Kinetic analysis of the three-substrate reaction mechanism of an NRPS-independent siderophore (NIS) synthetase.

The biosynthesis of many bacterial siderophores employs a member of a family of ligases that have been defined as NRPS-independent siderophore (NIS) synthetases. These NIS synthetases use a molecule of ATP to produce an amide linkage between a carboxylate and an amine. Commonly used carboxylate substrates include citrate or α-ketoglutarate, or derivatives thereof, while the amines are often hydroxamate derivatives of lysine or ornithine, or their decarboxylated forms cadaverine and putrescine. Enzymes that employ three substrates to catalyze a reaction may proceed through alternate mechanisms. Some enzymes use sequential mechanisms in which all three substrates bind prior to any chemical steps. In such mechanisms, substrates can bind in a random, ordered, or mixed fashion. Alternately, other enzymes employ a ping-pong mechanism in which a chemical step occurs prior to the binding of all three substrates. Here we describe an enzyme assay that will distinguish among these different mechanisms for the NIS synthetase, using IucA, an enzyme involved in the production of aerobactin, as the model system.

Methods for biochemical characterization of flavin-dependent N-monooxygenases involved in siderophore biosynthesis.

Siderophores are essential molecules released by some bacteria and fungi in iron-limiting environments to sequester ferric iron, satisfying metabolic needs. Flavin-dependent N-hydroxylating monooxygenases (NMOs) catalyze the hydroxylation of nitrogen atoms to generate important siderophore functional groups such as hydroxamates. It has been demonstrated that the function of NMOs is essential for virulence, implicating these enzymes as potential drug targets. This chapter aims to serve as a resource for the characterization of NMO's enzymatic activities using several biochemical techniques. We describe assays that allow for the determination of steady-state kinetic parameters, detection of hydroxylated amine products, measurement of the rate-limiting step(s), and the application toward drug discovery efforts. While not exhaustive, this chapter will provide a foundation for the characterization of enzymes involved in siderophore biosynthesis, allowing for gaps in knowledge within the field to be addressed.

Predicting metallophore structure and function through genome mining.

Metallophores are small molecule chelators that many microbes use to obtain trace metals from their environment. Through genome mining, where genomes are scanned for metallophore biosynthesis genes, one can not only identify which organisms are likely to produce a metallophore, but also predict the metallophore structure, thus preventing undesired reisolation of known compounds and accelerating characterization. Furthermore, the presence of accessory genes for the transport, utilization, and regulation can suggest the biological function and fate of a metallophore. Modern, user-friendly tools have made powerful genomic analyses accessible to scientists with no bioinformatics experience, but these tools are often not utilized to their full potential. This chapter provides an introduction to metallophore genomics and demonstrates how to use the free, publicly available antiSMASH platform to infer metallophore function and structure.

Plant-microbe interactions in the rhizosphere for smarter and more sustainable crop fertilization: the case of PGPR-based biofertilizers.

Biofertilizers based on plant growth promoting rhizobacteria (PGPR) are nowadays gaining increasingly attention as a modern tool for a more sustainable agriculture due to their ability in ameliorating root nutrient acquisition. For many years, most research was focused on the screening and characterization of PGPR functioning as nitrogen (N) or phosphorus (P) biofertilizers. However, with the increasing demand for food using far fewer chemical inputs, new investigations have been carried out to explore the potential use of such bacteria also as potassium (K), sulfur (S), zinc (Zn), or iron (Fe) biofertilizers. In this review, we update the use of PGPR as biofertilizers for a smarter and more sustainable crop production and deliberate the prospects of using microbiome engineering-based methods as potential tools to shed new light on the improvement of plant mineral nutrition. The current era of omics revolution has enabled the design of synthetic microbial communities (named SynComs), which are emerging as a promising tool that can allow the formulation of biofertilizers based on PGPR strains displaying multifarious and synergistic traits, thus leading to an increasingly efficient root acquisition of more than a single essential nutrient at the same time. Additionally, host-mediated microbiome engineering (HMME) leverages advanced omics techniques to reintroduce alleles coding for beneficial compounds, reinforcing positive plant-microbiome interactions and creating plants capable of producing their own biofertilizers. We also discusses the current use of PGPR-based biofertilizers and point out possible avenues of research for the future development of more efficient biofertilizers for a smarter and more precise crop fertilization. Furthermore, concerns have been raised about the effectiveness of PGPR-based biofertilizers in real field conditions, as their success in controlled experiments often contrasts with inconsistent field results. This discrepancy highlights the need for standardized protocols to ensure consistent application and reliable outcomes.

Rhizobacterial diversity of Portuguese olive cultivars in the Douro valley and their potential as plant growth promoters.

AIMS: This study investigated the bacterial communities in the rhizosphere of two traditional Portuguese olive cultivars, Cobrançosa and Negrinha de Freixo in relation to soil properties. Additionally, we aimed to isolate and identify bacteria with potential for biocontrol and other plant growth promoting traits from these rhizosphere communities. METHODS AND RESULTS: Bacterial communities in the olive rhizosphere were investigated using a metabarcoding approach and the soil physicochemical properties of the olive groves were also analyzed. Higher bacterial richness was associated with Negrinha de Freixo growing in soil with high organic matter content and water-holding capacity. In contrast, the soils of the Cobrançosa grove presented higher pH and electric conductivity. Negrinha de Freixo rhizosphere was enriched with ASVs (Amplicon Sequence Variants) belonging to Bacillus, Gaiella, Acidothermus, Bradyrhizobium, and uncultured Xanthobacteraceae. On the other hand, the Cobrançosa rhizosphere was characterized by higher relative abundance of Streptomyces and Sphingomonas. Bacterial isolation from the rhizosphere and screening for plant growth promoting activities were also performed. Six bacteria strains, predominantly Bacillus isolated from Negrinha de Freixo, demonstrated antagonistic activities against the olive fungal pathogen C. gloeosporoides and other plant growth promotion (PGP) traits. CONCLUSIONS: Our findings demonstrate that the structure of rhizosphere bacterial communities associated with olive trees is shaped by both plant cultivar and soil-related factors. The higher number of bacterial species in the rhizosphere of Negrinha de Freixo was related to a higher organic matter content and a greater abundance of isolates with plant growth promotion traits, particularly Bacillus strains.

Identification and characterization of the siderochelin biosynthetic gene cluster via coculture.

Many microbial biosynthetic gene clusters (BGCs) are inactive under standard laboratory conditions, making characterization of their products difficult. Silent BGCs are likely activated by specific cues in their natural environment, such as the presence of competitors. Growth conditions such as coculture with other microbes, which more closely mimic natural environments, are practical strategies for inducing silent BGCs. Here, we utilize coculture to activate BGCs in nine actinobacteria strains. We observed increased production of the ferrous siderophores siderochelin A and B during coculture of Amycolatopsis strain WAC04611 and Tsukamurella strain WAC06889b. Furthermore, we identified the siderochelin BGC in WAC04611 and discovered that the GntR-family transcription factor sidR3 represses siderochelin production. Deletion of the predicted aminotransferase sidA abolished production of the carboxamides siderochelin A/B and led to the accumulation of the carboxylate siderochelin D. Finally, we deleted the predicted hydroxylase sidB and established that it is essential for siderochelin production. Our findings show that microbial coculture can successfully activate silent BGCs and lead to the discovery and characterization of unknown BGCs for molecules like siderochelin.IMPORTANCESiderophores are vital iron-acquisition elements required by microbes for survival in a variety of environments. Furthermore, many siderophores are essential for the virulence of various human pathogens, making them a possible target for antibacterials. The significance of our work is in the identification and characterization of the previously unknown BGC for the siderophore siderochelin. Our work adds to the growing knowledge of siderophore biosynthesis, which may aid in the future development of siderophore-targeting pharmaceuticals and inform on the ecological roles of these compounds. Furthermore, our work demonstrates that combining microbial coculture with metabolomics is a valuable strategy for identifying upregulated compounds and their BGCs.

The role of FoxA, FiuA, and FpvB in iron acquisition via hydroxamate-type siderophores in Pseudomonas aeruginosa.

Siderophores are specialized molecules produced by bacteria and fungi to scavenge iron, a crucial nutrient for growth and metabolism. Catecholate-type siderophores are mainly produced by bacteria, while hydroxamates are mostly from fungi. This study investigates the capacity of nine hydroxamate-type siderophores from fungi and Streptomyces to facilitate iron acquisition by the human pathogen Pseudomonas aeruginosa. Growth assays under iron limitation and 55Fe incorporation tests showed that all nine siderophores promoted bacterial growth and iron transport. The study also aimed to identify the TonB-dependent transporters (TBDTs) involved in iron import by these siderophores. Using mutant strains lacking specific TBDT genes, it was found that iron is imported into P. aeruginosa cells by FpvB for coprogen, triacetylfusarinine, fusigen, ferrirhodin, and ferrirubin. Iron complexed by desferioxamine G is transported by FpvB and FoxA, ferricrocin-Fe and ferrichrycin-Fe by FpvB and FiuA, and rhodotoluric acid-Fe by FpvB, FiuA, and another unidentified TBDT. These findings highlight the effectiveness of hydroxamate-type siderophores in iron transport into P. aeruginosa and provide insights into the complex molecular mechanisms involved, which are important for understanding microbial interactions and ecological balance.

Biotechnological application of Aureobasidium spp. as a promising chassis for biosynthesis of ornithine-urea cycle-derived bioproducts.

The ornithine-urea cycle (OUC) in fungal cells has biotechnological importance and many physiological functions and is closely related to the acetyl glutamate cycle (AGC). Fumarate can be released from argininosuccinate under the catalysis of argininosuccinate lyase in OUC which is regulated by the Ca2+ signaling pathway and over 93.9 ± 0.8 g/L fumarate can be yielded by the engineered strain of Aureobasidium pullulans var. aubasidani in the presence of CaCO3. Furthermore, 2.1 ± 0.02 mg of L-ornithine (L-Orn)/mg of the protein also can be synthesized via OUC by the engineered strains of Aureobasidum melanogenum. Fumarate can be transformed into many drugs and amino acids and L-Orn can be converted into siderophores (1.7 g/L), putrescine (33.4 g/L) and L-piperazic acid (L-Piz) (3.0 g/L), by different recombinant strains of A. melanogenum. All the fumarate, L-Orn, siderophore, putrescine and L-Piz have many applications. As the yeast-like fungi and the promising chassis, Aureobasidium spp, have many advantages over any other fungal strains. Further genetic manipulation and bioengineering will enhance the biosynthesis of fumarate and L-Orn and their derivates.

OUC in fungal cells has biotechnological importance and many physiological functions; OUC is closely related to acetyl glutamate cycle (AGC). Fumarate, L-Orn, siderophore, putrescine and L-Piz produced from OUC have many applications.

Siderophores: A Case Study in Translational Chemical Biology.

Siderophores are metal-binding secondary metabolites that assist in iron homeostasis and have been of interest to the scientific community for the last half century. Foundational siderophore research has enabled several translational applications including siderophore-antibiotic and siderophore-peptide conjugates, identification of new antimicrobial targets, advances in disease imaging, and novel therapeutics. This review aims to connect the basic science research (biosynthesis, cellular uptake, gene regulation, and effects on homeostasis) of well-known siderophores with the successive translational application that results. Intertwined throughout are connections to the career of Christopher T. Walsh, his impact on the field of chemical biology, and the legacy of his trainees who continue to innovate.

[68Ga]Ga-Schizokinen, a Potential Radiotracer for Selective Bacterial Infection Imaging.

Gallium-68-labeled siderophores as radiotracers have gained interest for the development of in situ infection-specific imaging diagnostics. Here, we report radiolabeling, in vitro screening, and in vivo pharmacokinetics (PK) of gallium-68-labeled schizokinen ([68Ga]Ga-SKN) as a new potential radiotracer for imaging bacterial infections. We radiolabeled SKN with ≥95% radiochemical purity. Our in vitro studies demonstrated its hydrophilic characteristics, neutral pH stability, and short-term stability in human serum and toward transchelation. In vitro uptake of [68Ga]Ga-SKN by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and S. epidermidis, but no uptake by Candida glabrata, C. albicans, or Aspergillus fumigatus, demonstrated its specificity to bacterial species. Whole-body [68Ga]Ga-SKN positron emission tomography (PET) combined with computerized tomography (CT) in healthy mice showed rapid renal excretion with no or minimal organ uptake. The subsequent ex vivo biodistribution resembled this fast PK with rapid renal excretion with minimal blood retention and no major organ uptake and showed some dissociation of the tracer in the urine after 60 min postinjection. These findings warrant further evaluation of [68Ga]Ga-SKN as a bacteria-specific radiotracer for infection imaging.

Enhancement of growth media for extreme iron limitation in Escherichia coli.

Iron is an essential nutrient for microbial growth and bacteria have evolved numerous routes to solubilize and scavenge this biometal, which is often present at very low concentrations in host tissue. We recently used a MOPS-based medium to induce iron limitation in Escherichia coli K-12 during the characterization of novel siderophore-conjugated antibiotics. In this study we confirm that growth media derived from commercially available M9 salts are unsuitable for studies of iron-limited growth, probably through the contamination of the sodium phosphate buffer components with over 100 µM iron. In contrast, MOPS-based media that are treated with metal-binding Chelex resin allow the free iron concentration to be reduced to growth-limiting levels. Despite these measures a small amount of E. coli growth is still observed in these iron-depleted media. By growing E. coli in conditions that theoretically increase the demand for iron-dependent enzymes, namely by replacing the glucose carbon source for acetate and by switching to a microaerobic atmosphere, we can reduce background growth even further. Finally, we demonstrate that by adding an exogeneous siderophore to the growth media which is poorly used by E. coli, we can completely prevent growth, perhaps mimicking the situation in host tissue. In conclusion, this short study provides practical experimental insight into low iron media and how to augment the growth conditions of E. coli for extreme iron-limited growth.

Recently developed radiopharmaceuticals for bacterial infection imaging.

BACKGROUND: Infection remains a major cause of morbidity and mortality, regardless of advances in antimicrobial therapy and improved knowledge of microorganisms. With the major global threat posed by antimicrobial resistance, fast and accurate diagnosis of infections, and the reliable identification of intractable infection, are becoming more crucial for effective treatment and the application of antibiotic stewardship. Molecular imaging with the use of nuclear medicine allows early detection and localisation of infection and inflammatory processes, as well as accurate monitoring of treatment response. There has been a continuous search for more specific radiopharmaceuticals to be utilised for infection imaging. This review summarises the most prominent discoveries in specifically bacterial infection imaging agents over the last five years, since 2019. MAIN BODY: Some promising new radiopharmaceuticals evaluated in patient studies are reported here, including radiolabelled bacterial siderophores like [68Ga]Ga-DFO-B, radiolabelled antimicrobial peptide/peptide fragments like [68Ga]Ga-NOTA-UBI29-41, and agents that target bacterial synthesis pathways (folic acid and peptidoglycan) like [11C]para-aminobenzoic acid and D-methyl-[11C]-methionine, with clinical trials underway for [18F]fluorodeoxy-sorbitol, as well as for 11C- and 18F-labelled trimethoprim. CONCLUSION: It is evident that a great deal of effort has gone into the development of new radiopharmaceuticals for infection imaging over the last few years, with remarkable progress in preclinical investigations. However, translation to clinical trials, and eventually clinical Nuclear Medicine practice, is apparently slow. It is the authors' opinion that a more structured and harmonised preclinical setting and well-designed clinical investigations are the key to reliably evaluate the true potential of the newly proposed infection imaging agents.

The iron(III) coordinating properties of citrate and α-hydroxycarboxylate containing siderophores.

The iron(III) binding properties of citrate and rhizoferrin, a citrate containing siderophore, are compared. Citrate forms many oligonuclear complexes, whereas rhizoferrin forms a single mononuclear complex. The α-hydroxycarboxylate functional group, which is present in both citrate, and rhizoferrin, has a high affinity and selectivity for iron(III) under most biological conditions. The nature of the toxic form of iron found in the blood of patients suffering from many haemoglobinopathies and haemochromatosis is identified as a mixture of iron(III)citrate complexes. The significance of the presence of this iron pool to patients suffering from systemic iron overload is discussed. The wide utilisation of the α-hydroxycarboxylate functional group in siderophore structures is described, as is their photo-induced decarboxylation leading to the release of iron(II) ions. The importance of this facile dissociation to algal iron uptake is discussed.

Siderophores and metallophores: Metal complexation weapons to fight environmental pollution.

Siderophores are small molecules of organic nature, released by bacteria to chelate iron from the surrounding environment and subsequently incorporate it into the cytoplasm. In addition to iron, these secondary metabolites can complex with a wide variety of metals, which is why they are commonly studied in the environment. Heavy metals can be very toxic when present in large amounts on the planet, affecting public health and all living organisms. The pollution caused by these toxic metals is increasing, and therefore it is urgent to find practical, sustainable, and economical solutions for remediation. One of the strategies is siderophore-assisted bioremediation, an innovative and advantageous alternative for various environmental applications. This research highlights the various uses of siderophores and metallophores in the environment, underscoring their significance to ecosystems. The study delves into the utilization of siderophores and metallophores in both marine and terrestrial settings (e.g. bioremediation, biocontrol of pathogens, and plant growth promotion), such as bioremediation, biocontrol of pathogens, and plant growth promotion, providing context for the different instances outlined in the existing literature and highlighting their relevance in each field. The study delves into the structures and types of siderophores focusing on their singular characteristics for each application and methodologies used. Focusing on recent developments over the last two decades, the opportunities and challenges associated with siderophores and metallophores applications in the environment were mapped to arm researchers in the fight against environmental pollution.

Exploring the Biological Pathways of Siderophores and Their Multidisciplinary Applications: A Comprehensive Review.

Siderophores are a class of small molecules renowned for their high iron binding capacity, essential for all life forms requiring iron. This article provides a detailed review of the diverse classifications, and biosynthetic pathways of siderophores, with a particular emphasis on siderophores synthesized via nonribosomal peptide synthetase (NRPS) and non-NRPS pathways. We further explore the secretion mechanisms of siderophores in microbes and plants, and their role in regulating bioavailable iron levels. Beyond biological functions, the applications of siderophores in medicine, agriculture, and environmental sciences are extensively discussed. These applications include biological pest control, disease treatment, ecological pollution remediation, and heavy metal ion removal. Through a comprehensive analysis of the chemical properties and biological activities of siderophores, this paper demonstrates their wide prospects in scientific research and practical applications, while also highlighting current research gaps and potential future directions.

Overview of Proteus mirabilis pathogenicity and virulence. Insights into the role of metals.

Proteus mirabilis is a Gram-negative bacterium with exclusive molecular and biological features. It is a versatile pathogen acclaimed for its distinct urease production, swarming behavior, and rapid multicellular activity. Clinically, P. mirabilis is a frequent pathogen of the human urinary system where it causes urinary tract infections (UTIs) and catheter-associated urinary tract infections (CAUTIs). This review explores the epidemiology, risk factors, clinical manifestations, and treatment of P. mirabilis infections, emphasizing its association with UTIs. The bacterium's genome analysis revealed the presence of resistance genes against commonly used antibiotics, an antibiotic-resistant phenotype that poses a serious clinical challenge. Particularly, the emergence of extended-spectrum ß-lactamases (ESBLs) and carbapenemases resistant P. mirabilis strains. On a molecular level, P. mirabilis possesses a wide array of virulence factors including the production of fimbriae, urease, hemolysins, metallophores, and biofilm formation. This review thoroughly tackles a substantial gap in understanding the role of metallophores in shaping the virulence factors of P. mirabilis virulence. Siderophores, iron metal chelating and transporting metallophores, particularly contribute to the complex pathogenic strategies, displaying a potential target for therapeutic intervention.

Plants, Microorganisms and Their Metabolites in Supporting Asbestos Detoxification-A Biological Perspective in Asbestos Treatment.

Many countries banned asbestos due to its toxicity, but considering its colossal use, especially in the 1960s and 1970s, disposing of waste containing asbestos is the current problem. Today, many asbestos disposal technologies are known, but they usually involve colossal investment and operating expenses, and the end- and by-products of these methods negatively impact the environment. This paper identifies a unique modern direction in detoxifying asbestos minerals, which involves using microorganisms and plants and their metabolites. The work comprehensively focuses on the interactions between asbestos and plants, bacteria and fungi, including lichens and, for the first time, yeast. Biological treatment is a prospect for in situ land reclamation and under industrial conditions, which can be a viable alternative to landfilling and an environmentally friendly substitute or supplement to thermal, mechanical, and chemical methods, often characterized by high cost intensity. Plant and microbial metabolism products are part of the green chemistry trend, a central strategic pillar of global industrial and environmental development.

Siderophore-mediated iron acquisition by Klebsiella pneumoniae.

Microbes synthesize and secrete siderophores, that bind and solubilize precipitated or otherwise unavailable iron in their microenvironments. Gram (-) bacterial TonB-dependent outer membrane receptors capture the resulting ferric siderophores to begin the uptake process. From their similarity to fepA, the structural gene for the Escherichia coli ferric enterobactin (FeEnt) receptor, we identified four homologous genes in the human and animal ESKAPE pathogen Klebsiella pneumoniae (strain Kp52.145). One locus encodes IroN (locus 0027 on plasmid pII), and three other loci encode other FepA orthologs/paralogs (chromosomal loci 1658, 2380, and 4984). Based on the crystal structure of E. coli FepA (1FEP), we modeled the tertiary structures of the K. pneumoniae FepA homologs and genetically engineered individual Cys substitutions in their predicted surface loops. We subjected bacteria expressing the Cys mutant proteins to modification with extrinsic fluorescein maleimide (FM) and used the resulting fluorescently labeled cells to spectroscopically monitor the binding and transport of catecholate ferric siderophores by the four different receptors. The FM-modified FepA homologs were nanosensors that defined the ferric catecholate uptake pathways in pathogenic strains of K. pneumoniae. In Kp52.145, loci 1658 and 4984 encoded receptors that primarily recognized and transported FeEnt; locus 0027 produced a receptor that principally bound and transported FeEnt and glucosylated FeEnt (FeGEnt); locus 2380 encoded a protein that bound ferric catecholate compounds but did not detectably transport them. The sensors also characterized the uptake of iron complexes, including FeGEnt, by the hypervirulent, hypermucoviscous K. pneumoniae strain hvKp1. IMPORTANCE: Both commensal and pathogenic bacteria produce small organic chelators, called siderophores, that avidly bind iron and increase its bioavailability. Klebsiella pneumoniae variably produces four siderophores that antagonize host iron sequestration: enterobactin, glucosylated enterobactin (also termed salmochelin), aerobactin, and yersiniabactin, which promote colonization of different host tissues. Abundant evidence links bacterial iron acquisition to virulence and infectious diseases. The data we report explain the recognition and transport of ferric catecholates and other siderophores, which are crucial to iron acquisition by K. pneumoniae.

Siderophore-producing bacteria from Spitsbergen soils-novel agents assisted in bioremediation of the metal-polluted soils.

Siderophores are molecules that exhibit a high specificity for iron (Fe), and their synthesis is induced by a deficiency of bioavailable Fe. Complexes of Fe-siderophore are formed extracellularly and diffuse through porins across membranes into bacterial cells. Siderophores can bind heavy metals facilitating their influx into cells via the same mechanism. The aim of the studies was to determine the ability of siderophore-producing bacteria isolated from soils in the north-west part of Wedel Jarlsberg Land (Spitsbergen) to chelate non-Fe metals (Al, Cd, Co, Cu, Hg, Mn, Sn, and Zn). Specially modified blue agar plates were used, where Fe was substituted by Al, Cd, Co, Cu, Hg, Mn, Sn, or Zn in metal-chrome azurol S (CAS) complex, which retained the blue color. It has been proven that 31 out of 33 strains were capable of producing siderophores that bind to Fe, as well as other metals. Siderophores from Pantoea sp. 24 bound only Fe and Zn, and O. anthropi 55 did not produce any siderophores in pure culture. The average efficiency of Cd, Co, Cu, Mn, Sn, and Zn chelation was either comparable or higher than that of Fe, while Al and Hg showed significantly lower efficiency. Siderophores produced by S. maltophilia 54, P. luteola 27, P. luteola 46, and P. putida 49 exhibited the highest non-Fe metal chelation activity. It can be concluded that the siderophores of these bacteria may constitute an integral part of the metal bioleaching preparation, and this fact will be the subject of further research.