Insights Into The Maintanence Mechanism Of Myxococcus Plasmid PMF1

Myxobacteria are a group of Gram-negative bacteria that exhibit complex multicellular social behavior,which makes them to be among the model organisms for the study of cell cooperation and communication in bacteria.Myxobacteria are also able to produce lots of secondary metabolites.The plasmid pMFl,which was discovered in Myxococcus fulvus 124B02,is the only autonomously replicating plasmid found in myxobacteria.Based on the replication origin region of pMF1,Myxococcus-Escherichia coli shuttle plasmids have been constructed,which provide us a new and efficient tool for the genetic manipulation of myxobacteria.However,pZJY41,a shuttle plasmid with only the replication origin region of pMFl,disappeared quickly from its Myxococcus host without selection of antibiotics,and the addition of pMF1.21-pMF1.23 fragment into the shuttle plasmid improved the plasmid stability.The pMFl.21-pMF1.23 region was proved to be the partitioning(par)system of pMF1.Compared with the typical par system,the par system of pMF1 contain an unknown gene pMF1.21,named parC.In this study,we determined that parC plays a significant role in plasmid stability.The parC gene encoded proteins for its functions.The ParC proteins could not bind to either DNA or ParB.We performed pull-down experiments,which indicated that there was a binding interaction between ParA and ParC.The modeling study showed that the ParA surface was positive charged while the ParC surface was negative.Protein-protein interaction predicted that ParA could form a dimer.There were two binding modes between the ParA and ParC proteins,and the binding energy were-37.56 kJ/mol and-31.15 kJ/mol,respectively.In this way,the ParA homodimer is capable of gathering together via ParC to form ParA-ParA-ParC-ParA-ParA pentamer.Thus,the ParA dimer and ParC proteins were alternately linked to form filaments to participate in the plasmid segregation.The previous plasmid stability experiment showed that the loss rate of pZJY4111 was approximately 50%after 168 hours cultivation without positive selection of antibiotics.We speculate there were some other factors involved in the pMF1 inheritance.In the third part of this thesis,a post-killing segregational system was found,which was encoded by pMF1.19-pMF1.20.We found that pMF1.20 encoded a novel nuclease,and pMF1.19 encoded the corresponding antitoxin protein.The pMF1.19 protein inhibited the toxicity of pMF1.20 via protein-protein interaction.Thus,the stable maintenance of pMF1 relies on several plasmid maintenance mechanisms.