Functional Genetic Diversity and Plant Growth Promoting Potential of Polyphosphate Accumulating Bacteria in Soil.

Polyphosphate (polyP) accumulation is an important trait of microorganisms. Implication of polyP accumulating bacteria (PAB) in enhanced biological phosphate removal, heavy metal sequestration, and dissolution of dental enamel is well studied. Phosphorous (P) accumulated within microbial biomass also regulates labile P in soil; however, abundance and diversity of the PAB in soil is still unexplored. Present study investigated the genetic and functional diversity of PAB in rhizosphere soil. Here, we report the abundance of Pseudomonas spp. as high PAB in soil, suggesting their contribution to global P cycling. Additional subset analysis of functional genes i.e., polyphosphate kinase (ppk) and exopolyphosphatase (ppx) in all PAB, indicates their significance in bacterial growth and metabolism. Distribution of functional genes in phylogenetic tree represent a more biologically realistic discrimination for the two genes. Distribution of ppx gene disclosed its phylogenetic conservation at species level, however, clustering of ppk gene of similar species in different clades illustrated its environmental condition mediated modifications. Selected PAB showed tolerance to abiotic stress and strong correlation with plant growth promotary (PGP) traits viz. phosphate solubilization, auxin and siderophore production. Interaction of PAB with A. thaliana enhanced the growth and phosphate status of the plant under salinity stress, suggestive of their importance in P cycling and stress alleviation. IMPORTANCE Study discovered the abundance of Pseudomonas genera as a high phosphate accumulator in soil. The presence of functional genes (polyphosphate kinase [ppk] and exopolyphosphatase [ppx]) in all PAB depicts their importance in polyphosphate metabolism in bacteria. Genetic and functional diversity reveals conservation of the ppx gene at species level. Furthermore, we found a positive correlation between PAB and plant growth promotary traits, stress tolerance, and salinity stress alleviation in A. thaliana.

Deciphering Trifolium pratense L. holobiont reveals a microbiome resilient to future climate changes.

The plant microbiome supports plant growth, fitness, and resistance against climate change. Trifolium pratense (red clover), an important forage legume crop, positively contributes to ecosystem sustainability. However, T. pratense is known to have limited adaptive ability toward climate change. Here, the T. pratense microbiomes (including both bacteria and fungi) of the rhizosphere and the root, shoot, and flower endospheres were comparatively examined using metabarcoding in a field located in Central Germany that mimics the climate conditions projected for the next 50-70 years in comparison with the current climate conditions. Additionally, the ecological functions and metabolic genes of the microbial communities colonizing each plant compartment were predicted using FUNGuild, FAPROTAX, and Tax4Fun annotation tools. Our results showed that the individual plant compartments were colonized by specific microbes. The bacterial and fungal community compositions of the belowground plant compartments did not vary under future climate conditions. However, future climate conditions slightly altered the relative abundances of specific fungal classes of the aboveground compartments. We predicted several microbial functional genes of the T. pratense microbiome involved in plant growth processes, such as biofertilization (nitrogen fixation, phosphorus solubilization, and siderophore biosynthesis) and biostimulation (phytohormone and auxin production). Our findings indicated that T. pratense microbiomes show a degree of resilience to future climate changes. Additionally, microbes inhabiting T. pratense may not only contribute to plant growth promotion but also to ecosystem sustainability.

Plant growth promoting Pseudomonas aeruginosa from Valeriana wallichii displays antagonistic potential against three phytopathogenic fungi.

The soil nature and characterstics are directly related to the micro-organisms present, bio-mineralization process, plant type and thus having harmonius and interdependent relationships. Soil bacteria having antagonistic activity against phytopathogens, play an important role in root growth, overall plant growth and also their composition depends upon the plant species. Population explosion across globe has resulted in indiscriminate use of chemical fertilizers, fungicides and pesticides, thus posing serious risk to plant productivity and soil flora. Plant growth promoting rhizobacteria (PGPRs) are considered safer than chemical fertilizers as they are eco-friendly and sustain longer after colonization in rhizospheric soil. PGPRs are preferred as a green choice and acts as a superior biocontrol agents against phytopathogens. In the present study, a potential rhizobacteria, Pseudomonas aeruginosa (isolate-2) was isolated from the rhizosphere of a medicinal plant, Valeriana wallichi. The bacterial isolate exhibited qualitative tests for plant growth promoting determinatives. It was also subjected to in-vitro biocontrol activity against potential phytopathogens viz. Alternaria alternata, Aspergillus flavus and F. oxysporum. The antagonistic efficacy against F. oxysporum was 56.2% followed by Alternaria alternata to be 51.02%. The maximum inhibition of radial growth of F. oxysporum was 69.2%, Alternaria alternata (46.4%) and Aspergillus flavus (15%). The Pseudomonas aeruginosa exhibited plant growth promotion rhizobacterial activity which can be expoited as biofertilizers. This study deals with microbial revitalization strategy and offers promising solution as a biocontrol agent to enhance crop yield. Further, PGPRs research using the interdisciplinary approaches like biotechnology, nanotechnology etc. will unravel the molecular mechanisms which may be helpful for maximizing its potential in sustainable agriculture.

A Framework for the Selection of Plant Growth-Promoting Rhizobacteria Based on Bacterial Competence Mechanisms.

The use of plant growth-promoting rhizobacteria (PGPR) is increasingly meaningful for the development of more environmentally friendly agricultural practices. However, often the PGPR strains selected in the laboratory fail to confer the expected beneficial effects when evaluated in plant experiments. Insufficient rhizosphere colonization is pointed out as one of the causes. With the aim of minimizing this inconsistency, we propose that besides studying plant growth promotion traits (PGP), the screening strategy should include evaluation of the microbial phenotypes required for colonization and persistence. As a model, we carried out this strategy in three Rhizobium sp. strains that showed phosphorus solubilization ability and production of siderophores. All strains displayed colonization phenotypes like surface spreading, resistance to hydrogen peroxide, and formed biofilms. Regarding their ability to persist, biofilm formation was observed to be influenced by pH and the phosphorus nutrient provided in the growth media. Differences in the competence of the strains to use several carbon substrates were also detected. As part of our framework, we compared the phenotypic characteristics of the strains in a quantitative manner. The data analysis was integrated using a multicriteria decision analysis (MCDA). All our results were scored, weighted, and grouped as relevant for PGP, colonization, or persistence. MCDA demonstrated that, when the phenotypes related to PGP and colonization are weighted over those for persistence, strain B02 performs better than the other two Rhizobium sp. strains. The use of our framework could assist the selection of more competent strains to be tested in greenhouse and field trials.IMPORTANCE Numerous plant growth-promoting rhizobacteria (PGPR) have been inoculated into the soil with the aim of improving the supply of nutrients to crop plants and decreasing the requirement of chemical fertilizers. However, sometimes these microbes fail to competitively colonize the plant roots and rhizosphere. Hence, the plant growth promotion effect is not observed. Here, we describe a new screening strategy aiming at the selection of more competent PGPR. We evaluated bacterial phenotypes related to plant growth promotion, colonization, and persistence. Our results demonstrated that despite the fact that our Rhizobium sp. strains successfully solubilized phosphorus and produced siderophores, their abilities to spread over surfaces, resist hydrogen peroxide, and form biofilms varied. Additionally, a multicriteria decision analysis was used to analyze the data that originated from bacterial physiological characterizations. This analysis allowed us to innovatively evaluate each strain as a whole and compare the performances of the strains under hypothetical scenarios of bacterial-trait requirements.

Citryl Ornithine Is an Intermediate in a Three-Step Biosynthetic Pathway for Rhizoferrin in Francisella.

The Gram-negative bacterium Francisella tularensis secretes the siderophore rhizoferrin to scavenge necessary iron from the environment. Rhizoferrin, also produced by a variety of fungi and bacteria, comprises two citrate molecules linked by amide bonds to a central putrescine (diaminobutane) moiety. Genetic analysis has determined that rhizoferrin production in F. tularensis requires two enzymes: FslA, a siderophore synthetase of the nonribosomal peptide synthetase-independent siderophore synthetase (NIS) family, and FslC, a pyridoxal-phosphate-dependent decarboxylase. To discern the steps in the biosynthetic pathway, we tested F. tularensis strain LVS and its ΔfslA and ΔfslC mutants for the ability to incorporate potential precursors into rhizoferrin. Unlike putrescine supplementation, supplementation with ornithine greatly enhanced siderophore production by LVS. Radioactivity from L-[U-14C] ornithine, but not from L-[1-14C] ornithine, was efficiently incorporated into rhizoferrin by LVS. Although neither the ΔfslA nor the ΔfslC mutant produced rhizoferrin, a putative siderophore intermediate labeled by both [U-14C] ornithine and [1-14C] ornithine was secreted by the ΔfslC mutant. Rhizoferrin was identified by liquid chromatography and mass spectrometry in LVS culture supernatants, while citryl-ornithine was detected as the siderophore intermediate in the culture supernatant of the ΔfslC mutant. Our findings support a three-step pathway for rhizoferrin production in Francisella; unlike the fungus Rhizopus delemar, where putrescine functions as a primary precursor for rhizoferrin, biosynthesis in Francisella preferentially starts with ornithine as the substrate for FslA-mediated condensation with citrate. Decarboxylation of this citryl ornithine intermediate by FslC is necessary for a second condensation reaction with citrate to produce rhizoferrin.

Iron Homeostasis in Bacillus subtilis Requires Siderophore Production and Biofilm Formation.

Iron (Fe) is the most important metal in biology. Despite its abundance, Fe is mostly present as a ferric form in soils, strongly limiting its bioavailability. To overcome the challenge of Fe acquisition, many microorganisms produce siderophores to retrieve Fe from natural sources. Another ubiquitous feature of bacteria in natural environments is biofilm formation. Previous studies showed that external Fe strongly influenced biofilm formation in several bacteria, suggesting that this microenvironment plays a mechanistic role in micronutrient acquisition for bacteria. Here, we applied a complementary set of analytical methods and deletion mutants to evaluate the role of biofilm formation, siderophore production, and their interaction in Fe homeostasis in Bacillus subtilis We observed that Fe homeostasis, i.e., active growth at a constant intracellular Fe concentration, requires both siderophore production and biofilm formation. Also, we report that in B. subtilis, both biofilm formation and siderophore production are required to achieve active Fe acquisition from the medium and to sustain normal growth. Furthermore, we provide evidence that the formation of biofilm slightly enhances the kinetics of Fe complexation by catechol siderophores and markedly improves siderophore use efficiency. These results provide new perspectives on the mechanism underlying siderophore-based acquisition of Fe in biofilm-forming bacteria.IMPORTANCE Iron acquisition is of fundamental importance for microorganisms, since this metal is generally poorly bioavailable under natural conditions. In the environment, most bacteria are found tightly packed within multicellular communities named biofilms. Here, using the soil Gram-positive bacterium Bacillus subtilis, we show that biofilm formation and the production of siderophores, i.e., small molecules specifically binding metals, are both essential to ensure Fe uptake from the medium and maintain cellular Fe homeostasis. The biofilm matrix appears to play an important role favoring the efficient usage of siderophores. Taken together, our results demonstrate a close link between biofilm formation and iron acquisition in B. subtilis, allowing a better comprehension of how bacteria can cope with metal limitation under environmental conditions.

Zinc Ions Affect Siderophore Production by Fungi Isolated from the Panax ginseng Rhizosphere.

Although siderophore compounds are mainly biosynthesized as a response to iron deficiency in the environment, they also bind with other metals. A few studies have been conducted on the impact of heavy metals on the siderophore-mediated iron uptake by microbiome. Here, we investigated siderophore production by a variety of rhizosphere fungi under different concentrations of Zn²âº ion. These strains were specifically isolated from the rhizosphere of Panax ginseng (Korean ginseng). The siderophore production of isolated fungi was investigated with chrome azurol S (CAS) assay liquid media amended with different concentrations of Zn²âº (50 to 250 µg/ml). The percentage of siderophore units was quantified using the ultra-violet (UV) irradiation method. The results indicated that high concentrations of Zn²âº ion increase the production of siderophore in iron-limited cultures. Maximum siderophore production by the fungal strains was detected at Zn²âº ion concentration of 150 µg/ml except for Mortierella sp., which had the highest siderophore production at 200 µg/ml. One potent siderophore-producing strain (Penicillium sp. JJHO) was strongly influenced by the presence of Zn²âº ions and showed high identity to P. commune (100% using 18S-rRNA sequencing). The purified siderophores of the Penicillium sp. JJHO strain were chemically identified using UV, Fourier-transform infrared spectroscopy (FTIR), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) spectra.

Engineering a cleavable disulfide bond into a natural product siderophore using precursor-directed biosynthesis.

An analogue of the bacterial siderophore desferrioxamine B (DFOB) containing a disulfide motif in the backbone was produced from Streptomyces pilosus cultures supplemented with cystamine. Cystamine competed against native 1,5-diaminopentane during assembly. DFOB-(SS)1[001] and its complexes with Fe(iii) or Ga(iii) were cleaved upon incubation with dithiothreitol. Compounds such as DFOB-(SS)1[001] and its thiol-containing cleavage products could expand antibiotic strategies and Au-S-based nanotechnologies.

Interplay between siderophores and colibactin genotoxin in Escherichia coli.

Highly pathogenic Escherichia coli strains that belong to the phylogenetic group B2 have developed a greater ability to acquire iron (heme receptor and numerous siderophores), to produce the genotoxin colibactin and to synthesize antimicrobial siderophore-microcins. There is an increased prevalence of these E. coli strains over the last 30 years in the intestinal microbiota in industrialized countries. Integrating the regulation of fitness/virulence factors, such as siderophores, colibactin and siderophore-microcins into networks that respond to specific environmental signals, such as the local iron concentration, could result in an accurate production of specific fitness/virulence factors, so that the E. coli can adapt to the competitive environment that is the gut and/or the blood. Iron deficiency is common in infancy, even in industrialized countries. Usual strategies for anemia correction are iron supplementation and iron fortification of foods. The long-term consequences and risks associated with high iron supply in the light of this iron-dependent network described in this review could explain at least in part the increased prevalence of E. coli B2 in the gut of people in industrialized countries. © 2017 IUBMB Life, 69(6):435-441, 2017.

Xanthoferrin, the α-hydroxycarboxylate-type siderophore of Xanthomonas campestris pv. campestris, is required for optimum virulence and growth inside cabbage.

Xanthomonas campestris pv. campestris causes black rot, a serious disease of crucifers. Xanthomonads encode a siderophore biosynthesis and uptake gene cluster xss (Xanthomonas siderophore synthesis) involved in the production of a vibrioferrin-type siderophore. However, little is known about the role of the siderophore in the iron uptake and virulence of X. campestris pv. campestris. In this study, we show that X. campestris pv. campestris produces an α-hydroxycarboxylate-type siderophore (named xanthoferrin), which is required for growth under low-iron conditions and for optimum virulence. A mutation in the siderophore synthesis xssA gene causes deficiency in siderophore production and growth under low-iron conditions. In contrast, the siderophore utilization ΔxsuA mutant is able to produce siderophore, but exhibits a defect in the utilization of the siderophore-iron complex. Our radiolabelled iron uptake studies confirm that the ΔxssA and ΔxsuA mutants exhibit defects in ferric iron (Fe3+ ) uptake. The ΔxssA mutant is able to utilize and transport the exogenous xanthoferrin-Fe3+ complex; in contrast, the siderophore utilization or uptake mutant ΔxsuA exhibits defects in siderophore uptake. Expression analysis of the xss operon using a chromosomal gusA fusion indicates that the xss operon is expressed during in planta growth and under low-iron conditions. Furthermore, exogenous iron supplementation in cabbage leaves rescues the in planta growth deficiency of ΔxssA and ΔxsuA mutants. Our study reveals that the siderophore xanthoferrin is an important virulence factor of X. campestris pv. campestris which promotes in planta growth by the sequestration of Fe3+ .

Identification and genomic analysis of antifungal property of a tomato root endophyte Pseudomonas sp. p21.

Pseudomonas sp., which occupy a variety of ecological niches, have been widely studied for their versatile metabolic capacity to promote plant growth, suppress microbial pathogens, and induce systemic resistance in plants. In this study, a Pseudomonas sp. strain p21, which was isolated from tomato root endophytes, was identified as having antagonism against Aspergillus niger. Further analysis showed that this strain had the ability to biosynthesise siderophores and was less effective in inhibiting the growth of A. niger with the supplementation of Fe3+ in the agar medium. Genomic sequencing and the secondary metabolite cluster analysis demonstrated that Pseudomonas sp. p21 harboured 2 pyoverdine biosynthetic gene clusters, which encode compounds with predicted core structures and two variable tetra-peptide or eleven-peptide chains. The results indicated that siderophore-mediated competition for iron might be an important mechanism in Pseudomonas suppression of the fungal pathogen A. niger and in microbe-pathogen-plant interactions.

Salad Leaf Juices Enhance Salmonella Growth, Colonization of Fresh Produce, and Virulence.

We show in this report that traces of juices released from salad leaves as they become damaged can significantly enhance colonization of salad leaves by Salmonella enterica Salad juices in water increased Salmonella growth by 110% over the level seen with the unsupplemented control and in host-like serum-based media by more than 2,400-fold over control levels. In serum-based media, salad juices induced growth of Salmonella via provision of Fe from transferrin, and siderophore production was found to be integral to the growth induction process. Other aspects relevant to salad leaf colonization and retention were enhanced, such as motility and biofilm formation, which were increased over control levels by >220% and 250%, respectively; direct attachment to salad leaves increased by >350% when a salad leaf juice was present. In terms of growth and biofilm formation, the endogenous salad leaf microbiota was largely unresponsive to leaf juice, suggesting that Salmonella gains a marked growth advantage from fluids released by salad leaf damage. Salad leaf juices also enhanced pathogen attachment to the salad bag plastic. Over 5 days of refrigeration (a typical storage time for bagged salad leaves), even traces of juice within the salad bag fluids increased Salmonella growth in water by up to 280-fold over control cultures, as well as enhancing salad bag colonization, which could be an unappreciated factor in retention of pathogens in fresh produce. Collectively, the study data show that exposure to salad leaf juice may contribute to the persistence of Salmonella on salad leaves and strongly emphasize the importance of ensuring the microbiological safety of fresh produce. IMPORTANCE: Salad leaves are an important part of a healthy diet but have been associated in recent years with a growing risk of food poisoning from bacterial pathogens such as Salmonella enterica Although this is considered a significant public health problem, very little is known about the behavior of Salmonella in the actual salad bag. We show that juices released from the cut ends of the salad leaves enabled the Salmonella cells to grow in water, even when it was refrigerated. Salad juice exposure also helped the Salmonella cells to attach to the salad leaves so strongly that washing could not remove them. Collectively, the results presented in this report show that exposure to even traces of salad leaf juice may contribute to the persistence of Salmonella on salad leaves as well as priming it for establishing an infection in the consumer.

Enterobactin as Part of the Oxidative Stress Response Repertoire.

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.

Burkholderia genome mining for nonribosomal peptide synthetases reveals a great potential for novel siderophores and lipopeptides synthesis.

Burkholderia is an important genus encompassing a variety of species, including pathogenic strains as well as strains that promote plant growth. We have carried out a global strategy, which combined two complementary approaches. The first one is genome guided with deep analysis of genome sequences and the second one is assay guided with experiments to support the predictions obtained in silico. This efficient screening for new secondary metabolites, performed on 48 gapless genomes of Burkholderia species, revealed a total of 161 clusters containing nonribosomal peptide synthetases (NRPSs), with the potential to synthesize at least 11 novel products. Most of them are siderophores or lipopeptides, two classes of products with potential application in biocontrol. The strategy led to the identification, for the first time, of the cluster for cepaciachelin biosynthesis in the genome of Burkholderia ambifaria AMMD and a cluster corresponding to a new malleobactin-like siderophore, called phymabactin, was identified in Burkholderia phymatum STM815 genome. In both cases, the siderophore was produced when the strain was grown in iron-limited conditions. Elsewhere, the cluster for the antifungal burkholdin was detected in the genome of B. ambifaria AMMD and also Burkholderia sp. KJ006. Burkholderia pseudomallei strains harbor the genetic potential to produce a novel lipopeptide called burkhomycin, containing a peptidyl moiety of 12 monomers. A mixture of lipopeptides produced by Burkholderia rhizoxinica lowered the surface tension of the supernatant from 70 to 27 mN·m(-1) . The production of nonribosomal secondary metabolites seems related to the three phylogenetic groups obtained from 16S rRNA sequences. Moreover, the genome-mining approach gave new insights into the nonribosomal synthesis exemplified by the identification of dual C/E domains in lipopeptide NRPSs, up to now essentially found in Pseudomonas strains.

Inhibition of marine Vibrio sp. by pyoverdine from Pseudomonas aeruginosa PA1.

Siderophores are low-molecular-weight chemicals that are secreted by many microorganisms to chelate iron from the external environment in order to facilitate their growth and diverse metabolisms. In this study, a fluorescent siderophore, pyoverdine, secreted by Pseudomonas aeruginosa PA1 was purified by affinity chromatography using Cu-sepharose. Pyoverdine was determined to have a molecular mass of 1333.54 Da, as determined by MALDI-TOF/TOF, and belong to type I pyoverdine, as determined by PCR analysis of its corresponding outer membrane ferri-pyoverdine receptor. Pyoverdine showed different degrees of inhibitory effects on the growth of marine Vibrio sp. strains. It was also shown that the biofilm developed by Vibrio parahaemolyticus WzW1 and Wz2121 and Vibrio cyclitrophicus HS12 was significantly reduced, alone with the repressed growth in the presence of pyoverdine. Siderophore production was determined in the strains of Vibrio sp. in response to the pyoverdine-induced iron-limited conditions. The siderophore production of most Vibrio sp. was up-regulated, with the exception of the bacteria that produced little siderophore. Furthermore, Apostichopus japonicus cultured in pyoverdine pretreated seawater showed a relative percent of survival of 89% when they were challenged by Vibrio splendidus. Our results demonstrated that pyoverdine may be a promising agent that could be potentially applied to treat vibriosis.

A novel siderophore system is essential for the growth of Pseudomonas aeruginosa in airway mucus.

Pseudomonas aeruginosa establishes airway infections in Cystic Fibrosis patients. Here, we investigate the molecular interactions between P. aeruginosa and airway mucus secretions (AMS) derived from the primary cultures of normal human tracheal epithelial (NHTE) cells. PAO1, a prototype strain of P. aeruginosa, was capable of proliferating during incubation with AMS, while all other tested bacterial species perished. A PAO1 mutant lacking PA4834 gene became susceptible to AMS treatment. The ΔPA4834 mutant was grown in AMS supplemented with 100 µM ferric iron, suggesting that the PA4834 gene product is involved in iron metabolism. Consistently, intracellular iron content was decreased in the mutant, but not in PAO1 after the AMS treatment. Importantly, a PAO1 mutant unable to produce both pyoverdine and pyochelin remained viable, suggesting that these two major siderophore molecules are dispensable for maintaining viability during incubation with AMS. The ΔPA4834 mutant was regrown in AMS amended with 100 µM nicotianamine, a phytosiderophore whose production is predicted to be mediated by the PA4836 gene. Infectivity of the ΔPA4834 mutant was also significantly compromised in vivo. Together, our results identify a genetic element encoding a novel iron acquisition system that plays a previously undiscovered role in P. aeruginosa airway infection.

Optimization of triacetylfusarinine C and ferricrocin productions in Aspergillus fumigatus.

Iron is an essential element for all microorganisms. Bacteria and fungi produce versatile siderophores for binding and storing this essential transition metal when its availability is limited in the environment. The aim of the study was to optimize the fermentation medium of Aspergillus fumigatus for siderophore production. Triacetyl-fusarinine C and ferricrocin yields were dependent on glucose and glycine supplementations as well as the initial pH of the culture media. The optimal fermentation medium for triacetylfusarinine C production contained 8% glucose, 0.4% glycine and the initial pH was set to 5.9. Meanwhile, maximal ferricrocin yields were recorded in the presence of 10% glucose, 0.5% glycine and at an initial pH of 7.4. Under optimized fermentation conditions, the yields for triacetylfusarinine C and ferricrocin increased up to 2.9 g/l culture medium and 18.9 mg/g mycelium, respectively.

Endogenous siderophore 2,5-dihydroxybenzoic acid deficiency promotes anemia and splenic iron overload in mice.

Eukaryotes produce a siderophore-like molecule via a remarkably conserved biosynthetic pathway. 3-OH butyrate dehydrogenase (BDH2), a member of the short-chain dehydrogenase (SDR) family of reductases, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA). Depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of intracellular iron and mitochondrial iron deficiency in cultured mammalian cells, as well as in yeast cells and zebrafish embryos We disrupted murine bdh2 by homologous recombination to analyze the effect of bdh2 deletion on erythropoiesis and iron metabolism. bdh2 null mice developed microcytic anemia and tissue iron overload, especially in the spleen. Exogenous supplementation with 2,5-DHBA alleviates splenic iron overload in bdh2 null mice. Additionally, bdh2 null mice exhibit reduced serum iron. Although BDH2 has been proposed to oxidize ketone bodies, we found that BDH2 deficiency did not alter ketone body metabolism in vivo. In sum, our findings demonstrate a key role for BDH2 in erythropoiesis.

The ssbL gene harbored by the ColV plasmid of an Escherichia coli neonatal meningitis strain is an auxiliary virulence factor boosting the production of siderophores through the shikimate pathway.

The ability to capture iron is a challenge for most bacteria. The neonatal meningitis Escherichia coli strain S88 possesses several iron uptake systems, notably including siderophores. Transcriptional analysis of the ColV plasmid pS88 has shown strong induction of a previously undescribed gene with low identity to three E. coli chromosomal genes encoding phospho-2-dehydro-3-deoxyheptonate aldolases involved in aromatic amino acid and catecholate/phenolate siderophore biosynthesis through the shikimate pathway. Here, we investigated the role of this gene, ssbLp (ssbL carried on the plasmid), in siderophore biosynthesis and, consequently, in S88 virulence. We constructed an S88 mutant designated S88 ΔssbLp, which exhibited reduced growth under low-iron conditions compared to the wild-type strain. Liquid chromatography-mass spectroscopy analysis of culture supernatants showed that the mutant secreted significantly smaller amounts of enterobactin, salmochelin SX, and yersiniabactin than the wild-type strain. The mutant was also less virulent in a neonatal rat sepsis model, with significantly lower bacteremia and mortality. Supplementation with chorismate, the final product of the shikimate pathway, restored the wild-type phenotype in vitro. In a collection of human extraintestinal E. coli isolates, we found that ssbL was present only in strains harboring the iro locus, encoding salmochelins, and was located either on the chromosome or on plasmids. Acquisition of the iro locus has been accompanied by acquisition of the auxiliary gene ssbL, which boosts the metabolic pathway essential for catecholate/phenolate siderophore biosynthesis and could represent potential therapeutic targets.

Towards high-siderophore-content foods: optimisation of coprogen production in submerged cultures of Penicillium nalgiovense.

BACKGROUND: Fungal siderophores are likely to possess atheroprotective effects in humans, and therefore studies are needed to develop siderophore-rich food additives or functional foods to increase the siderophore uptake in people prone to cardiovascular diseases. In this study the siderophore contents of mould-ripened cheeses and meat products were analysed and the coprogen production by Penicillium nalgiovense was characterised. RESULTS: High concentrations of hexadentate fungal siderophores were detected in penicillia-ripened Camembert- and Roquefort-type cheeses and also in some sausages. In one sausage fermented by P. nalgiovense, the siderophore content was comparable to those found in cheeses. Penicillium nalgiovense produced high concentrations of coprogen in submerged cultures, which were affected predominantly by the available carbon and nitrogen sources under iron starvation. Considerable coprogen yields were still detectable in the presence of iron when the fermentation medium was supplemented with the iron chelator Na2-EDTA or when P. nalgiovense was co-cultivated with Saccharomyces cerevisiae. CONCLUSION: These data may be exploitable in the future development of high-siderophore-content foods and/or food additives. Nevertheless, the use of P. nalgiovense fermentation broths for these purposes may be limited by the instability of coprogen in fermentation media and by the ß-lactam production by the fungus.