| The bacteria of Pseudomonas genus are capable of colonizing a wide range of ecological niches. The adaptability of these bacteria is mainly due to their highly elaborate iron-uptake systems. The fluorescent Pseudomonas species are able to produce yellow-greenishly fluorescent peptidic siderophore named pyoverdins, which are the primary iron uptake pathways in them. Besides, other iron-scavenging agents termed as secondary siderophores, have been isolated from certain fluorescent Pseudomonas species. Secondary siderophores typically have a lower affinity for iron than pyoverdins. In addition to iron-scavenging, many secondary siderophores possess properties like forming complexes with other metal ions or antimicrobial activity.P. donghuensis was a species isolated from the water samples taken from Donghu Lake, was highlighted with its ability of producting of large quantities of iron-chelators in low-iron medium. Compared with two closely related species, P. fluorescens and P. putida, P. donghuensis show remarkable advantage in iron-chelator production in the low-iron medium MKB. The former studies of P. donghuensis led to the fact that this bacterium produced the fluorescent siderophore, pyoverdins, along with a non-fluorescent iron-chelator, the latter one was the dominant contributor to the iron-chelating capacity of the culture supernatant of P. donghuensis. However, little was known about the non-fluorescent iron-chelator. In the present study, further investigation has been made on the non-fluorescent iron-chelator.The isolation of the non-fluorescent iron-chelator. In UV/visible spectra, the culture supernatant of P. donghuensis showed absorptions at around 327 and 394 nm which shifted and changed when the pH was adjusted to 3. When the acidified supernatant was extracted by ethyl acetate, heavily reductions in these absorption maxima were observed, accompanied by significant decrease in the iron-chelating capacity in the aqueous phase. These observations were distinguishing from those of pyoverdins, indicated that the dominant contributor to the iron-chelating capacity of the culture supernatant of P. donghuensis was an ethyl acetate-extractable iron-chelating non-fluorescent iron-chelator. The changes of the non-fluorescent iron-chelator in absorptions and polarity from pH 3 to pH 7 implied that this substance shared some characteristics of of phenolic compounds. The isolation of the non-fluorescent iron-chelator from the culture supernatant turned to the method of the ethyl acetate extraction and the Sephadex LH20 column chromatography separation. The optimization of the isolation process confirmed the method of extractions in two different pHs to remove inpurities, and the usage of ethanol as mobile phase in column chromatograph. Through this process some pale yellow needle-like crystals were obtained from the culture supernatant of P. donghuensis, these were the isolated non-fluorescent iron-chelator samples. High performance liquid chromatography and gas chromatography analyses both indicated the simplicity of these samples, which approved the effectiveness of the isolation method of the non-fluorescent iron-chelator.The identification of the non-fluorescent iron-chelator of P. donghuensis. The electron ionization mass spectra at 70eV and 20eV determined that the molecular weight of the non-fluorescent iron-chelator was 138. The 1H NMR spectra of the samples showed two multiplets at 7.442 and 7.163 ppm, and 13C NMR spectra showed four signals at 170,162,130, and 122 ppm, indicated two inactive hydrogen atoms and four carbon atoms in an aromatic ring, the simplicity of the hydrogen and carbon signals suggested that the molecular was structurally symmetric. The infrared spectrum of the non-fluorescent iron-chelator showed bands at 3237 and 1608 cm-1, indicated the presence of hydroxyl and carbonyl groups in the compound. According to these results the structure of the non-fluorescent iron-chelator was elucidated to be that of 7-hydroxytropolone. Furthermore, elemental analysis result of the isolated samples (C,60.7-61.17%; H,5.017-5.155%; O,29.946-32.236%) was close to the elemental composition of 7-hydroxytropolone (C,60.87%; H,4.348%; O,34.78%). The UV/visible spectra, the electron ionization mass spectra, HPLC analysis, and the 1H NMR and 13C NMR spectra of the non-fluorescent iron-chelator samples were also identical to those of the synthesized 7-hydroxytropolone standard. The physico-chemical characteristics of the non-fluorescent iron-chelator agree with published ones reported for 7-hydroxytropolone as well as its derivative. All these evidences verified that the non-fluorescent iron-chelator was 7-hydroxytropolone.7-hydroxytropolone could form complex with ferric ions. The stoichiometry of 7-hydroxytropolone ferric complex was determined to be 2:1 by the continuous variation method at pH 1.78, and the stability constant of the complex was calculated to be approximately 109. The 7-hydroxytropolone ferric complex in maximum stoichiometry was orange in color, when ferric ions were excessive, purple-complex occurred maybe due to the incomplete coordination. In the extraction experiment of 7-hydroxytropolone and ferric ions mixed solutions, the orange-complex was found to be extractable by ethyl acetate in acid (pH=1.72), while the purple-complex stayed in the aqueous phase, this could be applied to prepare the 7-hydroxytropolone ferric complex or the gallium complex. In electrospray ionization mass spectrum of the prepared 7-hydroxytropolone ferric complex and the gallium complex, the detected ions m/z= 330 and m/z= 343 corresponded to the 2:1 ferric complex and the 2:1 gallium complex respectively, these observations verified that 7-hydroxytropolone formed complex with ferric ions or gallium ions in the ratio of 2:1. The 1H NMR spectrum of the prepared 7-hydroxytropolone gallium complex in CDCI3 showed triplets at 7.19 and 7.39, doublets at 7.57 and 7.66 ppm, indicated that the symmetry of 7-hydroxytropolone molecular was disturbed in the complex, which meant the ligands site in the molecular could be the carbonyl and one of the adjacent hydroxyl groups.7-hydroxytropolone can be utilized by P. donghuensis as a siderophore. Firstly, the production of 7-hydroxytropolone was regulated by P. donghuensis according to concentration of ferrous ions in the medium, the least of which to repress the production to minimum was around 8 ?mol·L-1. Secondly, the deficient strain ApvdA of P. donghuensis could uptake the 7-hydroxytropolone ferric complex, and the uptake of the complex was a bit higher than that of free ferric ion. Thirdly, the inhibited growth of the double-deficient strain m16?pvdA could be partly restored by adding adequate 7-hydroxytropolone in an iron-restricted medium. However, the deficient strain m16 which produced only pyoverdins grew much better than the strain ApvdA produced only 7-hydroxytropolone in the iron-restricted medium, indicated that pyoverdins were functioned as primary siderophores, while 7-hydroxytropolone as a secondary one in P. donghuensis.Both pyoverdins and 7-hydroxytropolone could act as siderophores for P. donghuensis. These two kinds of siderophores were different in stages of production, sensitivities to iron, and affinities to ferric ion. These siderophores in P. donghuensis were presumed to cope with different environmental conditions.7-hydroxytropolone was mainly used in minor iron-limiting conditions, while pyoverdins were used in extremely iron-restricted conditions.In summary, in this study the non-fluorescent iron-chelator produced by P. donghuensis was identified to be 7-hydroxytropolone. The form of the 7-hydroxytropolone ferric complex was analyzed. Moreover, the siderophore function of 7-hydroxytropolone in P. donghuensis was confirmed. The meaning of this study was the revelation of the unreported secondary siderophore function of 7-hydroxytropolone in fluorescent Pseudomonas species, and the analysis of which iron-restricted conditions that 7-hydroxytropolone and pyoverdins P. donghuensis used to cope with. This study provide novel views of multiple siderophores cooperation of Pseudomonas. |
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