Currently, Lactococcus lactis contains four subspecies: L. lactis subsp. lactis , L. lactis subsp. hordniae , L. lactis subsp. cremoris and L. lactis subsp. tructae . In the study of Pérez et al., these four subspecies could be clearly divided into two groups based on recA sequence analysis: L. lactis subsp. lactis and L. lactis subsp. hordniae ; L. lactis subsp. cremoris and L. lactis subsp. tructae . The two groups had a relatively low DNA–DNA hybridization value (about 60 %). In the present study, the taxonomic position of L. lactis subsp. cremoris and L. lactis subsp. tructae was re-examined based on sequence analyses of 16S rRNA, rpoB, recA and pheS genes, average nucleotide identity (ANI) values and digital DNA–DNA hybridization (dDDH) values. The result of 16S rRNA gene sequence analysis indicated that L. lactis subsp. cremoris NCDO 607T and L. lactis subsp. tructae L105T were phylogenetically related to the type strains of L. lactis subsp. hordniae , L. lactis subsp. lactis , Lactococcus taiwanensis , Lactococcus kimchii , Lactococcus allomyrinae , Lactococcus protaetiae, Lactococcus hircilactis , Lactococcus fujiensis and Lactococcus nasutitermitis . The 16S rRNA gene, rpoB, recA, pheS and concatenated rpoB, recA and pheS sequence similarities, ANI values, and dDDH values between the type strains of L. lactis subsp. cremoris , L. lactis subsp. tructae and phylogenetically related species were 93.5–99.4 %, 83.3–97.6 %, 80.6–92.4 %, 79.7–92.7 %, 83.5–94.3 %, 72.4–86.9 % and 21.4–32.5 %, respectively. Lower than 95–96 % ANI values and lower than 70 % dDDH values confirmed that the type strains of L. lactis subsp. cremoris and L. lactis subsp. tructae represent a novel species in the genus Lactococcus . Because L. lactis subsp. cremoris was proposed and validated before L. lactis subsp. tructae , L. lactis subsp. cremoris is elevated to the species level as Lactococcus cremoris sp. nov. and L. lactis subsp. tructae is transferred to L. cremoris as L. cremoris subsp. tructae comb. nov. The type strain of L. cremoris sp. nov. is NCDO 607T (=ATCC 19257T=DSM 20069T=JCM 16167T=LMG 6897T=NBRC 100676T). The type strain of L. cremoris subsp. tructae comb. nov. is L105T (=NBRC 110453T=DSM 21502T=JCM 31125T=LMG 24662T).
Four Gram-stain-positive bacterial strains were isolated from the gut of honeybee (Apis mellifera) in China. These strains were characterized using a polyphasic taxonomic approach. The data demonstrated that three of the four strains represented two novel species of the genus Lactobacillus , strains F306-1T and F551-2T were designated as the type strains. Results of 16S rRNA gene sequence analysis indicated that strains F306-1T, F447 and F551-2T were phylogenetically related to the type strains of Lactobacillus kimbladii and Lactobacillus kullabergensis , having 99.1–99.7 % 16S rRNA gene sequence (about 1400 bp) similarities. The phylogenetic tree based on concatenated pheS, rpoA, gyrB, hsp60, recA, rpoB and tuf sequences (4114 bp) and the phylogenomic tree based on whole genome sequences indicated that strains F306-1T and F447 were most closely related to L. kullabergensis Biut2NT, and strain F551-2T was most closely related to L. kimbladii Hma2NT. Strains F306-1T and F447 shared 99.9 % average nucleotide identity (ANI), 99.7 % digital DNA–DNA hybridization (dDDH) and 99.9 % average amino acid identity (AAI) values, indicating that they belong to the same species. Strain F306-1T exhibited the highest ANI (94.4 %), dDDH (56.7 %) and AAI (94.7 %) values to L. kullabergensis Biut2NT. Strain F551-2T had the highest ANI (94.0 %), dDDH (54.3 %) and AAI (95.8 %) values with L. kimbladii Hma2NT. Acid production from amygdalin, maltose, starch, gentiobiose and turanose, activity of esterase (C4) and α-glucosidase, growth with 3 % NaCl at 37 °C under strict anaerobic condition (on mMRS agar plates), and growth with 1–6% NaCl at 37 °C under aerobic condition (on mMRS agar plates supplemented with 0.05 % cysteine or with 1 % cysteine and 2 % fructose) could differentiate strains F306-1T and F447 from L. kullabergensis DSM 26262T. Acid production from d-glucose, arbutin and gentiobiose, growth with 3 % NaCl at 37 °C under strict anaerobic condition (on mMRS agar plates), and growth at 45 °C under strict anaerobic condition (on mMRS agar plates) could differentiate strain F551-2T from L. kimbladii DSM 26263T. Based upon the data obtained in the present study, two novel species, Lactobacillus huangpiensis sp. nov. and Lactobacillus laiwuensis sp. nov., are proposed and the type strains are F306-1T (=LMG 32144T=JCM 34361T=CCTCC AB 2020300T) and F551-2T (=JCM 34502T=CCTCC AB 2021027T), respectively.
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