Open Access

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

  • Christian Rückert
  • , Andreas Albersmeier
  • , Arwa Al-Dilaimi
  • , Hanna Bednarz
  • , Karsten Niehaus
  • , Rafael Szczepanowski
  • and Jörn Kalinowski
Corresponding author

DOI: 10.4056/sigs.4588337

Received: 31 December 2013

Accepted: 31 December 2013

Published: 15 June 2014

Abstract

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.

Keywords:

aerobicnon-motileGram-positivenon-sporeformingnon-haemolyticheterotrophicmesophilicsqualene-degrading

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 Gram-positive 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.

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.

C. terpenotabidum Y-11T cells are Gram-positive non acid fast rods (1.0-1.5 μm x 0.5-0.8 μm wide) that grow strictly aerobically in rough, grayish-white colonies without diffusible pigments or aerial mycelia [1], [Table 1]. Cells grow with a wax-like 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).

Table 1

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

MIGS ID

    Property

    Term

   Evidence codea)

    Current classification

    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 [15-17,20,21]

    Species Corynebacterium terpenotabidum

   TAS [1]

    Type-strain Y-11T (=DSM 44721T)

   TAS [1]

    Gram stain

    positive

   TAS [1]

    Cell shape

    rod-shaped

   TAS [1]

    Motility

    non-motile

   TAS [1]

    Sporulation

    non-sporulating

   TAS [1]

    Temperature range

    mesophile

   TAS [1]

    Optimum temperature

    28°C

   TAS [1]

    Salinity

    0-8% (w/v) NaCl

   TAS [1]

MIGS-22

    Oxygen requirement

    aerobe

   TAS [1]

    Carbon source

    fructose, galactose, mannose, lactate, ethanol

   TAS [1]

    Energy metabolism

    chemoorganoheterotrophic

   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

    Isolation

    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 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 [22].

Figure 2

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

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, methylamine, ethylamine, 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 [1].

In C. terpenotabidum, cellular fatty acids are composed mainly of oleic acid (C18:1ω9c, 31%), palmitic acid (C16:0, 28%), and tuberculostearic acid 10-methyl (C18:0, 21%). The whole-cell methanolysate of strain Y-11 contained mycolic esters [1]. The predominant isoprenoid quinone is menaquinone MK-9(H2).

Genome sequencing and annotation

Genome project history

C. terpenotabidum Y-11T was selected for sequencing as part of a project to define the core genome and pan genome of the non-pathogenic corynebacteria. While not being part 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 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 genomic libraries: one 454 pyrosequencing PE library (3.4 kb insert sizes), one Illumina library

MIGS-29

   Sequencing platforms

    454 GS FLX Titanium, Illumina MiSeq

MIGS-31.2

   Sequencing coverage

    29.52× Pyrosequencing; 61.71 × 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-11T, 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 et al. 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 29.52× 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 [27-30] 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 91.2× 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.

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 3

Genome Statistics

Attribute

     Value

    % of totala

Genome size (bp)

     2,751,233

    100.00

DNA coding region (bp)

     2,441,394

    88.74

DNA G+C content (bp)

     1,843,810

    67.02

Total genes

     2,441

    100.00

RNA genes

     72

    2.96

rRNA operons

     5

tRNA genes

     57

    2.34

Protein-coding genes

     2,369

    97.04

Genes with function prediction (protein)

     1,306

    55.13

Genes assigned to COGs

     1,812

    74.23

Genes in paralog clusters

     910

    38.41

Genes with signal peptides

     224

    9.54

Genes with transmembrane helices

     606

    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.

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

   136

    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

   274

    11.57

    General function prediction only

S

   138

    5.83

    Function unknown

-

   557

    23.51

    Not in COGs

Declarations

Acknowledgements

Christian Rückert acknowledges funding through a grant by the Federal Ministry for Education and Research (0316017) within the BioIndustry2021 initiative.


This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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