Identification Of Leuconostoc Falkenbergense Sp.mov. And Reclassifacation Of Leuconostoc Gelidum Subsp. Gasicomitatum

There are many traditional lactic acid fermented foods in China,such as naturally fermented kimchi and naturally fermented yogurt,which not only have unique taste and are loved by consumers,but also provide us with abundant lactic acid bacteria resources.Our aim is to identify a potential new species in Leuconostoc and determine its taxonomic status.And reclassifying Leuconostoc gelidum.The identification methods mainly include morphological identification,molecular biological identification,physiological and biochemical characteristics identification,ANI and d DDH calculation and analysis based on whole genome sequencing,API50CH sugar fermentation experiment,cell wall amino acid analysis,fatty acid methyl ester analysis and so on.In the early stage of this study,a Leuconostoc C isolated from natural fermented dairy products has the same rpo A gene sequence as LMG 10779~T,LMG 18969,and the similarity of 16S rRNA gene sequence is 100%and 99.9%,and the similarity of phe S gene sequence is 99.1%and99.4%,indicating that LMG 10779~T,LMG 18969 and C.The strains LMG 10779~T,LMG 18969,and C are related to phylogenetic species Leuconostoc pseudomesenteroides,Leuconostoc litchi,Leuconostoc mesoenteroides subsp.mesoenteroides,The model strains of Leuconostoc mesenteroides subsp.cremoris,Leuconostoc mesenteroides subsp.dextranicum,Leuconostoc mesenteroides subsp.jonggajibkimchii and Leuconostoc suionicum form a branch.Sequence similarity of 16S rRNA,phe S and rpo A between strain C and phylogenetic species were98.7-99.7%,81.8-97.9%and 86.6-95.5%,respectively.sequence similarity of 16S rRNA gene between strain C and phylogenetic model strain was higher than 98.65%,but the sequence similarity of rpo A gene was lower than 95.6%.The values of ANI and d DDH are 75.3-92.6%and20.3-47.9%,which are lower than the threshold of interspecific classification.the above data indicate that strains LMG 10779~T,LMG 18969 and C are the same new species in Leuconostoc.Leuconostoc gelidum subsp.gelidum,Leuconostoc gelidum subsp.gasicomitum and Leuconostoc gelidum subsp.aenigmaticum are three subspecies in Leuconostoc,among which,Leuconostoc gelidum subsp.gelidum and Leuconostoc gelidum subsp.gasicomitum have genome-wide sequences,while Leuconostoc gelidum subsp.aenigmaticum has no genome-wide sequences.Based on their genome-wide sequence analysis,The ANI value and d DDH value between Leuconostoc gelidum subsp.gelidum and Leuconostoc gelidum subsp.gasicomitatum are94.2%and 56.3%,which are lower than the threshold of interspecific classification.The results indicated that Leuconostoc gelidum subsp.gasicomitum is different from Leuconostoc gelidum subsp.gelidum and Leuconostoc gelidum subsp.aenigmaticum.The similarity of 16S rRNA gene sequences between Leuconostoc gelidum subsp.gasicomitum and Leuconostoc gelidum subsp.gelidum and Leuconostoc gelidum subsp.aenigmaticum is 99.5%and 99.7%,the similarity of phe S gene sequences is 92.7%and 95.6%,and the similarity of rpo A gene sequences is 96.0%and 95.9.The Neighbour-joining tree based on the16S rRNA gene sequence of the model strain of Leuconostoc gelidum subsp.gasicomitum and phylogenetic related species shows that it is most related to the phylogeny of Leuconostoc inhae,and its 16S rRNA,phe S and rpo A gene sequences are similar to 99.1%,89.8%and 96.5%,respectively.The ANI and d DDH values are 87.5%and 34.3%,which are lower than the threshold of interspecific classification,indicating that Leuconostoc gelidum subsp.gasicomitum is not the same species as other known species in Leuconostoc,and Leuconostoc gelidum subsp.gasicomitum is an independent species in Leuconostoc.To sum up,we can confirm that strains LMG 10779~T,LMG 18969 and C are new species in the same Leuconostoc genus,and the new species is named Leuconostoc falkenbergense;.Leuconostoc gelidum subsp.gasicomitum is not the same species as Leuconostoc gelidum subsp.gelidum and Leuconostoc gelidum subsp.aenigmaticum.Leuconostoc gelidum subsp.gasicomitum is an independent species in Leuconostoc,and its taxonomic status is re-evaluated by multi-phase identification.