- Short genome report
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High quality draft genome of Nakamurella lactea type strain, a rock actinobacterium, and emended description of Nakamurella lactea
Standards in Genomic Sciencesvolume 12, Article number: 4 (2017)
Nakamurella lactea DLS-10T , isolated from rock in Korea, is one of the four type strains of the genus Nakamurella. In this study, we describe the high quality draft genome of N. lactea DLS-10T and its annotation. A summary of phenotypic data collected from previously published studies was also included. The genome of strain DLS-10T presents a size of 5.82 Mpb, 5100 protein coding genes, and a C + G content of 68.9%. Based on the genome analysis, emended description of N. lactea in terms of G + C content was also proposed.
The genus Nakamurella , belong to the order Nakamurellales  and is one of the rare genera in the class Actinobacteria . The genus Nakamurella is the sole and type genus of the family Nakamurellaceae , which replaced the family Microsphaeraceae  in 2004 . The genus and family names were assigned in honour of the microbiologist Kazonuri Nakamura .
Only four species with validly published names, Nakamurella multipartita [3, 5], Nakamurella panacisegetis [6, 7], Nakamurella flavida [6–8], and Nakamurella lactea [6, 7, 9], have been described, and only the genome of Nakamurella multipartita has been published .
N. lactea was originally described as Saxeibacter lacteus , which was the type species of one of the three genera comprising in the family Nakamurellaceae . Then, in the light of the 16S rRNA gene and rpoB gene sequences similarities and chemotaxonomic features , the species was reclassified into the genus Nakamurella . Nakamurella lactea is represented by the type strain DLS-10T (= DSM 19367 T = JCM 16024T = KCTC 19285T ).
The availability of the genome of one more species in the genus will provide vital baseline information for better understanding of the ecology of these rare actinobacteria and their potential as source of bioactive natural products. In the present study, we summarise the phenotypic, physiological and chemotaxonomic, features of N. lactea DLS-10T together with the genomic data.
Classification and features
N. lactea DLS-10T was isolated from a rock collected on the parasitic volcano Darangshi Oreum at 300 m above sea level in Jeju island, Republic of Korea (latitude 33.51, longitude 126.52) . It has been shown by Lee et al.  and Kim et al. [4, 6] that its cells are aerobic, non-motile, non-spore and non-mycelium forming short rods with 0.4–0.7 μm and 0.9–1.0 μm of cell diameter and length, respectively (Fig. 1), producing cream-coloured colonies on TSA medium. A summary of the classification and general features of N. lactea strain DLS-10T is presented in the Table 1. Additional phenotypic features can be found in Lee et al. and Kim et al. [6, 9].
Only four species isolated from soil ( N. panacisegetis and N. flavida ), rock ( N. lactea ) and sludge (N. mutipartita), respectively, are currently classified in the genus. Due to this limited number of the characterised species, the ecological diversity as well as the biotechnological potential of the members of the genus Nakamurella remain to be studied in depth.
Phylogenies based on 16S rRNA gene sequences included in this manuscript were performed using the GGDC web server  implementation of the DSMZ phylogenomics pipeline . The multiple alignment was created with MUSCLE  and maximum likelihood (ML) and maximum parsimony (MP) trees were inferred from it with RAxML  and TNT , respectively. For ML, rapid bootstrapping in conjunction with the autoMRE bootstopping criterion  and subsequent search for the best tree was used; for MP, 1000 bootstrapping replicates were used in conjunction with tree-bisection-and-reconnection branch swapping and ten random sequence addition replicates. This analysis shows the family Nakamurellaceae  as the sister group of the families Cryptosporangiaceae , Sporichthyaceae , and Geodermatophilaceae . The monophyly of the genus Nakamurella was supported by (close to) maximum bootstrap values under ML and MP (Fig. 2).
Chemotaxonomic data (optional, Heading 3)
Glucose, mannose, ribose and rhamnose were detected as the whole-cell sugars . The pattern of polar lipid contains diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, aminophospholipid, five unidentified phosphoglycolipids, and one unidentified glycolipid .
The diagnostic peptidoglican is the meso-diaminopimelic acid. The major fatty acids are anteiso-C15:0, C16:0, iso-C16:0, and anteiso-C17:0 . MK-8(H4) and MK-9(H4) are the predominant menaquinones but MK-7(H4) was also revealed in a low amount .
Genome sequencing information
Genome project history
N. lactea DLS-10T (DSM 19367 T) was selected for sequencing on the basis of its phylogenetic position [17, 18], and is part of Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes project , a follow-up of the Genomic Encyclopedia of Bacteria and Archaea pilot project , which aims at increasing the sequencing coverage of key reference microbial genomes and to generate a large genomic basis for the discovery of genes encoding novel enzymes . KMG-I is the first of the production phases of the “Genomic Encyclopedia of Bacteria and Archaea: sequencing a myriad of type strains” initiative  and a Genomic Standards Consortium project . The project and the genome sequence are deposited in the Genome OnLine Database  and Genbank under the accession number AUFT00000000.1. In Table 2, we summarize genome sequence project.
Growth conditions and genomic DNA preparation
A N. lactea DLS-10T culture was prepared in DSM medium 65  at 28 °C. Genomic DNA was extracted using MasterPure™ Gram Positive DNA Purification Kit (Epicentre MGP04100) following the standard protocol provided by the manufacturer but modified by the incubation on ice overnight on a shaker, the use of additional 1 μl proteinase K, and the addition of 7.5 units achromopeptidase, 7.5 μg/μl lysostaphine, 1050.0 units lysozyme, and 7.5 units mutanolysine. DNA is available from DSMZ through the DNA Bank Network .
Genome sequencing and assembly
The draft genome of N. lactea DLS-10T was generated at the DOE Joint genome Institute (JGI) using the Illumina technology . An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform, which generated 13,910,936 reads totalling 2,086.6 Mb. All general aspects of library construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov. All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artefacts (unpublished results). Following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet (version 1.1.04) , (2) 1–3 kb simulated paired end reads were created from Velvet contigs using wgsim (https://github.com/lh3/wgsim), (3) Illumina reads were assembled with simulated read pairs using Allpaths–LG (version r42328) . Parameters for assembly steps were: 1) Velvet (velveth:63 –shortPaired and velvetg: −very clean yes –exportFiltered yes –min contig lgth 500 –scaffolding no–cov cutoff 10) 2) wgsim (−e 0 –1 100 –2 100 –r 0 –R 0 –X 0) 3) Allpaths–LG (PrepareAllpathsInputs:PHRED 64 = 1 PLOIDY = 1 FRAG COVERAGE = 125 JUMP COVERAGE = 25 LONG JUMP COV = 50, RunAllpathsLG: THREADS = 8 RUN = std shredpairs TARGETS = standard VAPI WARN ONLY = True OVERWRITE = True). The final draft assembly contained 31 contigs in 27 scaffolds. The total size of the genome is 5.8 Mb and the final assembly is based on 712.8 Mb of Illumina data, which provides an average 122.5X coverage of the genome.
The complete genome sequence was annotated using the JGI Prokaryotic Automatic Annotation Pipeline  with additional manual review using the Integrated Microbial Genomes - Expert Review (IMG-ER) platform . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non redundant database, UniProt, TIGRFam, Pfam, KEGG, COG, and InterPro databases. The tRNAScanSE tool  was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA . Other non–coding RNAs such as the RNA components of the protein secretion complex and the RNase P were identified by searching the genome for the corresponding Rfam profiles using INFERNAL . Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG) platform [35, 36] developed by the Joint Genome Institute, Walnut Creek, CA, USA .
The genome of N. lactea will be used to study, for the first time, its potential as bioactive natural products source and the correlation between the rare soil bacteria and their habitat. According to , the within-species deviation in genomic G + C content is at most 1%. The range of 70.4–74.3% given in by Kim et al.  is thus too broad and too deviating from the 68.9% calculated in the genome sequence, much like the value 74.3% provided by Lee et al. . This calls for an emendation of the species description .
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We thank Katja Steenblock (DSMZ) for her help in preparing the culture of N. lactea DSM 19367 T and Evelyne Brambilla (DSMZ) for her contribution in the DNA extraction. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported under Contract No. DE-AC02-05CH11231.
IN and HPK conceived of the study and participated in its design and coordination. IN, LC and MCMC collaborated in acquisition of data, analysis of them and drafted the manuscript. MG and RM performed the phylogenetic analysis and SEM images, respectively. TW and NCK participated in genome sequencing, annotation and analysis. All authors contributed in improving the quality of the manuscript and approved the final version.
The authors declare that they have no competing interests.