Phenotypic and Biochemical Tests
Cells of strain YIM 132084T were Gram-stain-positive, aerobic, non-spore forming, non-motile, coccus-shaped and 0.7-0.9 µm in a diameter (Fig. S1). Colonies were round, convex, smooth and light orange yellow on YIM 38 agar at 28 °C for 3 days. The strain was found to growth on ISP 2, R2A, TSA, LB and YIM 38 agar, No growth occurs on ISP 4 agar. The growth range of strain YIM 132084T at 10-40 ℃ (optimum 28 ℃), at pH 6.0-11.0 (optimum pH 7.0) and at 0-4 % NaCl (optimum 0 %). Hydrolysis of starch, cellulose, tyrosine and casein, Tweens (40, 60 and 80), gelatin liquefaction, H2S production, coagulation and peptonisation of milk were negative, except for hydrolysis of Tween 20. Susceptibility to ofloxacin, vancomycin, ciprofloxacin, norfloxacin, polymyxin B, gentamicin, chloramphenicol, neomycin, kanamycin were positive, susceptibility to ampicillin, ceftriaxone, penicillin G, streptomycin, novobiocin, lincomycin and tetracycline were negative. In the API ZYM tests, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and α-glucosidase activities were positive, but lipase (C14), β-glucuronidase, N-acetyl-β-glucosaminidase and α-fucosidase activities were negative. In the API 20NE strips, hydrolysis of L-arginine, urease, esculine and PNPG were positive, but other tests were negative. In the API 50CH strips, acid was produced from D-glucose, D-fructose, D-mannose, esculin citrate, D-maltose, D-sucrose, D-mycose, D-turanose and D-lyxose. The detailed physiological and biochemical characteristics of strain YIM 132084T are shown in the species description and Table 1.
Phylogenetic Analysis and 16S rRNA Gene Sequencing
The almost-complete 16S rRNA gene sequence of strain YIM 132084T was 1480bp (GenBank accession number MZ050064). Phylogenetic analyses based on the 16S rRNA gene sequence of strain YIM 132084T indicated that it should be recognized as a member of the genus Nakamurella. Strain YIM 132084T showed a high level of similarity with Nakamurella flavida KCTC 19127T (97. 7 %) and Nakamurella flava CGMCC 4.7524T (97.7 %). Phylogenetic trees were constructed by the neighbour-joining, maximum-likelihood algorithms and maximum parsimony based on the 16S rRNA gene sequence (Fig. 1, Fig. S2 and Fig. S3). The results of three tree-making algorithms showed that strain YIM 132084T groups within the genus Nakamurella.
Genomic Analysis
Based on the draft genome sequencing, strain YIM 132084T contained 39 contigs, with a total length of 4,472,446 bp and an N50 length of 232,774 bp (GenBank accession number JAERWK000000000). Based on the genomic annotation, the genome of strain YIM 132084T contains 4,101 genes, included 4,009 protein-coding genes, 3 rRNA genes, 46 tRNA genes, 3 ncRNA genes and 40 pseudogenes. The DNA G+C content of strain YIM 132084T was determined to be 72.4 mol% based on the draft genome. The ANI value between strain YIM 132084T and the type strains of Nakamurella flavida KCTC 19127T and Nakamurella flava CGMCC 4.7524T were 76.1 and 74.9 %, respectively. The ANI value was lower than the 95.0% cut-off for species demarcation (Richter et al. 2009). The dDDH value between strain YIM 132084T and the type strain: Nakamurella flavida KCTC 19127T and Nakamurella flava CGMCC 4.7524T were 20.9 and 20.6 %, respectively, which were much lower than the threshold value (70 %) recommended for distinguishing novel prokaryotic species.
Chemotaxonomic Analysis
The polar lipids profile of strain YIM 132084T contained the predominant compounds diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), an unidentified aminophospholipid (APL), an unidentified glycolipid (GL) and two unidentified phosphoglycolipids (PGL1-2) (Fig. S4). The predominant menaquinone was MK-8(H4) in agreement with the genus Nakamurella (Chaudhary et al. 2021), in addition, MK-8(H2) and MK-7(H4) were detected in strain YIM 132084T. The major cellular fatty acids consist of anteiso-C15:0 (27.9 %), anteiso-C17:0 (20.7 %), iso-C15:0 (12.5 %) and iso-C16:0 (16.0 %), which were similar to other members of the genus Nakamurella. The fatty acids composition and content comparison between strain YIM 132084T and other closely related species of the genus Nakamurella are shown in Table 2. Strain YIM 132084T had meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan, which concurs with the members of the genus Nakamurella. The whole-cell sugars detected in strain YIM 132084T were mannose, ribose, glucose and rhamnose.
In conclusion, based on phenotypic, chemotaxonomic and phylogenetic analyses, strain YIM 132084T is considered to represent a novel species of genus Nakamurella, for which the name Nakamurella leprariae sp. nov. is proposed.
Description of Nakamurella leprariaesp. nov.
Nakamurella leprariae (le.pra’ri.ae. N.L. gen. n. leprariae referring to the isolation of the organism from the lichen genus Lepraria).
Cells are Gram-stain-positive, catalase-positive, oxidase-negative, aerobic, non-motile, non-spore-forming and coccus-shaped (0.7-0.9 µm in diameter). Colonies on YIM 38 medium are round, smooth and convex, light orange yellow in colour. Growth occurs at 10-40 ℃ (optimum 28 ℃), at pH 6.0-11.0 (optimum pH 7.0) and at 0-4 % NaCl (optimum 0 %). Hydrolysis of starch, cellulose, tyrosine and casein, Tweens (40, 60 and 80), gelatin liquefaction, H2S production, coagulation and peptonisation of milk are negative, except for hydrolysis of Tween 20. In the Biolog GEN III system, the following substrates are used as a source of energy: β-methyl-D-glucoside, N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, N-acetyl-D-galactosamine, D-mannose, D-fructose, D-galactose, D-mannitol, D-arabitol, myo-inositol, glycerol, D-glucose-6-phosphate, D-fructose-6-phosphate, D-aspartic acid, L-aspartic acid, L-glutamic acid, L-histidine, L-pyroglutamic acid, L-serine, D-glucuronic acid, D-saccharic acid, L-lactic acid, citric acid, α-keto-glutaric acid, D-malic acid, L-malic acid, bromo-succinic acid. The major polar lipids are diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The predominant menaquinone is MK-8(H4). The major fatty acids are anteiso-C15:0, anteiso-C17:0, iso-C15:0 and iso-C16:0. Strain YIM 132084T contain meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan, and mannose, ribose, glucose and rhamnose as whole-cell sugars. The G+C content of the genomic DNA is 72.4 mol%. The type strain, YIM 132084T (=CGMCC 4.7667T = NBRC 114280T=KCTC 49367T) was isolated from Lepraria sp. lichen collected from Yunnan province, south-west PR China. The GenBank accession number for the 16S rRNA gene sequence and draft genome sequence of strain YIM 132084T are MZ050064 and JAERWK000000000, respectively.