The Identification Of New Subspecies Of Lactobacillus Futsaii

During the research of the National Natural Science Foundation of China?31170007,2012.01-2015.12,Rapid detection of Lactobacillus in Chinese traditional pickles and identification of new species and plasmids?,one strain of Lactobacillus,named CQ16Z1,was found in the home-made jamiecosley in Chongqing City.The results of the comparative analyses of the 16S rRNA and rpoA genetic sequences show the strain CQ16Z1 belongs to Lactobacillus futsaii;However,strain CQ16Z1 and Lactobacillus futsaii were very different in homology?74.05%?directly.The result showed that the phes sequence of the strain had a high homology?98.35%?with that of W.cibaria CH2.According to the experimental data,we speculate that CQ16Z1 may be initially identified as the subspecies of Lactobacillus futsaii.ObjectPolyphasic taxonomy approach was used for the identification of the CQ16Z1.Materials and Methods1.Separations and identifications of LactobacillusMRS semi selective medium,Gram staining method and Rapid detection technology of Lactobacillus by PCR technology were used for screening the Lactobacillus strains.2.The phenotype characteristics of the strainsThe strains'shapes characteristics were observed by scanning electron microscope then the Physiological and biochemical experiments of genus Lactobacillus were carried out;the widely used API 50 CHL system in the Identification of Lactobacillus was used for the identification of the Lactobacillus's utilization status of carbohydrates.3.System development characteristicssThe 16S rRNA,rpoA and pheS cloning and sequencing of conserved genes and sequence analyses were conducted and the evolutionary tree constructed.4.Genotype characteristicsThe G+C mol%identification,DNA-DNA hybridization experiment as well as DNA amplified fragment length polymorphism analysis technique were carried out during this phase.5.Identification of chemical classificationThin layer chromatography?TLC?identification method was used for the identification of the Chemical composition of cell wall;Gas chromatography?GC?wasused for the identification of the total fatty acid composition of cells.Results1.Through the Gram staining and Rapid detection technology of Lactobacillus by PCR,a Lactobacillus strain was screened and named CQ16Z1.2.From the phenotype of the strain,the cell size of CQ16Z1was?0.5-0.6?�?2.2-4.6??m,and the arrangement mode was single or paired arrangements under scanning electron microscope after culturing in the MRS mediun at 37?for 24h.It is a gram-positive bacteria,non-liquefied gelatin,non-production of hydrogen sulfideand indole and catalase negative,and this experiement result conforms to the characteristic phenotypic characteristics of the Lactobacillus.3.From the aspects of the phylogenetic,CQ16Z1 belongs to Lactobacillus futsaii,according to the phylogenetic tree of 16S rRNA.Meanwhile,Base on the evolution tree which was made constructed by 16S rRNA,rpoA and pheS,the strain can be classified as a novel subspecies of Lactobacillus futsaii.4.From the aspects of genotype characteristics,the DNA G+C content of the strain is 39.1%,within the 32-53%range of Lactobacillus.The DNA-DNA relatedness values between CQ16Z1 and Lactobacillus futsaii JCM 17355^T was 91.9%.Combing with the result of AFLP,strain CQ16Z1 can be viewed as a novel subspecies of Lactobacillus futsaii.5.For the view of the chemotaxonomy,TLC results showed that LL,meso and DD diaminopimelic acid isomers were all absent from cell wall peptidoglycan of the strian CQ16Z1.Cell wall of CQ16Z1 contained ribose,glucose and.And the major cellular fatty acids of the strain are C_16:0,C_18:1?9c and Summed feature 7(C_19:09:0 cyclo?10c/C_19:1?6c/C_19:1?7c).Comparedwith the result of the strians which relate species shows that strain CQ16Z1 is classified as representing a novel subspecies of Lactobacillus futsaii.ConclusionBased on the feature from those aspects,it is proposed that the new isolated can be classified as a a novel subspecies of Lactobacillus futsaii and the named Lactobacillus futsaii subsp.chongqingii subsp.nov.?type strain CQ16Z1^T=CCTCC AB 2017187^T=KCTC 21089^T?.