Biodegradation Mechanism Of Microcystin-LR By Rhizobium Sp.TH And The Evolutionary Origin Of The MlrA Gene

Microbial degradation has been extensivelyinvestigated as a main pathway for Microcystins?MCs?attenuation.Investigating the biodegradation process and mechanism of MCs is of great significance to recognize the fate of MCs in enviroment.Currently,approximately 60strains of MCs-degrading bacteria have been isolated,andthemajoritybelongstoclass?-proteobacteria,especiallySphingomonadales.Most of these?-proteobacterial MCs-degrading bacteria harbormlrA gene,suggesting thatthey degradedMCs via themlr-dependent mechanism.However,the evolutionary origin and the distribution of mlr genes in MCs-degrading bacteriais poorly understooddue to the lack of sufficient mlr data,especially that from?-proteobacteria other than Sphingomonadales.In previous study,a novelRhizobium sp.strainTHwas isolated,which is the first?-proteobacterialMCs-degrading bacterium to be found outside Sphingomonadales.Although,the mlrA gene was not detectedin strain TH,other three genes,mlrB,mlrC and mlrDhave been amplified,indicating that strain TH might anchor an mlr gene cluster.Therefore,strain THprovidesan ideal material for investigatingthe evolutionary origin of mlrA genes andtheirdistribution among the MCs-degrading bacteria.In this thesis,a pair of primerswere designed andused to determine themlrA gene.The fulllength of the mlr gene cluster was sequenced,and the function of genesin it were verified by heterologous expression.The evolutionary origin of mlrA was investigated bycomparing the phylogenetic trees ofmlrAand the associated 16S rDNA sequences.The main results are as follows:?1?Based on the mlrA genes sequenced thus far,a pair of primersmlrAtf/mlrAtrwere designed andused to determine themlrA gene in strain THsuccessfully.According to blastn analysis results,the primersmlrAtf/mlrAtr match well with the mlrA genes fromdifferent genus of MCs-degrading bacteria,implying that these primers may havewider application in mlrA detection.Hence,mlrAtf/mlrAtr could be used to confirm the MCs-degrading mechanism of novel isolates or to screen for MCs-degrading ability among environmental samples.?2?The partialsequenceof the mlr gene cluster in strain TH wasobtained through amplifying and sequencing the regions spanning mlr genes.Thefull-length mlr cluster?6673 bp?in strain TH was assembled after thethe flanking sequences of it were amplified and sequenced by genome walking PCR.The gene cluster consists ofmlrC,mlrA,mlrDandmlrB,successively.The transcriptionaldirectionofthemlrAand mlrD gene are reverse tothat of themlrB andmlrC gene.?3?The MlrAin strain TH was predicted to possess aH²⁶⁰AIH²⁶³NE²⁶⁵motif,which may be the active site domain.BothMlrAand MlrD have transmembrane domains and MlrA also has asignal polypeptide,whereas the MlrBand MlrC haveno signal polypeptide ortransmembrane domain.Furthermore,the MlrB contains aBeta-lactamasedomain,implying thatit belongs to the serineprotease.?4?The threeproteases,MlrA,MlrB and MlrC were proved to attend the degradationofMCLRsequentially.AlinearMCLR,tetrapeptideand Addaweresequentially produced through the breakage ofthe peptide bondsAdda-Arg,Ala-Leu and Adda-Glu.In addition,MlrCcan also degradethe linear MCLR to Addadirectly.These results demonstrate that strain TH degrades MCLR via themlr-dependent mechanism.?5?To elucidate the evolutionary origin of mlrA genes,phylogenetic trees were inferred using mlrAandthe associated 16S rDNA sequences,respectively.The?-proteobacterial MCs-degrading bacteria occupied similar phylogenetic positions in both trees,with taxonomically close species clustering together,implying that the mlrA gene initially occurred in?-proteobacteria by vertical evolution.For?-and?-proteobacterialMCs-degrading bacteria,however,the mlrA genes have high similar to Sphingopyxis sp.C-1,suggesting that these bacteria acquired mlrA gene by horizontal transfer.According tothe evolutionary origin of mlrA gene,it can be predictedthat themlrA genes may be mainly distributed among?-proteobacterial MCs-degrading bacteria,and only a few other classes or phyla of bacteria might have obtained the mlrA gene through lateral gene transfer.