Simplification Of The Myxococcus Xanthus DK1622Genome

Myxobacteria are Gram-negative and unicellular gliding bacteria, which exhibit complicated multicellular social behavior. The microbes are capable of producing large numbers of bioactive compounds that show anticancer, antibacterial, fungicidal or immune-modulating activities.Myxococcus xanthus DK1622, Myxococcales order, Cystobacterineae suborder, Myxococcaceae class, Myxococcus genus, is the type strain for the study of myxobacteria social behavior and hereditary or evolution, which has a single circle double-strand DNA but not endogenous plasmids, and its genome is about9.14Mb. The multicellular social behavior includes dual motility, fruiting body formation and sporulation, predation and so on.Sorangium cellulosum, Myxococcales order. Sorangineae suborder, Polyangiaceae class. Sorangium genus, has the largest prokaryotic genome hitherto. As reported, almost half of these bioactive secondary metabolites of myxobacteria were identified from Sorangium, and nearly all of Sorangium strains could produce these bioactive compounds.Hpothilones, which are anticancer mechanisms of paclitaxel (i.e. Microtubule stabilization) are produced by the Sorangium cellulosum. The polyketide compounds-epothilones has generated substantial interest over the last few years in the areas of chemistry, biology and medicine due to their interesting structure and more important activity against numerous cancer cell lines. Up to now. there are at least five kinds of epothilones or chemically modified derivatives of epothilone undergoing evaluation in clinical trials and one has already been authorized for clinical use and sale by the U.S. Food and Drug Administratio. However, the most serious problem is that the Sorangium cellulosum have the poorly developed genetic manipulation system and thus lack flexible genetic tools. So the studies of epothilones and other secondary metabolites are lagging. Because of those multiple limitations, the whole epothilone gene cluster was introduced into E. coli, Streptomyces and M. xanthus to obtain epothilone products which provide required substrates and auliliary enzyme. And Myxococcus xanthus DK1622is a suitable host that has the related substrate and enzyme for epothiloncs production.At present, structure modification and analogue synthesis of natural products are the main methods for screening new drugs. And new biosynthesis systems need to be expressed in suitable cell vectors, which have simple genomic structure-"minimum genome" to reduce noise and complication and to increase the controllability and maneuverability of systems. To make up the suitable vectors for new secondary metabolites synthesis, we begin to simplify myxococcus xanihus DK1622network via searching nonessential genes for deletion to form minimum genome.The research firstly tried to identify the genes that can be removed from the genome without seriously compromising cell viability by comparative genomics approach and the prediction of genomic islands and secondary metabolism gene clusters.We found an estimate of5223genes for the minimal set by comparing the three Myxococcus strains (M.fulvus HW-1, M.fulvus124B02) and5054genes by comparing the Myxobacteria strains (M. fulvus HW-1, M. fulvus124B02, S. cellulosum So0157-2, S. cellulosum So ce56, S. aurantiaca DW4/3-1, Haliangium ochraceum DSM14365, Anaeromyxobacter sp. Fw109-5, Anaeromyxobacter sp. K, Anaeromyxobacter dehalogenans2CP-C, Anaeromyxobacter dehalogenans2CP-1).Then, using IslandPath we predicted and found19GIs whose length is about400kb,4.5%of DK1622genome sequence length.What’s more, using antiSMASH we predicted and found22secondary metabolism gene clusters whose length is about1,283,071bp,14%of DK1622genome sequence length.In order to analyze whether the GIs and secondary metabolism gene clusters are essential for cellular life, we tried to inactivate these genes and analyze the function of the mutain strains. As a result, we got12mutant strains with GI deletion and4mutant strains with secondary metabolism gene clusters deletion, which could grow normally.This work provided us that the GIs and secondary metablolism gene clusters are not essential for cellular life, and we could knock out these genes contiunouselty to get a minimum genome set for DK1622.After these experiments, we knock out the GIs contiunouselty to simplify myxococcus Xanthus DK1622genome. We got GI01-03、GI01-04、GIO1-03-05GI01-03-05-16contiunousely deletion mutant strains, whose deletion genome size respectively were74689bp,54842bp,98923bp,131251bp. Among them, the missing genome size of the GI01-03-05-16mutant was about34.91%of the total GI fragment, and were accounted for the entire DK1622genome of about1.44%.What’s more, the results of studying the phenotype of4GI continuouselty deletion mutant strains showed that,compared to the wild strain DK1622, the grow ability、swarming and predation of the mutants had no obvious changes,but the fruiting body formation and sporulation were affected by the deletions.