- Open Access
Complete genome sequence of Cryptobacterium curtum type strain (12-3T)
- Konstantinos Mavrommatis1,
- Rüdiger Pukall2,
- Christine Rohde2,
- Feng Chen1,
- David Sims1, 3,
- Thomas Brettin1, 3,
- Cheryl Kuske1, 3,
- John C. Detter1, 3,
- Cliff Han1, 3,
- Alla Lapidus1,
- Alex Copeland1,
- Tijana Glavina Del Rio1,
- Matt Nolan1,
- Susan Lucas1,
- Hope Tice1,
- Jan-Fang Cheng1,
- David Bruce1, 3,
- Lynne Goodwin1, 3,
- Sam Pitluck1,
- Galina Ovchinnikova1,
- Amrita Pati1,
- Natalia Ivanova1,
- Amy Chen4,
- Krishna Palaniappan4,
- Patrick Chain1, 5,
- Patrik D’haeseleer1, 5,
- Markus Göker2,
- Jim Bristow1,
- Jonathan A. Eisen1, 6,
- Victor Markowitz4,
- Philip Hugenholtz1,
- Manfred Rohde7,
- Hans-Peter Klenk2 and
- Nikos C. Kyrpides1
- Published: 29 September 2009
Abstract
Cryptobacterium curtum Nakazawa et al. 1999 is the type species of the genus, and is of phylogenetic interest because of its very distant and isolated position within the family Coriobacteriaceae. C. curtum is an asaccharolytic, opportunistic pathogen with a typical occurrence in the oral cavity, involved in dental and oral infections like periodontitis, inflammations and abscesses. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the actinobacterial family Coriobacteriaceae, and this 1,617,804 bp long single replicon genome with its 1364 protein-coding and 58 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
Keywords
- oral infections
- opportunistic pathogenic
- periodontitis
- non-spore-former
- anaerobic
- asaccharolytic
- Coriobacteriaceae
Introduction
Strain 12-3T (= DSM 15641 = ATCC 700683 = CCUG 43107) is the type strain of Cryptobacterium curtum, which is the sole species within the genus Cryptobacterium [1]. C. curtum was described by Nakazawa et al. in 1999 [1]. The organism is of significant interest because of its position in the tree of life where it was initially wrongly placed close to Eubacterium (Firmicutes) to be then relocated in the phylum Actinobacteria, close to the Coriobacteriaceae [1]. Here we present a summary classification and a set of features for C. curtum 12-3T, together with the description of the complete genomic sequencing and annotation.
Classification and features
Phylogenetic tree of C. curtum 12-3T and most type strains of the family Coriobacteriaceae, inferred from 1422 aligned 16S rRNA characters [3,4] under the maximum likelihood criterion [5]. The tree was rooted with type strains of the genera Collinsella and Coriobacterium. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1000 bootstrap replicates if larger than 60%. Strains with a genome sequencing project registered in GOLD [6] are printed in blue; published genomes in bold, including two of which are reported in this issue of SIGS [7,8]
Scanning electron micrograph of C. curtum 12-3T
Classification and general features of C. curtum 12-3T according to the MIGS recommendations [9]
MIGS ID | Property | Term | Evidence code |
---|---|---|---|
Current classification | Domain Bacteria | TAS [10] | |
Phylum Actinobacteria | TAS [11] | ||
Class Actinobacteria | TAS [12] | ||
Order Coriobacteriales | TAS [12] | ||
Family Coriobacteriaceae | TAS [12] | ||
Genus Cryptobacterium | TAS [1] | ||
Species Cryptobacterium curtum | TAS [1] | ||
Type strain 12-3 | TAS [1] | ||
Gram stain | positive | TAS [1] | |
Cell shape | very short rods | TAS [1] | |
Motility | nonmotile | TAS [1] | |
Sporulation | non-sporulating | TAS [1] | |
Temperature range | mesophile | TAS [1] | |
Optimum temperature | 37°C | NAS | |
Salinity | normal | TAS [1] | |
MIGS-22 | Oxygen requirement | obligate anaerobic | TAS [1] |
Carbon source | asaccharolytic | TAS [1] | |
Energy source | arginine, lysine | NAS | |
MIGS-6 | Habitat | human oral microflora | TAS [1] |
MIGS-15 | Biotic relationship | free living, growth on enzymatic degradation products of inflamed tissues | NAS |
MIGS-14 | Pathogenicity | periodontal infections | TAS [1] |
Biosafety level | 1 (+) | TAS [13] | |
Isolation | infected human oral cavity | TAS [1] | |
MIGS-4 | Geographic location | not reported | NAS |
MIGS-5 | Sample collection time | about 1995 | TAS [1] |
MIGS-4.1 | Latitude - Longitude | not reported | |
MIGS-4.2 | |||
MIGS-4.3 | Depth | not reported | |
MIGS-4.4 | Altitude | not reported |
Figure 1 shows the phylogenetic neighborhood of C. curtum strain 12-3T in a 16S rRNA based tree. Analysis of the three 16S rRNA gene sequences in the genome of strain 12-3T indicated that the genes differ by at most one nucleotide from each other, but differ by 15 nucleotides and eight ambiguities (1.1%) from the previously published 16S rRNA sequence generated from DSM 15641 (AB019260). The higher sequence coverage and overall improved level of sequence quality in whole-genome sequences, as compared to ordinary gene sequences, implies that the significant differences between the genome data and the reported 16S rRNA gene sequence might be due to sequencing errors in the previously reported sequence data.
Genome sequencing and annotation
Genome project history
Genome sequencing project information
MIGS ID | Property | Term |
---|---|---|
MIGS-31 | Finishing quality | Finished |
MIGS-28 | Libraries used | Three genomic libraries: two Sanger libraries - 8 kb pMCL200 and fosmid pcc1Fos - and one 454 pyrosequence standard library |
MIGS-29 | Sequencing platforms | ABI3730, 454 GS FLX |
MIGS-31.2 | Sequencing coverage | 12.9× Sanger; 20× pyrosequence |
MIGS-30 | Assemblers | Newbler version 1.1.02.15, phrap |
MIGS-32 | Gene calling method | Genemark 4.6b, tRNAScan-SE-1.23, infernal 0.81, GenePRIMP |
INSDC / Genbank ID | CP001682 | |
Genbank Date of Release | August 26, 2009 | |
GOLD ID | Gc01086 | |
NCBI Project ID | 20739 | |
Database: IMG-GEBA | 2500901758 | |
MIGS-13 | Source material identifier | DSM 15641 |
Project relevance | Tree of Life, GEBA |
Growth conditions and DNA isolation
C. curtum strain 12-3T, DSM 15641, was grown anaerobically in DSMZ medium 78 (Chopped Meat Medium) [17], supplemented with 1 g/l arginine, at 37°C. DNA was isolated from 1–1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with protocol modification st/FT [16] for cell lysis.
Genome sequencing and assembly
The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov/. 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 1,799 overlapping fragments of 1000bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the parallel phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher [18] 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. 47 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 sequence types provided 32.9x coverage of the genome.
Genome annotation
Genes were identified using GeneMark [19] as part of the genome annotation pipeline in the Integrated Microbial Genomes Expert Review (IMG-ER) system [20], followed by a round of manual curation using the JGI GenePRIMP pipeline [21]. 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. The tRNAScanSE tool [22] was used to find tRNA genes, whereas ribosomal RNAs were found by using the tool RNAmmer [23]. Other non coding RNAs were identified by searching the genome for the Rfam profiles using INFERNAL (v0.81) [24]. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG) platform (http://img.jgi.doe.gov) [25].
Metabolic network analysis
The metabolic Pathway/Genome Database (PGDB) was computationally generated using Pathway Tools software version 12.5 [26] and MetaCyc version 12.5 [27], based on annotated EC numbers and a customized enzyme name mapping file. It has undergone no subsequent manual curation and may contain errors, similar to a Tier 3 BioCyc PGDB [28].
Genome properties
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.
Schematic cellular overview diagram of all pathways of C. curtum 12-3T. Nodes represent metabolites, with shape indicating class of metabolite. Lines represent reactions.
Genome Statistics
Attribute | Value | % of Total |
---|---|---|
Genome size (bp) | 1,617,804 | |
DNA Coding region (bp) | 1,439,290 | 88.97% |
DNA G+C content (bp) | 823,649 | 50.91% |
Number of replicons | 1 | |
Extrachromosomal elements | 0 | |
Total genes | 1425 | 100.00% |
RNA genes | 58 | 2.37% |
rRNA operons | 3 | |
Protein-coding genes | 1364 | 95.92% |
Pseudo genes | 7 | 0.49% |
Genes with function prediction | 1117 | 78.55% |
Genes in paralog clusters | 77 | 5.41% |
Genes assigned to COGs | 1103 | 77.57% |
Genes assigned Pfam domains | 1104 | 77.64% |
Genes with signal peptides | 276 | 19.37% |
Genes with transmembrane helices | 206 | 14.46% |
CRISPR repeats | 0 |
Number of genes associated with the general COG functional categories
Code | Value | % | Description |
---|---|---|---|
J | 128 | 9.4 | Translation, ribosomal structure and biogenesis |
A | 1 | 0.1 | RNA processing and modification |
K | 94 | 6.9 | Transcription |
L | 74 | 5.5 | Replication, recombination and repair |
B | 1 | 0.1 | Chromatin structure and dynamics |
D | 15 | 1.1 | Cell cycle control, mitosis and meiosis |
Y | 0 | 0.0 | Nuclear structure |
V | 20 | 1.5 | Defense mechanisms |
T | 64 | 4.7 | Signal transduction mechanisms |
M | 70 | 5.1 | Cell wall/membrane biogenesis |
N | 1 | 0.1 | Cell motility |
Z | 1 | 0.1 | Cytoskeleton |
W | 0 | 0.0 | Extracellular structures |
U | 20 | 1.5 | Intracellular trafficking and secretion |
O | 55 | 4.0 | Posttranslational modification, protein turnover, chaperones |
C | 100 | 7.3 | Energy production and conversion |
G | 41 | 3.0 | Carbohydrate transport and metabolism |
E | 96 | 7.0 | Amino acid transport and metabolism |
F | 47 | 3.4 | Nucleotide transport and metabolism |
H | 69 | 5.1 | Coenzyme transport and metabolism |
I | 39 | 2.9 | Lipid transport and metabolism |
P | 70 | 5.1 | Inorganic ion transport and metabolism |
Q | 9 | 0.7 | Secondary metabolites biosynthesis, transport and catabolism |
R | 119 | 8.7 | General function prediction only |
S | 81 | 5.9 | Function unknown |
- | 261 | 19.1 | Not in COGs |
Metabolic Network Statistics
Attribute | Value |
---|---|
Total genes | 1422 |
Enzymes | 316 |
Enzymatic reactions | 606 |
Metabolic pathways | 115 |
Metabolites | 506 |
Declarations
Acknowledgements
We would like to gratefully acknowledge the help of Gabriele Gehrich-Schröter for growing C. curtum cultures 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 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, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, as well as German Research Foundation (DFG) INST 599/1-1.
Authors’ Affiliations
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