Discovery Of The Cryptic Plasmid PMF1 From Myxococcus Fulvus And Application Of A Gene Cloning System In Myxococcus Xanthus

Myxobacteria are rod-shaped,Gram-negative bacteria that are phylogenetically located in the delta division of Proteobacteria.One of the most intriguing characteristics of myxobacteria is their complicated multicellular social behavior. which provides an excellent model for the study of cell-to-cell communication and evolution.Myxobacteria could produce diverse and novel bioactive secondary metabolites,which makes it an important resource for potential new drugs.But the study and utilization of myxobacteria have been limited by the formidable isolation and culture techniques required and the difficulty of performing genetic manipulations. In the past few decades,genetic studies of myxobacteria were performed mainly with the model species Myxococcus xanthus using transduction and electroporation protocols and also with the Sorangium strains using conjugation protocols.Since the 1960s,there has been extensive screening of myxobacteria.but no naturally occurring self-replicating plasmid has been discovered previously,and no broad-host-range vectors can replicate in myxobacterial cells.All the genetic transfer systems used have been based on integration of introduced plasmids or phages into the recipient chromosomes,which hindered the genetic manipulation in myxobacteria for decades. We tried to reevaluate whether the myxobacterial cells isolated from soil samples in China possess cryptic plasmids.Following the protocols previously used for Streptomyces,approximate 150 myxobacterial strains mainly of the genus Myxococcus and Corallococcus.were screened to determine whether indigenous plasmids exist.A circular plasmid named pMF1 was isolated from a M.fulvus strain 124B02.Sequence analysis showed the plasmid was 18,634 bp in length with 68.7%GC content.Twenty-three open reading frames(ORFs)were found in the plasmid,with 21 on the sense strand and 2 on the complementary strand.Nine ORFs resembled known gene sequences in the GenBank database,and the remaining 14 ORFs were not homologous to any known sequence.But the analysis didn't reveal the replicon region of pMF1. A cloning vector pZJY1 with a kanamycin resistance gene was constructed to locate the replication locus in pMF1.Plasmids containing the gene named pMF1.14,which encodes a large unknown protein,were shown to transform M.xanthus DZ1 and DK1622 at high frequencies(～10⁵ CFU/μg DNA),suggesting the locus is responsible for the autonomous replication of pMF1.The gene pMF1.14 was expressed in Escherichia coli cells,and all the trans(?)ormants containing the constructed plasmids except pQE-30E,could produce the target protein in soluble pattern and inclusion bodies.Although the reconstructed plasmids were able to replicate autonomously in M. xanthus cells,almost all the plasmids isolated from the individual transformants were unstable and smaller than the original plasmids from E.coli.The analysis showed that all plasmids lacked the E.coli origin region of pSP72 and the bla sequence of the ampiciUin resistance gene.We screened lots of M.xanthus transformants and found two plasmids,designated pZJY41 and pZJY156,which had maintained their original sizes from E.coll.After several rounds of subculture in the presence of kanamycin. pZJY41 and pZJY156 were stable in M.xanthus.Sequence analysis showed that there were one base deletion in the pMF1 insert in the sequences of pZJY41 and pZJY 156 respectively.The copy number of plasmid pZJY41 was estimated to be 13 in M.xanthus DZ1 and the copy number of pZJY156 was lower than pZJY41 in DZ1,and both plasmids had a lower copy number in DK1622 than in DZ1.The two shuttle vector were easily lost without antibiotic selection in M.xanthus.The insertion of pMF1.16(putative recombinase gene)and pMF1.22(putative parA gene)in pZJY41 and pZJY156 couldn't help to increase their inheritance.The conjugation plasmid pZJY21 and pZJY22 containing the pMF1 replicon could transfer S.cellulosum So0157-2 at a high frequency(10^-5),but they were also easily lost.Myxobacterial gliding motility is studied in Myxococcus xanthus,which is controlled by two distinct motility systems,the adventurous system(A)and the social system(S). The two distinct motility types could be easily selec(?)on the plates with different agar concentrations.In M.xanthus,three cell surface components,typeâ…£pili.fibrils. and lipopolysaccharide O antigen,are required for social motility.Typeâ…£pili pull the cells forward by pilus extension,attachment,and retraction.The gene pilA. which is essential for pili formation and social motility in M.xanthus,was cloned into the shuttle vectors and introduced into the pilA-deficient mutant.M.xanthus DK10410.The transformants subsequently exhibited the ability to form pili and social motility.When the target gene lay in the same orientation as the pMF1.14 gene.the plasmid could stably replicate in M.xanthus.Aminoglycosidic antibiotics are important clinical medicines.The resistance mechanisms to these antibiotics include three ways:decreased intracellular accumulation of the antibiotic by alteration of outer membrane permeability. diminished inner membrane transport,or active efflux;modification of the target by mutation in ribosomal proteins or 16S RNA;and enzymatic modification of the drug. which is the most common.Microorganis(?)that produce aminoglycosides have developed an additional pathway to avoid suicide.This self-defense mechanism involves posttranscriptional methylation of rRNA by the actions of 16S rRNA methyltransferase and 16S rRNA methylase,which also results in the acquisition of aminoglycosides-resistanee in nosocomial pathogens in these years.A locus(kmr)responsible for arninoglycosides-resistance of S.cellulosum was cloned and characterized in M.xanthus using the pMF1 replicon region.The gene kmr encodes a putative rRNA methyltransferase.Expression of the complete ORF endowed the Myxococcus transformants with the resistance to aminoglycosidic antibiotics of kanamycin,apramycin,gentamycin,neomycin and tobramycin at an extraordinary high level(MIC,higher than 500μg/ml).However,the gene did not function in E.coli cells.In Sorangium genome,the gene kmr was followed by a putative integrase gene,and was highly homologous in different Sorangium strains. The Sorangium rRNA methyltranferase sequence was in low similarity to the reported 16S rRNA methyltransferases,and their resistance spectrums were also different.The results indicate that the rRNA methyltransferase(Kmr)in Sorangium strains is a new member of the rRNA methyltransferases family.