Identification of a system for hydroxamate xenosiderophore-mediated iron transport in Burkholderia cenocepacia.

One of the mechanisms employed by the opportunistic pathogen Burkholderia cenocepacia to acquire the essential element iron is the production and release of two ferric iron chelating compounds (siderophores), ornibactin and pyochelin. Here we show that B. cenocepacia is also able to take advantage of a range of siderophores produced by other bacteria and fungi ('xenosiderophores') that chelate iron exclusively by means of hydroxamate groups. These include the tris-hydroxamate siderophores ferrioxamine B, ferrichrome, ferricrocin and triacetylfusarinine C, the bis-hydroxamates alcaligin and rhodotorulic acid, and the monohydroxamate siderophore cepabactin. We also show that of the 24 TonB-dependent transporters encoded by the B. cenocepacia genome, two (FhuA and FeuA) are involved in the uptake of hydroxamate xenosiderophores, with FhuA serving as the exclusive transporter of iron-loaded ferrioxamine B, triacetylfusarinine C, alcaligin and rhodotorulic acid, while both FhuA and FeuA are able to translocate ferrichrome-type siderophores across the outer membrane. Finally, we identified FhuB, a putative cytoplasmic membrane-anchored ferric-siderophore reductase, as being obligatory for utilization of all of the tested bis- and tris-hydroxamate xenosiderophores apart from alcaligin.

The Bradyrhizobium japonicum exporter ExsFGH is involved in efflux of ferric xenosiderophores from the periplasm.

The gram-negative bacterium Bradyrhizobium japonicum can take up structurally dissimilar ferric siderophores from the environment (xenosiderophores) to meet its nutritional iron requirements. Siderophore-bound iron transported into the periplasm is reduced to the ferrous form by FsrB, dissociated from the siderophore and the free ion is then transported into the cytoplasm by the ferrous iron transporter FeoAB. Here, we identified the RND family exporter genes exsFG and exsH in a selection for secondary site suppressor mutants that restore growth of an fsrB mutant on the siderophores ferrichrome or ferrioxamine. The low level of radiolabel accumulation from 55Fe-labeled ferrichrome or ferrioxamine observed in the fsrB mutant was restored to wild type levels in the fsrB exsG mutant. Moreover, the exsG mutant accumulated more radiolabel from the 55Fe-labeled siderophores than the wild type, but radiolabel accumulation from inorganic 55Fe was similar in the two strains. Thus, ExsFGH exports siderophore-bound iron, but not inorganic iron. The rescued fsrB exsG mutant required feoB for growth, indicating that ExsFGH acts on those siderophores in the periplasm. The exsG mutant was more sensitive to the siderophore antibiotic albomycin than the wild type, whereas the fsrB mutant was more resistant. This suggests ExsFGH normally exports ferrated albomycin. B. japonicum is naturally resistant to many antibiotics. The exsG strain was very sensitive to tetracycline, but not to six other antibiotics tested. We conclude that ExsFGH is a broad substrate exporter that is needed to maintain siderophore homeostasis in the periplasm.

Clostridioides difficile utilizes siderophores as an iron source and FhuDBGC contributes to ferrichrome uptake.

IMPORTANCE: This study is the first example of C. difficile growing with siderophores as the sole iron source and describes the characterization of the ferric hydroxamate uptake ABC transporter (FhuDBGC). This transporter shows specificity to the siderophore ferrichrome. While not required for pathogenesis, this transporter highlights the redundancy in iron acquisition mechanisms that C. difficile uses to compete for iron during an infection.

Characterization of the Attachment of Three New Coliphages onto the Ferrichrome Transporter FhuA.

Receptor-binding proteins (RBPs) allow phages to dock onto their host and initiate infection through the recognition of proteinaceous or saccharidic receptors located on the cell surface. FhuA is the ferrichrome hydroxamate transporter in Escherichia coli and serves as a receptor for the well-characterized phages T1, T5, and phi80. To further characterize how other FhuA-dependent phages attach to FhuA, we isolated and published the genomes of three new FhuA-dependent coliphages: JLBYU37, JLBYU41, and JLBYU60. We identified the egions of FhuA involved in phage attachment by testing the effect of mutant fhuA alleles containing single-loop deletions of extracellular loops (L3, L4, L5, L8, L10, and L11) on phage infectivity. Deletion of loop 8 resulted in complete resistance to SO1-like phages JLBYU37 and JLBYU60 and the previously isolated vB_EcoD_Teewinot phage, but no single-loop deletions significantly altered the infection of T1-like JLBYU41. Additionally, lipopolysaccharide (LPS) truncation coupled with the L5 mutant significantly impaired the infectivity of JLBYU37 and JLBYU60. Moreover, significant reductions in the infectivity of JLBYU41 were observed upon LPS truncation in the L8 mutant strain. Analysis of the evolutionary relationships among FhuA-dependent phage RBPs highlights the conservation of L8 dependence in JLBYU37, JLBYU60, Teewinot, T5, and phi80, but also showcases how positive selective pressure and/or homologous recombination also selected for L4 dependence in T1 and even the lack of complete loop dependence in JLBYU41. IMPORTANCE Phage attachment is the first step of phage infection and plays a role in governing host specificity. Characterizing the interactions taking place between phage tail fibers and bacterial receptors that better equip bacteria to survive within the human body may provide insights to aid the development of phage therapeutics.

Elucidating the molecular programming of a nonlinear non-ribosomal peptide synthetase responsible for fungal siderophore biosynthesis.

Siderophores belonging to the ferrichrome family are essential for the viability of fungal species and play a key role for virulence of numerous pathogenic fungi. Despite their biological significance, our understanding of how these iron-chelating cyclic hexapeptides are assembled by non-ribosomal peptide synthetase (NRPS) enzymes remains poorly understood, primarily due to the nonlinearity exhibited by the domain architecture. Herein, we report the biochemical characterization of the SidC NRPS, responsible for construction of the intracellular siderophore ferricrocin. In vitro reconstitution of purified SidC reveals its ability to produce ferricrocin and its structural variant, ferrichrome. Application of intact protein mass spectrometry uncovers several non-canonical events during peptidyl siderophore biosynthesis, including inter-modular loading of amino acid substrates and an adenylation domain capable of poly-amide bond formation. This work expands the scope of NRPS programming, allows biosynthetic assignment of ferrichrome NRPSs, and sets the stage for reprogramming towards novel hydroxamate scaffolds.

The Siderophore Ferricrocin Mediates Iron Acquisition in Aspergillus fumigatus.

The opportunistic fungal pathogen Aspergillus fumigatus utilizes two high-affinity iron uptake mechanisms, termed reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA). The latter has been shown to be crucial for virulence of this fungus and is a target for development of novel strategies for diagnosis and treatment of fungal infections. So far, research on SIA in this mold focused mainly on the hyphal stage, revealing the importance of extracellular fusarinine-type siderophores in iron acquisition as well as of the siderophore ferricrocin in intracellular iron handling. The current study aimed to characterize iron acquisition during germination. High expression of genes involved in biosynthesis and uptake of ferricrocin in conidia and during germination, independent of iron availability, suggested a role of ferricrocin in iron acquisition during germination. In agreement, (i) bioassays indicated secretion of ferricrocin during growth on solid media during both iron sufficiency and limitation, (ii) ferricrocin was identified in the supernatant of conidia germinating in liquid media during both iron sufficiency and limitation, (iii) in contrast to mutants lacking all siderophores, mutants synthesizing ferricrocin but lacking fusarinine-type siderophores were able to grow under iron limitation in the absence of RIA, and (iv) genetic inactivation of the ferricrocin transporter Sit1 decreased germination in the absence of RIA. Taken together, this study revealed that ferricrocin has not only an intracellular role but also functions as an extracellular siderophore to support iron acquisition. The iron availability-independent ferricrocin secretion and uptake during early germination indicate developmental, rather than iron regulation. IMPORTANCE Aspergillus fumigatus is one of the most common airborne fungal pathogens for humans. Low-molecular-mass iron chelators, termed siderophores, have been shown to play a central role in iron homeostasis and, consequently, virulence of this mold. Previous studies demonstrated the crucial role of secreted fusarinine-type siderophores, such as triacetylfusarinine C, in iron acquisition, as well as of the ferrichrome-type siderophore ferricrocin in intracellular iron storage and transport. Here, we demonstrate that ferricrocin is also secreted to mediate iron acquisition during germination together with reductive iron assimilation. During early germination, ferricrocin secretion and uptake were not repressed by iron availability, indicating developmental regulation of this iron acquisition system in this growth phase.

Elucidation of the ferrichrome siderophore biosynthetic pathway in albomycin-producing Streptomyces sp. ATCC 700974.

Sideromycins are a unique subset of siderophores comprising of a siderophore conjugated to an antimicrobial agent. The "Trojan horse" antibiotic albomycins are unique sideromycins consisting of a ferrichrome-type siderophore conjugated to a peptidyl nucleoside antibiotic. They exhibit potent antibacterial activities against many model bacteria and a number of clinical pathogens. Earlier studies have provided significant insight into the biosynthetic pathway of the peptidyl nucleoside moiety. We herein decipher the biosynthetic pathway of the ferrichrome-type siderophore in Streptomyces sp. ATCC 700974. Our genetic studies suggested that abmA, abmB, and abmQ are involved in the formation of the ferrichrome-type siderophore. Additionally, we performed biochemical studies to demonstrate that a flavin-dependent monooxygenase AbmB and an N-acyltransferase AbmA catalyze sequential modifications of L-ornithine to generate N5-acetyl-N5-hydroxyornithine. Three molecules of N5-acetyl-N5-hydroxyornithine are then assembled to generate the tripeptide ferrichrome through the action of a nonribosomal peptide synthetase AbmQ. Of special note, we found out that orf05026 and orf03299, two genes scattered elsewhere in the chromosome of Streptomyces sp. ATCC 700974, have functional redundancy for abmA and abmB, respectively. Interestingly, both orf05026 and orf03299 are situated within gene clusters encoding putative siderophores. In summary, this study provided new insight into the siderophore moiety of albomycin biosynthesis and shed light on the contingency of multiple siderophores in albomycin-producing Streptomyces sp. ATCC 700974.

The Bradyrhizobium japonicum fsrB gene is essential for utilization of structurally diverse ferric siderophores to fulfill its nutritional iron requirement.

In Bradyrhizobium japonicum, iron uptake from ferric siderophores involves selective outer membrane proteins and non-selective periplasmic and cytoplasmic membrane components that accommodate numerous structurally diverse siderophores. Free iron traverses the cytoplasmic membrane through the ferrous (Fe2+ ) transporter system FeoAB, but the other non-selective components have not been described. Here, we identify fsrB as an iron-regulated gene required for growth on iron chelates of catecholate- and hydroxymate-type siderophores, but not on inorganic iron. Utilization of the non-physiological iron chelator EDDHA as an iron source was also dependent on fsrB. Uptake activities of 55 Fe3+ bound to ferrioxamine B, ferrichrome or enterobactin were severely diminished in the fsrB mutant compared with the wild type. Growth of the fsrB or feoB strains on ferrichrome were rescued with plasmid-borne E. coli fhuCDB ferrichrome transport genes, suggesting that FsrB activity occurs in the periplasm rather than the cytoplasm. Whole cells of an fsrB mutant are defective in ferric reductase activity. Both whole cells and spheroplasts catalyzed the demetallation of ferric siderophores that were defective in an fsrB mutant. Collectively, the data support a model whereby FsrB is required for reduction of iron and its dissociation from the siderophore in the periplasm, followed by transport of the ferrous ion into the cytoplasm by FeoAB.

Pseudomonas aeruginosa FpvB Is a High-Affinity Transporter for Xenosiderophores Ferrichrome and Ferrioxamine B.

Iron is essential for many biological functions in bacteria, but its poor solubility is a limiting factor for growth. Bacteria produce siderophores, soluble natural products that bind iron with high affinity, to overcome this challenge. Siderophore-iron complexes return to the cell through specific outer membrane transporters. The opportunistic pathogen Pseudomonas aeruginosa makes multiple transporters that recognize its own siderophores, pyoverdine and pyochelin, and xenosiderophores produced by other bacteria or fungi, which gives it a competitive advantage. Some antibiotics exploit these transporters to bypass the membrane to reach their intracellular targets-including the thiopeptide antibiotic, thiostrepton (TS), which uses the pyoverdine transporters FpvA and FpvB to cross the outer membrane. Here, we assessed TS susceptibility in the presence of various siderophores and discovered that ferrichrome and ferrioxamine B antagonized TS uptake via FpvB. Unexpectedly, we found that FpvB transports ferrichrome and ferrioxamine B with higher affinity than pyoverdine. Site-directed mutagenesis of FpvB coupled with competitive growth inhibition and affinity label quenching studies suggested that the siderophores and antibiotic share a binding site in an aromatic pocket formed by the plug and barrel domains but have differences in their binding mechanism and molecular determinants for uptake. This work describes an alternative uptake pathway for ferrichrome and ferrioxamine B in P. aeruginosa and emphasizes the promiscuity of siderophore transporters, with implications for Gram-negative antibiotic development via the Trojan horse approach. IMPORTANCE Gram-negative bacteria express a variety of outer membrane transporters to import critical nutrients such as iron. Due to its insolubility, iron is taken up while bound to small-molecule chelators called siderophores. Pseudomonas aeruginosa takes up its own siderophores pyoverdine and pyochelin but can also steal siderophores produced by other bacteria and fungi, giving it a competitive advantage in iron-limited environments. Here, we used whole-cell reporter assays to show that FpvB, originally identified as a secondary transporter for pyoverdine, transports the chemically distinct fungal siderophore ferrichrome and the bacterial siderophore ferrioxamine B with high affinity. FpvB is also used by thiopeptide antibiotic thiostrepton for uptake. We predicted that all of these ligands bind to a common hydrophobic pocket in FpvB and used site-directed mutagenesis coupled with phenotypic assays to identify residues required for uptake. These analyses showed that siderophore and antibiotic uptake could be uncoupled. Our data show that FpvB is a promiscuous transporter of multiple chemically distinct ligands and fills in missing details of ferrichrome transport by P. aeruginosa. A clearer picture of the spectrum of outer membrane transporter substrate specificity is useful for the design of novel siderophore-antibiotic conjugates that can exploit nutrient uptake pathways to kill challenging Gram-negative pathogens.

Pseudomonas aeruginosa and its multiple strategies to access iron.

Pseudomonas aeruginosa is a ubiquitous bacterium found in many natural and man-made environments. It is also a pathogen for plants, animals, and humans. As for almost all living organisms, iron is an essential nutrient for the growth of P. aeruginosa. The bacterium has evolved complex systems to access iron and maintain its homeostasis to survive in diverse natural and dynamic host environments. To access ferric iron, P. aeruginosa is able to produce two siderophores (pyoverdine and pyochelin), as well as use a variety of siderophores produced by other bacteria (mycobactins, enterobactin, ferrioxamine, ferrichrome, vibriobactin, aerobactin, rhizobactin and schizokinen). Furthermore, it can also use citrate, in addition to catecholamine neuromediators and plant-derived mono catechols, as siderophores. The P. aeruginosa genome also encodes three heme-uptake pathways (heme being an iron source) and one ferrous iron acquisition pathway. This review aims to summarize current knowledge concerning the molecular mechanisms involved in all the iron and heme acquisition strategies used by P. aeruginosa.

Reprogramming of pancreatic adenocarcinoma immunosurveillance by a microbial probiotic siderophore.

There is increasing evidence suggesting the role of microbiome alterations in relation to pancreatic adenocarcinoma and tumor immune functionality. However, molecular mechanisms of the interplay between microbiome signatures and/or their metabolites in pancreatic tumor immunosurveillance are not well understood. We have identified that a probiotic strain (Lactobacillus casei) derived siderophore (ferrichrome) efficiently reprograms tumor-associated macrophages (TAMs) and increases CD8 + T cell infiltration into tumors that paralleled a marked reduction in tumor burden in a syngeneic mouse model of pancreatic cancer. Interestingly, this altered immune response improved anti-PD-L1 therapy that suggests promise of a novel combination (ferrichrome and immune checkpoint inhibitors) therapy for pancreatic cancer treatment. Mechanistically, ferrichrome induced TAMs polarization via activation of the TLR4 pathway that represses the expression of iron export protein ferroportin (FPN1) in macrophages. This study describes a novel probiotic based molecular mechanism that can effectively induce anti-tumor immunosurveillance and improve immune checkpoint inhibitors therapy response in pancreatic cancer.

Subtractive genomic analysis for computational identification of putative immunogenic targets against clinical Enterobacter cloacae complex.

BACKGROUND: Enterobacter is a major nosocomial genus of Enterobacteriaceae responsible for a variety of nosocomial infections, particularly in prolonged hospitalized patients in the intensive care units. Since current antibiotics have failed treating colistin- and carbapenem-resistant Enterobacteriaceae, efforts are underway to find suitable alternative strategies. Therefore, this study conducted a reverse vaccinology (RV) to identify novel and putative immunogenic targets using core proteome of 20 different sequence types (STs) of clinical Enterobacter spp. Moreover, we introduced a structural-based approach for exploration of potential vaccine candidates against the Enterobacteriaceae family using their conserved domain analysis. RESULTS: A number of 2616 core coding sequences (CDSs) were retrieved from 20 clinical strains of Enterobacter spp. with a similarity of ≥ 50%. Nine proteins with a score of ≥ 20 considered as the shortlisted proteins based on the quartile scoring method, including three TonB-dependent receptors, WP_008500981.1, WP_058690971.1 and WP_058679571.1; one YjbH domain-containing protein, WP_110108068.1; three flagellar proteins, WP_088207510.1, WP_033145204.1 and WP_058679632.1; one spore-coat U domain-containing protein, WP_039266612.1; and one DD-metalloendopeptidase family protein, WP_025912449.1. In this study, proteins WP_058690971.1 and WP_110108068.1 were detected as the top candidates with regard to immune stimulation and interactions with TLRs. However, their efficacy is remaining to be evaluated experimentally. CONCLUSIONS: Our investigation introduced common ferrichrome porins with high sequence similarity as potential vaccine candidates against the Enterobacteriaceae family. These proteins belong to the iron acquisition system and possess all criteria of suitable vaccine targets. Therefore, they need to be specifically paid attention for vaccine development against clinically important members of Enterobacteriaceae family.

Ferrichrome, a fungal-type siderophore, confers high ammonium tolerance to fission yeast.

Microorganisms and plants produce siderophores, which function to transport environmental iron into cells as well as participate in cellular iron use and deposition. Their biological functions are diverse although their role in primary metabolism is poorly understood. Ferrichrome is a fungal-type siderophore synthesized by nonribosomal peptide synthetase (NRPS). Herein we show that ferrichrome induces adaptive growth of fission yeast on high ammonium media. Ammonium is a preferred nitrogen source as it suppresses uptake and catabolism of less preferred nitrogen sources such as leucine through a mechanism called nitrogen catabolite repression (NCR). Therefore, the growth of fission yeast mutant cells with leucine auxotrophy is suppressed in the presence of high concentrations of ammonium. This growth suppression was canceled by ferrichrome in a manner dependent on the amino acid transporter Cat1. Additionally, growth retardation of wild-type cells by excess ammonium was exacerbated by deleting the NRPS gene sib1, which is responsible for the biosynthesis of ferrichrome, suggesting that intrinsically produced ferrichrome functions in suppressing the metabolic action of ammonium. Furthermore, ferrichrome facilitated the growth of both wild-type and sib1-deficient cells under low glucose conditions. These results suggest that intracellular iron regulates primary metabolism, including NCR, which is mediated by siderophores.

FoxR is an AraC-like transcriptional regulator of ferrioxamine uptake in Salmonella enterica.

Salmonella enterica spp. produce siderophores to bind iron with high affinity and can also use three xenosiderophores secreted by other microorganisms, ferrichrome, coprogen, and ferrioxamine. Here we focused on FoxA, a TonB-dependent transporter of ferrioxamines. Adjacent to foxA is a gene annotated as a helix-turn-helix (HTH) domain-containing protein, SL0358 (foxR), in the Salmonella enterica serovar Typhimurium SL1344 genome. FoxR shares homology with transcriptional regulators belonging to the AraC/XylS family. foxR is syntenic with foxA in the Enterobacteriaceae family, suggesting their functional relatedness. Both foxA and foxR are repressed by the ferric uptake regulator (Fur) under iron-rich growth conditions. When iron is scarce, FoxR acts as a transcriptional activator of foxA by directly binding to its upstream regulatory region. A point mutation in the HTH domain of FoxR abolished this binding, as did mutation of a direct repeat motif in the foxA upstream regulatory region. Desferrioxamine (DFOE) enhanced FoxR protein stability and foxA transcription but did not affect the affinity of FoxR binding to the foxA regulatory region. In summary, we have identified FoxR as a new member of the AraC/XylS family that regulates xenosiderophore-mediated iron uptake by S. Typhimurium and likely other Enterobacteriaceae members.

De novo synthesis of ferrichrome by Fusarium oxysporum f. sp. cubense TR4 in response to iron starvation.

Manipulation of iron bioavailability in the banana rhizosphere may suppress Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc). However, iron starvation induced by application of synthetic iron chelators does not effectively suppress Fusarium wilt. It is unclear whether Foc can subvert iron chelators and thereby evade iron starvation through the synthesis of iron-scavenging secondary metabolites, called siderophores. In vitro studies were conducted using iron-deficient growth medium and medium supplemented with a synthetic iron chelator, 2,2'-dipyridyl, to mimic iron starvation in Foc Tropical Race 4 (Foc TR4). Concentration of extracellular siderophores increased three-fold (p < 0.05) in the absence of iron. Liquid chromatography-mass spectrometry analysis detected the hydroxamate siderophore, ferrichrome, only in the mycelia of iron-starved cultures. Moreover, iron-starved cultures exhibited a reduction in total cellular protein concentration. In contrast, out of the 20 proteinogenic amino acids, only arginine increased (p < 0.05) under iron starvation. Our findings suggest that iron starvation does not cause a remodelling of amino acid metabolism in Foc TR4, except for arginine, which is required for biosynthesis of ornithine, the precursor for siderophore biosynthesis. Collectively, our findings suggest that biosynthesis of siderophores, particularly ferrichrome, could be a counteractive mechanism for Foc TR4 to evade iron starvation.

Slow Kinetics of Iron Binding to Marine Ligands in Seawater Measured by Isotope Exchange Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry.

Current understanding of dissolved iron (Fe) speciation in the ocean is based on two fundamentally different approaches: electrochemical methods that measure bulk properties of a heterogeneous ligand pool and liquid chromatography mass spectrometry methods that characterize ligands at a molecular level. Here, we describe a method for simultaneously determining Fe-ligand dissociation rate constants (kd) of suites of naturally occurring ligands in seawater by monitoring the exchange of ligand-bound 56Fe with 57Fe using liquid chromatography-inductively coupled mass spectrometry. Values of kd were determined for solutions of ferrichrome and ferrioxamine E. In seawater, the dissociation rate constant of ferrichrome (kd = 10 × 10-8 s-1) was greater than that of ferrioxamine E (kd = 3.6 × 10-8 s-1). The rates for both compounds were over twice as fast in seawater compared with pure water, suggesting that seawater salts accelerate dissociation. Isotope exchange experiments on organic extracts of natural seawater indicated that ligand-binding sites associated with chromatographically unresolved dissolved organic matter exchanged Fe more quickly (kd = 1.8 × 10-5 s-1) than amphibactin siderophores (kd = 2.15 × 10-6 s-1) and an unidentified siderophore with m/z 709 (kd = 9.6 × 10-6 s-1). These findings demonstrate that our approach can bridge molecular-level ligand identification with kinetic and thermodynamic metal-binding properties.

Differential Biosynthesis and Roles of Two Ferrichrome-Type Siderophores, ASP2397/AS2488053 and Ferricrocin, in Acremonium persicinum.

Ferrichromes are a family of fungal siderophores with cyclic hexapeptide structures. Most fungi produce one or two ferrichrome-type siderophores. Acremonium persicinum MF-347833 produces ferrichrome-like potent Trojan horse antifungal antibiotics ASP2397 and AS2488053, the aluminum- and iron-chelating forms of AS2488059, respectively. Here, we show by gene sequencing followed by gene deletion experiments that A. persicinum MF-347833 possesses two nonribosomal peptide synthetase genes responsible for AS2488059 and ferricrocin assembly. AS2488059 was produced under iron starvation conditions and excreted into the media to serve as a defense metabolite and probably an iron courier. In contrast, ferricrocin was produced under iron-replete conditions and retained inside the cells, likely serving as an iron-sequestering molecule. Notably, the phylogenetic analyses suggest the different evolutionary origin of AS2488059 from that of conventional ferrichrome-type siderophores. Harnessing two ferrichrome-type siderophores with distinct biological properties may give A. persicinum a competitive advantage for surviving the natural environment.

Cryo-EM reveals unique structural features of the FhuCDB Escherichia coli ferrichrome importer.

As one of the most elegant biological processes developed in bacteria, the siderophore-mediated iron uptake demands the action of specific ATP-binding cassette (ABC) importers. Although extensive studies have been done on various ABC importers, the molecular basis of these iron-chelated-siderophore importers are still not fully understood. Here, we report the structure of a ferrichrome importer FhuCDB from Escherichia coli at 3.4 Å resolution determined by cryo electron microscopy. The structure revealed a monomeric membrane subunit of FhuB with a substrate translocation pathway in the middle. In the pathway, there were unique arrangements of residues, especially layers of methionines. Important residues found in the structure were interrogated by mutagenesis and functional studies. Surprisingly, the importer's ATPase activity was decreased upon FhuD binding, which deviated from the current understanding about bacterial ABC importers. In summary, to the best of our knowledge, these studies not only reveal a new structural twist in the type II ABC importer subfamily, but also provide biological insights in the transport of iron-chelated siderophores.

Iron homeostasis in the absence of ferricrocin and its consequences in fungal development and insect virulence in Beauveria bassiana.

The putative ferricrocin synthetase gene ferS in the fungal entomopathogen Beauveria bassiana BCC 2660 was identified and characterized. The 14,445-bp ferS encodes a multimodular nonribosomal siderophore synthetase tightly clustered with Fusarium graminearum ferricrocin synthetase. Functional analysis of this gene was performed by disruption with the bar cassette. ΔferS mutants were verified by Southern and PCR analyses. HPLC and TLC analyses of crude extracts indicated that biosynthesis of ferricrocin was abolished in ΔferS. Insect bioassays surprisingly indicated that ΔferS killed the Spodoptera exigua larvae faster (LT50 59 h) than wild type (66 h). Growth and developmental assays of the mutant and wild type demonstrated that ΔferS had a significant increase in germination under iron depletion and radial growth and a decrease in conidiation. Mitotracker staining showed that the mitochondrial activity was enriched in ΔferS under both iron excess and iron depletion. Comparative transcriptomes between wild type and ΔferS indicated that the mutant was increased in the expression of eight cytochrome P450 genes and those in iron homeostasis, ferroptosis, oxidative stress response, ergosterol biosynthesis, and TCA cycle, compared to wild type. Our data suggested that ΔferS sensed the iron excess and the oxidative stress and, in turn, was up-regulated in the antioxidant-related genes and those in ergosterol biosynthesis and TCA cycle. These increased biological pathways help ΔferS grow and germinate faster than the wild type and caused higher insect mortality than the wild type in the early phase of infection.

Synthesis and structural insights into the binding mode of the albomycin δ1 core and its analogues in complex with their target aminoacyl-tRNA synthetase.

Despite of proven efficacy and well tolerability, albomycin is not used clinically due to scarcity of material. Several attempts have been made to increase the production of albomycin by chemical or biochemical methods. In the current study, we have synthesized the active moiety of albomycin δ1 and investigated its binding mode to its molecular target seryl-trna synthetase (SerRS). In addition, isoleucyl and aspartyl congeners were prepared to investigate whether the albomycin scaffold can be extrapolated to target other aminoacyl-tRNA synthetases (aaRSs) from both class I and class II aaRSs, respectively. The synthesized analogues were evaluated for their ability to inhibit the corresponding aaRSs by an in vitro aminoacylation experiment using purified enzymes. It was observed that the diastereomer having the 5'S, 6'R-configuration (nucleoside numbering) as observed in the crystal structure, exhibits excellent inhibitory activity in contrast to poor activity of its companion 5'R,6'S-diasteromer obtained as byproduct during synthesis. Moreover, the albomycin core scaffold seems well tolerated for class II aaRSs inhibition compared with class I aaRSs. To understand this bias, we studied X-ray crystal structures of SerRS in complex with the albomycin δ1 core structure 14a, and AspRS in complex with compound 16a. Structural analysis clearly showed that diastereomer selectivity is attributed to the steric restraints of the active site of SerRS and AspRS.