CAS Assay Research Articles

Intracellular co-localization of the Escherichia coli enterobactin biosynthetic enzymes EntA, EntB, and EntE

Bacteria utilize small-molecule iron chelators called siderophores to support growth in low-iron environments. The Escherichia coli catecholate siderophore enterobactin is synthesized in the cytoplasm upon iron starvation. Seven enzymes are required for enterobactin biosynthesis: EntA-F, H. Given that EntB-EntE and EntA-EntE interactions have been reported, we investigated a possible EntA-EntB-EntE interaction in E. coli cells. We subcloned the E. coli entA and entB genes into bacterial adenylate cylase two-hybrid (BACTH) vectors allowing for co-expression of EntA and EntB with N-terminal fusions to the adenylate cyclase fragments T18 or T25. BACTH constructs were functionally validated using the CAS assay and growth studies. Co-transformants expressing T18/T25-EntA and T25/T18-EntB exhibited positive two-hybrid signals indicative of an intracellular EntA-EntB interaction. To gain further insights into the interaction interface, we performed computational docking in which an experimentally validated EntA-EntE model was docked to the EntB crystal structure. The resulting model of the EntA-EntB-EntE ternary complex predicted that the IC domain of EntB forms direct contacts with both EntA and EntE. BACTH constructs that expressed the isolated EntB IC domain fused to T18/T25 were prepared in order to investigate interactions with T25/T18-EntA and T25/T18-EntE. CAS assays and growth studies demonstrated that T25-IC co-expressed with the EntB ArCP domain could complement the E. coli entB− phenotype. In agreement with the ternary complex model, BACTH assays demonstrated that the EntB IC domain interacts with both EntA and EntE.

Digital image quantification of siderophores on agar plates.

This article presents visual image data and detailed methodology for the use of a new method for quantifying the exudation of siderophores during fungal growth. The data include images showing time series for calibration, fungal exudation, and negative controls, as well as replication accuracy information. In addition, we provide detailed protocols for making CAS assay layer plates, the digital analysis protocol for determining area of color change, and discuss growth media that do and do not work with the layer plate method. The results of these data, their interpretation, and further discussion can be found in Andrews et al., 2016 [1].

Comparative Genomic Analysis of Delftia tsuruhatensis MTQ3 and the Identification of Functional NRPS Genes for Siderophore Production.

Plant growth-promoting rhizobacteria (PGPR) are a group of rhizosphere bacteria that promote plant growth. Delftia tsuruhatensis MTQ3 is a member of PGPR that produces siderophores. The draft genome sequence of MTQ3 has been reported. Here, we analyzed the genome sequence of MTQ3 and performed a comparative genome analysis of four sequenced Delftia strains, revealing genetic relationships among these strains. In addition, genes responsible for bacteriocin and nonribosomal peptide synthesis were detected in the genomes of each strain. To reveal the functions of NRPS genes in siderophore production in D. tsuruhatensis MTQ3, three NRPS genes were knocked out to obtain the three mutants MTQ3-Δ1941, MTQ3-Δ1945, and MTQ3-Δ1946, which were compared with the wild-type strain. In qualitative and quantitative analyses using CAS assay, the mutants failed to produce siderophores. Accordingly, the NRPS genes in MTQ3 were functionally related to siderophore production. These results clarify one mechanism by which plant growth is promoted in MTQ3 and have important applications in agricultural production.

Plasticity of the malleobactin pathway and its impact on siderophore action in human pathogenic bacteria.

The human pathogenic bacteria Burkholderia mallei, Burkholderia pseudomallei, and Burkholderia thailandensis harbor a highly conserved gene cluster coding for the biosynthesis of the long sought-after malleobactins. Four new, unexpected congeners of the malleobactin family that were isolated and fully characterized in this study feature unusual deviations from the parent, ornibactin-like architecture. Thus, the malleobactin non-ribosomal peptide synthetase (NRPS) has a rare flexibility that yields diverse peptide backbones, of which one candidate confers pronounced siderophore activity (EC50: 8.4 μM, CAS assay). These findings not only unveil a highly diverse assembly line but also are an important addition to the knowledgebase of the pathogens' metabolomes.

Bifidobacteria strains isolated from stools of iron deficient infants can efficiently sequester iron.

BackgroundBifidobacteria is one of the major gut commensal groups found in infants. Their colonization is commonly associated with beneficial effects to the host through mechanisms like niche occupation and nutrient competition against pathogenic bacteria. Iron is an essential element necessary for most microorganisms, including bifidobacteria and efficient competition for this micronutrient is linked to proliferation and persistence. For this research we hypothesized that bifidobacteria in the gut of iron deficient infants can efficiently sequester iron. The aim of the present study was to isolate bifidobacteria in fecal samples of iron deficient Kenyan infants and to characterize siderophore production and iron internalization capacity.ResultsFifty-six bifidobacterial strains were isolated by streaking twenty-eight stool samples from Kenyan infants, in enrichment media. To target strains with high iron sequestration mechanisms, a strong iron chelator 2,2-dipyridyl was supplemented to the agar media. Bifidobacterial isolates were first identified to species level by 16S rRNA sequencing, yielding B. bifidum (19 isolates), B. longum (15), B. breve (11), B. kashiwanohense (7), B. pseudolongum (3) and B. pseudocatenulatum (1). While most isolated bifidobacterial species are commonly encountered in the infantile gut, B. kashiwanohense was not frequently reported in infant feces. Thirty strains from culture collections and 56 isolates were characterized for their siderophore production, tested by the CAS assay. Siderophore activity ranged from 3 to 89% siderophore units, with 35 strains (41%) exhibiting high siderophore activity, and 31 (36%) and 20 (23%) showing intermediate or low activity. The amount of internalized iron of 60 bifidobacteria strains selected for their siderophore activity, was in a broad range from 8 to118 μM Fe. Four strains, B. pseudolongum PV8-2, B. kashiwanohense PV20-2, B. bifidum PV28-2a and B. longum PV5-1 isolated from infant stool samples were selected for both high siderophore activity and iron internalization.ConclusionsA broad diversity of bifidobacteria were isolated in infant stools using iron limited conditions, with some strains exhibiting high iron sequestration properties. The ability of bifidobacteria to efficiently utilize iron sequestration mechanism such as siderophore production and iron internalization may confer an ecological advantage and be the basis for enhanced competition against enteropathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0334-z) contains supplementary material, which is available to authorized users.

Hyalachelins A-C, unusual siderophores isolated from the terrestrial myxobacterium Hyalangium minutum.

Three new siderophores, termed hyalachelins A-C (1-3), were isolated from the terrestrial myxobacterium Hyalangium minutum. Their structures were determined by 2D NMR and HR-MS/MS experiments, and their stereochemical configuration was established by a combination of NMR data, quantum mechanical calculations, and circular dichroism experiments. Hyalachelins are unusual catecholate-type siderophores that bear a 3,7,8-trihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Their iron chelating activities were evaluated in a CAS assay showing EC50 values of ∼30 μM.

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 mediated metal uptake by <i>Pseudomonas fluorescens</i> and its comparison to Iron (III) chelation

Siderophores are iron chelating substances that are also known to bind metals such as Molybdenum and Lead. The pigment produced by P. fluorescens is a siderophore whose ability to bind to different metal ions was investigated in this study. Pigment production was increased in the presence of Sodium, Potassium, Lead, Molybdenum and Cadmium. Siderophores were detected using CAS assay and catechol - siderophores by Arnow assay. Peak changes and changes in extinction coefficient values are indicators of complex formation. Maximum difference was observed in siderophore free Iron and Iron-siderophore complexes. Some changes were also noted for siderophore- metal complexes for all metals included in this study. Thus siderophore of P. fluorescens shows a tendency to form complexes with almost all metals tested. Key words: pigment; CAS assay; metal ions; complex formation; peak changes; molar extinction coefficient. DOI: 10.4038/cjsbs.v39i2.3001 Cey. J. Sci. (Bio. Sci.) 39 (2): 147-155, 2010

Characterization of pyoverdine and achromobactin in Pseudomonas syringae pv. phaseolicola 1448a.

BackgroundPseudomonas syringae pv. phaseolicola 1448a (P. syringae 1448a), the causative agent of bean halo blight, is a bacterium capable of occupying diverse biological niches. Under conditions of iron starvation P. syringae 1448a secretes siderophores for active uptake of iron. The primary siderophore of P. syringae 1448a is pyoverdine, a fluorescent molecule that is assembled from amino acid precursors by non-ribosomal peptide synthetase (NRPS) enzymes. Whereas other species of Pseudomonas often exhibit structural variations in the pyoverdine produced by different strains, all P. syringae pathovars previously tested have been found to make an identical pyoverdine molecule. P. syringae 1448a also appears to have the genetic potential to make two secondary siderophores, achromobactin and yersiniabactin, each of which has previously been detected in different P. syringae pathovars.ResultsFive putative pyoverdine NRPS genes in P. syringae 1448a were characterized in-silico and their role in pyoverdine biosynthesis was confirmed by gene knockout. Pyoverdine was purified from P. syringae 1448a and analyzed by MALDI-TOF and MS/MS spectroscopy. Peaks were detected corresponding to the expected sizes for the pyoverdine structure previously found in other P. syringae pathovars, but surprisingly P. syringae 1448a appears to also produce a variant pyoverdine species that has an additional 71 Da monomer incorporated into the peptide side chain. Creation of pyoverdine null mutants of P. syringae 1448a revealed that this strain also produces achromobactin as a temperature-regulated secondary siderophore, but does not appear to make yersiniabactin. Pyoverdine and achromobactin null mutants were characterized in regard to siderophore production, iron uptake, virulence and growth in iron limited conditions.ConclusionsThis study provides the first evidence of a P. syringae pathovar producing a side chain variant form of pyoverdine. We also describe novel IC50 and liquid CAS assays to quantify the contribution of different siderophores across a range of iron starvation conditions, and show that although achromobactin has potential to contribute to fitness its contribution is masked by the presence of pyoverdine, which is a significantly more effective siderophore. Neither pyoverdine nor achromobactin appear to be required for P. syringae 1448a to cause bean halo blight, indicating that these siderophores are not promising targets for crop protection strategies.

O-CAS, a fast and universal method for siderophore detection

In this work, the popular CAS assay for siderophore detection, based on the utilization of chrome azurol S, was redesigned and optimized to produce a new, fast, non-toxic, and easy method to determine a wide variety of microorganisms capable of siderophore production on a solid medium. Furthermore, this specific bioassay allows for the identification of more than one single siderophore-producing microorganism at the same time, using an overlay technique in which a modified CAS medium is cast upon culture agar plates (thus its name “O-CAS”, for overlaid CAS). Detection was optimized through adjustments to the medium's composition and a quantifying strategy. Specificity of the bioassay was tested on microorganisms known for siderophore production. As a result, a total of 48 microorganisms were isolated from three different types of samples (fresh water, salt water, and alkaline soil), of which 36 were determined as siderophore producers. The compounds identified through this method belonged to both hydroxamate and catechol-types, previously reported to cause color change of the CAS medium from blue to orange and purple, respectively. Some isolated microorganisms, however, caused a color change that differed from previous descriptions.

Iron Acquisition by Legionella pneumophila

For nearly 20 years, it was believed that Legionella pneumophila does not produce siderophores. Yet, we have now determined that L. pneumophila secretes a siderophore (legiobactin) that is detectable by the CAS assay. We have optimized conditions for legiobactin expression, shown its biological activity, and found genes (lbtAB) involved in its production and secretion. LbtA is homologous with siderophore synthetases from E. coli (aerobactin), Sinorhizobium (rhizobactin), and Bordetella (alcaligin), while LbtB is a member of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtAB produce 40-70% less CAS reactivity. The lbtA mutant is also defective for growth in deferrated media containing citrate, indicating that legiobactin is required in conditions of severe iron limitation. lbtAB mutants grow normally in macrophages and amoebae host cells as well as within the lungs of mice. L. pneumophila does express lbtA in macrophages, suggesting that legiobactin has a dispensable role in infection. Legiobactin is iron repressed and does not react in the Csáky and Arnow assays. Anion-exchange HPLC has been used to purify legiobactin, and thus far, structural analysis suggests that the molecule is similar but not identical to rhizobactin, rhizoferrin, and alcaligin. The residual CAS reactivity present in supernatants of the lbtAB mutants suggests that L. pneumophila might produce a second siderophore. Besides siderophores, we have determined that ferrous iron transport, encoded by feoB, is critical for L. pneumophila growth in low-iron conditions, in host cells, and in the mammalian lung. Some of our other studies have discovered a critical, yet undefined, role for the L. pneumophila cytochrome c maturation locus in low-iron growth, intracellular infection, and virulence.

A brackishwater isolate of Pseudomonas PS-102, a potential antagonistic bacterium against pathogenic vibrios in penaeid and non-penaeid rearing systems

A Pseudomonas sp PS-102 recovered from Muttukkadu brackish water lagoon, situated south of Chennai, showed significant activity against a number of shrimp pathogenic vibrios. Out of the 112 isolates of bacterial pathogens comprising Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. fluvialis, and Aeromonas spp, 73% were inhibited in vitro by the cell-free culture supernatant of Pseudomonas sp PS-102 isolate. The organism produced yellowish fluorescent pigment on King's B medium, hydrolysed starch and protein, and produced 36.4% siderophore units by CAS assay and 32 μM of catechol siderophores as estimated by Arnow's assay. The PS-102 isolate showed wide ranging environmental tolerance with, temperatures from 25 to 40 °C, pH from 6 to 8, salinity from 0 to 36 ppt, while the antagonistic activity peaked in cultures grown at 30 °C, pH 8.0 and at 5 ppt saline conditions. The antagonistic activity of the culture supernatant was evident even at 30% v / v dilution against V. harveyi. The preliminary studies on the nature of the antibacterial action indicated that the antagonistic principle as heat stable and resistant to proteolytic, lipolytic and amylolytic enzymes. Pseudomonas sp PS 102 was found to be safe to shrimp when PL-9 stage were challenged at 10 7 CFU ml − 1 and by intramuscular injection into of ∼5 g sub-adults shrimp at 10 5 to 10 8 CFU. Further, its safety in a mammalian system, tested by its pathogenicity to mice, was also determined and its LD 50 to BALB/c mice was found to be 10 9 CFU. The results of this study indicated that the organism Pseudomonas sp PS 102 could be employed as a potential probiont in shrimp and prawn aquaculture systems for management and control of bacterial infections.

A comparative study of siderophore production by fungi from marine and terrestrial habitats

Siderophore producing potential of 20 fungal isolates (same 10 species from each marine and terrestrial habitat) were examined and compared. Except marine Aspergillus flavus, all isolates produced siderophores as evidenced by positive reaction in FeCl 3 test, CAS assay and CAS agar plate test. The results indicated widespread occurrence of siderophores in both the habitats. Examination of the chemical nature of siderophores revealed that mucoraceous fungi produced carboxylate, while others produced hydroxamate siderophores. Thus, the nature of siderophore was found to be independent of habitat. Among all the isolates, Cunninghamella elegans (marine form) was maximum siderophore producer (1987.5 μg/ml) followed by terrestrial form of C. elegans (1248.75 μg/ml). There was no marked variation in siderophore concentration of Penicillium funiculosum strains. Comparison of quantification of siderophore production between marine and terrestrial revealed that four terrestrial isolates ( Aspergillus niger, Aspergillus ochraceous, Penicillium chrysogenum, Penicillium citrinum) were ahead in siderophore production, while, the other four marine isolates ( Aspergillus versicolor, C. elegans, Rhizopus sp., Syncephalastrum racemosum) were found to be more potent siderophore producers, indicating that they were equally competent.

Is Fe deficiency rather than P deficiency the cause of cluster root formation in Casuarina species?

When subjected, directly (through nutritional deficiencies) or indirectly (through alkaline constraints leading to such deficiencies) to nutrient deficiencies, certain plants respond by developing special root structures called cluster roots. This phenomenon can be considered as an ecophysiological response to a specific nutrient deficiency enabling plants to enhance nutrient uptake. Experiments conducted on an alkaline and an acid soil showed that Casuarina glauca (Sieber ex Spreng.) produced cluster roots only in the alkaline soil and not in the acid soil. In addition, iron (Fe) and phosphorus (P) deficiencies were examined separately or together to determine their effect on cluster root formation in C. glauca seedlings grown hydroponically. Results from experiments carried out on three Casuarina species (C. glauca, C. cunninghamiana Miq. and C. equisetifolia L.) indicated that Fe is involved in cluster root formation. In nutrient media lacking P but containing Fe, no cluster roots formed while seedlings receiving P and lacking Fe developed cluster roots. When incubated on chrome-azurol S-agar on blue plates (CAS assay), a technique used routinely to detect the production of siderophores by micro-organisms, the root system of Fe-deficient plants exhibited orange halos around cluster roots, indicating production of a ferric-chelating agent. It is concluded that the capacity of cluster roots of C. glauca to chelate Fe allows the plant to grow normally on alkaline soils.

Iron-acquisition Ability of Edwardsiella tarda with Involvement in Its Virulence.

Forty-one strains of Edwardsiella tarda including both virulent and avirulent strains were examined in their iron-acquisition ability. The strains were divided into two groups by growth under an iron-Iimiting condition (LB broth containing casamino acid in the presence of 6.25 μm EDDHA); one group was able to grow more than 102 times the initial bacterial density, but the other was not. The bioassay and the modified CAS assay for detecting bacterial siderophore production showed that the strains of the former group produced the siderophore with a higher ability for ironacquisition than those of the latter group. The siderophore-producing strains include all virulent ones. In addition, a natural mutant that showed lowered siderophore production had a remarkably reduced virulence. All of strains produced two outer membrane proteins, one of which is considered to be a receptor of the siderophore under the iron-limiting condition, indicating that these proteins are hardly related to the virulence of E. tarda. Our data in the present study suggest that the iron-acquisition ability of E. tarda by the siderophore is involved in its virulence.

Influence of iron depletion on growth and production of catechol siderophores by different Frankia strains

Nine strains of Frankia isolated from six Casuarinaceae (including four Casuarina sp., one Allocasuarina and one Gymnostoma) and one Elaeagnaceae (Hippophae¨ rhamnoides) were screened for growth and production of siderophores in an iron-deficient liquid medium. Siderophore production was detected only in four strains (Cj, G2, CH and G82) using the CAS and Arnow assays. Salicylates formed more than 90% and dihydroxybenzoates formed less than 10% of all catechol-type siderophores produced. Growth of the former strains was less affected by iron deficiency than that of strains Rif, Thr, URU, BR and RT which do not produce siderophores. Optimal siderophore production by strain Cj was noted when iron concentration reached 0.5μm and was completely inhibited at an iron concentration of 10μm. The kinetics of siderophore production by strain Cj showed that siderophore synthesis was detectable during the growth stationary phase. Growth of Cj (a siderophore-producing strain) and of RT (a non-siderophore-producing strain) differed when 2,2-dipyridyl or ethylene di(o-hydroxyphenyl) acetic acid (EDDHA) was added to the iron-deficient growth medium. Frankia strain RT was the most sensitive to the detrimental effect of both iron chelators.

Detection of siderophores in growing cultures ofPseudomonas spp.

The siderophores produced byPseudomonas fluorescens andP. chlororaphis were detected from the culture supernatants in MM9 and modified King's medium by the universal CAS assay at wavelengths 620–690 nm. The CAS assay was applied to detectPseudomonas siderophores directly in situ, during their production phase, in modified King's medium. Optimum results were detected with a final CAS concentration of 0.025 mM and an iron concentration of 1.25 μM. The problems of the method are discussed with respect to the absorbance spectrum, the toxicity of the HDTMA detergent, the influence of the iron concentration and the complexity of media for siderophore production.

Tn [formula omitted] mutagenesis of chinese Rhizobium fredii for siderophore overproduction

In the midwestem United States, members of USDA Bradyrhizobium japonicum serogroup 135 dominate in alkaline soils for nodulation of soybean [ Glycine max (L.) Merr.], whereas members of USDA serogroup 123 dominate in non-alkaline soils. A possible explanation for the dominance of 135 in alkaline soils is that native 135 strains can produce siderophores that assist in acquiring iron. Our objectives were to test six reference strains (three slow-growing B. japonicum and three fast-growing Rhizobium fredii) for siderophore production and to evaluate mutants of R.fredii (developed by Tn 5 -insertion for overproduction of siderophores) for their competitive abilities in an alkaline soil. B. japonicum reference strains USDA 110, 123 and 135 were tested for siderophore production in liquid culture by using Neilands' CAS Assay Solution. Mutants were developed for siderophore overproduction by introducing transposon Tn 5 (Km r) into a siderophore-producing Chinese R. fredii strain. Two siderophore overproducing mutants that produced mature and pink nodules on soybean in growth pouches were examined for the presence of Tn 5 by Southern hybridization with a Tn 5 probe. The Tn 5 -carrying mutants were further tested in the greenhouse for their competitiveness against native strains present in an alkaline soil of Iowa and for their symbiotic effectivity. Among the three slow-growing bradyrhizobia tested for siderophore production, USDA 135 was the only strain producing siderophore in a liquid medium low in iron. Nodule occupancies in the greenhouse by the two siderophore overproducing mutants of R. fredii were 3 and 4%, compared with 19% for the wild-type strain. Thus, either the wild-type was acquiring all the iron needed for maximum growth and nodulation, or the insertion of Tn 5 and the overproduction of siderophore resulted in less competitive strains.

Production of siderophore by Edwardsiella tarda.

Whether Edwardsiella tarda produces siderophore or not was investigated. CAS assay was used for detecting siderophore and Eagle's MEM was employed as a basal medium with a slight modification. Iron was eliminated from the basal medium by chloroform-8-hydroxyquinoline extraction. On this iron deficient agar medium, all the strains of E. tarda showed siderophore positive reactions. The siderophores were prepared from two virulent and two avirulent strains and examined their electrophoretic mobilities at pH 5.6. The siderophore mobilities against H2PO4-were all-0.05 and no differece was found among these 4 strains.