Polyphasic Identification Of 16 Novel Species Of Lactobacillus

Naturally fermented pickle and naturally fermented yoghurt are traditional fermented foods in China.They have a long history of brewing and eating,which contains rich resources of lactic acid bacteria.In this study,we aimed to identify 15 potential novel species of Lactobacillus isolated from pickles in Northeast China and 1 potential novel species of Lactobacillus isolated from yogurt in Tibet by multiphase identification and to determine their taxonomic status.The identification methods used mainly include 16S r RNA gene sequence analysis,phe S and rpo A gene sequence analysis,ANI and d DDH analysis based on whole genome sequencing,API50CH sugar fermentation experiment,cell wall amino acid analysis,physiological and biochemical experiments and fatty acid methyl ester analysis.Strains 159-4^T,47-3^T,73-4^T and 247-3^T were related to type strains of Lactobacillus porciane,Lactobacillus manihotivorans,Lactobacillus nasuensis,Lactobacillus camelliae,Lactobacillus pantheris,Lactobacillus thailandensis,Lactobacillus sharpeae and Lactobacillus songhuajiangensis.Among them,the 16S r RNA gene sequence similarity between strains 159-4^T,247-3^T and the type strains were less than 98.7%,which were determined as two novel species.The 16S r RNA gene sequence similarity of strain 47-3^T and L.porciane R-42633^T?98.9%?was higher than the cut-off threshold of 98.7%;phe S and rpo A gene sequence similarity of strain 47-3^Tand L.porciane R-42633^T was lower,74.0%and 90.3%,respectively;ANI value was 73.8%,lower than the cut-off threshold 95%,indicating that strain 47-3^T was a novel species.16S r RNA gene sequence similarity between strain 73-4^T and L.camelliae MCH3-1^T?98.9%?was higher than98.7%;phe S and rpo A gene sequence similarity of 73-4^T and L.camelliae MCH3-1^T was lower,85.5%and 92.8%,respectively;ANI value was 84.9%,lower than the critical value of fixed species,indicating that strain 73-4^T was a novel species.Strains 143-4?a?^T and 33-1^T were related to type strains of Lactobacillus pantheris,Lactobacillus thailandensis,Lactobacillus songhuajiangensis,Lactobacillus sharpeae,Lactobacillus rhamnosus,Lactobacillus paracasei,Lactobacillus casei and Lactobacillus zeae.the16S r RNA gene sequence similarity between the strains was 91.9-96.5%,which was lower than the cut-off threshold of 98.7%,identified strains 143-4?a?^T and 33-1^T are 2 novel species;phe S gene sequence similarity between the strains was 73.9-82.5%;rpo A gene sequence similarity between the strains was 68.9-81.2%.Strains 257-1^T and 187-3^T were related to type strains of Lactobacillus coryniformis,Lactobacillus iwatensis,Lactobacillus backii,Lactobacillus rennini and Lactobacillus bifermentans.The 16S r RNA gene sequence similarity between strains 257-1^T and 187-3^T?98.8%?,strains 257-1^T and L.coryniformis DSM 20253^T?99.3%?was higher than 98.7%,but phe S and rpo A gene sequence similarity of strains 257-1^T and 187-3^T?phe S-77.5%,rpo A-91.7%?,strains257-1^T and L.coryniformis DSM 20253^T?phe S-78.7%,rpo A-96.2%?was lower;ANI values between strains 257-1^T and 187-3^T,strains 257-1^T and L.coryniformis DSM 20253^T were 79.0%and 78.8%,lower than the critical value of 95%,Indicated that strain 257-1^T was a novel species.16S r RNA gene sequence similarity between strain 187-3^T and L.coryniformis DSM 20253^T?99.0%?was higher than 98.7%,but phe S and rpo A gene sequence similarity was lower,81.8%and92.1%,respectively;The ANI value was 79.3%,which was lower than the cut-off threshold of95%,indicated that strain 187-3^T was a novel species.Strains 220-4^T,151-2B^T,M1530-1^T,137-3^T,244-4^T,218-10^T,218-6^T and 112-3^T were related to type strains of Lactobacillus paucivorans,Lactobacillus brevis,Lactobacillus hammesii,Lactobacillus senmaizukei,Lactobacillus parabrevis,Lactobacillus yonginesis,Lactobacillus koreensis and Lactobacillus cerevisiae.Among them,strains 220-4^T and 151-2B^T?99.9%?,strains220-4^T and L.paucivorans TMW 1.1424^T?99.9%?,strains 151-2B^T and L.paucivorans TMW1.1424^T?99.7%?,strains M1530-1^T and L.brevis ATCC 14869^T?99.0%?,strains M1530-1^T and112-3^T?98.7%?,strains M1530-1^T and L.cerevisiae TUM BP 140423000-2250^T?98.7%?,strains137-3^T and 244-4^T?99.3%?,strains 137-3^T and 218-10^T?98.7%?,strains 137-3^T and L.parabrevis LMG 11984^T?99.6%?,strains 244-4^T and 218-10^T?98.8%?,strains 244-4^T and 112-3^T?98.9%?,strains 244-4^T and L.cerevisiae TUM BP 140423000-2250^T?98.9%?,strains 244-4^T and L.hammesii TMW 1.1236^T?99.0%?,strains 244-4^T and L.koreensis DCY50^T?98.7%?,strains244-4^T and L.parabrevis LMG 11984^T?99.7%?,strains 218-10^T and 218-6^T?98.7%?,strains218-10^T and 112-3^T?99.1%?,strains 218-10^T and L.cerevisiae TUM BP 140423000-2250^T?99.0%?,strains 218-10^T and L.koreensis DCY50^T?99.1%?,strains 218-10^T and L.parabrevis LMG 11984^T?98.7%?,strains 218-10^T and L.yonginesis THK-V8^T?98.9%?,strains 218-6^T and112-3^T?98.8%?,strains 218-6^T and L.cerevisiae TUM BP 140423000-2250^T?98.8%?,strains218-6^T and L.koreensis DCY50^T?99.3%?,strain 112-3^T and L.cerevisiae TUM BP140423000-2250^T?99.9%?,strain 112-3^T and L.koreensis DCY50^T?99.5%?,strain 112-3^T and L.parabrevis LMG 11984^T?98.8%?,strain 112-3^T and L.yonginesis THK-V8^T?98.7%?,The 16S r RNA gene sequence similarity of them were higher than 98.7%,but the phe S gene sequence similarity of 28 pairs of strains was lower,79.6-93.9%;The ANI value was 75.9-92.7%,which was lower than the critical value of fixed species 95%,indicated that strains 220-4^T,151-2B^T,M1530-1^T,137-3^T,244-4^T,218-10^T,218-6^T and 112-3^T were novel species.The DNAG+C mol%of 159-4^T,47-3^T,257-1^T,187-3^T,220-4^T,151-2B^T,137-3^T,244-4^T,218-10^T,218-6^T,112-3^T,73-4^T,247-3^T,143-4?a?^T,33-1^T and M1530-1^T were 58.3%,50.9%,42.6%,42.7%,49.2%,47.5%,49.8%,48.6%,47.7%,47.4%,50.3%,55.5%,53.0%,58.0%,55.4%and 50.5%,respectively.The major fatty acids of strains 159-4^T,247-3^T,143-4?a?^T and 33-1^T were C_16:0 and C_18:1?9c.The major fatty acids of strains 73-4^T,257-1^T,151-2B^T and 218-6^T were C_16:0,C_18:1?9c and C_19:1?6c/C_19:1cyclo?10c.The major fatty acids of strains 47-3^T,220-4^T,M1530-1^T,244-4^T,218-10^T and 112-3^T were C_16:0,C_18:1?9c,C_19:1?6c/C_19:0 cyclo?10c and C_18:1?6c/C_18:1?7c.The major fatty acids of strains 137-3^T was C_16:0,C_18:1?9c and C_18:1?6c/C_18:1?7c.The major fatty acids of strains 187-3^T was C_14:0,C_16:0,C_18:1?9c and C_19:1?6c/C_19:0 cyclo?10c.Based upon the data of polyphasic characterization obtained in the present study,16 novel species have been named 159-4^T?Lactobacillus jixianensis sp.nov.?,47-3^T?Lactobacillus baoqingensis sp.nov.?,257-1^T?Lactobacillus jiayinensis sp.nov.?,187-3^T?Lactobacillus zhaoyuanensis sp.nov.?,220-4^T?Lactobacillus lindianensis sp.nov.?,151-2B^T?Lactobacillus huananensis sp.nov.?,137-3^T?Lactobacillus tangyuanensis sp.nov.?,244-4^T?Lactobacillus fuyuanensis sp.nov.?,218-10^T?Lactobacillus tongjiangensis sp.nov.?,218-6^T?Lactobacillus fujinensis sp.nov.?,112-3^T?Lactobacillus mulengensis sp.nov.?,73-4^T?Lactobacillus hegangensis sp.nov.?,247-3^T?Lactobacillus suibinensis sp.nov.?,143-4?a?^T?Lactobacillus daqingensis sp.nov.?,33-1^T?Lactobacillus yichunensis sp.nov.?and M1530-1^T?Lactobacillus angrenensis sp.nov.?.