假单胞菌荧光与非荧光铁载体对铁离子的应答差异

假单胞菌既能产荧光铁载体也能产非荧光铁载体.通过对假单胞菌在不同铁离子浓度下,在通用CAS(Chrome azroul S)检测平板、改进的蔗糖-天冬氨酸(SA)平板(MSA)上以及通用液体CAS培养基和MSA培养基内的铁载体产生情况的比较,发现在通用CAS的液体培养基上产生的主要为非荧光铁载体(pyochelin),而在改进的MSA培养基上产生的主要为荧光铁载体(pyoverdine);在铁离子的应答方面,pyoverdine较pyochelin灵敏,较低的铁离子浓度即可抑制荧光铁载体的产生,但是不能抑制非荧光铁载体.     

lbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin

Under iron stress, Legionella pneumophila secretes legiobactin, a nonclassical siderophore that is reactive in the chrome azurol S (CAS) assay. Here, we have optimized conditions for legiobactin expression, shown its biological activity, and identified two genes, lbtA and lbtB, which are involved in legiobactin production. lbtA appears to be iron repressed and encodes a protein that has significant homology with siderophore synthetases, and FrgA, a previously described iron-regulated protein of L. pneumophila. lbtB encodes a protein homologous with members of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtA or lbtB were defective for legiobactin, producing 40 to 70% less CAS reactivity in deferrated chemically defined medium (CDM). In bioassays, mutant CDM culture supernatants, unlike those of the wild type, did not support growth of iron-limited wild-type bacteria in 2',2'-dipyridyl-containing buffered charcoal yeast extract (BCYE) agar and a ferrous iron transport mutant on BCYE agar without added iron. The lbtA mutant was modestly defective for growth in deferrated CDM containing the iron chelator citrate, indicating that legiobactin is required in conditions of severe iron limitation. Complementation of the lbt mutants restored both siderophore expression, as measured by the CAS assay and bioassays, and bacterial growth in deferrated, citrate-containing media. The lbtA mutant replicated as the wild type did in macrophages, amoebae, and the lungs of mice. However, L. pneumophila expresses lbtA in the macrophage, suggesting that legiobactin, though not required, may play a dispensable role in intracellular growth. The discovery of lbtAB represents the first identification of genes required for L. pneumophila siderophore expression.     

CAS平板覆盖法检测氢氧化细菌铁载体

【目的】用CAS平板覆盖法检测氢氧化细菌铁载体,解决通用CAS琼脂平板法中十六烷基三甲基溴化铵对真菌和某些细菌的生长抑制问题。【方法】将改良的CAS检测培养基覆盖在长满菌落的无铁培养基上,生长抑制问题因微生物未与十六烷基三甲基溴化铵直接接触而解决。【结果】3株氢氧化细菌SDW-5、SDW-9和AaP-13均能产生单菌落,加入CAS检测培养基1 h后,菌落周围产生明显的铁载体晕圈。【结论】本方法成功解决了生长抑制问题,可以作为检测微生物铁载体的通用方法。     

Siderophores of halophilic archaea and their chemical characterization

Nine halophilic archaea viz., Halobacterium salinarum, Halobacterium sp.1, Halobacterium sp.2, Halobaculum sp., Halococcus saccharolyticus, Halorubrum saccharovorum, Haloterrigena turkmenica, Halogeometricum sp. and Natrialba sp. isolated from marine salterns around Bhavnagar coast were screened for siderophore production. Five isolates viz., Halococcus saccharolyticus, Halorubrum saccharovorum, Haloterrigena turkmenica, Halogeometricum sp. and Natrialba sp. produced siderophores as evidenced by positive reaction in FeCl3 test, CAS assay and CAS agar plate test. Determination of chemical nature of siderophores by chemical assays and bioassays identified them as carboxylates. Quantification of siderophores indicated Halorubrum saccharovorum to be the maximum siderophore producer (2.62 RE mg/ml) and Halococcus saccharolyticus to be the least (1.33 RE mg/ml). The present study is the first report on siderophore production in Indian haloarchaeal strains. Mechanism of iron assimilation in four non-siderophore isolates still needs to be investigated further.     

Lead-Enhanced Siderophore Production and Alteration in Cell Morphology in a Pb-Resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Strain 4EA

A lead-resistant bacterial strain 4EA from soil contaminated with car battery waste from Goa, India was isolated and identified as Pseudomonas aeruginosa. This lead-resistant bacterial isolate interestingly revealed lead-enhanced siderophore (pyochelin and pyoverdine) production up to 0.5 mM lead nitrate whereas cells exhibit a significant decline in siderophore production above 0.5 mM lead nitrate. The bacterial cells also revealed significant alteration in cell morphology as size reduction when exposed to 0.8 mM lead nitrate. Enhanced production of siderophore was evidently detected by chrome azurol S agar diffusion (CASAD) assay as increase in diameter of orange halo, and reduction in bacterial size along with significant biosorption of lead was recorded by scanning electron microscopy coupled with energy dispersive X-ray spectrometry (SEM-EDX). Pseudomonas aeruginosa strain 4EA also exhibits cross tolerance to other toxic metals viz. cadmium, mercury, and zinc besides resistance to multiple antibiotics such as ampicillin, erythromycin, amikacin, cephalexin, co-trimoxazole, mecillinam, lincomycin, ciphaloridine, oleondamycin, and nalidixic acid.     

Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus

AIMS: To evaluate the suitability of chrome azurol S (CAS) agar plate assay as a quantitative methodology for siderophore production. METHODS AND RESULTS: Aspergillus species (A. flavus, A. niger, A. tamarii) were inoculated in the CAS-agar plates and the siderophores production was determined and expressed as CAS-reaction rate (mm per day). All the species showed positive CAS reaction with different rates depending on culture conditions and A. flavus showed the highest CAS-reaction rate. The siderophore production in solid medium expressed as CAS-reaction rate was correlated with siderophore production in liquid medium. CONCLUSIONS: The use of CAS-agar plate assay was modified and the evaluation of CAS reaction in mm per day made it possible to study and quantify the effect of several variables on the siderophore production by Aspergillus fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe the CAS-agar plate assay as a quantitative methodology, which make it possible to select and evaluate the siderophore production by several microorganisms (fungi and bacteria) according to different culture conditions.     

Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay.

A well-known and widely used method for detection of siderophore production by microorganisms in solid medium is the universal chrome azurol S (CAS)-agar plate assay. However, the high toxicity of CAS-blue agar medium caused by the presence of a detergent impedes its utilization with many varieties of fungi and Gram-positive bacteria. To solve this problem, a modification of the CAS-agar plate assay was made by incorporating the CAS-blue dye in a medium with no contact with the microorganisms tested. Half of each plate used in our experiments was filled with the most appropriate culture medium for each type of microorganism and the other half with CAS-blue agar. This modification allowed us to study several strains of fungi (basidiomycetes, deuteromycetes, ascomycetes and zygomycetes) and bacteria (Gram positive and negative), some of them appearing for the first time in the literature. All the microorganisms grew properly and reacted in different manners to the CAS assay. Some strains of wood-decaying basidiomycetes (mainly white-rot fungi) and Aspergillus species produced the fastest color-change reactions in the CAS-blue agar. This modified method could facilitate optimization of culture conditions, since both CAS-blue agar and growth medium were prepared and added in the Petri plate separately.     

高灵敏假单胞菌铁载体的平板检测方法

CAS蓝色检测平板是一种筛选、检测各类细菌铁载体的常用方法,而蔗糖-天冬酰氨培养基被用于假单胞菌产铁载体规律的研究。用天冬氨酸替代天冬酰氨,将CAS蓝色检测液与蔗糖-天冬氨酸培养基（MSA培养基）相结合,得到一种改进的MSA-CAS检测平板。通过对假单胞菌属7个种8个株进行荧光与非荧光铁载体检测方面的比较研究,结果表明MSA-CAS检测平板假单胞菌铁载体的检测灵敏度比通用CAS检测平板高,而且在检测荧光铁载体方面具有荧光背景低、荧光铁载体晕圈明显和晕圈与背景的对比度大的优点。     

Universal chemical assay for the detection and determination of siderophores

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.     

Siderophore production in relation to N2 fixation and iron uptake in pigeon pea-Rhizobium symbiosis

Thirty-one bradyrhizobial and rhizobial strains infecting pigeon pea were screened for siderophore production using Chrome Azurol S (CAS) agar plate as well as a CAS assay solution. Of a total of 31 strains only 23 showed siderophore production. Of the 23 siderophore-positive strains, 21 strains showed the production of hydroxamate while 6 strains showed the presence of catechol type of siderophore. A large variation in the quantity of hydroxamate and catechol produced by different rhizobial strains was observed (1.03–3.73 μg hydroxamate N per mg protein; 0.19–3.43 μmol/L of catechol per mg protein). Maximum nodule biomass was produced by strain PP-11 (CC-1020); strain G-14 formed minimum nodule biomass. Nitrogen contents of low, moderate and high siderophore-producing strains were 11.4, 15.4, 20.9 mg per plant, respectively, iron contents were 1445, 1768 and 2003 ppm, respectively. Siderophore production was related to N₂-fixing efficiency.     

双层平板法检测嗜盐古菌铁载体

采用双层平板法应用于嗜盐古菌铁载体的原位检测。双层平板的上层为不添加铁离子的嗜盐古菌培养基, 嗜盐古菌可在其上生长, 在缺铁胁迫下可向外界分泌铁载体; 下层为含有CAS检测液用于铁载体检测的琼脂。当上层平板生长的嗜盐古菌分泌的铁载体透过培养基渗透到下层检测琼脂后, 即可在下层检测平板上产生明显的特征性的铁载体螯合晕圈, 表明双层平板法在嗜盐古菌的铁载体检测中确实可行, 且较原有的嗜盐古菌铁载体检测方法简便、直接。     

双层平板法检测嗜盐古菌铁载体

采用双层平板法应用于嗜盐古菌铁载体的原位检测.双层平板的上层为不添加铁离子的嗜盐古菌培养基,嗜盐古菌可在其上生长,在缺铁胁迫下可向外界分泌铁载体;下层为含有CAS检测液用于铁载体检测的琼脂.当上层平板生长的嗜盐古菌分泌的铁载体透过培养基渗透到下层检测琼脂后,即可在下层检测平板上产生明显的特征性的铁载体螯合晕圈,表明双层平板法在嗜盐古菌的铁载体检测中确实可行,且较原有的嗜盐古菌铁载体检测方法简便、直接.     

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 FeCl₃ 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.     

A new approach to isolating siderophore-producing actinobacteria

Aims:  This study was conducted to investigate the application of 2,2′‐dipyridyl as a new approach to isolating siderophore‐producing actinobacteria. Methods and Results:  Isolation of actinobacteria from soil was conducted by a soil dilution plate technique using starch‐casein agar. Iron starvation was fostered by the incorporation of the iron chelator 2,2′‐dipyridyl in the isolation medium. Pretreatment of the samples at an elevated temperature (40°C) ensured that the majority of nonsporulating bacteria were excluded. The survivors of this treatment were largely actinobacteria. Of the viable cultures grown in the presence of 2,2′‐dipyridyl, more than 78–88% (average of three separate studies) were reported to produce siderophore‐like compounds compared to 13–18% (average of three separate studies) when grown on the basic media in the absence of the chelating agent. The most prolific producers as assessed by the chrome azurol sulphate (CAS) assay were further characterized and found to belong to the genus Streptomyces. Conclusions:  Selective pressure using 2,2′‐dipyridyl as an iron‐chelating agent in starch‐casein media increased the isolation of siderophore‐producing actinobacteria compared to the unamended medium. Significance and Impact of the Study:  The study described represents a new approach to the isolation of siderophore‐producing actinobacteria using a novel procedure that places a selection on cell population based upon the incorporation of a chelating agent in the medium.     

AmoA、AmoE和AmoF蛋白在嗜水气单胞菌铁载体合成中的作用研究

铁载体被认为是嗜水气单胞菌的毒力因子之一, 其由amoCEBFAGH七个基因编码, AmoCGH在前人的研究中已证实参与铁载体的合成。RT-PCR实验表明amoAEF基因的表达受到铁的调控。为进一步探究amoAEF基因的功能, 利用融合PCR和基因同源重组原理, 以自杀性质粒PRE112为载体构建基因缺失株ΔamoA、ΔamoE和ΔamoF。通过CAS平板检测实验以及arnow实验来检测野生株WT与各基因缺失突变株铁载体的合成情况, 并比较野生株与各缺失株在低铁培养基中的生长差异。结果显示, 成功构建了基因缺失株ΔamoA、ΔamoE和ΔamoF; 在富铁条件下, 基因缺失株ΔamoA、ΔamoE和ΔamoF的生长与野生株无显著性差异, 但在低铁条件下, 基因缺失株ΔamoA、ΔamoE和ΔamoF的生长能力、铁载体合成能力显著低于野生株。可见, amoA、amoE和amoF基因是嗜水气单胞菌铁载体合成的关键基因, 其缺失会导致细菌在低铁环境中的生长受到抑制。     

Bioassay for siderophore utilization by Candida albicans

A convenient plate assay which is sensitive to medium pH has been developed to evaluate potential siderophores of Candida albicans. Adding a siderophore to a filter paper disk on chemically defined Lee's agar (final pH 7.2) seeded with the test strain reversed the growth inhibitory effects of the supplemented (25-100 micrograms/ml) iron chelator ethylenediaminedi(o-hydroxyphenylacetic acid), to provide a zone of growth stimulation. This bioassay has been used to demonstrate the structure-activity relationships of ferrichrome and several water-soluble hydroxamate peptide building blocks of this natural siderophore. Of all compounds so evaluated, ferrichrome exhibited the best activity.     

Development and application of an assay for uranyl complexation by fungal metabolites,including siderophores

An assay to detect UO(2)(2+) complexation was developed based on the chrome azurol S (CAS) assay for siderophores (B. Schwyn and J. B. Neilands, Anal. Biochem. 160:47-56, 1987) and was used to investigate the ability of fungal metabolites to complex actinides. In this assay the discoloration of two dyed agars (one containing a CAS-Fe(3+) dye and the other containing a CAS-UO(2)(2+) dye) caused by ligands was quantified. The assay was tested by using the siderophore desferrioxamine B (DFO), and the results showed that there was a regular, reproducible relationship between discoloration and the amount of siderophore added. The ratio of the discoloration on the CAS-UO(2)(2+) agar to the discoloration on the CAS-Fe(3+) agar was independent of the amount of siderophore added. A total of 113 fungi and yeasts were isolated from three soil samples taken from the Peak District National Park. The fungi were screened for the production of UO(2)(2+) chelators by using the CAS-based assay and were also tested specifically for hydroxamate siderophore production by using the hydroxamate siderophore auxotroph Aureobacterium flavescens JG-9. This organism is highly sensitive to the presence of hydroxamate siderophores. However, the CAS-based assay was found to be less sensitive than the A. flavescens JG-9 assay. No significant difference between the results for each site for the two tests was found. Three isolates were selected for further study and were identified as two Pencillium species and a Mucor species. Our results show that the new assay can be effectively used to screen fungi for the production of UO(2)(2+) chelating ligands. We suggest that hydroxamate siderophores can be produced by mucoraceous fungi.     

Detection of siderophores in growing cultures ofPseudomonas spp.

Summary 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 M. 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.