Taxonomic Identification Of A Novel Species Of Massilia And Study On Violacein Production

Violacein is a natural purple secondary metabilite produced by bacteria,which has recently attracted much attention for its antibacterial and antitumor activities.However,Chromobacterium and Janthinobacterium producing violacein were considered as opportunistic pathogens.They offten caused serious diseases,and their pigment yields were not high.It is urgent to screen and isolate novel strains which could produce violacein.Recently,many new species of Massilia were isolated from different kinds of environmental sources.Massilia can not only synthesize multiple secondary metabolites and enzymes,but also have many functions such as phosphorus solubilization,degradation of phenantherene and resistance to heavy metals.However,there were few Massilia that can produce violacein.Also,few studies were available on the molecular regulatory mechanism of violacein and multi-omics of Massilia.A bacterial strain with producing violacein was isolated from soil and it represented a novel species of the genus Massilia based on polyphasic approach.The pigment was purified and its structure was identified by spectrum methods.Furthermore,the mechanism of pigment-production of strain SOD was studied.The main results are as follows:1.A dark purple-pigmented,Gram-stain-negative and motile rod-shaped bacterium,designated SOD^T,was isolated from soil by using dilution-plate method and soil extract medium.It was characterized using a polyphasic approach.Phylogenetic analysis based on 16S r RNA gene sequences indicated that strain SOD^Tbelonged to the genus Massilia and showed the highest similarities to Massilia violaceinigra B2^T（99.3%）,followed by Massilia glaciei B448-2^T（98.7%）,Massilia eurypsychrophila CGMCC 1.12828^T（98.6%）and Rugamonas rubra CCM3730^T（97.8%）.Average nucleotide identity and digital DNA–DNA hybridization values between genome sequences of strain SOD^T and the closely related species ranged from 77.1%to 89.3%and from 22.2%to 34.7%.The DNA G+C?content of strain SOD^T was 65.29?mol%.Strain SOD^T contained Q-8 as the major ubiquinone and the dominant fatty acids were summed feature 3(C_16:1ω7c and/or C_15:0iso 2-OH;58.5%),C_16:0（26.8%）and C_18:1ω7c（5.0%）.The major polar lipids were diphosphatidylglycerol,phosphatidylethanolamine and phosphatidylglycerol.On the basis of the evidences presented in this study,strain SOD^T represents a novel species of the genus Massilia,for which the name Massilia atriviolacea sp.nov.is proposed.The type strain is SOD^T（=KCTC 62720^T=LMG 30840^T）.2.Pigment produced by SOD^T was extracted and purified.Then the chemical structure of purified blue fraction I was analyzed by ultraviolet,HPLC,NMR and MS spectrum technology.It was shown that the purified blue fraction I was identified as violacein.The purple pigment was stabile under high temperature and p H6-10,but it was sensitive to light.The results of MTT,cell apoptosis,reactive oxygen species,mitochondrial membrane potential inditicated that the purified violacein had a good inhibitory capacity on He La cells and could induce cell apoptosis by changing mitochondrial membrane potential.3.The genome of strain SOD^T contained a 7 425 094bp circular chromosome including 6596 prdicted protein-coding genes,14 r RNA genes and 87 t RNA genes.The DNA G+C content of strain SOD^T was 65.29 mol%.Compared to the closely related species of the genus Massilia,genome size of strain SOD^T was not the biggest,but it contained the most CDSs,11 secondary metabolites gene clusters,12 genome islands,a level of about 31 Kb prophage fragments,79 CRISPRs,4 kinds of secretion systems,and many two-component signal transduction systems.It also contained all the five genes（Vio ABCDE）needed for the synthesis of violacein and related regulatory genes.4.To understand the temperature regulation mechanism of pigment production of strain SOD^T between 28℃and 33℃,RNA-seq transcriptome analysis and q RT-PCR was performed.When the Log₂（Fold Change）>1 or<-1 and at p<0.05level,the m RNAs were considered as differentially expressed.RNA-seq analysis indicated that 1997 genes displayed statistically significant m RNA level changes;804of the genes displayed increased transcript levels,and 1193 displayed decreased transcript levels.The most prominently up-regulated genes in H group（33℃）encoded stress proteins and transporter proteins,respectively.The most prominently down-regulated genes in H group encoded transcription factors,histidine kinase receptors,regulatory proteins,etc.And both of DEGs encoded a large number of hypothetical proteins.The functions of hypothetical proteins deserve further study.KEGG pathway analysis revealed that the DEGs were linked with flagllar assembly pathway（map02040）,staurosporine biosynthesis pathway（map00404）,nitrogen metabolism pathway（map00910）and quorum sensing pathway（map02024）.Furthermore,compared with the expression pattern of the gene Vio ABCDE in L group which are required for the production of violacein,it was interestingly found that the expression level of SOD＿4174（MFS transporter）in H group was down-regulated.The result of q RT-PCR indicated that the expression level of genes Vio A significantly decreased in H group（33℃）and was consistent with those from RNA-seq.The400bp upstream region of Vio A was cloned,and it exhibited cold inducible promoter activity.Thus,it was presumed potential as tools for molecular biotechnology.