- Short genome report
- Open Access
High quality draft genome of Nakamurella lactea type strain, a rock actinobacterium, and emended description of Nakamurella lactea
© The Author(s). 2017
- Received: 1 March 2016
- Accepted: 6 December 2016
- Published: 6 January 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.
- Rare actinobacteria
- Bioactive natural product
- Next generation sequencing
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
Species Nakamurella lactea
Type strain DLS-10
L-Arabinose, myo-inositol and methyl α-D-mannoside, D-cellobiose, D-fructose, D-glucose, D-galactose, lactose, D-maltose, D-mannitol, D-mannose, L-rhamnose, salicin, sucrose and D-trehalose, D- turanose
Up to 3% NaCl
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.
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 project history
Level 1: Standard Draft
Illumina, Illumina HiSeq 2000
Gene calling method
GenBank Date of Release
Source Material Identifier
GEBA-KMG, Tree of Life
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 .
% of Total
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, Cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
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 .
Emended description of Nakamurella lactea (Lee et al. ) Kim et al. 
The properties are as given in the species description by Kim et al.  with the following emendation. Based on the genomic data the G + C content is 68.9%.
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.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Sen A, Daubin V, Abrouk D, Gifford I, Berry AM, Normand P. Phylogeny of the class Actinobacteria revisited in the light of complete genomes. The orders 'Frankiales' and Micrococcales should be split into coherent entities: proposal of Frankiales ord. nov., Geodermatophilales ord. nov., Acidothermales ord. nov. and Nakamurellales ord. nov. Int J Syst Evol Microbiol. 2014;64:3821–32.View ArticlePubMedGoogle Scholar
- Stackebrandt E, Rainey FA, Ward-Rainey NL. Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol. 1997;47:479–91.View ArticleGoogle Scholar
- Tao TS, Yue YY, Chen WX, Chen WF. Proposal of Nakamurella gen. nov. as a substitute for the bacterial genus Microsphaera Yoshimi et al. 1996 and Nakamurellaceae fam. nov. as a substitute for the illegitimate bacterial family Microsphaeraceae. Rainey et al. 1997. Int J Syst Evol Microbiol. 2004;54:999–1000.View ArticlePubMedGoogle Scholar
- Kim KK, Lee JS. The Family Nakamurellaceae. In: Rosenberg E, Delong EF, Lory S, Stackebrandt E, Thompson F, editors. The Prokaryotes-Actinobacteria. Heidelberg: Springer-Verlag Berlin; 2014. doi:10.1007/978-3-642-30138-4_278.Google Scholar
- Yoshimi Y, Hiraishi A, Nakamura L. Isolation and characterization of Microsphaera multipartita gen. nov., sp. nov., a polysaccharide-accumulating Gram-positive bacterium from activated sludge. Int J Syst Bacteriol. 1996;46:519–25.View ArticleGoogle Scholar
- Kim KK, Lee KC, Lee JS. Nakamurella panacisegetis sp. nov. and proposal for reclassification of Humicoccus flavidus Yoon et al., 2007 and Saxeibacter lacteus Lee et al., 2008 as Nakamurella flavida comb. nov. and Nakamurella lactea comb. nov. Syst App Micro. 2012;35:291–6.View ArticleGoogle Scholar
- List Editor. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2012;62:2549–54.View ArticleGoogle Scholar
- Yoon JH, Kang SJ, Jung SY, Oh TK. Humicoccus flavidus gen. nov., sp. nov., isolated from soil. Int J Syst Evol Microbiol. 2007;57:56–9.View ArticlePubMedGoogle Scholar
- Lee SD, Park SK, Yun YW, Lee DW. Saxeibacter lacteus gen. nov., sp. nov., an actinobacterium isolated from rock. Int J Syst Evol Micro. 2008;58:906–9.View ArticleGoogle Scholar
- Tice H, Mayilra S, Sims D, Lapidus A, Nolan M, Lucas S, et al. Complete genome sequence of Nakamurella multipartita type strain (Y-104T). Stand Genomic Sci. 2010;2:168–75.View ArticlePubMedPubMed CentralGoogle Scholar
- Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics. 2013;14:60.View ArticlePubMedPubMed CentralGoogle Scholar
- Meier-Kolthoff JP, Hahnke RL, Petersen J, Scheuner C, Michael V, Fiebig A, et al. Complete genome sequence of DSM 30083T, the type strain (U5/41T) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy. Stand Genomic Sci. 2014;10:2.View ArticleGoogle Scholar
- Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312–3.View ArticlePubMedPubMed CentralGoogle Scholar
- Goloboff PA, Farris JS, Nixon KC. TNT, a free program for phylogenetic analysis. Cladistics. 2008;24:774–86.View ArticleGoogle Scholar
- Pattengale ND, Alipour M, Bininda-Emonds ORP, Moret BME, Stamatakis A. How many bootstrap replicates are necessary? J Comput Biol. 2010;17:337–54.View ArticlePubMedGoogle Scholar
- Göker M, Klenk HP. Phylogeny-driven target selection for large-scale genome-sequencing (and other) projects. Stand Genomic Sci. 2013;8:360–74.View ArticlePubMedPubMed CentralGoogle Scholar
- Klenk HP, Göker M. En route to a genome-based classification of Archaea and Bacteria? Syst Appl Microbiol. 2010;33:175–82.View ArticlePubMedGoogle Scholar
- Kyrpides NC, Woyke T, Eisen JA, Garrity G, Lilburn TG, Beck BJ, Whitman WB, Hugenholtz P, Klenk HP. Genomic Encyclopedia of Type Strains, Phase I: The one thousand microbial genomes (KMG-I) project. Stand Genomic Sci. 2013;17:9(3):1278–84.Google Scholar
- Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, et al. A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature. 2009;462:1056–60.View ArticlePubMedPubMed CentralGoogle Scholar
- Piao H, Froula J, Du C, Kim TW, Hawley ER, Bauer S, et al. Identification of novel biomass-degrading enzymes from genomic dark matter: Populating genomic sequence space with functional annotation. Biotechnol Bioeng. 2014;111:1550–65.View ArticlePubMedGoogle Scholar
- Kyrpides NC, Hugenholtz P, Eisen JA, Woyke T, Göker M, Parker CT, et al. Genomic Encyclopaedia of Bacteria and Archaea: sequencing a myriad of type strains. PLoS Biol. 2014;12. doi:10.1371/journal.pbio.1001920.Google Scholar
- Field D, Amaral-Zettler L, Cochrane G, Cole JR, Dawyndt P, Garrity GM, et al. The Genomic Standards Consortium. PLoS Biol. 2011;9:8–10.View ArticleGoogle Scholar
- Reddy TBK, Thomas AD, Stamatis D, Bertsch J, Isbandi M, Jansson J, et al. The Genomes OnLine Database (GOLD) v.5: a metadata management system based on a four level (meta) genome project classification. Nucleic Acids Res. 2015;43(Database issue):D1099–106.Google Scholar
- List of growth media used at DSMZ. http://www.dsmz.de/microorganisms/media_list.php.Google Scholar
- Gemeinholzer B, Dröge G, Zetzsche H, Haszprunar G, Klenk H-P, Güntsch A, et al. Bank Network: The start from a German initiative. Biopreserv Biobank. 2011;9:51–5.View ArticlePubMedGoogle Scholar
- Bennett S. Solexa Ltd. Pharmacogenomics. 2004;5:433–8.View ArticlePubMedGoogle Scholar
- Zerbino D, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18:821–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Gnerre S, MacCallum I. High–quality draft assemblies of mammalian genomes from massively parallel sequence data. Proc Natl Acad Sci U S A. 2011;108:1513–8.View ArticlePubMedGoogle Scholar
- Huntemann M, Ivanova NN, Mavromatis K, Tripp HJ, Paez-Espino D, Palaniappan K, et al. The Standard Operating Procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4). Stand Genomic Sci. 2015;10:86.View ArticlePubMedPubMed CentralGoogle Scholar
- Markowitz VM, Ivanova NN, Chen IMA, Chu K, Kyrpides NC. IMG-ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009;25:2271–8.View ArticlePubMedGoogle Scholar
- Lowe TM, Eddy SR. tRNAscan–SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997;25:955–64.View ArticlePubMedPubMed CentralGoogle Scholar
- Pruesse E, Quast C, Knittel, Fuchs B, Ludwig W, Peplies J, Glckner FO. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nuc Acids Res. 2007;35:2188–7196.View ArticleGoogle Scholar
- INFERNAL. Inference of RNA alignments. http://infernal.janelia.org.Google Scholar
- Chen IM, Markowitz VM, Palaniappan K, Szeto E, Chu K, Huang J, Ratner A, Pillay M, Hadjithomas M, Huntemann M, Mikhailova N, Ovchinnikova G, Ivanova NN, Kyrpides NC. BMC Genomics. 2016;17:307.View ArticlePubMedPubMed CentralGoogle Scholar
- Markowitz VM, Chen IM, Palaniappan K, Chu K, Szeto E, Pillay M, et al. IMG 4 version of the integrated microbial genomes comparative analysis system. Nucleic Acids Res. 2014;42:D560–7.View ArticlePubMedGoogle Scholar
- Joint Genome Institute. http://jgi.doe.gov/Google Scholar
- Meier-Kolthoff JP, Klenk HP, Göker M. Taxonomic use of the G + C content and DNA:DNA hybridization in the genomic age. Int J Syst Evol Microbiol. 2014;64:352–6.View ArticlePubMedGoogle Scholar
- Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990;87:4576–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Goodfellow M. Phylum XXVI Actinobacteria phyl. nov. In: Goodfellow M, Kämpfer P, Busse HJ, Trujillo ME, Suzuki K, Ludwig W, Whitman WB, editors. Bergey's Manual of Systematic Bacteriology. New York: Springer; 2012. 5, Part A:33.View ArticleGoogle Scholar
- Zhi XY, LiW J, Stackebrandt E. An update of the structure and 16S rRNAa gene sequence-based definition of higher ranks of the class Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher taxa. Int J Syst Evol Microbiol. 2009;59:589–608.View ArticlePubMedGoogle Scholar
- Ashburner M, Ball CA, Blake JA, Botstein D, But-ler H, Cherry JM, et al. Gene Ontology: tool for the unification of biology. Nat Genet. 2000;25:25–9.View ArticlePubMedPubMed CentralGoogle Scholar