Chrome Azurol Sulfonate Research Articles

New Strain of Bacillus amyloliquefaciens G1 as a Potential Downy Mildew Biocontrol Agent for Grape

To obtain effective biocontrol strains for downy mildew of grape, 38 endophytic bacteria were isolated from fruits, seeds, and old stems of six grape varieties. Using spot inoculation mixtures of sporangial suspensions of Plasmopara viticola and biocontrol bacterial suspension, this screen yielded three strains (G1, G5, and G9) with good antagonistic effects against P. viticola. The growth inhibition rate was 100%, which was comparable to the effect of the positive control Bacillus subtilis strain CN181. The enzyme activity and the metabolites of strain G1 were examined on casein hydrolysate medium, sodium carboxymethyl cellulose agar plates, and chrome azurol sulfonate (CAS) agar plates. The antifungal protein component was identified by liquid chromatography–mass spectrometry (LC–MS). The results showed that strain G1 was more effective against Plasmopara viticola after two field trials, and the inhibition rates of strain G1 on the seventh day of the two field trials were 47.5% and 36.9%, respectively. Strain G1 was identified as Bacillus amyloliquefaciens based on morphological examination and 16S rDNA sequence analysis. It produced proteases, cellulases, and siderophores. Crude protein of the strain mainly included the putative segregation protein SpoVG, which inhibited P. viticola.

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such mechanism is the scavenging of iron through siderophore production. P. aeruginosa produces two different siderophores: pyochelin, which has lower iron-chelating affinity, and pyoverdine, which has higher iron-chelating affinity. This report demonstrates that pyoverdine can be directly quantified from bacterial supernatants, while pyochelin needs to be extracted from supernatants before quantification. The primary method for qualitatively analyzing siderophore production is the Chrome Azurol Sulfonate (CAS) agar plate assay. In this assay, the release of CAS dye from the Fe3+-Dye complex leads to a color change from blue to orange, indicating siderophore production. For the quantification of total siderophores, bacterial supernatants were mixed in equal proportions with CAS dye in a microtiter plate, followed by spectrophotometric analysis at 630 nm. Pyoverdine was directly quantified from the bacterial supernatant by mixing it in equal proportions with 50 mM Tris-HCl, followed by spectrophotometric analysis. A peak at 380 nm confirmed the presence of pyoverdine. As for Pyochelin, direct quantification from the bacterial supernatant was not possible, so it had to be extracted first. Subsequent spectrophotometric analysis revealed the presence of pyochelin, with a peak at 313 nm.

Isolation and Characterization of Siderophores Producing Chemolithotrophic Bacteria from the Coal Samples of the Aluminum Industry

Siderophores are iron grabbers and are classified according to the ligands used to chelate the ferric iron. The role of siderophores is to scavenge iron and make it accessible to the cell. It has crucial applications in industries, medical, research, and environmental clean-up. In the current research work, the authors attempted to isolate and characterize a few siderophores producing bacteria from the coal samples, collected from the Bharat Aluminum Company Ltd. Chhattisgarh. The quantitative and qualitative assay for siderophores producing bacteria (SPB) was carried by Chrome Azurol Sulfonate agar test for all the ten samples and positive results were observed only in two strains, i.e., B6 and B7 when grown in iron-deficient succinate medium in an incubator shaker for 48 h at 120 rpm. The biochemical tests revealed that the B6 and B7 strains have similarities to Bacillus sp. and Vibrio sp., respectively. The study reflects the potential of these strains as effective siderophores producers and holds a noteworthy perspective in further industrial and research applications.

Paenibacillus arenosi sp. nov., a siderophore-producing bacterium isolated from coastal sediment.

In this study, strain CAU 1523T, a novel Gram-positive-positive bacterium isolated from marine sediment collected from the coast of Busan, Republic of Korea, was characterized using a polyphasic taxonomic approach. This strain showed growth at a temperature range of 20-37°C (optimum, 30°C), a pH range of 6.5-9.5 (optimum, 7.5), and in the presence of 0-3% (w/v) NaCl (optimum, 1%). Phylogenetic analysis based on 16S rRNA gene sequencing and 92 concatenated core genes indicated that CAU 1523T belonged to the genus Paenibacillus, sharing the highest sequence similarity with P. assamensis JCM 13186T (98.0%). CAU 1523T was differentiated from other Paenibacillus species by average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values, using cut-off values of 95-96%, 90%, and 70%, respectively, for closely related strains. The genome of CAU 1523T possessed various biosynthetic gene clusters, one of which encoded a putative siderophore-interacting protein. Siderophore production by the isolate was confirmed using the qualitative chrome azurol sulfonate (CAS) agar assay. Based on its phylogenetic and physiological characteristics, strain CAU 1523T represents a novel, siderophore-producing species within the genus Paenibacillus, for which the name Paenibacillus arenosi sp. nov. is proposed, with the type strain CAU 1523T (= KCTC 43108T = MCCC 1K04063T).

Induction of Iron Stress in Hepatocellular Carcinoma Cell Lines by Siderophore of Aspergillus nidulans Towards Promising Anticancer Effect.

Hepatocellular carcinoma is among the leading causes of cancer-related deaths worldwide and needs efficient and feasible approach of treatment. Present study focuses on exploring the anticancer activity of a secondary metabolite called siderophore of Aspergillus nidulans against hepatocellular carcinoma cell line HepG2. These small peptides are produced by microorganisms including fungi for scavenging iron from its surroundings. Fungi including Aspergillus spp. are known to produce siderophores under iron-limited conditions. Siderophores have high affinity towards iron and are classified into various types. In the present study, siderophore isolated and purified from fungal cultures was confirmed to be of hydroxamate type by chrome azurol sulfonate and Atkin's assay. HPLC analysis confirmed purity while LC-ESI-MS revealed that the siderophore is triacetyl fusigen. Cancerous cells, HepG2, grown under siderophore treatment showed inhibition in growth and proliferation in a dose- and time-dependent manner. Reduction in viability and metabolic activity was evident upon treatment as seen in trypan blue, MTT and WST assay. Fluorescent staining using PI and DAPI confirmed the same while DCFDA staining revealed increased reactive oxygen species production which might have led to cell death and deterioration. Such increase in ROS has been correlated with iron accumulation by assessing intracellular iron level through ICP-MS. To assess the effect of siderophore treatment on normal cells, WRL-68, same assays were carried out but the effect was mostly non-significant up to 48h. Thus, present work suggests that an optimum dose of siderophore purified from A. nidulans culture might prove a useful anticancer agent.

Easy detection of siderophore production in diluted growth media using an improved CAS reagent

Siderophores are low molecular weight organic compounds produced by various microorganisms, especially pathogenic bacteria including rhizobacteria, and have a high affinity for iron. Although most microorganisms are thought to secrete siderophores under iron-depleted conditions, it is unclear how many microorganisms produce siderophores in the natural environment. Also, the chrome azurol sulfonate (CAS) assay, which is widely used for the detection of siderophores, needs to be improved for wider applicability. We developed a simple, high-throughput CAS assay in a 96-well microplate with a concentrated CAS reagent and commonly used diluted growth media in the absence of artificial iron depletion. The improved microplate CAS shuttle assay revealed that it could easily detect siderophores released from Pseudomonas (P.) fluorescence, P. putida, Burlkholderia stabilis, and Ottowia oryzae, as models of siderophore-producing bacteria. This CAS shuttle assay employed along with diluted growth media is a promising tool to screen new siderophore-producing bacteria.

A Comparative Analysis of Virulence Factors in Klebsiella Species

Klebsiella is a genus of bacteria frequently associated with nosocomial infections due to their multi-drug resistant properties. Klebsiella pneumoniae accounts for a large majority of hospital-acquired pneumonia, urinary tract infections, and septicemia. The pathogenicity of K. pneumoniae clinical isolates has been greatly established to better understand and treat these infections. Klebsiella oxytoca is also primarily healthcare associated and is considered to be an emerging nosocomial pathogen, as it causes the second most frequent infections from the Klebsiella genus. Although it shares many characteristics with K. pneumoniae, its virulence factors have not been well studied due to its lesser degree of incidence. As an emerging pathogen, more information must be gathered in order to learn how to best treat this infectious, antibiotic-resistant bacteria. This study aims to first determine if the pathogenic factors found in clinical Klebsiella pneumoniae isolates are also present in Klebsiella oxytoca and then compare the production of these virulence factors under normal and inducing conditions. This work compares the expression of pathogenic factors in five isolates of K. oxytoca, three isolates of K. pneumoniae, and one strain of Escherichia coli. The pathogenic factors chosen for this study include antibiotics resistance, siderophore production, biofilm formation abilities, and reactive oxygen species (ROS) detoxification. To determine antibiotic resistance, the minimum inhibitory concentration of nine different antibiotics was calculated for each isolate. Siderophore production was measured using a Chrome Azurol Sulfonate colorimetric test in both the absence and presence of 2,2’-dipyridyl, an iron chelator. Biofilm formation was detected with Crystal Violet dye in both the absence and presence of acetaminophen and hydrochlorothiazide, both of which are commonly used therapeutic pharmaceuticals in nosocomial settings, as well as 2,2’-dipyridyl. ROS detoxification via superoxide dismutase enzyme activity was measured through native gel activity assays. Our findings indicate that K. pneumoniae has resistance to more antibiotics than K. oxytoca, but the two species have comparable siderophore production, biofilm formation capabilities, and ROS detoxification capabilities. In conclusion, Klebsiella pneumoniae and Klebsiella oxytoca share several mechanisms of pathogenicity under normal and inducing conditions and drug resistance is the most notable difference among the strains tested in this work.

Siderophore Production by Rhizosphere Inhabiting Bacteria and Fungi

The production of siderophores is greater in rhizosphere inhabiting fungi and bacteria. The present work was designed to isolate the rhizosphere inhabiting bacteria and fungi from the rhizosphere of Phaseolus Vulgaris, Pisum sativum, Vicia faba and Alfa alfa. The obtained isolates has been screened for siderophores production using Chrome Azurol Sulfonate assay (CAS). The highest siderophore producer bacterium and fungus has been molecularly identified as Bacillus MG214652 andAspergillus niger MH844535 respectively. They were able to produce 70.4% and 87% units of siderophore respectively. Bacillus MG214652 siderophores was characterized as catecholate type with maximum absorbance at 495 nm. Aspergillus niger MH844535 siderophore was characterized as hydroxymate type with maximum absorbance at 450 nm.

Phenotypic and genotypic characterization of Pseudomonas aeruginosa siderophores

Pseudomonas aeruginosa is an opportunistic human pathogen that causes many infections such as urinary tract, respiratory tract, burn infections in addition to septicemia. Iron (Fe) is an essential element for the growth of most living microorganisms. Siderophores are iron chelating compounds produced by bacteria. Ps. aeruginosa produces two major siderophores; pyoverdine and pyochelin.Ps. aeruginosa isolates were identified morphologically, biochemically and by culture characteristics. Siderophore production was assessed phenotypically by chrome azurol sulfonate (CAS) Shuttle assay and genotypically by PCR.Siderophore production varied among the clinical isolates. High production was found in 63.33% of isolates. Intermediate production of siderophore was detected in 30%, while 6.66% of isolates showed a low level of siderophores. Genotypic detection of genes responsible for the formation of siderophores and their receptors revealed the presence of PvdS, PtxR and FpvA genes that are responsible for pyoverdine synthesis and its receptor formation in all isolates. Whereas pchG and FptA encoding pyochelin and its receptor were detected in 85.7% of isolates.Siderophores are important iron chelators in Ps. aeruginosa. Substantial correlation was received from the genotypic and phenotypic investigations of siderophore production.

Bacterial cyclic β-(1,2)-glucans sequester iron to protect against iron-induced toxicity.

Cellular iron homeostasis is critical for survival and growth. Bacteria employ a variety of strategies to sequester iron from the environment and to store intracellular iron surplus that can be utilized in iron-restricted conditions while also limiting the potential for the production of iron-induced reactive oxygen species (ROS). Here, we report that membrane-derived oligosaccharide (mdo) glucan, an intrinsic component of Gram-negative bacteria, sequesters the ferrous form of iron. Iron-binding, uptake, and localization experiments indicated that both secreted and periplasmic β-(1,2)-glucans bind iron specifically and promote growth under iron-restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of β-(1,2)-glucan accumulate low amounts of intracellular iron under iron-restricted conditions, whereas they exhibit elevated ROS production and sensitivity under iron-replete conditions. Our results reveal a critical role of glucan in intracellular iron homeostasis conserved in Gram-negative bacteria.

Effects of two new siderophore-producing rhizobacteria on growth and iron content of maize and canola plants

ABSTRACTSiderophore-producing rhizobacteria beneficially affect plant growth by providing available iron to plants. In this study, bacteria were isolated from the rhizosphere of canola (Brassica napus L.) plants grown in the central fields in Iran, for the presence of siderophore-producing bacteria. A total of 45 distinct isolates were found to produce siderophore using qualitative chrome azurol sulfonate (CAS)-agar assay. Of them, ten isolates, based on the highest halo diameter/colony diameter ratios, were selected to quantify the rate of siderophore production using CAS-liquid assay. A variety of biochemical assays was used to determine the type(s) of siderophores produced by each of the ten isolates. The best isolates, based on production of the highest rates of either hydroxamates or carboxylates, were identified and used in further studies. Based on 16S ribosomal ribonucleic acid (rRNA) sequence analysis and a variety of phenotypic properties, the isolates were identified as Micrococcus yunnanensis YIM 65004 (T) and Stenotrophomonas chelatiphaga LPM-5 (T). We also studied the plant growth-promoting effect of the most promising isolates (YIM 65004 and LPM-5) on canola and maize plants under greenhouse conditions. The results of this study showed that M. yunnanensis and S. chelatiphaga increased gain weight and iron (Fe) content of roots and shoots significantly, in comparison with control, indicating beneficial effects of these rhizobacteria on plant growth and development. This study reports M. yunnanensis and S. chelatiphaga, as new records for Iran. The latter is reported for the first time from plant (canola) rhizosphere. Besides, the ability of both M. yunnanensis and S. chelatiphaga to produce siderophores is documented for the first time.

Bacterial siderophores promote plant growth: Screening of catechol and hydroxamate siderophores

ABSTRACTThe aim of the study was to determine the quality and quantity of siderophores produced by bacteria isolated from plants' roots. The second aim was to determine the effect of siderophores on plants growth (Festuca rubra L. and Brassica napus L.). The study was carried out using bacteria isolated from roots of: Arabidopsis thaliana L., F. rubra, and Agrostis capillaris L., growing on the heavy metals contaminated area. The chrome azurol sulfonate (CAS) test, Arnow's test for catechol siderophores, and Csaksy's test for hydroxamate siderophores were performed. Among the bacteria, 42 isolates (39%) had a positive result in the CAS. Endophytic bacteria were mostly producing the catechol siderophores. It was found that F. rubra is the plant which is linked with the highest number of siderophores producing bacteria. The highest concentration of siderophores was noted for ectorhizospheric bacteria associated with A. thaliana, hyperaccumulating plant. It was found that hydroxamate siderophores are mainly produced by ectorhizosphere and rhizoplane bacteria. The siderophores producing bacteria reduced the toxicity of metals and improved the phytoremediation. Siderophores treatment increased the growth of plants in the biological assay, growing on two different soils: one highly contaminated with heavy metals and the second strongly alkaline soil.

Complete Genome Sequence and Transcriptomic Analysis of the Novel Pathogen Elizabethkingia anophelis in Response to Oxidative Stress.

Elizabethkingia anophelis is an emerging pathogen that can cause life-threatening infections in neonates, severely immunocompromised and postoperative patients. The lack of genomic information on E. anophelis hinders our understanding of its mechanisms of pathogenesis. Here, we report the first complete genome sequence of E. anophelis NUHP1 and assess its response to oxidative stress. Elizabethkingia anophelis NUHP1 has a circular genome of 4,369,828 base pairs and 4,141 predicted coding sequences. Sequence analysis indicates that E. anophelis has well-developed systems for scavenging iron and stress response. Many putative virulence factors and antibiotic resistance genes were identified, underscoring potential host–pathogen interactions and antibiotic resistance. RNA-sequencing-based transcriptome profiling indicates that expressions of genes involved in synthesis of an yersiniabactin-like iron siderophore and heme utilization are highly induced as a protective mechanism toward oxidative stress caused by hydrogen peroxide treatment. Chrome azurol sulfonate assay verified that siderophore production of E. anophelis is increased in the presence of oxidative stress. We further showed that hemoglobin facilitates the growth, hydrogen peroxide tolerance, cell attachment, and biofilm formation of E. anophelis NUHP1. Our study suggests that siderophore production and heme uptake pathways might play essential roles in stress response and virulence of the emerging pathogen E. anophelis.

Comparison between Siderophores Production by Fungi Isolated from Heavy Metals Polluted and Rhizosphere Soils

Although siderophores are induced primarily in response to iron deficiency, soil and other ecological factors can affect on this process. This study was to evaluate the production of siderophores by different fungal species isolated from heavy metal contaminated and uncontaminated soils. More than thirty fungal strains were isolated from heavy metal contaminated and rhizosphere uncontaminated soils. Chrome azurol sulfonate (CAS) was used for both quantitative and qualitative evaluation of siderophores production. No significant correlations were observed between the tested variables such as ultraviolet (UV) irradiation method and CAS-agar plate and heavy metal concentration in both soils. The production of siderophores in rhizosphere fungi was higher than those isolated from the contaminated soil; however, the difference was not significant. The siderophore production (%) by fungi isolated from heavy metal contaminated soil using UV irradiation method was positively correlated with the qualitative values using CAS-plate method (P 〈0.05). Pearson correlation test indicated a positive correlation between the quantitative and qualitative methods of detection for fungi isolated from rhizosphere and also those isolated from heavy metal contaminated soil.

Streptobactin, a Tricatechol-Type Siderophore from Marine-Derived Streptomyces sp. YM5-799

A new catechol-type siderophore, streptobactin (1), was isolated from a culture broth of the marine-derived actinomycete Streptomyces sp. YM5-799. The structure of streptobactin was determined by NMR and MS analyses and ESIMS/MS experiments to be a cyclic trimer of benarthin. A dibenarthin (2), a tribenarthin (3), and benarthin (4) were also obtained. The production of 1 was regulated by an iron concentration in the culture. The iron-chelating activity of the compounds was evaluated by the chrome azurol sulfonate assay.

DETECTION, PURIFICATION, AND IDENTIFICATION OF SIDEROPHORES PRODUCED BY Pseudomonas fluorescens ISOLATES USING SDS-PAGE AND HPLC.

Three isolates of Pseudomonas fluorescens were isolated from the soil rhizosphere of potato growing fields and identification was confirmed by comparison of the morphological traits and physiological characteristics according to Murray et al., 1984 and Klement et aL,(1990). P. fluorescens isolates were tested to prove their ability in production of siderophores. Chrome Azurol Sulfonate assay (CAS assay) test was used to detect the presence of siderophores. Formation of an orange halo around a well bored into the CAS plate, was taken as an indication for the presence of siderophores. Concerning the optimization of conditions for highest production of siderophores on different media, the results revealed that kings B medium, exhibited the highest production of sidrophores. Also, data showed that pH 7 and 27 °C were the best in this respect while at pH 6 and 8 sidrophores production completely inhibited. One of the siderophores gene was detected by specific primer in P. fluorescens and one band at molecular weight 520 by was observed. SDS-PAGE analysis was used to detect the possible outer membrane receptor protein (OMRP), in both whole cell pellet and membrane pellet at no iron and high iron concentration. The results revealed the existence of one band at 84 KDa in case of no iron concentration. Purification of siderophores in culture supernatant was done by a sephadex G -25 gel filtration column. Nine fractions were obtained and they tested for siderophores activity by CAS assay. There were two types of siderophores in P. fluorescens culture supernatant (pyoverdin and pyochelin) and they were detected by high performance liquid chromatography (HPLC).

Siderophore production and utilization by milk spoilage Pseudomonas species

Many bacteria respond to potentially growth-limiting availability of iron by producing low-molecular-weight iron chelators (siderophores). The aim of this work was to examine the siderophores synthesized and utilized by Pseudomonas spp. implicated in milk spoilage. Twenty isolates of Pseudomonas spp. previously shown to have significant milk spoilage potential were tested for the ability to produce siderophores. Of these, 14 produced pyoverdin and 2 of these also produced pyochelin; 1 produced only pyochelin; 1 produced only salicylate; 2 produced non-pyoverdin, hydroxamate-containing siderophore; and 2 produced chrome azurol sulfonate reactive material that was neither pyoverdin nor pyochelin. There was considerable diversity among the pyoverdins produced. All isolates were shown to utilize iron complexed with exogenous pyoverdin, but usage of particular exogenous pyoverdins differed among isolates. Interference with the iron-uptake systems of the Pseudomonas spp. may be a means by which food spoilage can be slowed, and the pyoverdin system would appear to be a potential target. However, given the diversity of pyoverdins produced and utilized, and the presence of other siderophores, successful interference with bacterial iron acquisition in this context may be challenging.

The 58-Kilodalton Major Virulence Factor of Francisella tularensis Is Required for Efficient Utilization of Iron

We investigated the role of the 58-kDa FTT0918 protein in the iron metabolism of Francisella tularensis. The phenotypes of SCHU S4, a prototypic strain of F. tularensis subsp. tularensis, and the Delta FTT0918 and Delta fslA isogenic mutants were analyzed. The gene product missing in the Delta fslA mutant is responsible for synthesis of a siderophore. When grown in broth with various iron concentrations, the two deletion mutants generally reached lower maximal densities than SCHU S4. The Delta FTT0918 mutant, but not the Delta fslA mutant, upregulated the genes of the F. tularensis siderophore locus (fsl) operon even at high iron concentrations. A chrome azurol sulfonate plate assay confirmed siderophore production by all strains except the Delta fslA strain. In a cross-feeding experiment using medium devoid of free iron, SCHU S4 promoted growth of the Delta fslA strain but not of the Delta FTT0918 strain. The sensitivity of SCHU S4 and the Delta FTT0918 and Delta fslA strains to streptonigrin demonstrated that the Delta FTT0918 strain contained a smaller free intracellular iron pool and that the Delta fslA strain contained a larger one than SCHU S4. In contrast to the marked attenuation of the Delta FTT0918 strain, the Delta fslA strain was as virulent as SCHU S4 in a mouse model. Altogether, the data demonstrate that the FTT0918 protein is required for F. tularensis to utilize iron bound to siderophores and that it likely has a role also in siderophore-independent iron acquisition. We suggest that the FTT0918 protein be designated Fe utilization protein A, FupA.

Tenacibactins A−D, Hydroxamate Siderophores from a Marine-Derived Bacterium, Tenacibaculum sp. A4K-17

Four new hydroxamate siderophores, tenacibactins A-D (1-4), were isolated from a culture broth of the marine-derived bacterium Tenacibaculum sp. A4K-17. The structures of these tenacibactins were determined by NMR analyses and ESIMS/MS experiments. The iron-binding (chelating) activity of 1-4 was evaluated by the chrome azurol sulfonate (CAS) assay.

Rapid identification of siderophores by combined thin‐layer chromatography/matrix‐assisted laser desorption/ionization mass spectrometry

The investigation of a combined thin-layer chromatography/matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI-MS) method for the analysis of siderophores from microbial samples is described. The investigated siderophores were enterobactin, ferrioxamine B, ferrichrome, ferrirhodin, rhodotorulic acid and coprogen. Solid-phase extraction was employed to recover the siderophores from the microbial samples. After visualization of the spots via spraying with ferric chloride or chrome azurol sulfonate assay solution, the MALDI matrix was applied to the gel surface. Several TLC/MALDI experimental parameters were optimized, such as type and concentration of MALDI matrix, as well as the type and composition of solvent to facilitate analyte transport from the inside of the TLC gel to the surface. The impact of these parameters on sensitivity, precision and ion formation of the various siderophores was studied. The detection limits for the investigated siderophores were in the range 1-4 pmol. These values were about 4-24 times higher than the detection limits obtained directly from stainless steel MALDI targets. The differences were most likely due to incomplete transport of the 'trapped' analyte molecules from the deeper layers of the TLC gel to the surface and into the matrix layer. In addition, chromatographic band broadening spread the analyte further in TLC as compared with the steel plates, resulting in less analyte per surface area. The identification of the siderophores was aided by concurrently applying a Ga(III) nitrate solution to the TLC plate during the visualization step. The resulting formation of Ga(III) complexes lead to distinctive (69)Ga/(71)Ga isotope patterns in the mass spectra. The versatility of the TLC/MALDI-MS assay was demonstrated by using it to analyze siderophores in a Pseudomonas aeruginosa sample. An iron-binding compound was identified in the sample, namely pyochelin (2-(2-o-hydroxyphenyl-2-thiazolin-4-yl)-3-methylthiazolidine-4-carboxylic acid).