Investigation On The Siderophore From A Probiotics Bacterial Strain A18 And Cloning, Sequencing And Expression Of The Gene Of Ferric-uptake Regulator

A high siderophore-producing mutant strain named J61321 was obtained from the marine probiotic bacterial strain A18 (Alteromonas aurantia) by a series of chemical and physical mutagenesis with EMS, NaNO₂, EB and UV. A pathogenic strain W-1 (Vibrio anguillarum) was selected as an indicator bacterium to test the inhibitory activities of extracellular products of mutant J61321 and the parent strain A18. The results show that the growth of strain W-l was inhibited by the sterile supernatant of strain A18 and mutant J61321. Meanwhile, the results reveal that antibacterial activities of extracellular products from mutant J61321 were higher than those from strain A18. After the extracellular products were treated at 100℃ for10min to 50min, they still had inhibitory activity. The siderophores from strain A18, mutant J61321 and W-l were detected by non-specific assay (CAS method) and specific methods (Arnow assay for catechol-type and Csaky method for hydroxamate-type siderophore). The results indicate that the extracellular products of three strains of A18, J61321 and W-l showed a positive response to the CAS assay, while strain W-l produced catechol-type siderophore and both train A18 and mutant J61321 produced. Hydroxamate-type siderophore. The siderophores produced by mutant J61321 were more than those by strain A18. In order to determine the effect of synthetic iron chelator on the growth of strain A18 and mutant J61321, iron-deficient media supplemented with different concentration of 2,2-dipyridyl were used. The results show that both of them did not grow at 200μmol/L of 2,2-dipyridyl, however, mutant J61321 grew well at 100μmol/L of 2,2-dipyridyl while the strain A18 did not, suggesting that mutant J61321 could secrete more siderophore into media to chelate iron for growth in the iron-deficient media than its parent strain. Siderophores from the marine probiotic bacterial strainA18 could effectively inhibit growth of the pathogenic Vibrio, indicating that the chelating activity of hydroxamate-type siderophore for iron was higher than that of catechol-type siderophore.The optimal medium components (carbon and nitrogen sources) and growth conditions (initial pH, temperature and dissolved oxygen) for siderophore production by mutant J61321 were studied using "one-variable-at-a-time" method. The results show that the optimal medium components for the highest siderophore yield by mutant J61321 were yeast extraction 0.1%(W/V), peptone 0.2%(W/V), FePO4 0.001%(W/V) and the optimal growth conditions for the highest siderophore yield by mutant J61321 were rotation speed 180rpm, initial pH7.5, temperature 28°C. Under these conditions, the siderophore in the liquid medium reached the highest yields after 20 hours of fermentation. The biomass, siderophore yields and antibacterial activities of the mutant J61321 were compared with those of the parent A18. The results show that the biomass of the two strains has no difference (P>0.05) during the growth, whereas the siderophore yields and antibacterial activities from mutant J61321 were higher those from the parent strain (PO.05).In this study, two purification methods were used to get crude siderophore from supernatants of culture of mutant J61321 (Alteromona aurantid). One of them was that crude siderophore was extracted from the culture supernatants by using organic solvents. The other one was that the crude siderophore was eluted from resin saturated with supernatant including siderophoes. Pooled fractions containing the siderophores were partially purified by reverse phase high pressure liquid chromatography (RP-HPLC) using a linear gradient from 0 to 100% methanoi in distilled water. The fractions with both the antibacterial activity and the positive CAS reaction were collected. The siderophores with inhibitory activity were further purified by RP-HPLC / ESI-MS (electronspray ionization mass spectrometry) using photodiode array detection. The chromatographic conditions were as follows: a C18 intersil DDS-3 column (150mm X4.6mm i.d., 5[im), a mobile phase composed of water (containing 0.5%(v/v) formic acid) and acetonitrile, a flow rate of 0.8 ml/min and UV absorbance detection at 235nm. The eluted siderophores samples byRP-HPLC were immediately subjected to ESI-MS spectrometry analysis using atmosphere pressure ionization-electrospray interface and positive scan mode was set for MS data. The results show that the compounds with antibacterial activity appeared in two peaks. According to their molecular ion peak, they were identified to be ferrichrome A and ferrioxamine D, respectively 0Alteromonas aurantia A18 total DNA was used as a template DNA. The bidirectional degenerated oligonucleotide primers were designed and synthesized based on the highly identity sequences of ferric uptake regulator (fur) genes in gram-negative bacteria. The complete fur gene from A. aurantia A18 was amplified by PCR. The fur gene was gel purified, cloned into a pUCm-T cloning vector, transformed into E. coli (JM109). Nucleotide sequence of the cloned gene was analyzed using an automatic ABI prism 377sequencer in the Bioasia Company (shanghai) and the gene sequence analysis was performed using BLAST search software. The results indicate that the Fur protein of A. aurantia A18 was 98% identical to the Alteromonas sp. 0-7 Fur protein and 70% identical to that of the E.coli. The putative fur open reading frame codes for 148 amino acids representing a protein of 16.7 kDa. The structure of Fur protein of A. aurantia A18 modeled on line using SWISS-MODEL was similar to that of Fur proteins reported previously. Non-fused expression vector pQE-fur was constructed to confirm the function of the cloned fur gene. The plasmid pQE-fur was transformed into Escherichia coli fur mutant {E.coli strain QC1732) and the transformants with pQE-fur were selected on Luria- Bertani (LB) agar containing ampicillin and kanamycin (50 mg/ml). QC1732 and transformants bearing pQEAfur (induced by IPTG) were inoculated into LB liquid media with 50mg/ml of ampicillin, 50mg/ml of kanamycin and 150mmol/L of 2,2-dipyridyl or no, and cultivated aerobically over night at 37°C. Transformants carrying pQE-fur (not induced by IPTG) were cultivated in both types of LB medium as a negative control. We found that siderophore yields of the transformants bearing pQE-fur (induced by IPTG) were much lower than those of the other two strains. The results show that some enzymes responsible for synthesis and secretion of siderophores were repressed by Fur protein, encoded by fur gene from A. aurantia Al 8.