铁载体和铁离子对细菌生长过程的影响
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摘要
铁是生命体必需的一种营养元素,它可以参与众多的生物代谢过程,如光合作用、呼吸作用、氮的固定和DNA的生物合成等。铁是地壳中含量最多的一种过渡金属元素,它存在着还原型Fe2+和氧化型Fe3+两种可以可逆变化的化合态,在自然界的有氧条件下铁主要以三价的氧化物或氢氧化物的形式存在,其溶解性极低、生物可利用性低,游离Fe3+的浓度一般不超过10-18M。为了适应在低铁的限制性环境中生长代谢对铁的需要,微生物发展了多种铁吸收系统来从环境中摄取铁。主要包括合成和利用铁载体,还原三价铁为生物利用性更高的亚铁形式并吸收,降低环境pH值来增加铁的溶解性,利用宿主的铁蛋白,如乳铁蛋白和转铁蛋白等。合成和利用铁载体是微生物铁代谢的一种主要途径。铁载体是微生物在低铁的限制性条件的诱导下合成的一类低分子量化合物,对Fe3+有特异的高亲和力,在微生物界分布广泛,种类多样,生物活性多样。绝大多数微生物都可以合成铁载体,依据化学结构不同可基本分为三类：氧肟酸盐型、儿茶酚盐型和多羟基羧酸类。细菌中各种类型的铁载体都可以产生,而真菌只合成氧肟酸盐型铁载体。某些病原微生物的铁载体与它们的毒力有关,因为从宿主体内吸收铁是病原菌繁殖和感染的必要步骤。此外,铁载体在病原微生物的控制方面也扮演着重要的角色,由于铁的螯合作用可以抑制病原菌对铁的利用,从而抑制病原菌的生长和代谢活性。由于人体细胞没有铁载体合成的相关途径,所以它们的生物合成和吸收途径还可以应用于抗微生物治疗。
除此之外,Fe2+还可以与超氧阴离子、过氧化氢等氧化因子反应生成反应性更强的羟基自由基。羟基自由基是一种极强的氧化剂,它可以无选择性的破坏几乎所有的生物分子,包括糖、脂、DNA链等,对细胞具有极大的损害效应。因此,好氧和兼性厌氧微生物面临一种两难的境地：一方面微生物细胞必须利用有效的铁吸收机制来从环境中吸收铁以供其生长代谢的需要,另一方面又要严格控制细胞内游离铁的浓度,使细胞内催化性铁的浓度维持在无害的水平。对铁和铁载体的研究有助于阐明微生物铁代谢的途径、机理等问题,对医学、工业、农业和环境都有重要的应用价值。
论文通过对27株丝状真菌铁载体合成情况的比较研究,从中筛选到Aspergillus niger An76菌株作为生产菌株。对其在低铁的培养条件下合成的铁载体进行分离纯化,理化性质测定,并对该铁载体的细菌抑制活性进行了较深入的研究。同时对高浓度的Fe3+的抑菌活性和作用机理进行了探索研究。
论文取得了如下创新性的结果：
1.筛选到一株高产铁载体的菌株A. niger An76,其铁载体在浓度高达1 mM Fe3+的培养条件下仍然能够合成,为组成型表达。An76铁载体与典型的氧肟酸盐型和儿茶酚盐型铁载体不同,它具有羟基羧酸盐型铁载体的特性,为一种新的铁载体。它不含氨基酸成分,并且具有羟基自由基清除活性。An76铁载体具有显著的抑菌活性,这为筛选新的抗生素药物提供了新途径。采用2D/3D作图法表征实验数据的方法可用于研究药物的抗菌机理。
2.研究发现供试的G+/G细菌都可以吸附Fe3+,并可以迅速的将Fe3+还原为Fe2+。高浓度的Fe3+对细菌生长有显著的抑制作用,推测Fe3+对细菌的抑制作用是通过Fe2+催化羟基自由基的形成从而抑制细菌生长的。不同的铁载体对Fe3+的抑菌活性具有不同的影响,这为发展新的抗菌剂提供了线索。
一、铁载体生产菌的筛选、铁载体的分离纯化和性质鉴定,以及铁载体的细菌抑制活性。
通过对27株丝状真菌在三种不同培养基条件下的铁载体生产情况进行比较研究,我们发现Aspergillus niger An76铁载体合成能力的表达在浓度高达1 mMFe3+的培养条件下仍然不受抑制,表明它的铁载体合成不是低铁的限制性条件诱导表达的,而是组成型表达。然而其它的大多数供试真菌的铁载体合成在Fe3+浓度达到20μM时就会受到抑制。低浓度的铁更有利于铁载体的合成。
对A. niger An76在低铁的限制性条件下培养所产生的铁载体进行分离纯化,通过大量的探索性试验,确定了一可以将其部分纯化的分离方案。采用该方案可以在较短时间内制备足够量的样品来研究其理化性质和细菌抑制活性。通过超滤、真空冷冻干燥得到粗样品,采用醋酸铅沉淀法进行初步的分离,经过CMFast Flow离子交换层析得到较好的分离效果,如图1所示,Peak 1为铁载体活性峰。
经过离子交换层析得到的活性样品进一步采用TSK-GEL G2500PW液相色谱柱进行分离,结果如图2所示,Peak 4为活性峰。图1Aspergillus niger An76铁载体CM Fast Flow离子交换层析图谱图2经离子交换层析所得活性样品TSK-GEL G2500PW分离图谱
采用Arnow法和Csaky法对An76铁载体进行检测,结果显示它既不是儿茶酚盐也不是氧肟酸盐。An76铁载体与铜离子形成的络合物在250.50 nm有紫外吸收,具有羟基羧酸盐型铁载体的性质,为一种新的铁载体。考马斯亮蓝染色、茚三酮试验和双缩脲试验,以及氨基酸组成分析结果显示它不含氨基酸成分。同时试验中我们发现An76铁载体具有高的极性、高的热稳定性。
铁还原试验表明An76铁载体具有还原Fe3+能力,可以将Fe3+还原成溶解性、生物可利用性更高Fe2+,这为满足A. niger An76在低铁的限制性环境下生长代谢对铁的需求提供了有力的保障。
通过硫代巴比妥酸(TBA)法和电子自旋共振(ESR)谱测定,发现An76铁载体具有显著的羟基自由基清除活性。如图3所示,DMPO-HO信号随着An76铁载体浓度的增加而减弱。这为发展新的抗氧化剂、自由基清除剂提供了新的选择。图3Aspergillus niger An76铁载体羟基自由基清除活性电子自旋共振检测结果
通过对32株G+/G-细菌,包括植物病原菌、动物病原菌和人类病原菌的研究,发现An76铁载体具有广谱的细菌生长抑制活性,其抑菌活性随铁载体浓度的增加而增强,并随时间的延长成指数性增加。这为发展新的抗菌药物提供了新的选择。酸性pH条件是它发挥抑菌活性所必须依赖的。
采用Spline-Numerical-Gaussian (SNG)方法,对两株植物病原细菌Pseudomonas solanacearum和Ralstonia solanacearum在分批培养条件下的生长阶段进行划分。研究了An76铁载体在不同浓度和不同处理时间下,对处于不同生理时期的菌群存活率的影响。提出了采用二维等高线图和三维图谱来对试验数据进行分析的新方法。常规的动力学表征方法对不同药物浓度、不同处理时间条件下的结果的动态过程难以表征。利用二维、三维图形就可以把这一复杂的动态过程表征出来。它所显示的虽然仅是相对值,但对反应趋势、范围都可以清楚的显示出来,而且由三维图的曲线下总面积AUC (Area Under Curve)还可以做定量估算,而这些信息都是现在已有方法不能提供的,也表明了现在通用的药效检测方法存在的问题。2D/3D作图法对应用研究中分析药物抑菌/杀菌机理和确定用药方式等都会有重要的意义。图4、5、6分别用常规动力学曲线、二维和三维图谱显示了不同浓度An76铁载体对处于4个不同生理时期的P. solanacearum的抑制作用结果。图4铁载体浓度和菌群生长期对Pseudomonas solanacearum生长的影响(以CFU%表示)图5铁载体浓度和菌群生长期对Pseudomonas solanacearum生长的影响(以CFU%二维等高线图表示)图6铁载体浓度和菌群生长期对Pseudomonas solanacearum生长的影响(以CFU%三维图表示)
二、细菌对Fe3+的吸附还原作用,以及Fe3+对细菌生长的抑制作用。
通过对20株G+/G-细菌,包括植物病原菌和动物病原菌的研究,发现高浓度的Fe3+对细菌具有明显的抑制作用,这是一种普适性的抑菌能力。从中挑选出4株典型的细菌,2株G-细菌Escherichia coli CVCC 249和Ralstonia solanacearum和2株G+细菌Staphyloccocus aureus ATCC 25923和Bacillus subtilis,研究了它们对Fe3+的吸附还原作用,以及Fe3+对它们生长的抑制效应,初步分析了Fe3+的抑菌机理。
研究发现细菌菌体对Fe3+有明显的吸附作用,同时可以迅速的将Fe3+还原为Fe2+。高浓度Fe3+对细菌具有显著的抑制作用,并且抑制效果与Fe3+浓度成正相关。4株细菌对Fe3+的敏感性不同,但抑菌动力学曲线相同,表明Fe3+对细菌的抑制作用是一非选择性的作用机制。铁载体可以和Fe3+形成络合物,对Fe3+的抑菌活性有不同的影响,acetohydroxamic acid和2,3-dihydroxybenzoic acid与Fe3+形成的络合物可以催化羟基自由基的产生,增加Fe3+的抑菌作用。A. niger An76铁载体由于它具有羟基自由基清除活性,可以降低Fe3+的抑菌作用。推测Fe3。对细菌的抑制作用是通过羟基自由基的形成而起作用的。这为利用铁和铁载体生产新型抗菌剂提供了线索。
Iron is a nutritionally essential trace element. It has certain important functions in the metabolic processes of aerobic organisms, such as photosynthesis, respiration, oxygen transport, gene regulation and DNA biosynthesis. Under physiological conditions, iron mainly exists in two readily reversible redox states, the reduced Fe2+ form and the oxidized Fe3+ form. Although abundant in nature, iron tends to form highly insoluble hydroxides in the aerobic neutral pH environment. The concentration of free ferric ion in solution at biological pH is probably not greater than 10-18 M, a concentration too low to allow growth by aerobic and facultative anaerobic microorganisms. Microorganisms have evolved a range of strategies to acquire iron. The major strategies include production and utilization of siderophores, utilization of host iron proteins such as transferrin, and lactoferrin, and reduction of Fe3+ to Fe2+ with subsequent transport of Fe2+. Under such iron-limiting conditions, microorganisms synthesize and excrete a variety of high-affinity, low molecular weight (<1,000 Da), ferric-chelating compounds called siderophores, which specifically solubilize ferric iron in an extracellular aqueous environment and transport it into the cells. In general, siderophores can be divided into three major categories based on their chemical structure:catecholates, hydroxamates and carboxylates. Most fungal siderophores are hydroxamates, however zygomycetes can produce carboxylates (e.g., rhizoferrin produced by various Mucorales). Siderophores have often been suggested to play a role in pathogenic virulence by facilitating growth under iron-limiting conditions, because the acquisition of iron is a key step in the infection process. Moreover, siderophores are known to play a role in the biological control of pathogenic microorganisms by chelating iron, thereby inhibiting their growth or metabolic activity. Biosynthesis and uptake of siderophores represent possible targets for antifungal chemotherapy, because human cells do not have these biochemical pathways.
On the other hand, iron is a devastating metal. Fe2+ reacts with H2O2 to form a hydroxyl radical. HO·is known to be a highly reactive, indiscriminate oxidizing agent, which can damage proteins and nuclear acids. Thus, aerobic and facultative anaerobic organisms have a dilemma:they need a scavenging system that is effective in accumulating iron, without allowing too much to accumulate. Much attention has been given to the response of bacteria to iron-limited conditions, but not to how the bacteria respond to adequate or excess iron supply. The growth of bacteria usually requires 10-6 M of iron, and the proper amount of iron can stimulate the growth of bacteria, but under excessive concentrations, the microorganism's growth will be inhibited.
It is contribute to illustrate the way and mechanism of microbial iron metabolism that investigation of the effects of iron and siderophore on bacterial growth, which has the important application value to the medicine, industry, agriculture, and environment.
In this paper, we investigated siderophore production from 27 filamentous fungal strains, and the effects of ferric ion concentration on siderophore production. Selected Aspergillus niger An76 strain as the production strain, the siderophores produced by An76 were isolated and purified, and identified the physical and chemical properties of An76 siderophore. The antibacterial activities of An76 siderophore were conducted a more in-depth study. At the same time, the antibacterial effects of Fe3+ were investigated and we conducted a preliminary analysis of the inhibition mechanism of Fe3+.
The paper has obtained following innovative results:
1. Screened a high production siderophore strain, Aspergillus niger An76 which could still synthesize and excrete siderophores even at 1 mM FeCl3. The structural characteristics of An76 siderophore differed from typical siderophores and no amino acids or peptide bonds were detected. It represents a new type of siderophore structure. It has remarkable scavenging activity of hydroxyl free radicals in vitro. The An76 siderophore had broad-spectrum antibacterial activity, and may therefore provide more choices for biological control agents against pathogens. Using 2D/3D mapping method to character the experimental data can be used to study the mechanism of antibacterial drugs.
2. All tested Gram-negative and Gram-positive bacteria could absorb Fe3+ and reduce Fe3+ to Fe2+ at the same time. High concentrations of ferric iron had significant inhibitory effects on the bacterial growth, which were directly proportional to the concentrations of Fe3+. It presumed that the inhibitory effect of Fe3+ acts through the formation of hydroxyl free radicals which have a strong non-selective sterilization effect on the bacteria. The various siderophores have different effects on the Fe3+ bacteriostasis activities. The information should provide knowledge towards making a disinfectant using iron chelators and iron.
Screening Siderophore Production Strains, Isolation and Purification, Structural Characteristics, and Antibacterial Activities of An76 Siderophore
Among 27 filamentous fungi, a strain A. niger An76 gave the highest siderophore yield even when cultured on natural medium or minimal medium containing 1 mM Fe3+. Whereas for most other strains, the capacities of siderophore production was repressed as the concentrations of Fe3+ was higher than 20μM. Lower ferric iron concentrations were more conducive to siderophore synthesis for those strains.
The siderophore which produced by A. niger An76 under the iron limiting condition, were separated through the massive exploring experiments, determined one project which can partially purify the An76 siderophore. It might prepare enough quantity of sample for study its physical and chemical properties and antibacterial activities. The lead acetate precipitation method was used for preliminary isolation of An76 siderophore. The siderophore was partially purified by a series of chromatography steps. The ion-exchange chromatography was conducted using a CM-Sepharose Fast Flow column. Peak 1 showed in Figure 1 gave a CAS-positive reaction.
After ion-exchange chromatography, the active samples were purified further using TSK-GEL G2500PW column, the results shown in Figure 2, Peak 4 was active peak. Figure 1 CM-Sepharose Fast Flow chromatogram of Aspergillus niger An76 siderophore. Figure 2 TSK-GEL G2500PW chromatogram of the Aspergillus niger An76 siderophore.
The structure of An76 siderophore was determined by the Arnow test and the modified Csaky method. The results showed that it was neither a catecholate nor a hydroxamate. The An76 siderophore-Cu2+ complex had an ultraviolet absorption peak in 250.50 nn, the nature of hydroxyl carboxylate. Coomassie blue staining, ninhydrin test, and a biuret protein assay showed that there was no amino acid or peptide bond structure in An76 siderophore. Amino acid composition analysis also indicated that An76 siderophore did not contain amino acids in its structure. It may therefore represent a new type of siderophore structure. In the course of the study we found that An76 siderophore was a strong polar compound, which is further purified and identified.
The An76 siderophore has reducing ability. It can reduce Fe3+ to Fe2+ which has the higher soluble and bio-availability.
Thiobarbituric acid (TBA) assay and Electron-Spin Resonance (ESR) trapping studies showed that An76 siderophore had remarkable scavenging activity of hydroxyl free radicals in vitro. As showed in Figure 3, the DMPO-HO·signals decreased with increasing siderophore concentration, which confirmed the HO·scavenging activities of An76 siderophore. It provides a new choice for the development of new anti-oxidant and free radical scavenger.
Figure 3 ESR spectra of DMPO-OH·adducts formed by Fenton reaction with different concentrations of An76 siderophore.
The data acquisition parameters were:modulation frequency,25 KHz; modulation amplitude, 1 G; microwave power,10 mW; center field,3470 G; sweeping time,40 s.
The antibiotic activities of siderophores produced by An76 were tested against 32 bacterial strains, including plant, animal, and human pathogens. The results showed that the An76 siderophore had broad-spectrum antibacterial activity. Therefore, it may provide more choices for biological control agents against pathogens.
Previous studies in our lab demonstrated that the antibacterial characteristics for a bacterial species not only depend on the concentration of antibiotics and the treatment time, but also depend on the growth phase of the bacterial population. Using Spline-Numerical-Gaussian (SNG) method, the growth curves under batch culture conditions of two plant pathogenic bacteria, Pseudomonas solanacearum and Ralstonia solanacearum could be divided into four phases. Using these four different growth phases culture as samples, which were treated with A. niger An76 siderophore under different concentrations and different treatment time conditions. Those combined effects on bacterial survival rates were so complex and could not be clearly observed using general assay method, such as MIC and MBC methods, this also indicates the existence problems of the present common methods of drug efficacy examination.
As demonstrate in this paper, this complex dynamic process could be characterized as using 2-Dimensional and 3- Dimensional graphics. Although it was only shown the relative value, but both of the reaction tendency and the scope could be demonstrated clearly. Moreover, the total area AUC (Area Under Curve) of three dimensional graph could be used as a quantitatively estimate. The results of the present study provide evidences to support the isograms method as a useful tool in understanding of the reaction mechanisms of siderophore. Figure 4 Effects of Aspergillus niger An76 siderophore concentrations and treatment time on the antibiotic resistance for four growth phases of Pseudomonas solanacearum. Expressed as CFU.
Figure 5 Effects of Aspergillus niger An76 siderophore concentrations and treatment time on the antibiotic resistance for four growth phases of Pseudomonas solanacearum. Expressed as CFU and visualized by contour plots.
Figure 6 Effects of Aspergillus niger An76 siderophore concentrations and treatment time on the antibiotic resistance for four growth phases of Pseudomonas solanacearum. Expressed as CFU and clearly visualized by 3D graph
Figure 4,5 and 6 showed that the results of residual CFU, which the different growth phases population of P. solanacearum were treated by different concentrations of An76 siderophore and a series different of treatment time, characterized by three different methods respectively.
Bacterial Absorption and Reduction Capacities to Fe3+, and Antibacterial Activities of Fe3+
The antibiotic activities of Fe3+ were tested against 20 bacterial strains, including plant and animal pathogens. The results showed that high concentrations of Fe3+ had significant antibacterial activity, which is a kind of universality bacteriostasis ability.
From the investigation of two Gram-negative (Escherichia coli CVCC 249 and Ralstonia solanacearum) and two Gram-positive (Staphylococcus aureus ATCC 25923 and Bacillus subtilis) bacteria, it was determined that the bacteria could adsorb Fe3+ and reduce Fe3+ to Fe2+ at the same time. High concentrations of iron had. significant inhibitory effects on the bacterial growth, which were directly proportional to the concentrations of Fe3+. It presumed that the inhibitory effect of Fe3+ acts through the formation of hydroxyl free radicals which have a strong non-selective sterilization effect on the bacteria. The sensitivities of the various bacteria to the ferric iron were different, but the bacteriostasis dynamics curves were the same. This indicates that the antibacterial effect of Fe3+ is likely a non-selective mechanism.
Under differing pH conditions, the amount of iron hydrolysis varies, causing the free Fe3+ concentration to change, thus influencing the bacteriostatic effect of the iron.
The various siderophores have different effects on the Fe3+ bacteriostasis activities. The information should provide knowledge towards making a disinfectant using iron chelators.
除此之外,Fe2+还可以与超氧阴离子、过氧化氢等氧化因子反应生成反应性更强的羟基自由基。羟基自由基是一种极强的氧化剂,它可以无选择性的破坏几乎所有的生物分子,包括糖、脂、DNA链等,对细胞具有极大的损害效应。因此,好氧和兼性厌氧微生物面临一种两难的境地：一方面微生物细胞必须利用有效的铁吸收机制来从环境中吸收铁以供其生长代谢的需要,另一方面又要严格控制细胞内游离铁的浓度,使细胞内催化性铁的浓度维持在无害的水平。对铁和铁载体的研究有助于阐明微生物铁代谢的途径、机理等问题,对医学、工业、农业和环境都有重要的应用价值。
论文通过对27株丝状真菌铁载体合成情况的比较研究,从中筛选到Aspergillus niger An76菌株作为生产菌株。对其在低铁的培养条件下合成的铁载体进行分离纯化,理化性质测定,并对该铁载体的细菌抑制活性进行了较深入的研究。同时对高浓度的Fe3+的抑菌活性和作用机理进行了探索研究。
论文取得了如下创新性的结果：
1.筛选到一株高产铁载体的菌株A. niger An76,其铁载体在浓度高达1 mM Fe3+的培养条件下仍然能够合成,为组成型表达。An76铁载体与典型的氧肟酸盐型和儿茶酚盐型铁载体不同,它具有羟基羧酸盐型铁载体的特性,为一种新的铁载体。它不含氨基酸成分,并且具有羟基自由基清除活性。An76铁载体具有显著的抑菌活性,这为筛选新的抗生素药物提供了新途径。采用2D/3D作图法表征实验数据的方法可用于研究药物的抗菌机理。
2.研究发现供试的G+/G细菌都可以吸附Fe3+,并可以迅速的将Fe3+还原为Fe2+。高浓度的Fe3+对细菌生长有显著的抑制作用,推测Fe3+对细菌的抑制作用是通过Fe2+催化羟基自由基的形成从而抑制细菌生长的。不同的铁载体对Fe3+的抑菌活性具有不同的影响,这为发展新的抗菌剂提供了线索。
一、铁载体生产菌的筛选、铁载体的分离纯化和性质鉴定,以及铁载体的细菌抑制活性。
通过对27株丝状真菌在三种不同培养基条件下的铁载体生产情况进行比较研究,我们发现Aspergillus niger An76铁载体合成能力的表达在浓度高达1 mMFe3+的培养条件下仍然不受抑制,表明它的铁载体合成不是低铁的限制性条件诱导表达的,而是组成型表达。然而其它的大多数供试真菌的铁载体合成在Fe3+浓度达到20μM时就会受到抑制。低浓度的铁更有利于铁载体的合成。
对A. niger An76在低铁的限制性条件下培养所产生的铁载体进行分离纯化,通过大量的探索性试验,确定了一可以将其部分纯化的分离方案。采用该方案可以在较短时间内制备足够量的样品来研究其理化性质和细菌抑制活性。通过超滤、真空冷冻干燥得到粗样品,采用醋酸铅沉淀法进行初步的分离,经过CMFast Flow离子交换层析得到较好的分离效果,如图1所示,Peak 1为铁载体活性峰。
经过离子交换层析得到的活性样品进一步采用TSK-GEL G2500PW液相色谱柱进行分离,结果如图2所示,Peak 4为活性峰。图1Aspergillus niger An76铁载体CM Fast Flow离子交换层析图谱图2经离子交换层析所得活性样品TSK-GEL G2500PW分离图谱
采用Arnow法和Csaky法对An76铁载体进行检测,结果显示它既不是儿茶酚盐也不是氧肟酸盐。An76铁载体与铜离子形成的络合物在250.50 nm有紫外吸收,具有羟基羧酸盐型铁载体的性质,为一种新的铁载体。考马斯亮蓝染色、茚三酮试验和双缩脲试验,以及氨基酸组成分析结果显示它不含氨基酸成分。同时试验中我们发现An76铁载体具有高的极性、高的热稳定性。
铁还原试验表明An76铁载体具有还原Fe3+能力,可以将Fe3+还原成溶解性、生物可利用性更高Fe2+,这为满足A. niger An76在低铁的限制性环境下生长代谢对铁的需求提供了有力的保障。
通过硫代巴比妥酸(TBA)法和电子自旋共振(ESR)谱测定,发现An76铁载体具有显著的羟基自由基清除活性。如图3所示,DMPO-HO信号随着An76铁载体浓度的增加而减弱。这为发展新的抗氧化剂、自由基清除剂提供了新的选择。图3Aspergillus niger An76铁载体羟基自由基清除活性电子自旋共振检测结果
通过对32株G+/G-细菌,包括植物病原菌、动物病原菌和人类病原菌的研究,发现An76铁载体具有广谱的细菌生长抑制活性,其抑菌活性随铁载体浓度的增加而增强,并随时间的延长成指数性增加。这为发展新的抗菌药物提供了新的选择。酸性pH条件是它发挥抑菌活性所必须依赖的。
采用Spline-Numerical-Gaussian (SNG)方法,对两株植物病原细菌Pseudomonas solanacearum和Ralstonia solanacearum在分批培养条件下的生长阶段进行划分。研究了An76铁载体在不同浓度和不同处理时间下,对处于不同生理时期的菌群存活率的影响。提出了采用二维等高线图和三维图谱来对试验数据进行分析的新方法。常规的动力学表征方法对不同药物浓度、不同处理时间条件下的结果的动态过程难以表征。利用二维、三维图形就可以把这一复杂的动态过程表征出来。它所显示的虽然仅是相对值,但对反应趋势、范围都可以清楚的显示出来,而且由三维图的曲线下总面积AUC (Area Under Curve)还可以做定量估算,而这些信息都是现在已有方法不能提供的,也表明了现在通用的药效检测方法存在的问题。2D/3D作图法对应用研究中分析药物抑菌/杀菌机理和确定用药方式等都会有重要的意义。图4、5、6分别用常规动力学曲线、二维和三维图谱显示了不同浓度An76铁载体对处于4个不同生理时期的P. solanacearum的抑制作用结果。图4铁载体浓度和菌群生长期对Pseudomonas solanacearum生长的影响(以CFU%表示)图5铁载体浓度和菌群生长期对Pseudomonas solanacearum生长的影响(以CFU%二维等高线图表示)图6铁载体浓度和菌群生长期对Pseudomonas solanacearum生长的影响(以CFU%三维图表示)
二、细菌对Fe3+的吸附还原作用,以及Fe3+对细菌生长的抑制作用。
通过对20株G+/G-细菌,包括植物病原菌和动物病原菌的研究,发现高浓度的Fe3+对细菌具有明显的抑制作用,这是一种普适性的抑菌能力。从中挑选出4株典型的细菌,2株G-细菌Escherichia coli CVCC 249和Ralstonia solanacearum和2株G+细菌Staphyloccocus aureus ATCC 25923和Bacillus subtilis,研究了它们对Fe3+的吸附还原作用,以及Fe3+对它们生长的抑制效应,初步分析了Fe3+的抑菌机理。
研究发现细菌菌体对Fe3+有明显的吸附作用,同时可以迅速的将Fe3+还原为Fe2+。高浓度Fe3+对细菌具有显著的抑制作用,并且抑制效果与Fe3+浓度成正相关。4株细菌对Fe3+的敏感性不同,但抑菌动力学曲线相同,表明Fe3+对细菌的抑制作用是一非选择性的作用机制。铁载体可以和Fe3+形成络合物,对Fe3+的抑菌活性有不同的影响,acetohydroxamic acid和2,3-dihydroxybenzoic acid与Fe3+形成的络合物可以催化羟基自由基的产生,增加Fe3+的抑菌作用。A. niger An76铁载体由于它具有羟基自由基清除活性,可以降低Fe3+的抑菌作用。推测Fe3。对细菌的抑制作用是通过羟基自由基的形成而起作用的。这为利用铁和铁载体生产新型抗菌剂提供了线索。
Iron is a nutritionally essential trace element. It has certain important functions in the metabolic processes of aerobic organisms, such as photosynthesis, respiration, oxygen transport, gene regulation and DNA biosynthesis. Under physiological conditions, iron mainly exists in two readily reversible redox states, the reduced Fe2+ form and the oxidized Fe3+ form. Although abundant in nature, iron tends to form highly insoluble hydroxides in the aerobic neutral pH environment. The concentration of free ferric ion in solution at biological pH is probably not greater than 10-18 M, a concentration too low to allow growth by aerobic and facultative anaerobic microorganisms. Microorganisms have evolved a range of strategies to acquire iron. The major strategies include production and utilization of siderophores, utilization of host iron proteins such as transferrin, and lactoferrin, and reduction of Fe3+ to Fe2+ with subsequent transport of Fe2+. Under such iron-limiting conditions, microorganisms synthesize and excrete a variety of high-affinity, low molecular weight (<1,000 Da), ferric-chelating compounds called siderophores, which specifically solubilize ferric iron in an extracellular aqueous environment and transport it into the cells. In general, siderophores can be divided into three major categories based on their chemical structure:catecholates, hydroxamates and carboxylates. Most fungal siderophores are hydroxamates, however zygomycetes can produce carboxylates (e.g., rhizoferrin produced by various Mucorales). Siderophores have often been suggested to play a role in pathogenic virulence by facilitating growth under iron-limiting conditions, because the acquisition of iron is a key step in the infection process. Moreover, siderophores are known to play a role in the biological control of pathogenic microorganisms by chelating iron, thereby inhibiting their growth or metabolic activity. Biosynthesis and uptake of siderophores represent possible targets for antifungal chemotherapy, because human cells do not have these biochemical pathways.
On the other hand, iron is a devastating metal. Fe2+ reacts with H2O2 to form a hydroxyl radical. HO·is known to be a highly reactive, indiscriminate oxidizing agent, which can damage proteins and nuclear acids. Thus, aerobic and facultative anaerobic organisms have a dilemma:they need a scavenging system that is effective in accumulating iron, without allowing too much to accumulate. Much attention has been given to the response of bacteria to iron-limited conditions, but not to how the bacteria respond to adequate or excess iron supply. The growth of bacteria usually requires 10-6 M of iron, and the proper amount of iron can stimulate the growth of bacteria, but under excessive concentrations, the microorganism's growth will be inhibited.
It is contribute to illustrate the way and mechanism of microbial iron metabolism that investigation of the effects of iron and siderophore on bacterial growth, which has the important application value to the medicine, industry, agriculture, and environment.
In this paper, we investigated siderophore production from 27 filamentous fungal strains, and the effects of ferric ion concentration on siderophore production. Selected Aspergillus niger An76 strain as the production strain, the siderophores produced by An76 were isolated and purified, and identified the physical and chemical properties of An76 siderophore. The antibacterial activities of An76 siderophore were conducted a more in-depth study. At the same time, the antibacterial effects of Fe3+ were investigated and we conducted a preliminary analysis of the inhibition mechanism of Fe3+.
The paper has obtained following innovative results:
1. Screened a high production siderophore strain, Aspergillus niger An76 which could still synthesize and excrete siderophores even at 1 mM FeCl3. The structural characteristics of An76 siderophore differed from typical siderophores and no amino acids or peptide bonds were detected. It represents a new type of siderophore structure. It has remarkable scavenging activity of hydroxyl free radicals in vitro. The An76 siderophore had broad-spectrum antibacterial activity, and may therefore provide more choices for biological control agents against pathogens. Using 2D/3D mapping method to character the experimental data can be used to study the mechanism of antibacterial drugs.
2. All tested Gram-negative and Gram-positive bacteria could absorb Fe3+ and reduce Fe3+ to Fe2+ at the same time. High concentrations of ferric iron had significant inhibitory effects on the bacterial growth, which were directly proportional to the concentrations of Fe3+. It presumed that the inhibitory effect of Fe3+ acts through the formation of hydroxyl free radicals which have a strong non-selective sterilization effect on the bacteria. The various siderophores have different effects on the Fe3+ bacteriostasis activities. The information should provide knowledge towards making a disinfectant using iron chelators and iron.
Screening Siderophore Production Strains, Isolation and Purification, Structural Characteristics, and Antibacterial Activities of An76 Siderophore
Among 27 filamentous fungi, a strain A. niger An76 gave the highest siderophore yield even when cultured on natural medium or minimal medium containing 1 mM Fe3+. Whereas for most other strains, the capacities of siderophore production was repressed as the concentrations of Fe3+ was higher than 20μM. Lower ferric iron concentrations were more conducive to siderophore synthesis for those strains.
The siderophore which produced by A. niger An76 under the iron limiting condition, were separated through the massive exploring experiments, determined one project which can partially purify the An76 siderophore. It might prepare enough quantity of sample for study its physical and chemical properties and antibacterial activities. The lead acetate precipitation method was used for preliminary isolation of An76 siderophore. The siderophore was partially purified by a series of chromatography steps. The ion-exchange chromatography was conducted using a CM-Sepharose Fast Flow column. Peak 1 showed in Figure 1 gave a CAS-positive reaction.
After ion-exchange chromatography, the active samples were purified further using TSK-GEL G2500PW column, the results shown in Figure 2, Peak 4 was active peak. Figure 1 CM-Sepharose Fast Flow chromatogram of Aspergillus niger An76 siderophore. Figure 2 TSK-GEL G2500PW chromatogram of the Aspergillus niger An76 siderophore.
The structure of An76 siderophore was determined by the Arnow test and the modified Csaky method. The results showed that it was neither a catecholate nor a hydroxamate. The An76 siderophore-Cu2+ complex had an ultraviolet absorption peak in 250.50 nn, the nature of hydroxyl carboxylate. Coomassie blue staining, ninhydrin test, and a biuret protein assay showed that there was no amino acid or peptide bond structure in An76 siderophore. Amino acid composition analysis also indicated that An76 siderophore did not contain amino acids in its structure. It may therefore represent a new type of siderophore structure. In the course of the study we found that An76 siderophore was a strong polar compound, which is further purified and identified.
The An76 siderophore has reducing ability. It can reduce Fe3+ to Fe2+ which has the higher soluble and bio-availability.
Thiobarbituric acid (TBA) assay and Electron-Spin Resonance (ESR) trapping studies showed that An76 siderophore had remarkable scavenging activity of hydroxyl free radicals in vitro. As showed in Figure 3, the DMPO-HO·signals decreased with increasing siderophore concentration, which confirmed the HO·scavenging activities of An76 siderophore. It provides a new choice for the development of new anti-oxidant and free radical scavenger.
Figure 3 ESR spectra of DMPO-OH·adducts formed by Fenton reaction with different concentrations of An76 siderophore.
The data acquisition parameters were:modulation frequency,25 KHz; modulation amplitude, 1 G; microwave power,10 mW; center field,3470 G; sweeping time,40 s.
The antibiotic activities of siderophores produced by An76 were tested against 32 bacterial strains, including plant, animal, and human pathogens. The results showed that the An76 siderophore had broad-spectrum antibacterial activity. Therefore, it may provide more choices for biological control agents against pathogens.
Previous studies in our lab demonstrated that the antibacterial characteristics for a bacterial species not only depend on the concentration of antibiotics and the treatment time, but also depend on the growth phase of the bacterial population. Using Spline-Numerical-Gaussian (SNG) method, the growth curves under batch culture conditions of two plant pathogenic bacteria, Pseudomonas solanacearum and Ralstonia solanacearum could be divided into four phases. Using these four different growth phases culture as samples, which were treated with A. niger An76 siderophore under different concentrations and different treatment time conditions. Those combined effects on bacterial survival rates were so complex and could not be clearly observed using general assay method, such as MIC and MBC methods, this also indicates the existence problems of the present common methods of drug efficacy examination.
As demonstrate in this paper, this complex dynamic process could be characterized as using 2-Dimensional and 3- Dimensional graphics. Although it was only shown the relative value, but both of the reaction tendency and the scope could be demonstrated clearly. Moreover, the total area AUC (Area Under Curve) of three dimensional graph could be used as a quantitatively estimate. The results of the present study provide evidences to support the isograms method as a useful tool in understanding of the reaction mechanisms of siderophore. Figure 4 Effects of Aspergillus niger An76 siderophore concentrations and treatment time on the antibiotic resistance for four growth phases of Pseudomonas solanacearum. Expressed as CFU.
Figure 5 Effects of Aspergillus niger An76 siderophore concentrations and treatment time on the antibiotic resistance for four growth phases of Pseudomonas solanacearum. Expressed as CFU and visualized by contour plots.
Figure 6 Effects of Aspergillus niger An76 siderophore concentrations and treatment time on the antibiotic resistance for four growth phases of Pseudomonas solanacearum. Expressed as CFU and clearly visualized by 3D graph
Figure 4,5 and 6 showed that the results of residual CFU, which the different growth phases population of P. solanacearum were treated by different concentrations of An76 siderophore and a series different of treatment time, characterized by three different methods respectively.
Bacterial Absorption and Reduction Capacities to Fe3+, and Antibacterial Activities of Fe3+
The antibiotic activities of Fe3+ were tested against 20 bacterial strains, including plant and animal pathogens. The results showed that high concentrations of Fe3+ had significant antibacterial activity, which is a kind of universality bacteriostasis ability.
From the investigation of two Gram-negative (Escherichia coli CVCC 249 and Ralstonia solanacearum) and two Gram-positive (Staphylococcus aureus ATCC 25923 and Bacillus subtilis) bacteria, it was determined that the bacteria could adsorb Fe3+ and reduce Fe3+ to Fe2+ at the same time. High concentrations of iron had. significant inhibitory effects on the bacterial growth, which were directly proportional to the concentrations of Fe3+. It presumed that the inhibitory effect of Fe3+ acts through the formation of hydroxyl free radicals which have a strong non-selective sterilization effect on the bacteria. The sensitivities of the various bacteria to the ferric iron were different, but the bacteriostasis dynamics curves were the same. This indicates that the antibacterial effect of Fe3+ is likely a non-selective mechanism.
Under differing pH conditions, the amount of iron hydrolysis varies, causing the free Fe3+ concentration to change, thus influencing the bacteriostatic effect of the iron.
The various siderophores have different effects on the Fe3+ bacteriostasis activities. The information should provide knowledge towards making a disinfectant using iron chelators.
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