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Universal CAS assay for the detection and determination of siderophores
Abstract
A universal method to detect and determine siderophores was developed by using their high affinity for iron(III). The ternary complex chrome azurol S/iron(III)/hexadecyltrimethylammonium bromide, with an extinction coefficient of approximately 100,000 M-1 cm-1 at 630 nm, serves as an indicator. When a strong chelator removes the iron from the dye, its color turns from blue to orange. Because of the high sensitivity, determination of siderophores in solution and their characterization by paper electrophoresis chromatography can be performed directly on supernatants of culture fluids. The method is also applicable to agar plates. Orange halos around the colonies on blue agar are indicative of siderophore excretion. It was demonstrated with Escherichia coli strains that biosynthetic, transport, and regulatory mutations in the enterobactin system are clearly distinguishable. The method was successfully used to screen mutants in the iron uptake system of two Rhizobium meliloti strains, DM5 and 1021.
... Qualitative production of siderophore was determined on Chrome Azurol S (CAS) agar medium. The isolates were spotted onto CAS agar plates and incubated at 28 ± 2°C for 6 days to observe the yellow to orange halo zone around the colony (Schwyn and Neilands, 1987). ...
Rice false smut, which is caused by the soil-borne fungal pathogen Ustilaginoidea virens (U. virens), is one of the most threatening diseases in most of the rice-growing countries including India.
... Isolated strains were tested for plant growth-promoting (PGP) traits like hydrogen cyanide (HCN) production (Bakker and Schippers, 1987), tricalcium phosphate (TCP) solubilization (Mehta and Nautiyal, 2001), ammonia production (Cappuccino and Sherman, 1992), indole acetic acid (IAA) production (Rahman et al. 2010), and siderophore production (Schwyn and Neilands, 1987) using standard procedures. Furthermore, Henderson and Payne (1994)'s CAS shuttle assay was used for quantification. ...
... A log-phase bacterial culture was inoculated onto chrome azurol sulfonate (CAS) agar plates and incubated at 30 • C for 48 s. Following incubation, plates containing the inoculated colonies were inspected for the appearance of an orange halo surrounding the colonies on the blue agar medium [21]. The orange halo and colony diameters were measured to calculate the ratio of the orange halo to the colony diameter (D/d value). ...
The sclerotium of the edible mushroom Polyporus umbellatus (Zhuling) exhibits various medicinal properties. However, given its long growth cycle and overexploitation, wild resources are facing depletion. Macrofungal growth depends on diverse microbial communities; however, the impact of soil bacteria on P. umbellatus development is unknown. Here, we combined high-throughput sequencing and pure culturing to characterize the diversity and potential function of bacteria and fungi inhabiting the P. umbellatus sclerotium and tested the bioactivities of their isolates. Fungal operational taxonomic units (OTUs) were clustered and classified, revealing 1275 genera. Bacterial OTUs yielded 891 genera. Additionally, 81 bacterial and 15 fungal strains were isolated from P. umbellatus sclerotia. Antagonism assays revealed three bacterial strains (FN2, FL19, and CL15) promoting mycelial growth by producing indole-3-acetic acid, solubilizing phosphate, and producing siderophores, suggesting their role in regulating growth, development, and production of active compounds in P. umbellatus. FN2-CL15 combined with bacterial liquid promoted growth and increased the polysaccharide content of P. umbellatus mycelia. This study reports new bioactive microbial resources for fertilizers or pesticides to enhance the growth and polysaccharide accumulation of P. umbellatus mycelia and offers guidance for exploring the correlation between medicinal macrofungi and associated microbial communities.
... Siderophore production was tested using the chrome azurol sulfonate (CAS) assay (Schwyn & Neilands, 1987). The assay was performed according to the method described by Arora and Verma (2017) in which CAS agar plates were spotinoculated with bacterial strains and incubated at 30˚C for 5-7 days. ...
In this study, we investigated the bacterial diversity and plant growth‐promoting (PGP) properties of bulk soil bacteria of Vachellia tortilis subsp. raddiana collected from the Taidalt and Amazloug nature preserves area in southern Morocco. Using repetitive extragenic palindromic polymerase chain reaction fingerprinting and 16S rDNA sequencing, 93 strains were identified. They belonged mainly to the genera Bacillus and Peribacillus. In vitro tests for PGP traits showed high levels of activity for strains belonging to the genera Bacillus and Pseudomonas. It was relevant that several Bacillus strains produced auxin (maximum amount 262.61 µg mL⁻¹ for Bacillus sp. LMR1097), solubilized phosphate (maximum amount 22.10 µg mL⁻¹), or produced siderophores, while three strains were able to fix atmospheric nitrogen (LMR1145, LMR1013, and LMR1015). Inoculation of V. tortilis subsp. raddiana plants with selected bacterial strains (LMR881 and LMR1097) increased shoot and root growth parameters. The strain Pantoea sp. LMR881 had the highest mean values for shoot and root length (24.60–14.80 cm) and shoot dry weight (1.31 g), whereas Bacillus sp. LMR1097 showed the lowest mean shoot dry weight. Overall, these results highlight the potential of using selected native bacterial inoculants for improving growth of candidate tree plants to be used in restoration programs of arid degraded areas.
Wetlands, refer to the confluence with regards to land and water having a rich biological diversity with multiple purpose. As per the Ramsar Convention, framed in August 2002, East Kolkata Wetlands have been considered to be of global significance. Although inappropriate supervision of these wetlands, has led to the gradual deterioration of this wetland. In this present study, the soil sample was collected from these reclaimed agricultural wetlands of Chowbaga area, of East Kolkata areas with coordinates of 22.5296°N, 88.4207°E. The predominant or lingering bacterial flora was isolated from the soil surrounding the rhizospheric region of Cucurbita sp. and Zea mays and their plant growth promotion properties of the lingering bacterial flora were analyzed. These plant growth promoting rhizobacteria (PGPR) included some species of Bacillus sp. that could augment plant growth by different methodologies like nitrogen fixation, phosphate solubilization, phytohormone synthesis, and siderophore production. One of the bacterial isolates DPP(C) showed highest ability of siderophore production of 37.89% at 48 hours, whereas DCP(A) showed better capability in IAA production of 56.785 μg/mL than rest of the bacterial isolates. These reclaimed agricultural lands were found to have very high quantities of heavy metals, especially lead. All the bacterial isolates, were found to have great potential in lead mitigation, whereby they could adsorb up to 8.28% of lead. Some studies have shown the capability of microbial degradation for bioremediation of organic forms of lead and thereby can be removed from agricultural field for longer period of time. The prospective potential use of PGPR has gradually escalated, since it is one of the best substitutes against the constant usage of chemical fertilizers as well as pesticides. The use of these PGPR is one of the most reliable methods for ensuring sustainable agriculture. Furthermore, in future, the PGPR population will be correlated with the nutrient availability and productivity of the plants.
Stoichiometric or thermodynamic equilibrium constants for the binding of Fe3+ to transferrin were evaluated by the method of equilibrium dialysis using citrate as a competing complexing agent, near pH 6.7 and near pH 7.4. In each case K1 was substantially greater than K2, the effect being more marked at lower pH. These overall stability constants for the binding of iron, which take account of the participation of bicarbonate and protons in the reactions of binding, decrease with increasing pH. This may reflect the influence of the protein's negative charge, which increases with pH, on the critical role of the anion in metal binding. However, at any pH and pCO2 apparent stability constants may be defined, and these increase with increasing pH as a result of the inverse dependence of iron binding on the fourth power of the hydrogen ion concentration. At pH 7.4 and atmospheric pCO2, the apparent stability constants K'1 and K'2 are 4.7 x 1020 M-1 and 2.4 x 1019 M-1, respectively. Using the urea gel electrophoresis method of Makey and Seal (Makey, D.G., and Seal, U.S. (1976) Biochim. Biophys. Acta 453, 250-256), relative concentrations of the two species of monoferric transferrin were measured in each preparation at equilibrium. This made it possible to estimate the four intrinsic site constants for the binding of iron to transferrin. A slight negative cooperativity was evident in the binding of iron to each site. Although one site, designated the a-site, is more strongly binding than the b-site, it is not necessarily more accessible to all complexes of iron. Thus, iron as ferric citrate, ferric oxalate, ferrous ammonium sulfate and ferric chloride preferentially occupy the b-site when presented to transferrin under conditions in which an equilibrium distribution of iron between the binding sites of the protein would not be expected. Iron as ferric nitrilotriacetate, however, is directed toward the a-site. This is also the site which retains iron at low pH. On the basis of these observations single site transferrins were prepared, and a difference in their EPR spectra was demonstrated under near physiologic conditions.
Iron is required at a number of stages in the biological fixation of dinitrogen. Environmental iron occurs in an oxidized and polymerized state which is quantitatively insoluble at biological pH. Consequently, most aerobic and facultative anaerobic microorganisms have evolved specific carriers, termed siderophores, for the dissolution and uptake of iron (III). We have investigated the symbiotic nitrogen fixing bacterium, Rhizobium meliloti DM4, for the presence of a siderophore system of iron assimilation. The bacterium when cultured on low‐iron media was observed to form a substance which corrects iron starvation imposed by the synthetic chelating agent ethylenediamine‐di‐(o‐hydroxyphenyl acetic acid) (EDDA). The compound, designated rhizobactin, was extracted and obtained in crystalline form. A structural analysis of rhizobactin by means of mass and nuclear magnetic resonance spectroscopic techniques indicates that it is not a member of either the catechol or hydroxamate type families of siderophores. Of thirteen wild type isolates of R. meliloti, six vere stimulated by rhizobactin. In addition to rhizobactin, R. meliloti DM4 was stimulated by certain other siderophores, such as ferrichrome and ferrioxamine B, but not by entero‐bactin, aerobactin or mugeneic acid.
At moderate temperatures, a variety of sols, gels, amorphous or crystalline precipitates emerge from hydrolysis of iron(III) in aqueous solution. The product distribution reflects the interplay of fast proton transfer, olation and aggregation reactions. Polynuclear oxohydroxocomplexes with well-defined structures are formed under favorable circumstances in homogeneous solution. Polynuclear species with β-FeO(OH) structures have been identified in chloride solutions.
The complex formation of iron(IIl) with 3”-sulpho-2”,6”-dichloro-3,3'-dimethyl-4'-hydroxy-fuchson-5,5'-dicarboxylic acid (chrome azurol S) was studied by spectrophotometric, conductometric and potentiometric methods. The pure tetrabasic acid of the ligand was prepared from the impure trisodium salt (commercially availalile), and the dissociation constants of the ligand were redetermined. At 20° ± 1° and in the presence of 0.10 M potassium chloride the dissociation constants were: pk1 < 0.0, pk2 = 2.25 ± 0.05, pk3 = 4.71 ± 0.03 and pk4 = 11.81 ± 0.03.
The nitrogen-fixing Gram-negative bacterium Rhizobium meliloti DM4 when cultured on a low-iron media forms a siderophore which corrects iron starvation in the microorganism. The compound N2-[2-[(1-carboxyethyl)amino]ethyl]-N 6-(3-carboxy-3-hydroxy-1-oxopropyl)lysine, designated rhizobactin, contains ethylenediaminedicarboxyl and α-hydroxycarboxyl moieties as metal-coordinating groups. The ethylenediamine group is novel as a natural product and is unprecedented as a ligand in the siderophore series, which characteristically contain catechol or hydroxamate functional groups.
Four methods for the quantitative determination of hydroxamic acids have been evaluated. The Csaky test for bound hydroxylamine was shown to be the most sensitive of the methods; the molar absorptivity was calculated to be 0.394 m2 mol-1. The effect of hydrolysis on several hydroxamic acids of microbial origin was studied, and an explanation for the incomplete hydrolysis of certain hydroxamic acids is presented. Methods based on the condensation of hydroxylamine with 8-hydroxyquinoline, the reduction of triphenyltetrazolium chloride by hydroxylamine, and the oxidation of hydroxylamine derivatives with periodic acid were found to be less suitable for the determination of hydroxylamine. The limit of detection for the Csaky test was estimated to be 0.25 μg NH2OH-N ml.-1, which is equivalent to 5 mg L-1 (1 × 10-5 M) of a dihydroxamate siderophore with a molecular weight of 500 daltons.
A highly sensitive, simple determination of serum iron and binding capacity is described.FeIII/FeII reacts with chromazurol B (CAB) and cetyltrimethyl ammonium bromide (CTMA), the resulting substance being a highly coloured ternary complex. Maximal absorbance of the complex occurs at pH 4.6–5.5 at 630 nm. Lambert Beer's law holds between 0 and 80 μmol Fe/1. Molar absorptivity is 1.68 × 105 1 · mor−1 · cm−1 at 630 nm. Interference by other serum components is negligible even at high concentrations.
A cloned 8.3-kilobase-pair DNA fragment carrying all the genes (iucABCD iutA) of the aerobactin iron transport system of plasmid pColV-K30 was subjected to in vitro mutagenesis to afford mutant genes iucA, iucC, and iucA iucC. Complementation analyses and identification of aerobactin precursors accumulated by Escherichia coli cells harboring the different constructions allowed assignment of the iucA and iucC genes to discrete steps in biosynthesis of the siderophore from N epsilon-acetyl-N epsilon-hydroxylysine and citrate. Plasmid pVLN10, a derivative carrying a DNA fragment complementing an iucC mutation, expressed in a minicell system a single 62,000-dalton protein as the product of this gene.
An examination of 142 strains within 19 genera of yeasts and yeastlike organisms for formation of hydroxamic acids in low-iron culture showed production of hydroxamates by two unclassified strains and by 52 strains among the genera Aessosporon (3 of 3 strains), Cryptococcus (1 of 43), Leucosporidium (3 of 11), Rhodosporidium (4 of 4), Rhodotorula (27 of 39), Sporidiobolus (2 of 2), and Sporobolomyces (12 of 13). Crystalline rhodotorulic acid was isolated in amounts sufficient to account for most or all of the measured hydroxamate in culture supernatants of 16 strains representative of the five last-mentioned hydroxamate-producing genera. A new alanine-containing ferrichrome was isolated from one strain of Cryptococcus melibiosum. Rhodotorulic acid was a major metabolic product of many of the positive strains when grown in low-iron media, and iron was shown to repress its synthesis and excretion into the culture medium. The taxonomic significance of production of hydroxamic acids is described in connection with the position of these yeast species in the subclass Heterobasidiomycetidae.