放线菌中铁载体生物合成机制研究进展

铁载体是由微生物产生,对铁元素具有高亲和性的小分子化合物。这类天然产物所展现的结构多样性引起人们对其生物合成机制的极大兴趣。目前已有研究报道的铁载体生物合成途径主要有2种,一是直接由非核糖体肽合成酶(Nonribosomal peptide synthetases,NRPSs)家族的多酶复合体负责合成,另一种是以不依赖于NRPS(NRPS-independent,NIS)的方式,由一类特殊合成酶家族参与合成。在过去的十多年中,铁载体生物合成成为天然产物生物合成研究领域的热点之一,其中几种依赖于NRPS途径合成的铁载体生物合成机制已得到充分阐明,而对NIS方式合成的铁载体研究也获得了诸多进展。作为放线菌的一类重要次级代谢产物,通过遗传学、化学等手段对放线菌所产生铁载体生物合成途径的遗传学和生物化学研究,能够为发展新的抗菌药物提供契机,同时也能加深我们对这一类生物活性物质形成机制的认识。综述近期该研究方向的进展。     

Investigations of Salmonella strains from different clinical-epidemiological origin with phenolate and hydroxamate (aerobactin)--siderophore bioassays

By means of phenolate siderophore negative S. typhimurium mutants as indicators, a bioassay for the detection of phenolate production was applied in Salmonella species. Different Salmonella strains have a weak or normal phenolate siderophore production. Host-adapted Salmonella strains show weak, other strains of Salmonella show normal growth zones of the indicator strain. Besides phenolate siderophore production there are defined S. typhimurium strains, exhibiting phage type ut/ut, biochemical type a and some strains of S. wien, S. infantis and S. haifa from nosocomial infection producing hydroxamate siderophore (aerobactin) as a compotent of a second iron uptake system.     

四株碱性环境真菌铁载体活性筛选

【目的】铁载体(Siderophore)是由微生物产生的一类低分子量金属离子螯合物。在生物医药、环境修复、健康食品等领域均具有广泛应用前景。文献调查显示,针对碱性环境真核微生物铁载体研究尚无相关报道。从中筛选高活性铁载体真菌具有重要意义。【方法】采用铬天青S显色法对分离于云南省碱性湖泊-程海和个旧大屯碱性尾矿土壤的99株真菌进行筛选;利用"分光光度法"对菌株产铁载体能力和铁载体类型进行考察;采用固相萃取(SPE)手段对菌株铁载体富集效果进行考察;根据菌体形态电镜观察和ITS基因系统发育树构建,对菌株进行生物学鉴定。【结果】通过初筛和复筛,确定菌株FEDT-866、FEDT-145、FECH-998和FECH-595均为铁载体高产菌株;所产铁载体类型主要为异羟肟酸型和羧酸型。除菌株FEDT-866外,铁载体活性产物适合采用固相萃取(SPE)方法进行富集。经生物学鉴定,菌株FEDT-866和FECH-998属于曲霉属,分别与Aspergillus tubigensis和A.nomius相似性较高;菌株FECH-595和FEDT-145属于青霉属,分别与Penicillium svalbardense和P.chrysogenum相似性较高。【结论】P.chrysogenum是1种常见的产铁载体真菌。而A.tubigensis、A.nomius及P.svalbardense菌株尚无产铁载体相关报道,可作为铁载体研究良好材料。     

The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues

Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron-siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA-pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferric-enterobactin receptor. The example of these two enterobactin receptors probably illustrates a more general phenomenon of siderophore receptor redundancy in P. aeruginosa.     

Iron transport in the genus Marinobacter

Marinobacter belong to the class of Gammaproteobacteria and these motile, halophilic or halotolerent bacteria are widely distributed throughout the world’s oceans having been isolated from a wide variety of marine environments. They have also been identified as members of the bacterial flora associated with other marine organisms. Here, using a combination of natural products chemistry and genomic analysis, we assess the nature of the siderophores produced by this genus and their potential relationship to phylogeny and lifestyle/ecological niche of this diverse group of organisms. Our analysis shows a wide level of diversity in siderophore based iron uptake systems among this genus with three general strategies: (1) production and utilization of native siderophores in addition to utilization of a variety of exogenous ones, (2) production and utilization of native siderophores only, (3) lack of siderophore production but utilization of exogenous ones. They all share the presence of at least one siderophore-independent iron uptake ABC transport systems of the FbpABC iron metal type and lack the ability for direct transport of ferrous iron. Siderophore production and utilization can be correlated with phylogeny and thus it forms a type of chemotaxonomic marker for this genus.     

Xanthoferrin,the α‐hydroxycarboxylate‐type siderophore of Xanthomonas campestris pv. campestris,is required for optimum virulence and growth inside cabbage

Xanthomonas campestris pv. campestris causes black rot, a serious disease of crucifers. Xanthomonads encode a siderophore biosynthesis and uptake gene cluster xss (Xanthomonas siderophore synthesis) involved in the production of a vibrioferrin‐type siderophore. However, little is known about the role of the siderophore in the iron uptake and virulence of X. campestris pv. campestris. In this study, we show that X. campestris pv. campestris produces an α‐hydroxycarboxylate‐type siderophore (named xanthoferrin), which is required for growth under low‐iron conditions and for optimum virulence. A mutation in the siderophore synthesis xssA gene causes deficiency in siderophore production and growth under low‐iron conditions. In contrast, the siderophore utilization ΔxsuA mutant is able to produce siderophore, but exhibits a defect in the utilization of the siderophore–iron complex. Our radiolabelled iron uptake studies confirm that the ΔxssA and ΔxsuA mutants exhibit defects in ferric iron (Fe³⁺) uptake. The ΔxssA mutant is able to utilize and transport the exogenous xanthoferrin–Fe³⁺ complex; in contrast, the siderophore utilization or uptake mutant ΔxsuA exhibits defects in siderophore uptake. Expression analysis of the xss operon using a chromosomal gusA fusion indicates that the xss operon is expressed during in planta growth and under low‐iron conditions. Furthermore, exogenous iron supplementation in cabbage leaves rescues the in planta growth deficiency of ΔxssA and ΔxsuA mutants. Our study reveals that the siderophore xanthoferrin is an important virulence factor of X. campestris pv. campestris which promotes in planta growth by the sequestration of Fe³⁺.     

Characterization and Fungal Inhibition Activity of Siderophore from Wheat Rhizosphere Associated Acinetobacter calcoaceticus Strain HIRFA32

Acinetobacter calcoaceticus HIRFA32 from wheat rhizosphere produced catecholate type of siderophore with optimum siderophore (ca. 92 % siderophore units) in succinic acid medium without FeSO₄ at 28 °C and 24 h of incubation. HPLC purified siderophore appeared as pale yellow crystals with molecular weight [M⁺¹] m/z 347.18 estimated by LCMS. The structure elucidated by ¹H NMR, ¹³C NMR, HMQC, HMBC, NOESY and decoupling studies, revealed that siderophore composed of 2,3-dihydroxybenzoic acid with hydroxyhistamine and threonine as amino acid subunits. In vitro study demonstrated siderophore mediated mycelium growth inhibition (ca. 46.87 ± 0.5 %) of Fusarium oxysporum. This study accounts to first report on biosynthesis of acinetobactin-like siderophore by the rhizospheric strain of A. calcoaceticus and its significance in inhibition of F. oxysporum.     

Social evolution of toxic metal bioremediation in Pseudomonas aeruginosa

Bacteria are often iron-limited, and hence produce extracellular iron-scavenging siderophores. A crucial feature of siderophore production is that it can be an altruistic behaviour (individually costly but benefitting neighbouring cells), thus siderophore producers can be invaded by non-producing social ‘cheats’. Recent studies have shown that siderophores can also bind other heavy metals (such as Cu and Zn), but in this case siderophore chelation actually reduces metal uptake by bacteria. These complexes reduce heavy metal toxicity, hence siderophore production may contribute to toxic metal bioremediation. Here, we show that siderophore production in the context of bioremediation is also an altruistic trait and can be exploited by cheating phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. Specifically, we show that in toxic copper concentrations (i) siderophore non-producers evolve de novo and reach high frequencies, and (ii) producing strains are fitter than isogenic non-producing strains in monoculture, and vice versa in co-culture. Moreover, we show that the evolutionary effect copper has on reducing siderophore production is greater than the reduction observed under iron-limited conditions. We discuss the relevance of these results to the evolution of siderophore production in natural communities and heavy metal bioremediation.     

Isolation and characterization of siderophore from cowpeaRhizobium (peanut isolate)

In an iron-depleted broth culture of cowpeaRhizobium (a peanut isolate), phenolate type of compounds were detected. Chemical characterization showed the presence of 2,3-dihydroxy benzoic acid (DHBA) and 3,4-DHBA in the siderophore extract. Lysine and alanine were identified as conjugated amino acids of the siderophore. Maximum concentration of the siderophore in the culture supernatant was found after 24 h of growth. The compounds in the extracted siderophore induced growth ofRhizobium in a medium containing EDTA. Addition of lysine and alanine in the growth medium (20 mM each) led to a fourfold increase in siderophore production.     

Optimization of MM9 Medium Constituents for Enhancement of Siderophoregenesis in Marine Pseudomonas putida Using Response Surface Methodology

Pseudomonasputida (CMMB2) was isolated from open ocean water of Gulf of Mannar. The isolate was identified based on 16S rRNA gene sequencing and phylogenetic analysis. Chrome azurol sulphonate assay confirms siderophore production by the isolate. Nature of siderophore produced by the isolate was found to be of mixed type. Siderophore production was found to be inversely proportional to iron concentration of the medium. Maximum siderophore production was observed with MM9 medium. Siderophore production was found to be influenced by different carbon, nitrogen and amino acid sources. Optimization of MM9 medium nutrient composition by response surface methodology (RSM) enhances siderophore production. Application of RSM is one of the strategic attempts in cost effective siderophore production process. Presence of aromatic ring in the siderophore with (C–O) and (C=C) stretching was ascertained by FTIR spectral analysis. Mass spectral analysis revealed the presence of chromophore in the pyoverdine siderophore. Cell free supernatant and purified siderophore was found to inhibit the growth of bacterial and fungal pathogens.     

DENSITY DEPENDENCE AND COOPERATION: THEORY AND A TEST WITH BACTERIA

Although cooperative systems can persist in nature despite the potential for exploitation by noncooperators, it is often observed that small changes in population demography can tip the balance of selective forces for or against cooperation. Here we consider the role of population density in the context of microbial cooperation. First, we account for conflicting results from recent studies by demonstrating theoretically that: (1) for public goods cooperation, higher densities are relatively unfavorable for cooperation; (2) in contrast, for self-restraint–type cooperation, higher densities can be either favorable or unfavorable for cooperation, depending on the details of the system. We then test our predictions concerning public goods cooperation using strains of the pathogenic bacterium Pseudomonas aeruginosa that produce variable levels of a public good—iron-scavenging siderophore molecules. As predicted, we found that the relative fitness of cheats (under-producers) was greatest at higher population densities. Furthermore, as assumed by theory, we show that this occurs because cheats are better able to exploit the cooperative siderophore production of other cells when they are physically closer to them.     

Isolation and partial characterization of phenolate siderophore from Rhizobium leguminosarum IARI 102

Abstract Rhizobium leguminosarum IARI 102 produced 2,3-dihydroxy benzoic acid, a type of phenolate siderophore, under iron-starved conditions. Hydroxamic acids were not detected. Maximum production of siderophore was found at 26 h of growth in a chemically defined medium at 28°C with shaking. Threonine was detected as the amino acid conjugate of the siderophore. Addition of Fe³⁺ to the culture medium increased the growth yield significantly, but depressed the production of the iron chelating compound.     

A simple agar plate assay for screening siderophore producer yeasts.

Yeasts produce hydroxamate-type siderophores (iron-binding compounds) in response to Fe-stress conditions. Because these siderophores are important to the biocontrol of postharvest diseases of apple and pears, a method for screening siderophore producer yeast was developed.The screening method was carried out in special Petri dishes with eight or nine wells (25-mm diameter). These wells were filled with siderophore production medium and seeded with yeasts isolated from epiphytic apple microflora. After yeasts grew (24-48 h), holes (2-mm diameter) were made in the agar of each well. Holes were filled with an acid solution of ferric perchlorate. After 10-15 min, reddish halos appeared in the bottom of the plate and their intensities were compared with standards. Standards were prepared in the same special dish with rhodotorulic acid solutions (concentrations between 0.05 and 1 g/l) plus 2% agar. When agar solidified into wells, holes were made and filled with ferric perchlorate solution. Color intensities of reddish halos were proportional to siderophore concentration and the detection limit was 0.1 g/l. It was possible to correlate the production of siderophore in solid medium with the results obtained in liquid medium. The methodology was also a useful tool for making a preliminary assessment of the influence of different factors on the siderophore production.     

Increased synthesis of dihydroxybenzoic acid in the presence of aluminum by Rhizobium MO1

Rhizobizum sp. MO1, a mung bean (Vigna radiata) symbiont, produces a catecholate type of siderophore, 2,3-dihydroxy benzoic acid (DHBA), in iron depleted medium. Addition of aluminum to the medium decreased the growth but increased the production of the siderophore.     

Regulation of the cholesterol efflux transporters ABCA1 and ABCG1 in retina in hemochromatosis and by the endogenous siderophore 2,5-dihydroxybenzoic acid

Hypercholesterolemia and polymorphisms in the cholesterol exporter ABCA1 are linked to age-related macular degeneration (AMD). Excessive iron in retina also has a link to AMD pathogenesis. Whether these findings mean a biological/molecular connection between iron and cholesterol is not known. Here we examined the relationship between retinal iron and cholesterol using a mouse model (Hfe^−/−) of hemochromatosis, a genetic disorder of iron overload. We compared the expression of the cholesterol efflux transporters ABCA1 and ABCG1 and cholesterol content in wild type and Hfe^−/− mouse retinas. We also investigated the expression of Bdh2, the rate-limiting enzyme in the synthesis of the endogenous siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA) in wild type and Hfe^−/− mouse retinas, and the influence of this siderophore on ABCA1/ABCG1 expression in retinal pigment epithelium. We found that ABCA1 and ABCG1 were expressed in all retinal cell types, and that their expression was decreased in Hfe^−/− retina. This was accompanied with an increase in retinal cholesterol content. Bdh2 was also expressed in all retinal cell types, and its expression was decreased in hemochromatosis. In ARPE-19 cells, 2,5-DHBA increased ABCA1/ABCG1 expression and decreased cholesterol content. This was not due to depletion of free iron because 2,5-DHBA (a siderophore) and deferiprone (an iron chelator) had opposite effects on transferrin receptor expression and ferritin levels. We conclude that iron is a regulator of cholesterol homeostasis in retina and that removal of cholesterol from retinal cells is impaired in hemochromatosis. Since excessive cholesterol is pro-inflammatory, hemochromatosis might promote retinal inflammation via cholesterol in AMD.     

Evolution of a novel lysine decarboxylase in siderophore biosynthesis

Structural backbones of iron‐scavenging siderophore molecules include polyamines 1,3‐diaminopropane and 1,5‐diaminopentane (cadaverine). For the cadaverine‐based desferroxiamine E siderophore in Streptomyces coelicolor, the corresponding biosynthetic gene cluster contains an ORF encoded by desA that was suspected of producing the cadaverine (decarboxylated lysine) backbone. However, desA encodes an l ‐2,4‐diaminobutyrate decarboxylase (DABA DC) homologue and not any known form of lysine decarboxylase (LDC). The only known function of DABA DC is, together with l ‐2,4‐aminobutyrate aminotransferase (DABA AT), to synthesize 1,3‐diaminopropane. We show here that S. coelicolor desA encodes a novel LDC and we hypothesized that DABA DC homologues present in siderophore biosynthetic clusters in the absence of DABA AT ORFs would be novel LDCs. We confirmed this by correctly predicting the LDC activity of a DABA DC homologue from a Yersinia pestis siderophore biosynthetic pathway. The corollary was confirmed for a DABA DC homologue, adjacent to a DABA AT ORF in a siderophore pathway in the cyanobacterium Anabaena variabilis, which was shown to be a bona fide DABA DC. These findings enable prediction of whether a siderophore pathway will utilize 1,3‐diaminopropane or cadaverine, and suggest that the majority of bacteria use DABA AT and DABA DC for siderophore, rather than norspermidine/polyamine biosynthesis.     

Directing the Biosynthesis of Putrebactin or Desferrioxamine B in Shewanella putrefaciens through the Upstream Inhibition of Ornithine Decarboxylase

To manage iron acquisition in an oxic environment, Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (PB) as its native siderophore. In this work, we have established the siderophore profile of S. putrefaciens in cultures augmented with the native PB precursor putrescine and in putrescine-depleted cultures. Compared to base medium, PB increased by two-fold in cultures of S. putrefaciens with 10?mM NaCl and 20?mM exogenous putrescine. In cultures augmented with 1,4-diaminobutan-2-one (DAB), PB decreased with only 0.02-fold PB detectable at 10?mM DAB. As an ornithine decarboxylase (ODC) inhibitor, DAB depleted levels of endogenous putrescine which attenuated downstream PB assembly. Under putrescine-depleted conditions, S. putrefaciens produced as its replacement siderophore the cadaverine-based desferrioxamine B (DFO-B), as characterised by ESI-MS of the Fe(III) -loaded form (m/z(obs) 614.13; m/z(calc) 614.27). A third siderophore, independent of DAB, was observed in low levels. LC/MS Analysis of the Fe(III) -loaded extract gave m/z(obs) 440.93, which, formulated as a 1?:?1 Fe(III) complex with a macrocyclic dihydroxamic acid, comprising one putrescine- and one cadaverine-based precursor (m/z(calc) 440.14). These results show that the production of native PB or non-native DFO-B by S. putrefaciens can be directed though upstream inhibition of ODC. This approach could be used to increase the molecular diversity of siderophores produced by S. putrefaciens and to map alternative diamine-dependent metabolites.     

Purification and characterization of rhodobactin: a mixed ligand siderophore from Rhodococcus rhodochrous strain OFS

The siderophore produced by Rhodococcus rhodochrous strain OFS, rhodobactin, was isolated from iron-deficient cultures and purified by a combination of XAD-7 absorptive/partition resin column and semi-preparative HPLC. The siderophore structure was characterized using 1D and 2D ¹H, ¹³C and ¹⁵N NMR techniques (DQFCOSY, TOCSY, NOESY, HSQC and LR-HSQC) and was confirmed using ESI-MS and MS/MS experiments. The structural characterization revealed that the siderophore, rhodobactin, is a mixed ligand hexadentate siderophore with two catecholate and one hydroxamate moieties for iron chelation. We further investigated the effects of Fe concentrations on siderophore production and found that Fe limiting conditions (Fe concentrations from 0.1 μM to 2.0 μM) facilitated siderophore excretion. Our interests lie in the role that siderophores may have in binding metals at mixed contamination sites (containing metals/radionuclides and organics). Given the broad metabolic capacity of this microbe and its Fe scavenging ability, R. rhodochrous OFS may have a competitive advantage over other organisms employed in bioremediation. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.