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。     

Studies on siderophore exchange properties between staphylococci and various species of gram-positive and gram-negative bacteria

The ability of iron utilizing by means of staphylococcal siderophores by bacteria belonging to genera: Acinetobacter, Corynebacterium, Curtobacterium, Clavibacter, Bacillus and Mycobacterium was investigated. The staphylococcal donor strains (18 species) used in these experiments were characterized by the ability to utilize siderophores produced by various strains belonging to aforenamed genera. The utilization of staphylococcal siderophores was studied on agar media in which minimally effective concentrations of ethylenediaminedi-ortho-hydroxyphenylacetic acid (EDDA) were used to inhibit indicator strains. Test colonies (staphylococcal) were applied to the surface of the media to determine whether the indicator organisms could obtain the required iron for growth by utilizing chelators from the test colony. The growth inhibition by EDDA of most strains from the Acinetobacter rods and from the coryneform-organisms (plant pathogen) genera, and strains from the species: B. subtilis, M. phlei, M. smegmatis, M. fortuitum was reversed by staphylococcal siderophores. None of the staphylococcal strains investigated, had the ability to exchange siderophores with strains from the species: C. pseudodiphtheriticum, Corynebacterium ANF group, B. megaterium, M. vaccae, M. chitae and M. parafortuitum.     

Iron transport-mediated antagonism between plant growth-promoting and plant-deleterious Pseudomonas strains

Both plant growth-promoting Pseudomonas B10 and its yellow-green, fluorescent iron transport agent (siderophore) pseudobactin enhance potato growth and biologically control certain soil-borne fungal diseases in part by depriving specific root-colonizing endemic microorganisms including phytopathogens of iron(III), thus inhibiting their growth. The present study examines this mode of iron deprivation. The growth inhibition of certain bean-deleterious fluorescent pseudomonads by specific bean-beneficial fluorescent pseudomonads is due in part to the inability of susceptible strains to utilize siderophores from beneficial strains to transport iron(III). Conversely, deleterious strains which were able to utilize siderophores from beneficial strains were not inhibited. The ability of a given pseudomonad to utilize another pseudomonad's siderophore may depend upon its possessing a specific outer membrane receptor protein for that pseudomonad's ferric siderophore. Siderophore-mediated competition for iron in microbial systems appears to be a widespread phenomenon.     

Growth of Actinobacillus pleuropneumoniae is promoted by exogenous hydroxamate and catechol siderophores.

Siderophores bind ferric ions and are involved in receptor-specific iron transport into bacteria. Six types of siderophores were tested against strains representing the 12 different serotypes of Actinobacillus pleuropneumoniae. Ferrichrome and bis-catechol-based siderophores showed strong growth-promoting activities for A. pleuropneumoniae in a disk diffusion assay. Most strains of A. pleuropneumoniae tested were able to use ferrichrome (21 of 22 or 95%), ferrichrome A (20 of 22 or 90%), and lysine-based bis-catechol (20 of 22 or 90%), while growth of 36% (8 of 22) was promoted by a synthetic hydroxamate, N5-acetyl-N5-hydroxy-L-ornithine tripeptide. A. pleuropneumoniae serotype 1 (strain FMV 87-682) and serotype 5 (strain 2245) exhibited a distinct yellow halo around colonies on Chrome Azurol S agar plates, suggesting that both strains can produce an iron chelator (siderophore) in response to iron stress. The siderophore was found to be neither a phenolate nor a hydroxamate by the chemical tests of Arnow and Csaky, respectively. This is the first report demonstrating the production of an iron chelator and the use of exogenous siderophores by A. pleuropneumoniae. A spermidine-based bis-catechol siderophore conjugated to a carbacephalosporin was shown to inhibit growth of A. pleuropneumoniae. A siderophore-antibiotic-resistant strain was isolated and shown to have lost the ability to use ferrichrome, synthetic hydroxamate, or catechol-based siderophores when grown under conditions of iron restriction. This observation indicated that a common iron uptake pathway, or a common intermediate, for hydroxamate- and catechol-based siderophores may exist in A. pleuropneumoniae.     

Iron-binding compounds and related outer membrane proteins in Vibrio cholerae non-O1 strains from aquatic environments

A total of 156 strains of Vibrio cholerae non-O1 from aquatic origins were examined for the presence of iron uptake mechanisms and compared with O1 strains and other Vibrio species. All non-O1 strains were able to grow in iron-limiting conditions, with MICs of ethylenediaminedi (O-hydroxyphenylacetic acid) ranging from 20 microM to 2 mM. The production of siderophores was demonstrated by growth in chrome azurol S agar and cross-feeding assays. All strains produced phenolate-type compounds, as assessed by the chemical tests and by bioassays with Salmonella typhimurium enb-7. Some of the strains also promoted the growth of S. typhimurium enb-1 (which can use only enterobactin as a siderophore) as well as some strains of Vibrio anguillarum deficient in the anguibactin-mediated system. The chromatographic analyses and absorption spectra of siderophores extracted from culture supernatants suggest that vibriobactin may be produced by the strains examined. Interestingly, some strains also produced hydroxamate-type compounds, as determined by chemical tests, and were able to promote the growth of an aerobactin-deficient strain of Escherichia coli. These results were confirmed by the absorption spectra and chromatographic analyses of the culture extracts. The synthesis of iron-regulated outer membrane proteins in representative strains was also examined. The molecular sizes of the main induced proteins ranged from 70 to 78 kilodaltons. These results indicate that several iron uptake mechanisms which could be involved in environmental survival and pathogenicity are present in environmental V. cholerae non-O1 strains.     

Iron-binding compounds and related outer membrane proteins in Vibrio cholerae non-O1 strains from aquatic environments.

A total of 156 strains of Vibrio cholerae non-O1 from aquatic origins were examined for the presence of iron uptake mechanisms and compared with O1 strains and other Vibrio species. All non-O1 strains were able to grow in iron-limiting conditions, with MICs of ethylenediaminedi (O-hydroxyphenylacetic acid) ranging from 20 microM to 2 mM. The production of siderophores was demonstrated by growth in chrome azurol S agar and cross-feeding assays. All strains produced phenolate-type compounds, as assessed by the chemical tests and by bioassays with Salmonella typhimurium enb-7. Some of the strains also promoted the growth of S. typhimurium enb-1 (which can use only enterobactin as a siderophore) as well as some strains of Vibrio anguillarum deficient in the anguibactin-mediated system. The chromatographic analyses and absorption spectra of siderophores extracted from culture supernatants suggest that vibriobactin may be produced by the strains examined. Interestingly, some strains also produced hydroxamate-type compounds, as determined by chemical tests, and were able to promote the growth of an aerobactin-deficient strain of Escherichia coli. These results were confirmed by the absorption spectra and chromatographic analyses of the culture extracts. The synthesis of iron-regulated outer membrane proteins in representative strains was also examined. The molecular sizes of the main induced proteins ranged from 70 to 78 kilodaltons. These results indicate that several iron uptake mechanisms which could be involved in environmental survival and pathogenicity are present in environmental V. cholerae non-O1 strains.     

Utilization of siderophores from mycobacteria by Staphylococcus

The ability of iron utilizing by means of siderophores (extracellular exochelins and cell-associated mycobactins) produced by mycobacteria (7 stains) by 24 staphylococcal strains was investigated. The mycobacterial donor strains belonged to rapid growing species: M. fortuitum, M. smegmatis, M. aurum, M. vaccae, M. chitae, M. phlei, M. parafortuitum. The utilization of mycobacterial siderophores was studied on agar or liquid media in which minimally effective concentrations of ethylene diamine di-ortho-hydroxyphenyl acetic acid (EDDA) were used to inhibit indicator staphylococcal strains. Mycobacterial siderophores (exochelins or mycobactins) reversed the inhibition of the majority (22/24) staphylococcal strains. Most of strains utilized exochelins from M. phlei as well as mycobactins from M. parafortuitum and M. chitae.     

A novel bioassay for the detection of siderophores containing keto-hydroxy bidentate ligands

Abstract A convenient and sensitive pour-plate Petri dish bioassay for the detection of siderophores containing monoprotic keto-hydroxy bidentate ligands (KHBL) has been developed. The bioassay is based on the fact that bacteria of the Proteus-Providencia-Morganella group (Proteeae) utilize various ferric α-hydroxy- or α-ketocarboxylate complexes very efficiently. While P. vulgaris and P. rettgeri were able to utilize virtually all iron complexes supplied, Morganella morganii SBK3 was unable to utilize trihydroxamate type siderophores and was therefore selected as an indicator strain for iron complexes containing keto-hydroxy bidentate ligands (KHBL-siderophores). Filter paper disks containing the ferric complexes of siderophores were tested on tryptone or Luria broth agar, seeded with the indicator strains and supplemented with the ferrous iron chelator 2,2-dipyridyl (300 μM) to reduce the bioavailable iron. In the presence of siderophores, growth inhibition was reversed to provide a zone of growth stimulation. Ferric complexes of α-hydroxycarboxylates, α-ketocarboxylates, salicylic acid, tropolonederivatives, α-hydroxypyridinones, cepabactin, citrate, rhizoferrin and even epihydroxymugineic acid showed significant growth stimulation. From the results with the trihydroxamate-non-utilizing strain, M. morganii SBK3 , it may be inferred that the Proteeae prossess an iron transport system which recognizes ferric α-hydroxycarboxylates, α-ketocarboxylates as well as aromatic and heteroaromatic keto-hydroxy compounds, collectively named keto-hydroxy bidentate ligands. The bioassay is especially suited for detection of new siderophores from low-iron cultures of fungi and bacteria.     

The evolution of siderophore production as a competitive trait

Microbes have the potential to be highly cooperative organisms. The archetype of microbial cooperation is often considered to be the secretion of siderophores, molecules scavenging iron, where cooperation is threatened by “cheater” genotypes that use siderophores without making them. Here, we show that this view neglects a key piece of biology: siderophores are imported by specific receptors that constrain their use by competing strains. We study the effect of this specificity in an ecoevolutionary model, in which we vary siderophore sharing among strains, and compare fully shared siderophores with private siderophores. We show that privatizing siderophores fundamentally alters their evolution. Rather than a canonical cooperative good, siderophores become a competitive trait used to pillage iron from other strains. We also study the physiological regulation of siderophores using in silico long‐term evolution. Although shared siderophores evolve to be downregulated in the presence of a competitor, as expected for a cooperative trait, privatized siderophores evolve to be upregulated. We evaluate these predictions using published experimental work, which suggests that some siderophores are upregulated in response to competition akin to competitive traits like antibiotics. Although siderophores can act as a cooperative good for single genotypes, we argue that their role in competition is fundamental to understanding their biology.     

Iron requirement and siderophore production in Rhizobium ciceri during growth on an iron-deficient medium

Under conditions of iron limitation many rhizospheric bacteria produce siderophores, ferric iron-specific ligands, which may enhance plant growth by increasing the availability of iron near the roots. Thirty-five strains of Rhizobium ciceri, specific to chickpea (Cicer arietinum L.), were screened for their ability to grow on iron-deficient medium and to produce siderophores. Maximal growth of all strains previously depleted in iron was obtained in medium containing 5 to 10 m of ferric iron. When iron limitation was achieved by the addition of 2,2-bipyridyl or EDDHA [ethylene diamine di(o-hydroxyphenyl) acetic acid] to the medium, only two strains were able to scavenge iron and grow. Siderophore production by these two strains was detected by the Chrome Azurol S assay (CAS), a universal test for siderophores. No hydroxamate-type siderophores were detected in the supernatants of Rhizobium ciceri cultures. However, some strains secreted salicylic acid and 2,3-dihydroxybenzoic acid as phenolate-type siderophores. Addition of ferric iron to the culture medium increased growth yield significantly but depressed the production of siderophores. Although these compounds are produced in response to iron deficiency, nutritive components of the culture medium significantly affected their production. It seems that CuII, MoVI and MnII ions bound competitively with iron to siderophores, resulting in a 34 to 100% increase in production.     

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.     

Genome-wide screen for genes with effects on distinct iron uptake activities in Saccharomyces cerevisiae

We screened a collection of 4847 haploid knockout strains (EUROSCARF collection) of Saccharomyces cerevisiae for iron uptake from the siderophore ferrioxamine B (FOB). A large number of mutants showed altered uptake activities, and a few turned yellow when grown on agar plates with added FOB, indicating increased intracellular accumulation of undissociated siderophores. A subset consisting of 197 knockouts with altered uptake was examined further for regulated activities that mediate cellular uptake of iron from other siderophores or from iron salts. Hierarchical clustering analysis grouped the data according to iron sources and according to mutant categories. In the first analysis, siderophores grouped together with the exception of enterobactin, which grouped with iron salts, suggesting a reductive pathway of iron uptake for this siderophore. Mutant groupings included three categories: (i) high-FOB uptake, high reductase, low-ferrous transport; (ii) isolated high- or low-FOB transport; and (iii) induction of all activities. Mutants with statistically altered uptake activities included genes encoding proteins with predominant localization in the secretory pathway, nucleus, and mitochondria. Measurements of different iron-uptake activities in the yeast knockout collection make possible distinctions between genes with general effects on iron metabolism and those with pathway-specific effects.     

Antibiotic activity and siderophores of Pseudomonas cepacia

The ability of 46 strains of Pseudomonas cepacia to inhibit phytopathogenic fungi and the effect of iron on their antifungal activity were studied. The antifungal effect of the bacteria and the antimicrobial activity of their crude yellow and violet pigments showed a 4-5-fold decrease in the presence of Fe(III). The addition of 100 micrograms/ml of FeCl3 to the medium decreased the biosynthesis of violet and yellow pigments; the complex of the yellow pigment with Fe(III) promoted the growth of the P. cepacia producing strain under iron-deficient conditions. The data obtained suggest a participation of some P. cepacia pigments in iron transport. The resistance of the P. cepacia strains to the synthetic chelating agents hydroxyethylenediphosphonic and diethylenediaminepentaacetic acids was demonstrated, which may indicate a high Fe(III)-binding constant of P. cepacia siderophores.     

Effects of iron level on the anatagonistic action of siderophores from non-pathogenicStaphylococcus spp

The production, detection and the effects of iron concentration on the siderophore production ofStaphylococcus strains used as meat starter cultures were studied. Non-pathogenicStaphylococcus strains produce extracellular low molecular weight compounds which exhibited positive reactivity when measured by a universal detection method for siderophores. The production of siderophores was very closely associated with the iron concentration in the medium, and very low additions considerably reduced siderophore production. Although the production of siderophores was highly iron-dependent, the antimicrobial activity of spent medium fromStaphylococcus cultures against selected yeasts and moulds remained considerable under high iron concentrations.     

鱼腥蓝细菌PCC7120铁吸收机制

铁离子是鱼腥蓝细菌PCC7120进行呼吸作用、光合作用和固氮作用中相关酶的重要辅基之一,缺铁将严重影响蓝细菌的生存.富氧的生态环境中铁通常以不溶的Fe3+形式存在,不易被细胞吸收利用.低铁条件下,鱼腥蓝细菌PCC7120分泌能螯合铁离子的嗜铁素,通过外膜上相应的转运体将嗜铁素-铁复合物转运到细胞内.综述了近年来在嗜铁素的种类及其生物合成途径、铁吸收系统的组成和功能等方面的最新进展,分析了铁吸收系统的调控机制,为进一步开展鱼腥蓝细菌铁吸收机制的研究提供依据.     

Iron uptake is essential for Escherichia coli survival in drinking water

AIMS: The aim of this study was to elucidate if the need for iron for Escherichia coli to remain cultivable in a poorly nutritive medium such as the drinking water uses the iron transport system via the siderophores. METHODS AND RESULTS: Environmental strains of E. coli (isolated from a drinking water network), referenced strains of E. coli and mutants deficient in TonB, an essential protein for iron(III) acquisition, were incubated for 3 weeks at 25 degrees C, in sterile drinking water with and without lepidocrocite (gamma-FeOOH), an insoluble iron corrosion product. Only cells with a functional iron transport system were able to survive throughout the weeks. CONCLUSIONS: The iron transport system via protein TonB plays an essential role on the survival of E. coli in a weakly nutritive medium like drinking water. SIGNIFICANCE AND IMPACTS OF THE STUDY: Iron is a key parameter involved in coliform persistence in drinking water distribution systems.     

Effect of exogenous siderophores on iron uptake activity of marine bacteria under iron-limited conditions

More than 60% of species examined from a total of 421 strains of heterotrophic marine bacteria which were isolated from marine sponges and seawater were observed to have no detectable siderophore production even when Fe(III) was present in the culture medium at a concentration of 1.0 pM. The growth of one such non-siderophore-producing strain, alpha proteobacterium V0210, was stimulated under iron-limited conditions with the addition of an isolated exogenous siderophore, N,N'-bis (2,3-dihydroxybenzoyl)-O-serylserine from a Vibrio sp. Growth was also stimulated by the addition of three exogenous siderophore extracts from siderophore-producing bacteria. Radioisotope studies using (59)Fe showed that the iron uptake ability of V0210 increased only with the addition of exogenous siderophores. Biosynthesis of a hydroxamate siderophore by V0210 was shown by paper electrophoresis and chemical assays for the detection of hydroxamates and catechols. An 85-kDa iron-regulated outer membrane protein was induced only under iron-limited conditions in the presence of exogenous siderophores. This is the first report of bacterial iron uptake through an induced siderophore in response to exogenous siderophores. Our results suggest that siderophores are necessary signaling compounds for growth and for iron uptake by some non-siderophore-producing marine bacteria under iron-limited conditions.     

Identification and characterization of a novel-type ferric siderophore reductase from a gram-positive extremophile

Iron limitation is one major constraint of microbial life, and a plethora of microbes use siderophores for high affinity iron acquisition. Because specific enzymes for reductive iron release in gram-positives are not known, we searched Firmicute genomes and found a novel association pattern of putative ferric siderophore reductases and uptake genes. The reductase from the schizokinen-producing alkaliphile Bacillus halodurans was found to cluster with a ferric citrate-hydroxamate uptake system and to catalyze iron release efficiently from Fe[III]-dicitrate, Fe[III]-schizokinen, Fe[III]-aerobactin, and ferrichrome. The gene was hence named fchR for ferric citrate and hydroxamate reductase. The tightly bound [2Fe-2S] cofactor of FchR was identified by UV-visible, EPR, CD spectroscopy, and mass spectrometry. Iron release kinetics were determined with several substrates by using ferredoxin as electron donor. Catalytic efficiencies were strongly enhanced in the presence of an iron-sulfur scaffold protein scavenging the released ferrous iron. Competitive inhibition of FchR was observed with Ga(III)-charged siderophores with K(i) values in the micromolar range. The principal catalytic mechanism was found to couple increasing K(m) and K(D) values of substrate binding with increasing k(cat) values, resulting in high catalytic efficiencies over a wide redox range. Physiologically, a chromosomal fchR deletion led to strongly impaired growth during iron limitation even in the presence of ferric siderophores. Inductively coupled plasma-MS analysis of ΔfchR revealed intracellular iron accumulation, indicating that the ferric substrates were not efficiently metabolized. We further show that FchR can be efficiently inhibited by redox-inert siderophore mimics in vivo, suggesting that substrate-specific ferric siderophore reductases may present future targets for microbial pathogen control.     

Reliable use of green fluorescent protein in fluorescent pseudomonads.

When fluorescent pseudomonads are cultured on standard solid media under iron limiting conditions, they produce fluorescent, pigmented iron collating agents (siderophores). Siderophores can be readily identified by strong fluorescence seen under UV/blue light. The application of the eukaryotic green fluorescent protein (GFP) as a bacterial marker in microbial ecology is increasingly being used, particularly as it is a powerful method for non-destructive monitoring in situ. As gfp expressing bacteria have to be detected under UV/blue light, the fluorescence of siderophore-producing Pseudomonas spp. masks normal levels of GFP fluorescence when colonies are viewed on standard bacterial agar. Here, we describe a simple but effective way of identifying gfp-expressing Pseudomonas fluorescens using media supplemented with 0.45 mM FeSO(4).7H(2)O. This is of relevance for the screening of insertion libraries and in the application of GFP transposons as promoter probes.