Effects Of Siderophore And Ferric Ion On Bacterial Growth Process

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 SiderophoreAmong 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.