DETECTION, PURIFICATION, AND IDENTIFICATION OF SIDEROPHORES PRODUCED BY Pseudomonas fluorescens ISOLATES USING SDS-PAGE AND HPLC.

Three isolates of Pseudomonas fluorescens were isolated from the soil rhizosphere of potato growing fields and identification was confirmed by comparison of the morphological traits and physiological characteristics according to Murray et al., 1984 and Klement et al.,(1990). P. fluorescens isolates were tested to prove their ability in production of siderophores. Chrome Azurol Sulfonate assay (CAS assay) test was used to detect the presence of siderophores. Formation of an orange halo around a well bored into the CAS plate, was taken as an indication for the presence of siderophores. Concerning the optimization of conditions for highest production of siderophores on different media, the results revealed that king’s B medium, exhibited the highest production of sidrophores. Also, data showed that pH 7 and 27 °C were the best in this respect while at pH 6 and 8 sidrophores production completely inhibited. One of the siderophores gene was detected by specific primer in P. fluorescens and one band at molecular weight 520 bp was observed. SDS–PAGE analysis was used to detect the possible outer membrane receptor protein (OMRP), in both whole cell pellet and membrane pellet at no iron and high iron concentration. The results revealed the existence of one band at 84 KDa in case of no iron concentration. Purification of siderophores in culture supernatant was done by a sephadex G -25 gel filtration column. Nine fractions were obtained and they tested for siderophores activity by CAS assay. There were two types of siderophores in P. fluorescens culture supernatant (pyoverdin and pyochelin) and they were detected by high performance liquid chromatography (HPLC).