Resources Diversity And Predation Mechanism Of Myxobacteria And The Genus Chitinophaga Of Dinghushan

Myxobacteria,which are a kind of rod-shaped and gram-negative bacteria,belong to the order Myxococcales and classδ-Deltaproteobacteria.Myxobacteria have complex multicellular behaviors,such as gliding motility,forming fruiting bodies and dry-resistant myxospores,predating other bacteria and fungi,providing abundant materials for fundamental research.Myxobacteria are the third-dominant bacteria of producing prospective new bioactive secondary metabolites after actinomycetes and bacillus.In addition,myxobacteria play a major role on biological control of plant diseases.Therefore,the isolation of in particular new groups of myxobacteria is of great interest.But myxobacteria are one of the most difficult microorganisms to be isolated and cultivated because of their slow and density dependent growth and extraordinary morphological development features.To date more than 200 years,myxobacteria identificated had only 30genera and 66 species.It is a way to solve the difficulty of isolation with innovation isolation techniques.Using prey bacteria inducing myxobacteria is the main way to isolate myxobacteria.However,the valid prey group were limit and the mechanism used was poorly understood.Moreover,myxobacterial diversity on acidic soil had not many researches and the biotic factors(bacteria and fungi)influencing on the diversity of myxobacteria,to the best of our knowledge,are not reported so far.Dinghushan Biosphere Reserve is a famous ecological research establishment of ecological system in domestic and overseas,in which there are abundance sources of plants,animals and microorganisms.Based on the reasons,we conducted researchs about resources diversity and predation mechanism of myxobacteria and the genus Chitinophaga of Dinghushan,and draw the following conclusions:(1)To obtain more myxobacteria,we evaluated the diversity of myxobacteria in Dinghushan forest soil using cultivation-based(with E.coli and filter papers)and cultivation-independent analyses based on high-throughput sequencing of 16S r DNA.The results indicated that myxobacteria including Sorangium(60%),Haliangium(18.8%),Anaeromyxobacter(4.1%)and some other bacteria,comprised 0.92-2.24%of all bacteria community.The p H of soil samples was 3.6-4.5 and organic matter contents were26.6-143.4 g/kg.Mantel test and Spearman analysis reveals that soil p H,AK and bacterial diversity had a significant influence on myxobacterial community structure and diversity.The network analysis predicted that Proteobacteria had the most interactions with myxobacteria,indicating there are many prey groups in the Proteobacteria.A total of 21cultures were isolated using standard cultivation methods,9 strains from the genus Myxococcus,11 strains from the genus Corallococus and 1 strain from the genus Archangium.Based on 16S r RNA gene sequences and morphological features,there are three potential novel species found in 21 cultures.Strain H22C18031201 and K23C18031201-2 belonged to new species in the genus Corallococcus and 12S042801belonged to a new species in the genus Archangium.In addition,a novel genus of Chitinophagaceae K23C18032701~T and a new species of Chitinophaga K20C18050901~Twere obtained during isolation of myxobacteria.(2)Polyphysic taxonomic analysis revealed that strain H22C18031201 belongs to a novel species of Corallococcus genus.The proposed generic name is Corallococcus flavoviridis.Cells of strain H22C18031201 were greenyellow,of ellipsoidal fruiting bodies with abundant stress-resistant myxospores and moved by gliding motility.The genome size of strain H22C18031201 was 9.07 Mbp with a DNA G+C content of 69.5 mol%.The strain contains a large number of secondary metabolite gene clusters by anti SMASH database predicted.Comparative genomics analysis for average nucleotide identity and the digital DNA-DNA hybtidization values between Myxococcus xanthus and Myxococcus virescens of the genus Myxococcus were 97%and 73.1%,respectively,which surpassed the estimated thresholds of species definition for both ANI(95-96%)and d DDH(70%).Therefore,they should be merged to a single species.Meanwhile,Mauve analysis indicated that members of the genus Myxococcus have genetic diversity.(3)On the basis of phenotypic,genotypic and phylogenetic analysis,strain K23C18032701~T represents a novel species of a new genus in the family Chitinophagaceae,for which the name Deminuibacter soli is proposed.The type species K23C18032701~T=GDMCC 1.1403~T=KCTC 62913~T.Cells of strain K23C18032701~T were gliding motility and underwent a shape change where rod-shaped transformed into coccobacilli.In addition,we proposed the reclassification of Filimonas aurantiibacter as Arvibacter aurantiibacter comb.nov.and emended the description of the strain.Meanwhile,based on polyphasic taxonomic research,strain K20C18050901~T represents a novel species of Chitinophaga,for which the name Chitinophaga silvisoli is proposed.The type strain K20C18050901~T=GDMCC 1.1411~T=KCTC 62860~T.The cells of strain K20C18050901~Twere yellow,rod-shaped,gliding motility and secreting mucus.(4)We isolated many cultures of the genus Chitinophaga earlier when isolated myxobacteria.We studied the interaction of myxobacteria with strain K23C18032701~T in the family Chitinophagaceae and 11 strains in the genus Chitinophaga.Our results revealed that strain K23C18032701~T was preyed by myxobacteria quickly and easily,indicating potential ability to isolate new groups of myxobacteria.Myxobacteria exhibited different predition activities on the genus Chitinophaga.On rich nutrition media MD1,extracellular mucus secreted by Chitinophaga could protect themselves from predating by myxobacteria.Anti SMASH database predicted that the less secondary metabolite gene clusters of genus Chitinophaga and strain K23C18032701 had,the stronger ability of predation was shown,and vice versa.The results revealed that secondary metabolites produced by prey bacteria could protect themselves from predation by myxobacteria.We also found that C.eiseniae KACC 13774~T,C.flava K3CV102501~T,C.dinghuensis DHOC24~T and C.qingshengii JCM30026~T have a wide range of secondary metabolite gene clusters that are good medicinal microorganisms that have broad application prospects.