| Myxobacteria are a group of Gram-negative bacteria which own complicated intercellular cooperative behaviors. They are able to form multicellular fruiting bodies and development into resistant myxospores under adverse environment. They can glide on the solid surface by the cooperation of dual motility systems and prey on other bacteria in the nature conditions. Therefore, myxobacteria are regarded as an important research material in signal transduction and evolution of prokaryotic cells. On the other side, myxobacteria produce various kinds of second metabolites which have novel struture and special activity, thus as an important source of microbiol drug just behind Actinomycetes.Apart from the myxobacteria, theδ-proteobacterial genomes range from 3.66 to 5.01 Mb. Because the M. xanthus genome is 9.14 Mb, there has been a genome enlargement of 4-5 Mb from its inferredδ-proteobacterial ancestor. M. xanthus vegetative cells have 1-2 copies of genome, the copy number drops to 1 during the stable phase, and the myxospore has two complete chromosomes. The development process will be delayed if the DNA replication is inhibited, nevertheless, the regulation and mechanism between genome duplication and development remain unclear.Myxobacterial genetic manipulation system is limited, during the past half century, several methods, such as general transduction, conjugation, and electroporation, have been applied to introduce plasmid into myxobacterial cells. However, plamids have to integrate into chromosome after introduced into cells because of the lack of self replicative plamid, which to a large extent impeded the myxobacterial research and application. Until 2006, Institute of Plant Physiology & Ecology (Shanghai Institute for Biological Science, CAS) and our lab jointly discovered and isolated a self replicative circular plamid pMF1 from Myxococcus fulvus 124B02, and constructed Escherichia coli-M. xanthus shuttle vectors pZJY41 and pZJY156 based on the pMFl origin of replication region. This discovery opened a new page in the field of myxobacterial genetics.Circular plasmid replication contains three types, theta replication, strand displacement replication and rolling circle replication. Rolling circle replication-type and strand displacement replication-type plasmids produce single-stranded DNA intermediates, and theta-type plasmids do not. Generally, plamid replication initiation requires (ⅰ)Rep initiator protein, which is encoded by the plasmid, (ⅱ)an adjacent AT-rich region containing sequence repeats, where opening of the strands and assembly of host initiation factors occur, (ⅲ) one or more dnaA boxes, and (ⅳ) multiple Dam methylation sequences. The latter two are not necessary for all plasmids. Many plasmids harbor several directly repeated sequences, termed iterons, which are the binding sites for the Rep proteins, and have the role of replication regulation. In addition, some plasmids could produce antisense RNA, which is complementary to target RNA (sense RNA) and negatively regulates the plasmid replication in-cis or-trans.rep ABC plamids contain at least three protein-encoding genes:rep A, repB and repC. The first two genes encode proteins involved in plasmid segregation and copy number control, whereas repC encodes a protein crucial for replication. Those genes are co-transcribed by a common upstream promoter, which is negatively regulated by RepA, with RepB under some conditions.In this research, based on pMFl origin region in pZJY156 and pZJY41, the pMF1 replication mode, replication region organization, and its property are studied.First, replication intermediate of pZJY41 from M. xanthus DZ1 were extracted by PEG6000 precipitation method, and isolated by gel electrophoresis. one piece of gels was treated by alkaline denaturation and the other was not, and the DNA in the gels was transferred to nylon membrane. DIG-labeled pMF1.14 gene fragment was used as the hybridization probe, and the Southern Blot did not detect any single-stranded DNA in the M. xanthus DZ1 pZJY41 replication intermediate, in other words, in M. xanthus DZ1, single-stranded DNA is not produced in the process of pZJY41 replication. That is to say, in Myxococcus, pMF1 replicates via a theta mode, which is adopted by most Gram-negative bacteria.The 2.2 kb inserted sequence (11645-13853) in pZJY156 contains only one complete ORF,pMF1.14, so pMF1.14 is proposed to encode the replication initiation protein necessary for replication.Bioinformatics analysis implies that pMF1.13, pMF1.14, and pMF1.15 might be organized in an operon. We isolated the total RNA from M. fulvus 124B02, applied reverse transcription assay using a downstream primer, and performed PCR specific to the three genes using the reverse transcript as the template. The result showed that pMF1.13,pMF1.14, and pMF1.15 genes are located in an operon, and their transcripts are a same strand of mRNA, and they are named rep A, repB and repC, respectively. This operon is different from the known repABC model, and might possess its own special replication and regulation pattern.We studied the property of pMF1 ori based on E. coli-M. xanthus shuttle vector pZJY156. First, we attempted to heterelogously express pMF1.14 protein in E. coli expression systems. We can not find homologous proteins of pMF1.14 in the GenBank database, and this protein do not contain any DNA-binding motifs. Its theoretical pI is 9.85, classified a basic protein. We tried to express its full-length protein with his-tag, but failed to obtain its soluble purified protein. Then we tried to increase its solubility via GST tag using pGEX-6p-1 vector and via Trigger Factor tag using pCold TF vector, and the soluble part were not able to fully combine with the affinity chromatography beads, and the purification result was not perfect. We also tried to truncate the protein into several parts in order to increase its solubility, but unfortunately failed.In the circumstances of unsuccessful studying the DNA binding property of pMF1.14 protein in vitro, we determined the replication initiation property of pMF1.14, and located plasmid cis-acting region by complementation assay in vivo. The results showed that pMF1.14 is the indispensable replication initiator and functions in trans, the cis-acting region of pMF1 is located within the open reading frame of pMF1.14, and its minimal region is from 12894 to 13263. Within the～380 bp region, there are more repeat sequences than other region, and these repeats are not typical iteron structures. Considering the specificity of pMF1.14 protein, we propose that the interaction of pMF1.14 and the plamid cis-acting region DNA may be in a special way. Besides, with the co-exiestence of pMFl.13, the plasmid transformation efficiency and copy numbers are elevated, inferring that pMFl.13 might play a role in promoting pMF1.14 expression or replication function. |
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