Study On The Quorum Sensing System And The Algicidal Function Of Marine Bacterium Ponticoccus Sp.PD-2

Algicidal bacteria living in the marine environment possess inhibiting effects on marine microalgae.They could lyse or kill the microalgae directly.The reserch on the algicidal function and mechanism of these bacteria is a hot issue for interaction between microalgae and bacteria.This study has important guiding value in some key scientific issues such as biological treatment against red tide.In this paper,we studied the quorum sensing system of a microalgae-associated bacterium Ponticoccus sp.PD-2.The quorum sensing homologous genes zlaI and zlbI were knocked out using CRISPR/CAS9 technology.Finally,we illuminated the relationship between the algicidal activity and quorum sensing of PD-2.Through analyzing the genome sequence of PD-2,we find two pairs of quorum sensing homologous gene(luxI/R)named zlaI/R and zlbI/R.By constructing phylogenetic tree and analyzing multiple sequence alignments,we found that these two pairs of quorum sensing regulation proteins have homology similarity more than 69%.The amino acid sequence of lux I homologous protein are highly conservative.The types of AHLs produced by PD-2 based on biosensor and GC-MS were detected.The result showed that PD-2 could produce two kinds of AHLs molecules,OC8-HSL and OC10-HSL.PCR and cloning method were employed to clone zlaI and zlbI which were later connected to pGEX-6P-1.We expressed zlaI gene and zlbI gene in E scherichia coli with high efficiency and used bioautography to prove that both of these two luxI homologous genes could produce OC8-HSL and OC10-HSL and OC8-HSL is produced mainly under the control of zlbI.According to our algicidal experiment,PD-2 has obvious inhibiting effects on three microalgae(Prorocentrum donghaiense,Phaeocystis globaosa,Alexandrium tamarense),with the significant inhibition rate on its host Prorocentrum donghaiense.After adding β-cyclodextrin into the PD-2 culture medium,we found a great decrease of the concentration of AHLs.As well as,the algicidal activity of PD-2 was reduced by 50%-80%.This implying that the restraint of QS system could inhibit the production of algicidal substance of PD-2.By constructing CRISPR/Cas9 gene editing system,we knocked out two synthetase genes zlaI and zlbI from PD-2,applying CRISPR/CAS9 to marine bacteria.Detections on A HLs and algal inhibition effects of zlaI and zlbI deletion mutants showed that PD-2 without homologous gene of luxI could not generate AHLs signal molecules any longer;also,the deficiency of algal inhibition effects indicated the control of quorum sensing on in the molecular level.