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Complete genome sequence of Cryptobacterium curtum type strain (12-3T)
Standards in Genomic Sciencesvolume 1, pages93–100 (2009)
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.
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 . C. curtum was described by Nakazawa et al. in 1999 . 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 . 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
The type strain 12-3T and a second strain of the species, KV43-B, both classified in C. curtum were isolated from a periodontal pocket sample of an adult patient and from necrotic dental pulp, respectively . C. curtum can also be isolated from human oral and dental infections like pulpal inflammations, advanced caries , dental abscesses or periodontitis . 16S rRNA gene sequence analysis revealed that the two isolates represent a distinct lineage within the family Coriobacteriaceae, between the neighboring genera Eggerthella and Slackia (Figure 1). No significant matches with any 16S rRNA sequences from environmental genomic samples and surveys are reported at the NCBI BLAST server (February 2009).
The very short and non-motile rods form tiny translucent colonies of less than 1 mm in diameter on BHI-blood agar without hemolysis after prolonged incubation under strictly anaerobic conditions (Table 1). Transmission electron micrographs of ultrathin sections of C. curtum 12-3T showed a single-layered Gram-positive cell wall of approximately 10 nm thickness (Figure 2) . Carbohydrates are not metabolized; the species is asaccharolytic . C. curtum is non-reactive in most biochemical tests. The human oral cavity contains arginine and other amino acids and oligopeptides due to proteinase and peptidase activities. C. curtum degrades arginine through arginine deiminase pathway . Like Slackia exigua, a closely related species, these bacteria are very difficult to cultivate. Optimal doubling time is 12 hours . There are no chemotaxonomic data available to C. curtium strain 12-3T.
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
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  and the complete genome sequence 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.
Growth conditions and DNA isolation
C. curtum strain 12-3T, DSM 15641, was grown anaerobically in DSMZ medium 78 (Chopped Meat Medium) , 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  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  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.
Genes were identified using GeneMark  as part of the genome annotation pipeline in the Integrated Microbial Genomes Expert Review (IMG-ER) system , followed by a round of manual curation using the JGI GenePRIMP pipeline . 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  was used to find tRNA genes, whereas ribosomal RNAs were found by using the tool RNAmmer . Other non coding RNAs were identified by searching the genome for the Rfam profiles using INFERNAL (v0.81) . Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG) platform (http://img.jgi.doe.gov) .
Metabolic network analysis
The metabolic Pathway/Genome Database (PGDB) was computationally generated using Pathway Tools software version 12.5  and MetaCyc version 12.5 , 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 .
The genome is 1,617,804 bp long and comprises one main circular chromosome with a 50.9% GC content (Table 3 and Figure 3). Of the 1422 genes predicted, 1364 were protein coding genes, and 58 RNAs. A total of 7 pseudogenes were also identified. Among the majority of protein coding genes (78.5%) were assigned with a putative function while the remaining were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 3. The distribution of genes into COG functional categories is presented in Table 4, and a cellular overview diagram is presented in Figure 4, followed by a summary of metabolic network statistics shown in Table 5.
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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.