Studies On The Melanin Production Of Pseudomonas Sp. BIp-2Isolated From Deep-sea Sediment And The Methods Of HPPD Inhibitor Screening In Vivo

Melanin is the most widely found pigment throughout all thephylogenetic scale, including bacteria, fungi, plants, and animals.Although its nature is not well-defined, melanin can be described aspolymers presenting dark color, generally insoluble in aqueous media,amorphous structure, and formed from phenolic, diphenolic and quinoniccompounds. Melanin synthesis is not essential for fungal growth anddevelopment, but confers a broad spectrum of biological functions suchas increased virulence in many fungi pathogenic to human and plants, andenhanced survival under different stress conditions like extremetemperatures, desiccation, protection from UV irradiation, metals andcell-wall degrading enzymes.4-Hydroxyphenylpyruvate dioxygenase (4-HPPD, EC1.13.11.27) is anFe(II)-dependent, non-heme oxygenase that catalyzes the conversion of4-hydroxyphenylpyruvate (4-HPP) to homogentisate (HGA). Thetransformation catalyzed by4-HPPD has both agricultural and therapeuticsignificance.4-HPPD catalyzes the second step in the pathway for thecatabolism of tyrosine; in plants this pathway has an anabolic branch fromhomogentisate that forms essential isoprenoid redox cofactors such asplastoquinone and tocopherol. This has been the basis for the developmentof a range of very effective herbicides that are currently usedcommercially.A pigment-prouding strain was isolated from the Atlantic Oceandeep-sea sediment and identified as Pseudomonas stutzeri (99%identity)by16S rDNA sequencing. The study of growth and melanin production withvaried culture conditions show that: The melanin production in strainbIp-2is growth-associated; the maximum yield of pigment was obtained when the culture attained the stationary growth phase. When the strain bIp-2was cultured at different temperatures, maximum melanin production wasobserved at28°C wheras its optimal temperature for growth was37°C.Melanin formation was found to be maximum at pH7.0and favoured byalkaline pHs. Strain bIp-2was capable of growing in the medium with1%-9%NaCl concentrations and producing maximum melanin at5%NaClconcentration. A degenerate priemer pair was designed for the full-lengthhppD gene cloning and a1087bp fragment was amplified from the strainbIp-2. The hppD gene encoded amino acid sequence shared a97%similaritywith the hppD gene from Pseudomonas stutzeri RCH2. The hppD gene clonedfrom bIp-2was over-expressed in Escherichia coli BL21and the recombinantproduced an oxidized red-brown pigment on plate.A novel HPPD assay was developed to screen large numbers of inhibitors.By culture of pigment-prouding strain or its hppD gene recombinant inliquid media supplemented with different candidate compounds andobservation of the color change of the culture,7potential inhibitorswere selected by their good bleaching effects and no blockage in bacteriagrowth. The identification of new herbicides usually requires multipleenzymatic assays, which hinders the selection procedure and does notensure that the molecules selected will be effective in vivo. The designof new strategies for the rapid and effective identification of thesekinds of inhibitors could facilitate the isolation of new therapeuticherbicides.