Stable Maintaining Region Location Of The Entogenous Plasmid PMF1 In Myxococcus

Myxobacteria are rod-shaped,Gram-negative bacteria that are phylogenetically located in the delta division of Proteobacteria.Myxobacteria are the most sophisticated prokaryotes with regard to morphogenesis,which provides an important model for the research in communication and evolution.On the other side, myxobacteria have rich secondary metabolism and could produce various kinds of bioactive secondary metabolites,which makes it an important resource for potential new drugs.But the researches in myxobacteria have been limited because of the difficulty in performing genetic manipulations.In the past years,no entogenous plasmid was discovered in Myxobacteria cells, and broad-host-range plasmids from orther bacteria were all found not to be able to replicate in Myxobacteria cells,and the plasmids normally seem to survive in myxobacterial cells only when inserted into the chromosomal DNA,which immensely restrict the genetic manipulation in myxobacteria cells.Under the cooperation of Shanghai Institute of Plant Physiology and our lab,a circular plasmid named pMF1was discovered and isolated from Myxococcus fulvus 124B02.The replicon of pMF1 was determined and shuttle vectors named pZJY41 and pZJY156 were constructed based on the pMF1 replicon and E.coli cloning-vector pSP72. However,when the inheritance of pZJY41 and pZJY156 was investigated in Myxococcus xanthus DZ1 and DK1622,the transformants will quickly lose the plasmid in the absence of antibiotic selection.The unstable inheritance of pZJY41 and pZJY156 in M.xanthus DZ1 leads to insufficient application of the shuttle vectors in myxobacteria.Several factors influence the genetic stability of prokaryotic plasmids,mainly include the copy number control of the plasmid,site-specific resolution systems, post-segregational killing(PSK) systems and plasmid partition loci.In general,the stable inheritance of the low-copy-plasmid mostly depends on the partitioning(par) loci that actively segregate plasmid copies to daughter cells before cell division.In addition,many low-copy-number plasmids encode so-called post-segregational killing (PSK)systems that increase plasmid maintenance by killing plasmid-free cells.These systems may be viewed as providing a backup stabilization mechanism of last resort that is executed when a true par locus fails to function properly.All known plasmid-encoded partitioning loci encode two trans-acting proteins expressed from an operon and one or more cis-acting centromere-like sites,at which the proteins act.All three components of a par locus,i.e.,two proteins and a centromerelike cis-acting site,are essential.Generally,par loci function as cassettes independently of the replicon on which they reside.The first gene of a par operon encodes an ATPase,The second gene of a par operon encodes a DNA-binding protein that recognizes varying numbers of direct or inverted repeats within a cognate centromere-like site.Binding of the protein to these sites results in the formation of a nucleoprotein complex,also known as the partitioning complex.The partitioning complex is the substrate for plasmid segregation,in which replicated plasmid molecules,often located at the mid-cell position,are moved in opposite directions.According to the research in the maintance of other natural plasmid and the anlysis of the sequence of pMF1,the predicted plasmid partitioning loci was further characterized and analysised following the former work.The predicted plasmid partitioning loci consists of 3 genes named pMF1.21,pMF1.22 and pMF1.23.It may also includes several kinds of low-copy imperfect short direct repeats as well as several copies of long direct repeats,which locate upside and downstream of the 3 ORFs.Three DNA segments in different length were amplified from pMF1 including 17242-50 of pMF1,17242-1136 of pMF1 and 16604-1136 of pMF1,each of which involves all the 3 ORFs and different range of the predicted non-coding region.Each of the segments was cloned into pZJY41 and 3 reconstructed vectors named pZJY4111,pZJY4112 and pZJY4114 were constructed.The inheritance stability of these vectors in Myxococcus xanthus DZ1 were investigated respectively in the absence of antibiotic selection.The results show that the inheritance of pZJY4112 in DZ1 was improved to a certain extent compared with pZJY41.The analysis showed that the predicted plasmid patitioning loci surely played an essential role in the maintenance of the plasmid.As a shuttle vector more stable than pZJY41,pZJY4112 should greatly facilitate genetic manipulations in myxobacteria.At the same time,other method was used to determine the region for inheritance of pMF1.Without regarding to the information of the pMF1 sequence,the inheritance related region was screened from pMF1 using the method of restricting the pMF1 subclone,pSQ37 and pSQ38 to get the pMF1 segments and then cloning these segments into pZJY41 respectively to investigate the inheritance of the reconstructed vectors.Firstly,pSQ37 and pSQ38 were restricted with EcoRI and the pMF1 segments at the length of 10.8kb and 7.8kb were cloned into pZJY41 respectively. The reconstructed shuttle vector named pZJY4116 and pZJY4117 were transformed into Myxococcus xanthus DZ1.The plasmid pZJY4117 isolated from DZ1 transformants became smaller than the original plasmids from E.col,showing that pZJY4117 was unstable in myxobacteria.Although the shuttle vector pZJY4116 maintained the size in Myxococcus xanthus DZ1,it was lost quickly in DZ1 when segregating into the daughter cells without selection pressure.It was inferred that the segments insered into pZJY41 was too long to make the reconstructed vector unstable. Secondly,the pMF1 segments in pSQ37 and pSQ38 were restricted with 2 kinds of restriction endonucleases respectively.The segments were cloned into pZJY41 and shuttle vectors pZJY4101 to pZJY4108 were constructed.As to the inheritance in DZ1 of these plasmids,pZJY4102 was more stable than pZJY41 and as stable as pZJY4112 while pZJY4103 was much more stable than pZJY41 and pZJY4112 without selection,pZJY4101 and other reconstructed vectors above-mentioned were unstable in DZ1.These results implied that pZJY4102 and pZJY4103 were more stable because of the partitioning loci in the reconstructed vectors while pZJY4103 was more stable than pZJY4102 because of other mechanism and region in pMF1-maybe the 16382-17242 of pMF1-rather than the par loci.As to further improve the stability of the shuttle vectors,the predicted gene encoding recombinase pMF1.16 and 971-3680 of pMF1 were cloned into pZJY4103 respectively and pZJY4109 and pZJY4110 were constructed.When the inheritance was investigated,pZJY4109 and pZJY4110 were no more stable than pZJY4103.The results inferred that the predicted recombinase coden gene possibly not played a role in pMF1 inheritance maintenance in myxobacteria.In this thesis,3 M.xanthus-E.coli shuttle vectors pZJY4102,pZJY4103 and pZJY4109 which were more stable than pZJY41 were constructed.They have great application value and should greatly facilitate genetic manipulations in myxobacteria. Compared with pZJY41,both pZJY4102 and pZJY4103 contain the predicted plasmid partirioning loci of pMF1,indicating that surely played an essential role in the maintenance of the plasmid.The insert fragment of pZJY4103 was longer than that of pZJY4102 and pZJY4103 is more stable than pZJY4102.It is implied that other mechanism and region in pMF1-maybe the 16382-17242 of pMF1-rather than the par loci.The predicted par loci contains 3 ORFs which is first found in natural plasmids suggesting that the plasmid pMF1 possibly maintain the inheritance in other mechanism.