Nakamurella leprariae sp. nov., isolated from a lichen sample

A novel actinobacterium, YIM 132084T, was isolated from Lepraria sp. lichen collected from Yunnan province, south-west PR China and identified by a polyphasic taxonomic approach. The strain was Gram-stain-positive, aerobic, catalase-positive, oxidase-negative, non-motile and coccus-shaped. Colonies were round, convex, smooth and light orange yellow in color. It grew at 10–40 °C (optimum 28 °C), at pH 6.0–11.0 (optimum pH 7.0) and in the presence of 0–4% NaCl (optimum 0%). Strain YIM 132084T comprised diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol as the major polar lipids, MK-8(H4) as the predominant menaquinone, and anteiso-C15:0, anteiso-C17:0, iso-C15:0 and iso-C16:0 as major fatty acids. Strain YIM 132084T had meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan, and mannose, ribose, glucose and rhamnose as whole-cell sugars. The 16S rRNA gene sequence showed high level of similarity with Nakamurella flavida KCTC 19127T (97.7%) and Nakamurella flava CGMCC 4.7524T (97.7%). The G + C content of the genomic DNA was 72.4 mol%. Based on draft genome sequences, strain YIM 132084T showed an average nucleotide identity value of 76.1% and 74.9%, a digital DNA–DNA hybridization value of 20.9% and 20.6% with the reference strains Nakamurella flavida and Nakamurella flava, respectively. The results of the phenotypic, chemotaxonomic and phylogenetic analyses showed that strain YIM 132084T represents a novel species of the genus Nakamurella, for which the name Nakamurella leprariae sp. nov. is proposed. The type strain is YIM 132084T (= CGMCC 4.7667T = NBRC 114280T = KCTC 49367T).

De-Feng An and Shao-Juan Yang equally contributed to this work.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM132084 T is MZ050064 and the genome sequence is JAERWK000000000.

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19 Page 2 of 7 lichen (Jiang et al. 2020), automobile air conditioning system (Chaudhary et al. 2021), plant (Yan et al. 2020) and bark (Tuo et al. 2016). At the time of writing, the genus Nakamurella is composed of 11 species with validly published names and 2 species with not validly published names (https:// www. bacte rio. net/ genus/ nakam urella), and Nakamurella multipartita is the type species of this genus. During an investigation on the diversity of cultivable actinobacteria from lichen samples collected in Yunnan province, southwest PR China, a new actinobacterium strain YIM 132084 T was isolated from Lepraria sp.. The strain was identified by a polyphasic approach, which indicated that it represented a new species of the genus Nakamurella.

Isolation and culture of strains
The lichen Lepraria sp. sample was collected from Yunnan province (99° 39′ E, 22° 23′ N), south-west PR China. The lichen sample was transferred into a sterile paper bag and air-dried at 28 °C for 1 week, then washed three times with sterile water and homogenized with 18 ml of sterile 0.1% Na 4 P 2 O 7 using a sterile glass homogenizer. Strain YIM 132084 T was isolated using a standard dilution plate method on humic acid-vitamin (HV) agar (Hayakawa and Nonomura 1987). An isolated colony was selected and further purified on YIM 38 medium ). Strain YIM 132084 T was stored in tubes of aqueous glycerol (20%, v/v) and then in a -80 ℃ refrigerator. The reference strain, Nakamurella flavida KCTC 19127 T was obtained from the Korean Collection for Type Cultures (KCTC), Republic of Korea. Nakamurella flava CGMCC 4.7524 T was obtained from the China General Microbiological Culture Collection Centre (CGMCC).

Phylogenetic analysis and 16S rRNA gene sequencing
Extraction of genomic DNA and PCR amplification of the 16S rRNA gene were done as described by Li et al. (2007). The purified product was cloned using the pEASY-T1 sample cloning kit to obtain the almost-complete 16S rRNA gene sequence. The sequence obtained was compared with available 16S rRNA gene sequences of validly named species using the EzBioCloud server databases (https:// www. ezbio cloud. net/) (Yoon et al. 2017). Phylogenetic trees were constructed with neighbor-joining (Saitou and Nei 1987), maximum-likelihood (Tamura et al. 2011) and maximum parsimony (Fitch 1971) methods using the software package MEGA version 7.0 (Kumar et al. 2016). Kimura's twoparameter model was used to calculate evolutionary distance matrices (Kimura 1980). Bootstrap values were calculated based on 1000 replications (Felsenstein 1985).

Genomic analysis
The draft genome sequence of strain YIM 132084 T and Nakamurella flavida KCTC 19127 T were determined using the Illumina NovaSeq PE150 sequencing platform. The processed reads data were assembled using SOAPdenovo version 2.04 short sequence group assembly software (Li et al. 2008). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were determined based on the genome sequences of YIM 132084 T and closely related species of Nakamurella using the EzBio-Cloud server databases and formula 2 Genome-to-Genome Distance Calculator website (http:// ggdc. dsmz. de/ ggdc. php) (Meier-Kolthoff et al. 2013), respectively. Gene annotations were conducted through the NCBI prokaryotic genome annotation pipeline.

Chemotaxonomic analysis
The strain YIM 132084 T and the reference strains were cultured on YIM 38 agar at 28 °C for 3 days to obtain the amount needed for chemotaxonomic characterization. Polar lipids were extracted and analyzed by the method of Minnikin et al. (1984). Menaquinones were extracted by the method of Collins et al. (1977) and detected by HPLC (Tamaoka et al. 1983). The composition of cellular fatty acids were extracted and analyzed according to the standard protocol of the Microbial Identification System (MIDI) (Sasser 1990;Kämpfer and Kroppenstedt 1996). Cell wall amino acids and whole-cell sugars were extracted, detected and analyzed according to procedures described by Schleifer and Kandler (1972) and Tang et al. (2009).

Phylogenetic analysis and 16S rRNA gene sequencing
The almost-complete 16S rRNA gene sequence of strain YIM 132084 T was 1480 bp (GenBank accession number MZ050064). Phylogenetic analyses based on the 16S rRNA gene sequence of strain YIM 132084 T indicated that it should be recognized as a member of the genus Nakamurella. Strain YIM 132084 T showed a high level of similarity with Nakamurella flavida KCTC 19127 T (97.7%) and Nakamurella flava CGMCC 4.7524 T (97.7%). Phylogenetic trees were constructed by the neighbour-joining, maximumlikelihood and maximum parsimony algorithms 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 132084 T groups within the genus Nakamurella.

Genomic analysis
Based on the draft genome sequencing, strain YIM 132084 T 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 132084 T 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 132084 T was determined to be 72.4 mol% based on the draft genome. The ANI values between strain YIM 132084 T and the type strains of Nakamurella flavida KCTC 19127 T and Nakamurella flava CGMCC 4.7524 T were 76.1 and 74.9%, respectively. The ANI value was lower than the 95.0% cutoff for species demarcation (Richter and Rossello-Mora 2009). The dDDH values between strain YIM 132084 T and the type strains: Nakamurella flavida KCTC 19127 T and Nakamurella flava CGMCC 4.7524 T were 20.9 and 20.6%, respectively, which were much lower than the threshold value (70%) recommended for distinguishing novel prokaryotic species (Elnar et al. 2020).
In conclusion, based on phenotypic, chemotaxonomic and phylogenetic analyses, strain YIM 132084 T is considered to represent a novel species of genus Nakamurella, for which the name Nakamurella leprariae sp. nov. is proposed.
China. The GenBank accession number for the 16S rRNA gene sequence and draft genome sequence of strain YIM 132084 T are MZ050064 and JAERWK000000000, respectively.