Preliminary Study On Function Of Replication-related Genes On Myxobacterial Plasmid

Myxobacteria are a group of Gram-negative bacteria which own complicated intercellular cooperative behaviors. They are able to form multicellular fruiting bodies and develop into resistant myxospores under adverse environment. Myxobacteria are regarded as an important research material in signal transduction and evolution of prokaryotic cells. Myxobacteria can produce various kinds of second metabolites which have novel strutures and special activities, thus as an important source of microbiol drug. The genetic manipulation system of myxobacteria is limited, which to a large extent impeded their research and application. Until2006, Shanghai Institute of Plant Physiology and our lab jointly discovered and isolated a self replicative circular plasmid pMFl from Myxococcus fulvus124B02, and constructed Escherichia coli-M. xanthus shuttle vectors pZJY41and pZJY156based on the pMF1replication region. This discovery opened a new page in the field of myxobacterial genetics.Replication intermediate of pZJY41from M. xanthus DZ1were extracted by PEG6000precipitation method, and isolated by gel electrophoresis. There was no single strand DNA in the M. xanthus DZ1pZJY41replication intermediate, that is to say, in Myxococcus, pMF1replicates via a theta mode. The pZJY41contains pMF1.13and pMF1.14. pMFl.14is proposed to encode the replication initiation protein which is necessary for replication. Bioinformatics analysis implied that pMF1.13,pMF1.14, and pMF1.15might be organized in an operon, which is different from the other common theta model. So we believe that the origin of replication would be useful and significant.This paper aims to study replication initiation region-pMFl.13and pMF1.14.Generally, plasmid replication initiation requires rep initiator protein, and many plasmids harbor several directly repeated sequences, termed iterons, which are the binding sites for the Rep proteins. There maybe have an adjacent AT-rich region containing sequence repeats, where opening of the strands and assembly of host initiation factors occur. It is predicted that the pMF1.14don’t have a DNA binding domain, but the minimum cis-acting district12894～13263contained repetitive DNA sequences, and it’s speculated that the replication initiator protein with DNA replication initiation has special way. Firstly, we tried to express pMF1.14protein in E. coli. We cannot find homologous proteins of pMF1.14in the GenBank database. It’s theoretical pi is9.85, and it’s classified as a basic protein. We tried to express its protein with his-tag, resulted as inclusion bodies, and failed to obtain its soluble purified protein. Then we tried to increase its solubility via GST tag and pCold TF vector, and soluble part were not able to fully combined with the affinity chromatography beads, and the purification result was not perfect.Bioinformatics analysis implies that pMF1.13, pMF1.14, pMF1.15and pMF1.16might be organized in an operon. We isolated the total RNA from M. fulvus124B02, and obtained cDNA by reverse transcription assay using a downstream primer, and performed PCR specific to inter-district of the four genes using the reverse transcript as the template. The result showed that the four genes are located in an operon, and their transcripts are a same strand of mRNA. They are likely to exercise the same function. Then what is the role of the four genes in the plasmid replication? Shuttle vector pZJY41which has one complete pMF1.13gene more than pZJY156, has more copies and more stronger stability. Further, by qPCR experiments, we found that the presence of the gene pMF1.13can improve the level of transcription of pMF1.14. There are many kinds of regulating ways on how regulatory factors affect replication. Maybe effect the activity of promoter though binding DNA, or influence the gene transcription level by combining enzymes to form complex impact. For further studying on the function of pMF1.13, we need to purify the protein. We tried to express pMF1.13protein in E. coli, but the purification result was not perfect. Later, we used labeled recombinant proteins to proceed DNA binding experiments and DNA pull-down in vitro, and we got some positive results. According to the results, we would further study the role of pMF1.13.