Study On Quorum Sensing Properties And Meloculor Regulation Of Pseudoalteromonas Spp.

Quorum sensing(QS)is an extremely important mechanism for microbial regulation of physiological metabolic activities of a population.A large number of studies have shown that the production of bacterial bioactive substances and the exertion of physiological metabolic functions are often regulated by quorum sensing.In this study,based on the 16 S rRNA gene sequence,the diverse bacteria isolated were studied.Twelve strains were found to be Pseudoalteromonas spp.Among them,11 strains were found to secret quorum sensing signals based on sensing of bacterial indicator.In order to explore the correlation between quorum sensing and physiological metabolism in Pseudoalteromonas,antibiotic susceptibility,motility and biofilm formation of these 12 Pseudoalteromonas isolates were studied.It was found that their quorum sensing features and physiological characteristics were different.The draft genomes sequences of 12 Pseudoalteromonas isolates were sequenced and their genomics were analyzed.It was found that Pseudoalteromonas has sophisticated quorum sensing signals and regulatory systems.Its networked system of quorum sensing includes the structures of three typical bacteria: Vibrio,Pseudoalteromonas and E.coli.Pseudoalteromonas sp.T1lg65 with the strongest AHL-based QS trait among isolates was selected to carry out random insertion mutagensis by using transposon mini-Tn10.More than two thousand individual transposon inserted mutants were gained.Through the screening of resistance,based on the phenotypic differences at colony color,size and morphology,approximately 300 strains with altered quorum-sensing signal molecules were obtained.After seven rounds of subculture,31 mutant strains that lost the ability to produce signaling molecules were found to be stable.After molecular identification,14 mutants were found to be inserted by transposons,resulting in the loss of quorum sensing signal molecule synthesis ability.We used TAIL-PCR to localizethe inserted gene.It was found that the insertion sites of the 6 mutants(T4,T6,T8,T18,T26 and T27)were the genes encoding unknown function.The site-directed mutant gene of mini-Tn10 in T1 encodes Threonine/homoserine exporter RhtA,which may be related to the transport of acyl serine and serine.The site-directed mutant gene of mini-Tn10 in T2 encodes the MarR family transcriptional regualtor.In T5,the mini-Tn10 inserted gene encodes TonB,the mini-Tn10 inserted gene in T15 encodes the outer membrane protein A precursor,and the mini-Tn10 inserted gene in T30 encodes the right origin-binding protein.The mini-Tn10 inserted gene in T32 encodes the HDOD domain-containing protein,and the mini-Tn10 inserted gene in T38 encodes the capsid biosynthesis protein CapB,which is associated with decidua synthesis.The mini-Tn10 inserted gene in T52 encodes a linear gramicidin synthase subunit D,and the mini-Tn10 inserted gene in T65 encodes VgrG protein.The mini-Tn10 inserted genes were separately complemented in the corresponding mutants with abolished ability to produce quorum signaling molecules through the tri-parental.The results showed that the complementation of either gene can restore the ability to produce the quorum-sensing signal molecule,indicating their positively regulatory role in production of quorum sensing signal.The relative expression levels of robP and acyl homoserine lactone synthase gene ahl were compared within wildtype,mutant and complon.The results demonstrated that robP positively regulates formation of quorum sensing signal.