Scholarly article on topic 'Genome sequence of the squalene-degrading bacterium Corynebacterium terpenotabidum type strain Y-11T (= DSM 44721T)'

Genome sequence of the squalene-degrading bacterium Corynebacterium terpenotabidum type strain Y-11T (= DSM 44721T) Academic research paper on "Biological sciences"

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Academic research paper on topic "Genome sequence of the squalene-degrading bacterium Corynebacterium terpenotabidum type strain Y-11T (= DSM 44721T)"

Standards in Genomic Sciences (2014) 9:505-513

DOI:10.4056/sigs.4588337

Genome sequence of the squalene-degrading bacterium Corynebacterium terpenotabidum type strain Y-11T (= DSM 44721T)

Christian Rückert1, Andreas Albersmeier1, Arwa Al-Dilaimi1, Hanna Bednarz2, Karsten Niehaus2, Rafael Szczepanowski1, Jörn Kalinowski1

1 Technology Platform Genomics, CeBiTec, Bielefeld University, Bielefeld, Germany

2 Proteome and Metabolome Research, Bielefeld University, Bielefeld, Germany

Correspondence: Jörn Kalinowski (Joern@CeBiTec.Uni-Bielefeld.DE)

Keywords: aerobic, non-motile, Gram-positive, non-sporeforming, non-haemolytic, heterotrophic, mesophilic, squalene-degrading

Corynebacterium terpenotabidum Takeuchi et. al 1999 is a member of the genus Corynebacterium, which contains Gram-positive and non-spore forming bacteria with a high G+C content. C. terpenotabidum was isolated from soil based on its ability to degrade squalene and belongs to the aerobic and non-hemolytic Corynebacteria. It displays tolerance to salts (up to 8%) and is related to Corynebacterium variabile involved in cheese ripening. As this is a type strain of Corynebacterium, this project describing the 2.75 Mbp long chromosome with its 2,369 protein-coding and 72 RNA genes will aid the Genomic Encyclopedia of Bacteria and Archaea project.

Introduction

Strain Y-11T (= DSM 444721T] is the type strain of the species Corynebacterium terpenotabidum [1]. It was originally isolated from soil, although the exact source has not been published [2,3]. The genus Corynebacterium is comprised of Grampositive bacteria with a high G+C content. It currently contains over 80 members [4] isolated from diverse backgrounds like human clinical samples [5] and animals [6], but also from soil [7] and ripening cheese [8].

Within this diverse genus, C. terpenotabidum has been proposed to form a subclade together with C. variabile DSM 20132T and C. nuruki S6-4T, demonstrating 97.4% and 95.9% similarity respectively between the 16S rRNA gene sequences. Information on the strain is scarce. It was isolated for its ability to metabolize the linear triterpene squalene and classified as an Arthrobacter species [2,3], but no further information on the strain was supplied. Neither the origin nor the exact isolation procedures were reported. C. terpenotabidum can cleave squalene yielding geranylacetone [2] but also accepts some squalene derivatives [3].

Here we present a summary classification and a set of features for C. terpenotabidum DSM 44721T,

together with the description of the genomic sequencing and annotation.

Classification and features

A representative genomic 16S rRNA sequence of C. terpenotabidum DSM 44721T was compared to the Ribosomal Database Project database [9]. C. terpenotabidum shows highest similarity to C. variabile (97.4%].

Figure 1 shows the phylogenetic neighborhood of C. terpenotabidum in a 16S rRNA based tree. Within the genus Corynebacterium, C. terpenotabidum forms a distinct subclade together with C. variabile and C. nuruki.

C. terpenotabidum Y-11T cells are Gram-positive non acid fast rods (1.0-1.5 [j.m x 0.5-0.8 [j.m wide] that grow strictly aerobically in rough, grayish-white colonies without diffusible pigments or aerial mycelia [1], [Table 1]. Cells grow with a waxlike quality on solid medium and tend to clot in liquid culture. Scanning electron micrograph pictures of liquid grown cultures revealed slight morphological differences between free-floating cells and clotted cells (Figure 2].

The Genomic Standards Consortium

Scale:

-Corynebacterium diphtheriae NCTC 11397T (X84248)

— Corynebacterium pseudotuberculosis CIP 102968T (X81916)

-Corynebacterium vitaeruminis NCTC 20294T (X84680)

-Corynebacterium aquilae CECT 5993T (AJ496733)

-Corynebacterium argentoratense CIP 104296T (X83955)

-Corynebacterium felinum CCUG399431 (AJ401282)

-Corynebacterium renale CIP 103421T (X81909)

Corynebacterium singulare CCUG 37330T (Y10999) Corynebacterium aurimucosum IMMIB D-1488T (AJ309207)

-Corynebacterium striatum NCTC 764T (X84442)

-Corynebacterium fiavescens NCDO 1320' (X84441)

-Corynebacterium thomssenii DSM 44276T (AF010474)

I-Corynebacterium cas ei LMG S-19264T (AF267152)

loo'-----1---i--.1.— - — -----''ID Arnttn'! IVOUMI

Corynebacterium ammoniagenes CIP 101283 (X84440)

Corynebacterium pilosum ATCC 29592T (X81908)

Corynebacterium halotolerans YIM700931 (AY226509)

Corynebacterium maris Coryn-1T (FJ423600)

-Corynebacterium humireducens MFC-5T (GQ421281 )

-Corynebacterium marinum 7015T (DQ219354)

-Corynebacterium efficiens YS-314T (AB055963)

-Corynebacterium glutamicum ATCC 13032T (AF314192)

Corynebacterium callunae NCFB 10338T (X84251)

■Corynebacterium xerosis ATCC 373T (X81914) 101)1 Corynebacterium freneyi 20695110T {AJ292762) - Corynebacterium terpenotabidum IFO 14764T (AB004730)

i-uoryneoacrerium lerpenoiaDiaum ii-u (A

J™-Corynebacterium variabile DSM 20132T (AJ222815)

1-Corynebacterium nuruki S6-4T (HM165487)

-Rhodococcus equi DSM 203071 (X80614)

Figure 1. Phylogenetic tree highlighting the position of C. terpenotabidum relative to type strains of other species within the genus Corynebacterium. Species with at least one publicly available genome sequence (not necessarily the type strain) are highlighted in bold face. The tree is based on sequences aligned by the RDP aligner and utilizes the Jukes-Cantor corrected distance model to construct a distance matrix based on alignment model positions without alignment inserts, using a minimum comparable position of 200. The tree is built with RDP Tree Builder, which utilizes the Weighbor method [10] with an alphabet size of 4 and length size of 1,000. The building of the tree also involves a bootstrapping process repeated 100 times to generate a majority consensus tree [11]. Rhodococcus equi (X80614) was used as an outgroup.

Figure 2. Scanning electron micrograph of C. terpenotabidum Y-11T. A) Free-floating cells. B) Aggregated cells.

Table 1. Classification and general features of C. terpenotabidum Y-11T according to the MIGS recommendations [12]._

MIGS ID Property_

MIGS-22

Current classification

Gram stain

Cell shape

Motility

Sporulation

Temperature range

Optimum temperature

Salinity

Oxygen requirement Carbon source

Term Evidence code«

Domain Bacteria TAS [13]

Phylum Actinobacteria TAS [14]

Class Actinobacteria TAS [15]

Order Actinomycetales TAS [15-18]

Family Corynebacteriaceae TAS [15-17,19]

Genus Corynebacterium TAS [1517,20,21]

Species Corynebacterium terpenotabidum TAS [1]

Type-strain Y-11 (=DSM 44721) TAS [1]

positive TAS [1]

rod-shaped TAS [1]

non-motile TAS [1]

non-sporulating TAS [1]

mesophile TAS [1]

28°C TAS [1]

0-8% (w/v) NaCl TAS [1]

aerobe TAS [1]

fructose, galactose, mannose, lactate, ethanol TAS [1

Energy metabolism chemoorganoheterotrop hic NAS

Terminal electron acceptor oxygen NAS

MIGS-6 Habitat soil TAS [2]

MIGS-15 Biotic relationship free-living NAS

MIGS-14 Pathogenicity non-pathogenic NAS

Biosafety level 1 NAS

MIGS-23.1 I solati on not reported

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

a) Evidence codes - TAS: Traceable Author Statement (i.e., a direct report exists in the traceable Author Statement (i.e., not directly observed for the living, isolated sample, ally accepted property for the species, or anecdotal evidence). These evidence codes Ontology project [22].

literature); NAS: Non-but based on a generare from of the Gene

C. terpenotabidum was found to be able to utilize fructose, galactose, mannose, lactate, and ethanol as carbon source, while many others like arginine, aspartate, histidine, methyl amine, ethyl amine, methanol, galactose, lactose, maltose, sucrose, glycerol, sorbitol, mannitol, inositol, citrate, succinate, malonate, pimelate, m-hydroxybenzoate and p-hydroxybenzoate cannot be used. Optimal growth of strain Y-11T is reported at 28°C. C. terpenotabidum was shown to grow with a salinity

between 0 and 8.0% (w/v NaCl], with no growth at 10% [1]. The biochemical characterization revealed positive signals for urease, catalase, and hydrolysis of Tween 80.

Chemotaxonomy

The cell wall of C. terpenotabidum Y-11T contains alanine, glutamic acid, and meso-diaminopimelic acid in a molar ratio of 2.12: 1.00: 0.97. The main components of the cell wall sugars are described

to be arabinose, galactose, and mannose in a molar ratio of 2.47: 1.71: 1.00. The glycan moiety of the cell wall was found to contain acetyl residues [!]■

In C. terpenotabidum, cellular fatty acids are composed mainly of oleic acid (Cmico9c, 31%], palmitic acid (Ci6:o, 28%], and tuberculostearic acid 10-methyl (Cmo, 21%]. The whole-cell methanolysate of strain Y-ll contained mycolic esters [1]. The predominant isoprenoid quinone is menaquinone MK-9(H2].

Genome sequencing and annotation

Genome project history

C. terpenotabidum Y-l 1T was selected for sequencing as part of a project to define the core genome and pan genome of the non-pathogenic coiynebacteria. While not being pail of the Genomic Encyclopedia of Bacteria and Archaea (GEBA] project [23], sequencing of the type strain will nonetheless aid the GEBA effort. The genome project is deposited in the Genomes OnLine Database [24] and the complete genome sequence is deposited in GenBank Sequencing finishing and annotation were performed by the Center of Biotechnology (CeBiTec], A summaiy 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 genomic libraries: one 454 pyrosequencing PE library (3.4 kb insert sizes), one lllumina library

MIGS-29 Sequencing platforms 454 GS FLX Titanium, lllumina MiSeq

MIGS-31.2 Sequencing coverage 29.52x Pyrosequencing; 61.71 x SBS

MIGS-30 Assemblers Newbler version 2.3

MIGS-32 Gene calling method GeneMark, Glimmer

INSDC ID CP003696

GenBank Date of Release September 1, 2013/ after publication

GOLD ID Gi18852

NCBI project ID 168617

MIGS-13 Source material identifier DSM 44721

Project relevance Industrial, GEBA

Growth conditions and DNA isolation

C. terpenotabidum strain Y-l 1T, DSM 44721, was grown aerobically in LB broth (Carl Roth GmbH, Karlsruhe,Germany] at 30 °C. DNA was isolated from ~ 108 cells using the protocol described by Tauch etal. 1995 [25].

Genome sequencing and assembly

The genome was sequenced using a 454 sequencing platform. A standard 3k paired end sequencing library was prepared according to the manufacturers protocol (Roche], The genome was sequenced using the GS-FLX platform with Titanium chemistry, yielding 384,252 total reads, providing 29.52x coverage of the genome. Pyrosequencing reads were assembled using the Newbler assembler v2.3 (Roche], The initial Newbler assembly consisted of 22 contigs in six scaffolds. Analysis of the six scaffolds revealed five that made up the

chromosome, while the remaining one contained five copies of the RRN operon that caused the scaffold breaks. The scaffolds were ordered based on alignments to the complete genomes of C. variabile [26] and subsequent verification by restriction digestion, Southern blotting and hybridization with a 16S rDNA specific probe.

The Phred/Phrap/Consed software package [2730] was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, gaps between contigs were closed by editing in Consed (for repetitive elements] and by PCR with subsequent Sanger sequencing (IIT Biotech GmbH, Bielefeld, Germany], A total of 12 additional reactions were necessary to close gaps not caused by repetitive elements.

To raise the quality of the assembled sequence, Illumina reads were used to correct potential base errors and increase consensus quality. A WGS library was prepared using the Illumina-Compatible Nextera DNA Sample Prep Kit (Epicentre, WI, U.S.A) according to the manufacturer's protocol. The library was sequenced in a 2x 120 bp paired read run on the MiSeq platform, yielding 2,307,926 total reads. Together, the combination of the Illumina and 454 sequencing platforms provided 91.2x coverage of the genome.

Genome annotation

Gene prediction and annotation were done using the PGAAP pipeline [31]. Genes were identified using GeneMark [32], GLIMMER [33], and Prodigal [34]. For annotation, BLAST searches against the NCBI Protein Clusters Database [35] are performed and the annotation is enriched by searches against the Conserved Domain Database [36] and subsequent assignment of coding sequences to

COGs. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [37], Infernal [38], RNAMMer [39], Rfam [40], TMHMM [41], and SignalP [42].

Genome properties

The genome consists of one circular chromosome of 2,751,233 bp (67.02% G+C content] with no additional extrachromosomal elements present. A total of 2,441 genes were predicted, 2,369 of which are protein coding genes. 1,306 (55.13%] of the protein coding genes were assigned to a putative function with the remaining annotated as hypothetical proteins. In addition, 910 protein coding genes belong to 281 paralogous families in this genome, corresponding to a gene content redundancy of 38.41% [Figure 3]. The properties and the statistics of the genome are summarized in Table 3, and Table 4.

Table 3. Genome Statistic

Attribute

% of totala

Genome size (bp) 2,751,233

DNA coding region (bp) 2,441,394

DNA G+C content (bp) 1,843,810

Total genes 2,441

RNA genes 72

rRNA operons 5

tRNA genes 57

Protein-coding genes 2,369

Genes with function prediction (protein) 1,306

Genes assigned to COGs 1,812

Genes in paralog clusters 910

Genes with signal peptides 224

Genes with transmembrane helices 606

100.00 88.74 67.02 100.00 2.96

2.34 97.04 55.13 74.23 38.41 9.54 25.58

a) The total is based on either the size of the genome in base pairs or the total number of genes in the annotated genome.

1500 kbp 1250 kbp

Figure 3. Graphical map of the chromosome. From the outside in: Genes on forward strand (colored according to COG categories), Genes on reverse strand (colored according to COG categories), GC content, GC skew.

Table 4. Number of genes associated with the general COG functional categories Code Value %age Description_

J 151 6. 37 Translation, ribosomal structure and biogenesis

A 1 0.04 RNA processing and modification

K 152 6.42 Transcription

L 1 36 5.74 Replication, recombination and repair

B 0 0.00 Chromatin structure and dynamics

D 20 0.84 Cell cycle control, cell division, chromosome partitioning

Y 0 0.00 Nuclear structure

V 32 1. 35 Defense mechanisms

T 58 2.45 Signal transduction mechanisms

M 81 3. 42 Cell wall/membrane biogenesis

N 1 0.04 Cell motility

Z 0 0.00 Cytoskeleton

W 0 0.00 Extracellular structures

U 26 1.10 Intracellular trafficking and secretion, and vesicular transport

O 72 3. 04 Posttranslational modification, protein turnover, chaperones

C 127 5.36 Energy production and conversion

G 115 4.85 Carbohydrate transport and metabolism

E 218 9.20 Amino acid transport and metabolism

F 68 2.87 Nucleotide transport and metabolism

H 97 4.09 Coenzyme transport and metabolism

I 121 5.11 Lipid transport and metabolism

P 151 6. 37 Inorganic ion transport and metabolism

Q 76 3.21 Secondary metabolites biosynthesis, transport and catabolism

R 2 74 11.57 General function prediction only

S 1 38 5.83 Function unknown

- 557 23.51 Not in COGs

Acknowledgements

Christian Rückert acknowledges funding through a

grant by the Federal Ministry for Education and Re-

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