Open Access

Complete genome sequence of Nakamurella multipartita type strain (Y-104T)

  • Hope Tice1,
  • Shanmugam Mayilraj2, 3,
  • David Sims4,
  • Alla Lapidus1,
  • Matt Nolan1,
  • Susan Lucas1,
  • Tijana Glavina Del Rio1,
  • Alex Copeland1,
  • Jan-Fang Cheng1,
  • Linda Meincke4,
  • David Bruce1, 4,
  • Lynne Goodwin1, 4,
  • Sam Pitluck1,
  • Natalia Ivanova1,
  • Konstantinos Mavromatis1,
  • Galina Ovchinnikova1,
  • Amrita Pati1,
  • Amy Chen5,
  • Krishna Palaniappan5,
  • Miriam Land1, 6,
  • Loren Hauser1, 6,
  • Yun-Juan Chang1, 6,
  • Cynthia D. Jeffries1, 6,
  • John C. Detter1, 4,
  • Thomas Brettin1, 4,
  • Manfred Rohde7,
  • Markus Göker2,
  • Jim Bristow1,
  • Jonathan A. Eisen1, 8,
  • Victor Markowitz4,
  • Philip Hugenholtz1,
  • Nikos C. Kyrpides1,
  • Hans-Peter Klenk2 and
  • Feng Chen1
Standards in Genomic Sciences20102:2020168

https://doi.org/10.4056/sigs.721316

Published: 30 April 2010

Abstract

Nakamurella multipartita (Yoshimi et al. 1996) Tao et al. 2004 is the type species of the monospecific genus Nakamurella in the actinobacterial suborder Frankineae. The nonmotile, coccus-shaped strain was isolated from activated sludge acclimated with sugar-containing synthetic wastewater, and is capable of accumulating large amounts of polysaccharides in its cells. Here we describe the features of the organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of a member of the family Nakamurellaceae. The 6,060,298 bp long single replicon genome with its 5415 protein-coding and 56 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Keywords

polysaccharide-accumulatingsepta-formingnonmotileGram-positiveMK-8 (H4)Microsphaeraceae Frankineae GEBA

Introduction

Strain Y-104T [1] (DSM 44233 = ATCC 700099 = JCM 9533) is the type strain of the species Nakamurella multipartita, which is the sole member and type species of the genus Nakamurella [2], the type genus of the family Nacamurellaceae [2]. N. multipartita was first described in 1996 by Yoshimi et al. as polysaccharide-accumulating ‘Microsphaera multipartita’ and type species of the genus ‘Microsphaera’ [1]. Unfortunately, Yoshimi et al. [1] overlooked the priority of the named fungal genus Microsphaera described 145 years earlier [3]. Principle 1(2) of the International Code of Nomenclature of Bacteria (1990 Revision) recommends avoiding the use of names which might cause confusion and therefore grants priority of the fungal genus Microsphaera in the family Erysiphaceae [4], Stackebrandt et al. maintained the illegitimate name when creating the likewise illegitimate family ‘Microsphaeraceae’ in 1997 [5]. In 2004 Tao et al. replaced the illegitimate genus and family names with the legitimate and validly published names Nakamurella and Nakamurellaceae, respectively, in honor of the Japanese microbiologist Kazonuri Nakamura, who also discovered strain Y-104T [2]. Here we present a summary classification and a set of features for N. multipartita strain Y-104T, together with the description of the complete genomic sequencing and annotation.

Classification and features of organism

The environmental diversity of the members of the species N. multipartita appears to be limited. Only one 16S rRNA gene sequence from a Finish indoor isolate (BF0001B070, 96.2% sequence identity) is reported in Genbank [6], as well as two Finish indoor phylotypes (FM872655, 98.2%; FM873571, 96.2%) by Taubel et al., and a phylotype from fresh water sediment of the high altitude Andean Altiplano (northern Chile) with 96.6% sequence identity (EF632902). None of the sequences generated from large scale environmental samplings and genome surveys surpassed 93% sequence identity and were thereby significantly less similar to strain Y-104T than the closest related type strain, DS-52 T of Humicoccus flavidus (95.9%) [7] (status November 2009).

Figure 1 shows the phylogenetic neighborhood of N. multipartita strain Y-104T in a 16S rRNA based tree. The sequences of the two identical 16S rRNA gene copies differ by one nucleotide (C-homopolymer close to 3’-end) from the previously published 16S rRNA sequence generated from JCM 9543 (Y08541).
Figure 1.

Phylogenetic tree highlighting the position of N. multipartita Y-104T relative to the other type strains within the Frankineae. The tree was inferred from 1362 aligned characters [8,9] of the 16S rRNA gene sequence under the maximum likelihood criterion [10] and rooted with the type strain of the order. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1,000 bootstrap replicates if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [11] such as the GEBA organism Geodermatophilus obscurus [12] are shown in blue. Important non-type strains are shown in green [13], and published genomes in bold.

N. multipartita strain Y-104T is aerobic and chemoorganotrophic. Cells are non-motile, non-spore forming, Gram-positive (Table 1) and coccus-shaped [1]. The cells are 0.8 to 3.0 µm in diameter; depending on the growth stage. They occur as singles, in pairs or in small irregular clusters (Figure 2). A rod-coccus cycle was not observed at any stage of the growth. Strain Y-104T has a characteristic cell division in which a cell wall-like structure occurs in the middle of each cell during their early growth phase. Such structures, also called septa, were frequently observed during the late log phase of the growth cycle [1]. The doubling time was reported to be approximately 11 hours in a liquid medium at pH 7.0 and at 25°C [1]. Colonies on agar plates are circular, smooth, convex and white at the early stage of growth and cream-colored at later stage of growth. The polysaccharide content of the cells is very high, sometimes more than 50% (wt/wt) depending on the culture conditions. Growth of strain Y-104T occurs at a temperature range of 10–35°C and a pH range of 5.0 to 9.0 and in the presence of up to 6% NaCl. N. multipartita is positive for catalase production and negative for oxidase activity [1]. It is capable of utilizing glucose, fructose, mannose, galactose, xylose, sucrose, maltose, lactose, mannitol, sorbitol, ethanol, propanol, glycerol, starch, pyruvate, aranine, glutamate, glutamine and histidine as carbon and energy sources [1]. The strain cannot utilize acetate, malate, succinate, arginine, asparagine, methanol or glycogen as carbon and energy sources [1]. Strain Y-104T is able to accumulate large amounts of polysaccharides in its cells [1].
Figure 2.

Scanning electron micrograph of N. multipartita strain Y-104T

Table 1.

Classification and general features of N. multipartita strain Y-104T according to the MIGS recommendations [14]

MIGS ID

Property

Term

Evidence code

 

Current classification

Domain Bacteria

TAS [15]

 

Phylum Actinobacteria

TAS [16]

 

Class Actinobacteria

TAS [5]

 

OrderActinomycetales

TAS [5]

 

Suborder Frankineae

TAS [2]

 

Family Nakamurellaceae

TAS [2]

 

Genus Nakamurella

TAS [2]

 

Species Nakamurella multipartita

TAS [2]

 

Type strain Y-104

TAS [1]

 

Gram stain

positive

TAS [1]

 

Cell shape

coccus

TAS [1]

 

Motility

non-motile

TAS [1]

 

Sporulation

non-sporulating

TAS [1]

 

Temperature range

10-35°C

TAS [1]

 

Optimum temperature

24°C

TAS [1]

 

Salinity

up to 6g NaCl/L

TAS [1]

MIGS-22

Oxygen requirement

aerobic chemoorganotroph

TAS [1]

 

Carbon source

sugars, alcohols, glucose, maltose, mannose, fructose, starch

TAS [1]

 

Energy source

starch, ethanol, propanol

TAS [1]

MIGS-6

Habitat

activated sludge cultured in fed-batch reactors

TAS [1,8]

MIGS-15

Biotic relationship

free-living

NAS

MIGS-14

Pathogenicity

none

NAS

 

Biosafety level

1

TAS [17]

 

Isolation

activated sludge

TAS [1]

MIGS-4

Geographic location

not reported

 

MIGS-5

Sample collection time

not reported

 

MIGS-4.1

Latitude

not reported

 

MIGS-4.2

Longitude

 

MIGS-4.3

Depth

not reported

 

MIGS-4.4

Altitude

not reported

 

Evidence codes - IDA: Inferred from Direct Assay (first time in publication); TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project [18]. If the evidence code is IDA, then the property was directly observed for a live isolate by one of the authors, or an expert mentioned in the acknowledgements.

Chemotaxonomy

The murein of N. multipartita strain Y-104T contains meso-diaminopimelic acid as the diagnostic diamino acid [1]. The fatty acid pattern of Y-104T is dominated by iso-C16:0 (19.7%), iso-C15:0 (15.7%) and C18:1 (14.0%) and substantial amounts of C16:0 (10.3%), anteiso-C15:0 (9.2%), iso-C17:0 (8.5%) and anteiso-C17:0 (5.2%) were detected [1]. The predominant menaquinones are MK-8 (H4), approximately 97.0%, and minor amounts of MK-7 (H4), MK-8 (H2) and MK-9 (H4) were present [1]. Mycolic acids are absent [1].

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of each phylogenetic position, and is part of the Genomic Encyclopedia of Bacteria and Archaea project. The genome project is deposited in the Genome OnLine Database [14] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
Table 2.

Genome sequencing project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

Finished

MIGS-28

Libraries used

Two Sanger genomic libraries: 8kb pMCL200 and fosmid pcc1Fos

MIGS-29

Sequencing platforms

ABI3730

MIGS-31.2

Sequencing coverage

15.4× Sanger

MIGS-30

Assemblers

Arachne, phrap

MIGS-32

Gene calling method

Prodigal, GenePRIMP

 

INSDC ID

CP001737

 

Genbank date of release

September 18, 2009

 

GOLD ID

Gi02230

 

NCBI project ID

29537

 

Database: IMG-GEBA

2501939634

MIGS-13

Source material identifier

DSM 44233

 

Project relevance

Tree of Life, GEBA

Growth conditions and DNA isolation

N. multipartita Y-104T, DSM 44233, was grown in DSMZ 553 medium [19] at 28°C. DNA was isolated from 1–1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions with modification st/FT for cell lysis according to Wu et al. [20].

Genome sequencing and assembly

The genome was sequenced using Sanger sequencing platform. All general aspects of library construction and sequencing can be found on the JGI website. Optimal raft assembly was produced using Arachne assembler. Finishing assemblies were made using the parallel phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher [21] or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 2,596 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. Together all Sanger reads provided 15.4× coverage of the genome. The final assembly contains 118,931 Sanger reads.

Genome annotation

Genes were identified using Prodigal [22] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePrimp pipeline [23]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform [24].

Genome properties

The genome is 6,060,298 bp long and comprises one main circular chromosome with a 70.9% G+C content (Table 3 and Figure 3). Of the 5,471 genes predicted, 5,415 were protein coding genes, and 56 RNAs; 175 pseudo genes were also identified. The majority of the protein-coding genes (66.5%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.
Figure 3.

Graphical circular map of the genome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

Table 3.

Genome Statistics

Attribute

Value

% of Total

Genome size (bp)

6,060,298

100.00%

DNA coding region (bp)

5,526,464

91.19%

DNA G+C content (bp)

4,297,749

70.92%

Number of replicons

1

 

Extrachromosomal elements

0

 

Total genes

5,471

100.00%

RNA genes

56

1.02%

rRNA operons

2

 

Protein-coding genes

5,415

98.98%

Pseudo genes

175

3.20%

Genes with function prediction

3,638

66.50%

Genes in paralog clusters

3,319

60.67%

Genes assigned to COGs

3,673

67.14%

Genes assigned Pfam domains

4,054

74.10%

Genes with signal peptides

1,713

31.31%

Genes with transmembrane helices

1,258

22.99%

CRISPR repeats

9

 
Table 4.

Number of genes associated with the general COG functional categories

Code

Value

%age

Description

J

166

3.9

Translation, ribosomal structure and biogenesis

A

2

0.0

RNA processing and modification

K

400

9.7

Transcription

L

324

7.8

Replication, recombination and repair

D

31

0.8

Cell cycle control, mitosis and meiosis

V

81

2.0

Defense mechanisms

T

238

5.8

Signal transduction mechanisms

M

173

4.2

Cell wall/membrane biogenesis

Z

1

0.0

Cytoskeleton

U

44

1.1

Intracellular trafficking and secretion

O

113

2.7

Posttranslational modification, protein turnover, chaperones

C

308

7.5

Energy production and conversion

G

341

8.3

Carbohydrate transport and metabolism

E

334

8.1

Amino acid transport and metabolism

F

97

2.4

Nucleotide transport and metabolism

H

190

4.6

Coenzyme transport and metabolism

I

160

3.9

Lipid transport and metabolism

P

182

4.4

Inorganic ion transport and metabolism

Q

117

2.8

Secondary metabolites biosynthesis, transport and catabolism

R

506

12.2

General function prediction only

S

330

8.0

Function unknown

-

1,773

32.4

Not in COGs

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Marlen Jando for growing N. multipartita cells, and Susanne Schneider for DNA extraction and quality analysis (both at DSMZ). This work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-1 and the Indian Council of Scientific and Industrial Research provided a Raman Research Fellow to Shanmugam Mayilraj.

Authors’ Affiliations

(1)
DOE Joint Genome Institute
(2)
DSMZ - German Collection of Microorganisms and Cell Cultures GmbH
(3)
MTCC - Microbial Type Culture Collection, Institute of Microbial Technology
(4)
Bioscience Division, Los Alamos National Laboratory
(5)
Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory
(6)
Oak Ridge National Laboratory
(7)
HZI - Helmholtz Centre for Infection Research
(8)
University of California Davis Genome Center

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© The Author(s) 2010