Study On Algicidal Activity Of Microalgae-associated Bacterium Rhodobacteraceae Pd-2and Gene Knockout Method Of Quorum Sensing Regulating Gene Zlb I

Algicidal bacteria possess inhibiting effect towards marine microalgae in marineenvironment. They could lyse or kill the algal cell directly. The research about algicidalfunction and mechanism of these bacteria is a hot issue in interaction between microalgaeand bacteria. This study has great guiding value in some key scientific issues such asbiological treatment against red tide.In this work, one strain of marine microalgae-associated bacteriumRhodobacteraceae PD-2was studied. The chemical structure of algicidal compound waselucidated. The algicidal style also was investigated. Two pairs of quorum sensingregulating gene were analyzed and the gene knock out methods for one quorum sensinghomologue gene based on recombination was established. The main researches werelisted below:The culture condition including incubation temperature and media was optimized bycomparing the bacterial density and algicidal activity. The optimum culture conditionwith highest bacterial density and algicidal activity was obtained when PD-2cultured inthe medium with Zobell2216E (add glucose), at30C. The preliminary research on theprofile of signal molecules AHLs produced by PD-2was studied based on biosensoroverlay assay. The result showed the bacterium could produce at least three AHLs andsome of them were new type of AHLs comparing with AHLs standards.Fermentation of PD-2was carried out under the optimal culture conditions.. Analgicidal compound in PD-2metabolites was separated and purified tracing bybioautography method. The molecular weight and structure of this algicidal compoundwere elucidated by mass spectrum (EI/MS) and one dimensional NMR (1H NMR,13C NMR and DEPT) analysis. The results indicated that molecular formula of the compoundis C12H10N2OS with molecular weight of230.051383and named as(4,5-dihydrothiazol-2-yl)(1H-indol-3-yl)methanone. Through observation of scanningelectron microscopy, algal cells of the host Prorocentrum donghaiense were found to belysed under the effection of algicidal compound.Two pairs of quorum sensing homologous gene (lux I/R) which named zla I/R andzlb I/R were founded in PD-2genome through analyzing the complete genome sequence.In order to confirm the function regulated by quorum sensing gene, gene knockoutmethod was established based on homologous recombination technology. Through theexperiment of PD-2-Rif-R-001mating with E. coli S17-1pSRK Gm, PD-2was foundhad conjugate ability and the mating condition was optimized. According to the genomesequence, primers were designed to amplify the up and down stream fragments with thesize around500bp in flank of zlb I gene. Overlap extension for up and down streamfragments was carried out by two-step PCR. Through restricted digestion and connectionmethod, fusion fragment of up and down stream was inserted into suicide vector pNPTS138Kan. Recombinant plasmid was transformed into E. coli S17-1. The E. coli S17-1pNPTS138Kan constructed strain was isolated and confirmed. Due to the low efficiencyof homologous recombination, the work of conjugation for PD-2-Rif-R-001mating withE. coli S17-1pNPTS138Kan construct strain was still on going.