CAS Assay Research Articles

Deciphering FUR-Regulated Gene Networks in Klebsiella pneumoniae Using FUR Knockout mutant

Klebsiella pneumoniae is a nosocomial pathogen causing life-threatening infections worldwide. It possesses many virulence properties and the ferric uptake regulator (FUR) is the transcription factor that regulates the expression of various genes in K. pneumoniae. FUR binds to a conserved 19bp sequence, called the FUR box, for either positive or negative regulation of the gene. FUR alters gene expression according to iron availability and also participates in virulence, colonization & toxin secretion in K. pneumoniae. FUR deletion mutant is an interesting model to understand the virulence factors. In the present study, we identified the genes containing putative FUR boxes (>50% sequence homology) from the genome of K. pneumoniae M33. FUR deletion mutant for K. pneumoniae M33 was created using lambda red recombinase system using the plasmids- pACBSR-hyg, pMDIAI, and pFLP-hyg. The mutant (M33∆FUR) was characterized using various assays like CAS assay, string test, crystal violet assay to access siderophore production, capsule synthesis and biofilm formation respectively. FUR box was present upstream of 18 genes with distinct functions. In the FUR knockout mutant, M33∆FUR an increase in expression of iron transport and siderophore related genes feoC, fhuA, fepB and fes was observed whereas genes feoA, cirA, fecA, fepA and entC were down-regulated. Genes related to biofilm (fimA and mrkD) were downregulated whereas genes related to capsular polysaccharide (rcsA and rcsB) were upregulated upon FUR deletion. Among other genes murG and sucA were downregulated and priB and srlB were upregulated upon FUR deletion. This study shows that FUR regulates many genes involved in virulence, either positively or negatively.

Buffer-free CAS assay using a diluted growth medium efficiently detects siderophore production and microbial growth.

Siderophores are iron chelators and low-molecular-weight compounds secreted by various microorganisms under low-iron conditions. Many microorganisms produce siderophores in the natural environment as iron is an essential element for many of them. CAS assays are widely used to detect siderophores in cultures of various microorganisms; however, it is necessary to improve their sensitivity for the efficient application to fastidious microorganisms. We developed a simple, high-throughput CAS assay employing a buffer-free CAS reagent and diluted growth medium (10% dR2A) in a 96-well microplate. Using a diluted growth medium in agar plates suitable for iron-restricted conditions supported siderophore production by microorganisms from activated sludge. A buffer-free CAS reagent combined with a diluted growth medium revealed that these microorganisms tended to produce more siderophores or iron chelators than microorganisms under iron-rich conditions. Moreover, this buffer-free CAS assay easily and efficiently detected not only siderophore production but also the growth of fastidious microorganisms.

Two distinct non-ribosomal peptide synthetase-independent siderophore synthetase gene clusters identified in Armillaria and other species in the Physalacriaceae.

Siderophores are important for ferric iron solubilization, sequestration, transportation, and storage, especially under iron-limiting conditions such as aerobic conditions at high pH. Siderophores are mainly produced by non-ribosomal peptide synthetase-dependent siderophore pathway, non-ribosomal peptide synthetase-independent siderophore synthetase pathway, or the hybrid non-ribosomal peptide synthetases/non-ribosomal peptide synthetases-independent siderophore pathway. Outcompeting or inhibition of plant pathogens, alteration of host defense mechanisms, and alteration of plant-fungal interactions have been associated with fungal siderophores. To understand these mechanisms in fungi, studies have been conducted on siderophore biosynthesis by ascomycetes with limited focus on the basidiomycetes. Armillaria includes several species that are pathogens of woody plants and trees important to agriculture, horticulture, and forestry. The aim of this study was to investigate the presence of non-ribosomal peptide synthetases-independent siderophore synthetase gene cluster(s) in genomes of Armillaria species using a comparative genomics approach. Iron-dependent growth and siderophore biosynthesis in strains of selected Armillaria spp. were also evaluated in vitro. Two distinct non-ribosomal peptide synthetases-independent siderophore synthetase gene clusters were identified in all the genomes. All non-ribosomal peptide synthetases-independent siderophore synthetase genes identified putatively encode Type A' non-ribosomal peptide synthetases-independent siderophore synthetases, most of which have IucA_IucC and FhuF-like transporter domains at their N- and C-terminals, respectively. The effect of iron on culture growth varied among the strains studied. Bioassays using the CAS assay on selected Armillaria spp. revealed in vitro siderophore biosynthesis by all strains irrespective of added FeCl3 concentration. This study highlights some of the tools that Armillaria species allocate to iron homeostasis. The information generated from this study may in future aid in developing molecular based methods to control these phytopathogens.

Siderophore-producing Bacillus amyloliquefaciens BM3 mitigate arsenic contamination and suppress Fusarium wilt in brinjal plants.

Arsenic contamination in agricultural soils poses a serious health risk for humans. Bacteria that produce siderophores, primarily for iron acquisition, can be relevant in combating arsenic toxicity in agricultural soils and simultaneously act as biocontrol agents against plant diseases. We evaluated the arsenic bioremediation and biocontrol potential of the rhizosphere isolate Bacillus amyloliquefaciens BM3 and studied the interaction between the purified siderophore bacillibactin and arsenic. BM3 showed high arsenic resistance [MIC value 475 and 24mM against As(V) and As(III), respectively] and broad spectrum in-vitro antagonism against several phytopathogenic fungi. BM3 was identified by biochemical characterization and 16S rRNA gene sequencing. Scanning electron microscopy (SEM) analysis revealed increased cell size of BM3 when grown in presence of sub-lethal arsenic concentrations. Bioremediation assays showed a 74% and 88.1% reduction in As(V) and As(III) concentrations, respectively. Genetic determinants for arsenic resistance (arsC and aoxB) and antifungal traits (bacAB and chiA) were detected by PCR. Arsenic chelating ability of bacillibactin, the siderophore purified from culture filtrate of BM3 and identified through spectroscopic data analysis, was observed in CAS assay and fluorescence spectrometry. In-vivo application of talc-based formulation of BM3 in brinjal seedlings showed significant reduction in Fusarium wilt disease. Strain B. amyloliquefaciens BM3 may be useful in arsenic bioremediation and may be considered for large field trials as an alternative to chemical fungicides by inhibiting soil borne pathogens.

Heterologous Production and Biosynthesis of Threonine-16:0dioic acids with a Hydroxamate Moiety.

Dereplication and genome mining in Streptomyces aureus LU18118 combined with heterologous expression of selected biosynthetic gene clusters (BGCs) led to the discovery of various threonine-16:0dioic acids named lipothrenins. Lipothrenins consist of the core elements l-Thr, d-allo-Thr, or Dhb, which are linked to hexadecanedioic acid by an amide bond. The main compound lipothrenin A (1) carries the N-hydroxylated d-allo form of threonine and expresses a siderophore activity. The lipothrenin BGC was analyzed by a series of deletion experiments. As a result, a variety of interesting genes involved in the recruitment and selective activation of linear 16:0dioic acids, amide bond formation, and the epimerization of l-Thr were revealed. Furthermore, a diiron N-oxygenase was identified that may be directly involved in the monooxygenation of the amide bond. This is divergent from the usual hydroxamate formation mechanism in siderophores, which involves hydroxylation of the free amine prior to amide bond formation. Siderophore activity was observed for all N-hydroxylated lipothrenins by application of the CAS assay method.

Nonribosomal peptide synthetase gene clusters and characteristics of predicted NRPS-dependent siderophore synthetases in Armillaria and other species in the Physalacriaceae.

Fungal secondary metabolites are often pathogenicity or virulence factors synthesized by genes contained in secondary metabolite gene clusters (SMGCs). Nonribosomal polypeptide synthetase (NRPS) clusters are SMGCs which produce peptides such as siderophores, the high affinity ferric iron chelating compounds required for iron uptake under aerobic conditions. Armillaria spp. are mostly facultative necrotrophs of woody plants. NRPS-dependent siderophore synthetase (NDSS) clusters of Armillaria spp. and selected Physalacriaceae were investigated using a comparative genomics approach. Siderophore biosynthesis by strains of selected Armillaria spp. was evaluated using CAS and split-CAS assays. At least one NRPS cluster and other clusters were detected in the genomes studied. No correlation was observed between the number and types of SMGCs and reported pathogenicity of the species studied. The genomes contained one NDSS cluster each. All NDSSs were multi-modular with the domain architecture (ATC)3(TC)2. NDSS clusters of the Armillaria spp. showed a high degree of microsynteny. In the genomes of Desarmillaria spp. and Guyanagaster necrorhizus, NDSS clusters were more syntenic with NDSS clusters of Armillaria spp. than to those of the other Physalacriaceae species studied. Three A-domain orthologous groups were identified in the NDSSs, and atypical Stachelhaus codes were predicted for the A3 orthologous group. In vitro biosynthesis of mainly hydroxamate and some catecholate siderophores was observed. Hence, Armillaria spp. generally contain one highly conserved, NDSS cluster although some interspecific variations in the products of these clusters is expected. Results from this study lays the groundwork for future studies to elucidate the molecular biology of fungal phyto-pathogenicity.

Aspergillus oryzae attenuates quorum sensing -associated virulence factors and biofilm formation in Klebsiella pneumoniae extended-spectrum beta-lactamases

Background: Klebsiella pneumoniae communicate between and among species using quorum sensing (QS). Biofilm formation and virulence factors are regulated by QS. This QS is indirectly responsible for K. pneumoniae pathogenicity. Inhibiting QS is a novel and highly effective method for controlling K. pneumoniae extended-spectrum beta-lactamases (KP-ESBL) infections. This study aimed to investigate how Aspergillus oryzae extracellular protein (AOEP) affected QS and KP-ESBL virulence factors. Methods: Methods used included minimal inhibitory concentration (MIC) through the microdilution method, biofilms with crystal violet staining, extracellular polysaccharides using the Congo Red assay, quantifying the expression of genes coding for capsular polysaccharide (wzI gene) and adhesion (mrkA gene) through quantitative reverse-transcription polymerase chain reaction (RT-qPCR), siderophore level measurement using Chrome Azurol sulphonate assay (CAS assay), biofilm morphology using a scanning electron microscope (SEM), and confirmation using the life span killing assay method on Caenorhabditis elegans (C. elegans). Results: In vitro studies revealed that AOEP inhibited biofilms and exopolysaccharides (EPS) in KP-ESBL at the sub-MIC level. In addition, AOEP inhibited the expression of the mrkA gene, which is involved in the adhesion process. Furthermore, an in vivo study revealed that AOEP levels of 75 and 150 µg/mL respectively increased C. elegans survival rates by 72.67% and 80.76% against K. pneumoniae infection. Conclusions: Our findings suggest that the extracellular protein of A. oryzae may be an effective QS inhibitor and a novel anti-virulence agent to control bacterial pathogens.

New Quinoline-Urea-Benzothiazole Hybrids as Promising Antitubercular Agents: Synthesis, In Vitro Antitubercular Activity, Cytotoxicity Studies, and In Silico ADME Profiling.

A series of 25 new benzothiazole–urea–quinoline hybrid compounds were synthesized successfully via a three-step synthetic sequence involving an amidation coupling reaction as a critical step. The structures of the synthesized compounds were confirmed by routine spectroscopic tools (1H and 13C NMR and IR) and by mass spectrometry (HRMS). In vitro evaluation of these hybrid compounds for their antitubercular inhibitory activity against the Mycobacterium tuberculosis H37Rv pMSp12::GPF bioreporter strain was undertaken. Of the 25 tested compounds, 17 exhibited promising anti-TB activities of less than 62.5 µM (MIC90). Specifically, 13 compounds (6b, 6g, 6i–j, 6l, 6o–p, 6r–t, and 6x–y) showed promising activity with MIC90 values in the range of 1–10 µM, while compound 6u, being the most active, exhibited sub-micromolar activity (0.968 µM) in the CAS assay. In addition, minimal cytotoxicity against the HepG2 cell line (cell viability above 75%) in 11 of the 17 compounds, at their respective MIC90 concentrations, was observed, with 6u exhibiting 100% cell viability. The hybridization of the quinoline, urea, and benzothiazole scaffolds demonstrated a synergistic relationship because the activities of resultant hybrids were vastly improved compared to the individual entities. In silico ADME predictions showed that the majority of these compounds have drug-like properties and are less likely to potentially cause cardiotoxicity (QPlogHERG > −5). The results obtained in this study indicate that the majority of the synthesized compounds could serve as valuable starting points for future optimizations as new antimycobacterial agents.

Antibacterial activity of siderophores from marine actinobacteria against Gram negative bacterial pathogens

Aim: To study the siderophore production and antibacterial activity of actinobacteria isolated from mangrove plants and sediments. Methodology: In the present study, 112 actinobacterial strains were recovered from mangrove rhizosphere sediments and plants collected from Andaman Islands and mangrove zones at Vellar estuary, Parangipettai, South India. All the strains were screened for antibacterial activity against Escherichia coli, Aeromonas hydrophila, Providencia stuartii, Salmonella paratyphi and Klebsiella pneumoniae and also studied for siderophore production. Results: In total, 13 actinobacterial strains showed broad spectrum of activity against all the five Gram negative bacterial pathogens tested with zone of inhibition ranging between 9.2 to 18.7 mm. In CAS assay,14 actinobacterial strains were found to produce siderophore. Among them, strain MEA 11 was found to produce 88% siderophore in iron free succinate broth. Results of CAS plate assay and FeCl3 test revealed that the siderophore produced by the strain MEA11 as hydroxamate type. In optimization study, variables such as glucose (17.7±0.9 and 17.7±0.3), malt extract (14.7±0.9 and 15.0±0.6) and MgSO4 (12.0±0.6 and 12.3±0.3) were found to influence siderophore production and antimicrobial activity. Further, this multi potentialstrain MEA 11 was characterized and identified as Streptomyces tendae. Interpretation: These results revealed that the marine ecosystem of Andaman coastal area and Parangipettai region harbored a rich consortium of many bioactive actinobacteria, which could synthesize novel bioactive compounds of pharmacological significance.

Isolation and characterization of arsenic-binding siderophores from Rhodococcus erythropolis S43: role of heterobactin B and other heterobactin variants.

Rhodococcus erythropolis S43 is an arsenic-tolerant actinobacterium isolated from an arsenic contaminated soil. It has been shown to produce siderophores when exposed to iron-depleting conditions. In this work, strain S43 was shown to have the putative heterobactin production cluster htbABCDEFGHIJ(K). To induce siderophore production, the strain was cultured in iron-depleted medium in presence and absence of sodium arsenite. The metabolites produced by S43 in the colorimetric CAS and As-mCAS assays, respectively, showed iron- and arsenic-binding properties reaching a chelating activity equivalent to 1.6 mM of desferroxamine B in the supernatant of the culture without arsenite. By solid-phase extraction and two subsequent HPLC separations from both cultures, several fractions were obtained, which contained CAS and As-mCAS activity and which were submitted to LC-MS analyses including fragmentation of the major peaks. The mixed-type siderophore heterobactin B occurred in all analyzed fractions, and the mass of the "Carrano heterobactin A" was detected as well. In addition, generation of a molecular network based on fragment spectra revealed the occurrence of several other compounds with heterobactin-like structures, among them a heterobactin B variant with an additional CH2O moiety. 1H NMR analyses obtained for preparations from the first HPLC step showed signals of heterobactin B and of "Carrano heterobactin A" with different relative amounts in all three samples. In summary, our results reveal that in R. erythropolis S43, a pool of heterobactin variants is responsible for the iron- and arsenic-binding activities. KEY POINTS: • Several heterobactin variants are the arsenic-binding compounds in Rhodococcus erythropolis S43. • Heterobactin B and the compound designated heterobactin A by Carrano are of importance. • In addition, other heterobactins with ornithine in the backbone exist, e.g., the new heterobactin C.

Talaromyces pinophilus strain M13: a portrayal of novel groundbreaking fungal strain for phytointensification.

The aim of current research was to explore different fungi as plant growth promoting fungi (PGPF). Strains of Trichoderma are well explored till now. But there are few other fungal strains that are better than Trichoderma strains. The study involves the isolation of different fungi from the rhizosphere of various agriculture farms. After isolation, 18S rRNA identification was carried out. Isolated fungi belonged to genus Penicillium, Talaromyces, Trichoderma, and Aspergillus. Isolate M13 belonging to genus Talaromyces was screened for its plant growth promoting (PGP) activity as it is a novel strain and still to be explored. Isolate M13 was identified as Talaromyces pinophilus (MG011365). Indole acetic acid (IAA) estimation was carried out using Salkowski reagent. Isolate was allowed to grow in cultivation media (Potato Dextrose Broth, PDB) in which one was supplemented with tryptophan (TRP) and one was without TRP. Phosphate solubilization was assessed using Pikovskaya's media and latter estimated using stannous chloride method, showing decent solubilization of phosphate. Siderophore production was assessed using CAS assay that indicated decent extent of siderophore production. Further for biocontrol, enzymatic assay for β-glucanase and chitinase was carried out. For β-glucanase enzyme production 1% carboxymethylcellulose (CMC) and for chitinase enzyme production 10% v/v colloidal chitin (as a sole carbon source) supplemented in solid minimal-medium-9 (MM9) were used. Antagonism effect of isolate M13 was carried out against Aspergillus niger by dual cultural method. As the strain M13 showed several PGP traits, liquid bioformulation was prepared to perform seed germination assay and pot trials on chickpea, followed by field trial on banana plant. All the experimental data of biochemical assessment with pot and field trials suggest T. pinophilus M13 as a novel fungus that can be used as biointensifier.

Siderophores from the Entomopathogenic Fungus Beauveria bassiana.

We report NMR- and MS-based structural characterizations of siderophores and related compounds from Beauveria bassiana (Balsamo-Crivelli) Vuillemin, including ten new chemical entities (2-4, 6-9, 11-12, and 15) and five known compounds, (1, 5, 10, 13, and 14). The siderophore mixture from ARSEF strain #2680 included two compounds in which N5-mevalonyl-N5-hydroxyornithine replaces both (2) or one (3) of the N5-anhydromevalonyl-N5-hydroxyornithine units of dimerumic acid (1). Mevalonolactone (14) was present as a degradation product of 2 and 3. ARSEF #2860 also produced compounds that have mannopyranose (5, 6) or 4-O-methyl-mannopyranose units (4, 7), two compounds (8, 9) that can be rationalized as 4-O-methyl-mannopyranosyl analogues of the esterifying acid moieties of metachelins A and B, respectively, and two probable decomposition products of 1, a nitro compound (11) and a formate (12). Beauverichelin A (15), a coprogen-type siderophore that represents the di-4-O-methyl-mannopyranosyl analogue of metachelin A, was detected in crude extracts of ARSEF #2860, but only in trace amounts. ARSEF strains #252 and #1955 yielded beauverichelin A in quantities that were sufficient for NMR analysis. Only the di- (1-7) and trihydroxamate (15) siderophores showed iron-binding activity in the CAS assay and, when ferrated, showed strong ESIMS signals consistent with 1:1 ligand/iron complexes.

The influence of pH on Staphylococcus saprophyticus iron metabolism and the production of siderophores

Staphylococcus saprophyticus is a gram-positive coagulase negative bacteria which shows clinical importance due to its capability of causing urinary tract infections (UTI), as well as its ability to persist in this environment. Little is known about how S. saprophyticus adapts to the pH shift that occurs during infection. Thus, in this study we aim to use a proteomic approach to analyze the metabolic adaptations which occur as a response by S. saprophyticus when exposed to acid (5.5) and alkaline (9.0) pH environments. Proteins related to iron storage are overexpressed in acid pH, whilst iron acquisition proteins are overexpressed in alkaline pH. It likely occurs because iron is soluble at acid pH and insoluble at alkaline pH. To evaluate if S. saprophyticus synthesizes siderophores, CAS assays were performed, and the results confirmed their production. The chemical characterization of siderophores demonstrates that S. saprophyticus produces carboxylates derived from citrate. Of special note is the fact that citrate synthase (CS) is down-regulated during incubation at acid pH, corroborating this result. This data was also confirmed by enzymatic assay. Our results demonstrate that iron metabolism regulation is influenced by different pH levels, and show, for the first time, the production of siderophores by S. saprophyticus. Enzymatic assays suggest that citrate from the tricarboxylic acid cycle (TCA) is used as substrate for siderophore production.

2D Image Quantification of Microbial Iron Chelators (Siderophores) Using Diffusive Equilibrium in Thin Films Method.

Siderophores are natural metal chelating agents that strongly control the biogeochemical metal cycles such as Fe in the environment. This article describes a new methodology to detect and quantify at the micromolar concentration the spatial distribution at millimeter scale of siderophores within the root's system. The "universal" CAS assay originally designed for bacterial siderophores detection and later designed for fungus was adapted here for diffusive equilibrium in thin film gel techniques (DET). The method was calibrated against the marketed desferrioxamine mesylate (DFOM) siderophore and applied with experiments performed with sunflower ( Helianthus annuus) and wheat ( Triticum aestivum) cultivated on free iron agar medium plates. We present here the first results with 2D images of the siderophores distribution in the vicinity of the root system of plants. With this technique we detected (i) the production of siderophores on bacteria inoculated ( Pseudomonas fluorescens) environments and (ii) hotspots of natural iron binding ligands production up to 50 μM in the wheat rhizosphere. The lower detection limit in our experiment was 2.5 μmol/L. This new technique offers a unique opportunity to investigate the siderophore production in two dimensions in a wide range of applications from laboratory experiments to natural systems very likely using an in situ and nondestructive tool.

Analysis of desferrioxamine-like siderophores and their capability to selectively bind metals and metalloids: development of a robust analytical RP-HPLC method

The Actinobacterium Gordonia rubripertincta CWB2 (DSM 46758) produces hydroxamate-type siderophores (188 mg L−1) under iron limitation. Analytical reversed-phase HPLC allowed determining a single peak of ferric iron chelating compounds from culture broth which was confirmed by the Fe-CAS assay. Elution profile and its absorbance spectrum were similar to those of commercial (des)ferrioxamine B which was used as reference compound. This confirms previously made assumptions and shows for the first time that the genus Gordonia produces desferrioxamine-like siderophores. The reversed-phase HPLC protocol was optimized to separate metal-free and -loaded oxamines. This allowed to determine siderophore concentrations in solutions as well as metal affinity. The metal loading of oxamines was confirmed by ICP-MS. As a result, it was demonstrated that desferrioxamine prefers trivalent metal ions (Fe3+ > Ga3+ > V3+ > Al3+) over divalent ones. In addition, we aimed to show the applicability of the newly established reversed-phase HPLC protocol and to increase the re-usability of desferrioxamines as metal chelators by immobilization on mesocellular silica foam carriers. The siderophores obtained from strain CWB2 and commercial desferrioxamine B were successfully linked to the carrier with a high yield (up to 95%) which was verified by the HPLC method. Metal binding studies demonstrated that metals can be bound to non-immobilized and to the covalently linked desferrioxamines, but also to the carrier material itself. The latter was found to be unspecific and, therefore, the effect of the carrier material remains a field of future research. By means of a reversed CAS assay for various elements (Nd, Gd, La, Er, Al, Ga, V, Au, Fe, As) it was possible to demonstrate improved Ga3+- and Nd3+-binding to desferrioxamine loaded mesoporous silica carriers. The combination of the robust reversed-phase HPLC method and various CAS assays provides new avenues to screen for siderophore producing strains, and to control purification and immobilization of siderophores.

Putative Iron Acquisition Systems in Stenotrophomonas maltophilia.

Iron has been shown to regulate biofilm formation, oxidative stress response and several pathogenic mechanisms in Stenotrophomonas maltophilia. Thus, the present study is aimed at identifying various iron acquisition systems and iron sources utilized during iron starvation in S. maltophilia. The annotations of the complete genome of strains K279a, R551-3, D457 and JV3 through Rapid Annotations using Subsystems Technology (RAST) revealed two putative subsystems to be involved in iron acquisition: the iron siderophore sensor and receptor system and the heme, hemin uptake and utilization systems/hemin transport system. Screening for these acquisition systems in S. maltophilia showed the presence of all tested functional genes in clinical isolates, but only a few in environmental isolates. NanoString nCounter Elements technology, applied to determine the expression pattern of the genes under iron-depleted condition, showed significant expression for FeSR (6.15-fold), HmuT (12.21-fold), Hup (5.46-fold), ETFb (2.28-fold), TonB (2.03-fold) and Fur (3.30-fold). The isolates, when further screened for the production and chemical nature of siderophores using CAS agar diffusion (CASAD) and Arnows’s colorimetric assay, revealed S. maltophilia to produce catechol-type siderophore. Siderophore production was also tested through liquid CAS assay and was found to be greater in the clinical isolate (30.8%) compared to environmental isolates (4%). Both clinical and environmental isolates utilized hemoglobin, hemin, transferrin and lactoferrin as iron sources. All data put together indicates that S. maltophilia utilizes siderophore-mediated and heme-mediated systems for iron acquisition during iron starvation. These data need to be further confirmed through several knockout studies.

Detection of arsenic-binding siderophores in arsenic-tolerating Actinobacteria by a modified CAS assay

The metalloid arsenic is highly toxic to all forms of life, and in many countries decontamination of water and soil is still required. Some bacteria have mechanisms to detoxify arsenic and can live in its presence. Actinobacteria are well known for their ability to produce a myriad of biologically-active compounds. In the present study, we isolated arsenic-tolerant Actinobacteria from contaminated water in Saxony, Germany, and determined their ability to produce siderophores able to bind arsenic. The binding capacity of different siderophore-like compounds was determined by a modified chrome azurol S (As-mCAS) assay with As(III) at high pH and using CAS decolorization as a readout. Arsenic-tolerant isolates from three actinobacterial genera were identified by 16 S rRNA gene sequence analysis: Rhodococcus, Arthrobacter and Kocuria. The isolated Actinobacteria showed a high As(III)-binding activity by siderophore-like compounds, resulting in 82–100% CAS decolorization, as compared to the results with EDTA. The interaction between As(III) and siderophore-like compounds was also detected at neutral pH. In summary, our results suggest that the isolated arsenic-tolerant Actinobacteria produce siderophores that bind arsenic, and open new perspectives on potential candidates for decontaminating environments with arsenic and for other biotechnological applications.

Comparison of virulence determinants among Acinetobacter baumannii Clinical isolates obtained from Spain and Lebanon.

Acinetobacter baumannii is a pathogen that is causing concern due to its high genetic elasticity, allowing it to show high rates of antibiotic resistance and to express a wide range of virulence determinants. Several studies are aimed towards targeting the virulence of A. baumannii as an adjunct to antibiotic therapy. In this study, we investigate the difference in virulence determinants between A. baumannii isolates obtained from Spain and Lebanon METHODOLOGY: Fifty-nine A. baumannii isolates were collected from La Paz Hospital, Spain, and 90 from St. George Hospital, Lebanon. The isolates were identified using VITEK-2. Biofilm formation was detected by crystal violet staining, hemolysis by blood agars, motility by surface motility assays, siderophore production by CAS assays, and proteolytic activity by azoalbumin assays. The expression of virulence determinants was highly variable among the isolates. Among the Spanish isolates, 84.4% produced biofilms, 54.2% showed hemolysis, and 69.5% produced siderophores. Among the Lebanese isolates, 85.6% produced strong biofilms, 47.8% showed hemolysis, and 57.8% produced siderophores. Proteolytic activity for the Spanish isolates (26.6 ± 8.4 U/L) was slightly higher than that of the Lebanese isolates (17.7 ± 9.5 U/L). Very few Spanish isolates (3) showed surface motility, as opposed to the majority of the Lebanese isolates (80) that showed surface motility. The genomic plasticity of A. baumannii is demonstrated by its ability to differentially express virulence determinants. This highlights the need to treat each isolate as a unique case when attempting to use anti-virulence agents to treat A. baumannii infections.

Root endophytic Chaetomium cupreum promotes plant growth and detoxifies aluminum in Miscanthus sinensisAndersson growing at the acidic mine site

Abstract Miscanthus sinensis Andersson grows naturally at the Hitachi mine. The root‐zone soil was acidic and contained high concentrations of Cu, Pb, Zn and exchangeable Al. Adventitious roots accumulated high concentrations of Al and Fe, but not other heavy metals. The purpose of this study was to elucidate the mechanism of tolerance of Al in M. sinensis, focusing on its chemical interaction with root endophytes. We isolated Chaetomium cupreum, which produced siderophores, from adventitious roots of M. sinensis via CAS assay. In inoculation tests, C. cupreum promoted M. sinensis seedling growth and increased Al and Fe uptake in the roots, although C. cupreum did not stimulate M. sinensis to produce Al detoxicants, such as citric and malic acids. Observation of the pattern of Al localization in the roots clarified that C. cupreum reduced Al toxicity in M. sinensis via compartmentalizing Al into fungal mycelia surrounding the roots and creating a less toxic Al‐localization pattern, allocating Al to the epidermis, endodermis and stele of roots. In conclusion, our results indicated that C. cupreum increases Al tolerance in M. sinensis growing at the acidic mine site.

A universal assay for the detection of siderophore activity in natural waters.

Siderophores, a family of biogenic metal chelating agents, play critical roles in the biogeochemical cycling of Fe and other metals by facilitating their solubilization and uptake in circumneutral to alkaline oxic environments. However, because of their small concentrations (ca. nM) and large number of molecular structures, siderophore detection and quantification in environmental samples requires specialized equipment and expertise, and often requires pre-concentration of samples, which may introduce significant bias. The "universal" CAS assay, which was originally designed for use in bacterial cultures, quantifies the iron chelating function of a pool of siderophores but only at concentrations (>2µM) well above the concentrations estimated to be present in marine, freshwater, and soil samples. In this manuscript, we present a high sensitivity modification of this universal assay (HS-CAS) suitable for detecting and quantifying siderophore activity in the nM concentration range, allowing for direct quantitation of siderophore reactivity in transparent aqueous samples.