urinary tract infection (UTI), deletion of fur alone did not attenuate virulence, whereas a ?ryhB mutant and a ?fur ?ryhB double mutant showed significantly reduced bladder colonization. The ?fur mutant was more sensitive to oxidative stress and produced more of the siderophores enterobactin, salmochelins, and aerobactin than the wild-type strain. In contrast, while RyhB was not implicated in oxidative stress resistance, the ?ryhB mutant produced lower levels of siderophores. This decrease was correlated with the downregulation of shiA (encoding a transporter of shikimate, a precursor of enterobactin and salmochelin biosynthesis) and iucD (involved in aerobactin biosynthesis) in this mutant grown in minimal medium or in human urine. iucD was also downregulated in bladders infected with the ?ryhB mutant compared to those infected with the wild-type strain. Our results thus demonstrate that the sRNA RyhB is involved in production of iron acquisition systems and colonization of the urinary tract by pathogenic E. coli.
Project description:Bacteria have aggressive acquisition processes for iron, an essential nutrient. Siderophores are small iron chelators that facilitate cellular iron transport. The siderophore enterobactin is a triscatechol derivative of a cyclic triserine lactone. Studies of the chemistry, regulation, synthesis, recognition, and transport of enterobactin make it perhaps the best understood of the siderophore-mediated iron uptake systems, displaying a lot of function packed into this small molecule. However, recent surprises include the isolation of corynebactin, a closely related trithreonine triscatechol derivative lactone first found in Gram-positive bacteria, and the crystal structure of a ferric enterobactin complex of a protein identified as an antibacterial component of the human innate immune system.
Project description:When iron is scarce, Bacillus subtilis expresses genes involved in the synthesis and uptake of the siderophore bacillibactin (BB) and uptake systems to pirate other microbial siderophores. Here, we demonstrate that transcriptional induction of the feuABCybbA operon, encoding the Fe-BB uptake system, is mediated by Btr (formerly YbbB), which is encoded by the immediately upstream gene. Btr contains an AraC-type DNA binding domain fused to a substrate binding protein (SBP) domain related to FeuA, the SBP for Fe-BB uptake. When cells are iron-limited, the Fur-mediated repression of btr is relieved and Btr binds to a conserved direct repeat sequence adjacent to feuA to activate transcription. If BB is present, Btr further activates feuA expression. Btr binds with high affinity to both apo-BB and Fe-BB, and the resulting complex displays a significantly increased efficacy as a transcriptional activator relative to Btr alone. Btr can also activate transcription in response to the structurally similar siderophore enterobactin, although genetic analyses indicate that the two siderophores make distinct interactions with the Btr substrate binding domain. Thus, the FeuABC transporter is optimally expressed under conditions of iron starvation, when Fur-mediated repression is relieved, and in the presence of its cognate substrate.
Project description:The secretion of small Fe-binding molecules called siderophores is an important microbial strategy for survival in Fe-limited environments. Siderophore production is often regulated by quorum sensing (QS), a microbial counting technique that allows organisms to alter gene expression based on cell density. However, the identity and quantities of siderophores produced under QS regulation are rarely studied in the context of their roles in Fe uptake. We investigated the link between QS, siderophores, and Fe uptake in the model marine organism <i>Vibrio harveyi</i> where QS is thought to repress siderophore production. We find that <i>V. harveyi</i> uses a single QS- and Fe-repressed gene cluster to produce both cell-associated siderophores (amphiphilic enterobactins) as well as several related soluble siderophores, which we identify and quantify using liquid chromatography-coupled (LC)-MS as well as tandem high-resolution MS (LC-HR-MS/MS). Measurements of siderophore production show that soluble siderophores are present at ?100× higher concentrations than amphi-enterobactin and that over the course of growth <i>V. harveyi</i> decreases amphi-enterobactin concentrations but accumulates soluble siderophores. <sup>55</sup>Fe radio-tracer uptake experiments demonstrate that these soluble siderophores play a significant role in Fe uptake and that the QS-dictated concentrations of soluble siderophores in stationary phase are near the limit of cellular uptake capacities. We propose that cell-associated and soluble siderophores are beneficial to <i>V. harveyi</i> in different environmental and growth contexts and that QS allows <i>V. harveyi</i> to exploit "knowledge" of its population size to avoid unnecessary siderophore production.
Project description:Bordetella pertussis is the causative agent of whooping cough. This pathogenic bacterium can obtain the essential nutrient iron using its native alcaligin siderophore and by utilizing xeno-siderophores such as desferrioxamine B, ferrichrome, and enterobactin. Previous genome-wide expression profiling identified an iron repressible B. pertussis gene encoding a periplasmic protein (FbpABp). A previously reported crystal structure shows significant similarity between FbpABp and previously characterized bacterial iron binding proteins, and established its iron-binding ability. Bordetella growth studies determined that FbpABp was required for utilization of not only unchelated iron, but also utilization of iron bound to both native and xeno-siderophores. In this in vitro solution study, we quantified the binding of unchelated ferric iron to FbpABp in the presence of various anions and importantly, we demonstrated that FbpABp binds all the ferric siderophores tested (native and xeno) with ?M affinity. In silico modeling augmented solution data. FbpABp was incapable of iron removal from ferric xeno-siderophores in vitro. However, when FbpABp was reacted with native ferric-alcaligin, it elicited a pronounced change in the iron coordination environment, which may signify an early step in FbpABp-mediated iron removal from the native siderophore. To our knowledge, this is the first time the periplasmic component of an iron uptake system has been shown to bind iron directly as Fe(3+) and indirectly as a ferric siderophore complex.
Project description:The molecular mechanisms that define asymptomatic bacteriuria (ABU) Escherichia coli colonization of the human urinary tract remain to be properly elucidated. Here, we utilize ABU E. coli strain 83972 as a model to dissect the contribution of siderophores to iron acquisition, growth, fitness, and colonization of the urinary tract. We show that E. coli 83972 produces enterobactin, salmochelin, aerobactin, and yersiniabactin and examine the role of these systems using mutants defective in siderophore biosynthesis and uptake. Enterobactin and aerobactin contributed most to total siderophore activity and growth in defined iron-deficient medium. No siderophores were detected in an 83972 quadruple mutant deficient in all four siderophore biosynthesis pathways; this mutant did not grow in defined iron-deficient medium but grew in iron-limited pooled human urine due to iron uptake via the FecA ferric citrate receptor. In a mixed 1:1 growth assay with strain 83972, there was no fitness disadvantage of the 83972 quadruple biosynthetic mutant, demonstrating its capacity to act as a "cheater" and utilize siderophores produced by the wild-type strain for iron uptake. An 83972 enterobactin/salmochelin double receptor mutant was outcompeted by 83972 in human urine and the mouse urinary tract, indicating a role for catecholate receptors in urinary tract colonization.
Project description:Iron acquisition systems are essential for the in vivo growth of bacterial pathogens. Despite the epidemiological importance of Klebsiella pneumoniae, few experiments have examined the importance of siderophores in the pathogenesis of this species. A previously reported signature-tagged mutagenesis screen identified an attenuated strain that featured an insertional disruption in ybtQ, which encodes a transporter for the siderophore yersiniabactin. We used this finding as a starting point to evaluate the importance of siderophores in the physiology and pathogenesis of K. pneumoniae. Isogenic strains carrying in-frame deletions in genes required for the synthesis of either enterobactin or yersiniabactin were constructed, and the growth of these mutants was examined both in vitro and in vivo using an intranasal infection model. The results suggest divergent functions for each siderophore in different environments, with enterobactin being more important for growth in vitro under iron limitation than in vivo and the reverse being true for the yersiniabactin locus. These observations represent the first examination of isogenic mutants in iron acquisition systems for K. pneumoniae and may indicate that the acquisition of nonenterobactin siderophores is an important step in the evolution of virulent enterobacterial strains.