Complete genome sequence of Capnocytophaga ochracea type strain (VPI 2845T)

Capnocytophaga ochracea (Prévot et al. 1956) Leadbetter et al. 1982 is the type species of the genus Capnocytophaga. It is of interest because of its location in the Flavobacteriaceae, a genomically not yet charted family within the order Flavobacteriales. The species grows as fusiform to rod shaped cells which tend to form clumps and are able to move by gliding. C. ochracea is known as a capnophilic (CO2-requiring) organism with the ability to grow under anaerobic as well as aerobic conditions (oxygen concentration larger than 15%), here only in the presence of 5% CO2. Strain VPI 2845T, the type strain of the species, is portrayed in this report as a gliding, Gram-negative bacterium, originally isolated from a human oral cavity. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence from the flavobacterial genus Capnocytophaga, and the 2,612,925 bp long single replicon genome with its 2193 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain VPI 2845 T (= DSM 7271 = ATCC 27872 =JCM 1296) is the type strain of Capnocytophaga ochracea, and the type species of the genus Capnocytophaga. C. ochracea was first described by Prévot et al. [1] as 'Fusiformis nucleatus var. ochraceus' and later renamed by Leadbetter et al [2]. Other synonyms for C. ochracea are 'Bacteroides oralis var. elongatus' [3],'Bacteroides ochraceus' (basonym) [4] and "Ristella ochraceus" (sic) [5]. The organism is of significant interest for its posi-tion in the tree of life where the genus Capnocytophaga (8 species) is located within the large family of the Flavobacteriaceae. First, Leadbetter et al. placed the genus Capnocytophaga in the family of the Cytophagaceae within the order Cytophagales [6] which was emended in 2002 by the Subcommittee on the Taxonomy of Flavobacterium and Cytophaga-like bacteria of the International Committee on Systematics of Prokaryotes [7]. C. ochracea is most often found in association with animal and human hosts. In general, it is a normal inhabitant of the human mouth and other non-oral sites. C. ochracea is associated with juvenile and adult periodontitis [8,9] and may cause severe infections in immunocompromised as well as in immunocompetent patients [10][11][12]. Among these are endocarditis, endometritis, osteomyelitis, abscesses, peritonitis, and keratitis. Here we present a summary classification and a set of features for C. ochracea VPI 2845 T together with the description of the complete genomic sequence and annotation.

Classification and features
Genbank lists 16S rRNA sequences for only a few small number of cultivated strains belonging to C. ochraceae, all of them isolated from human oral cavity(e.g. U41351, U41353, DQ012332). Phylotypes (sequences from uncultivated bacteria) closely linked to C. ochracea also originate in almost exclusively from human oral samples col-lected from European, American, Asian and African samples (AF543292, AF543298, AY278613, AM420149, AY429469, FJ470418). Only two bacterial clones are reported from non-human sources. One was isolated from Strongylocentrotus intermedius (sea urchin) in the Sea of Japan (EU432412, EU432438), and the second from Oncorhynchus mykiss (rainbow trout) caught in Scotland (AM179907). Screening of environmental genomic samples and surveys reported at the NCBI BLAST server indicated no closely related phylotypes (>91% sequence identity) that can be linked to the species or genus. Figure 1 shows the phylogenetic neighborhood of C. ocharcea VPI 2845 T in a 16S rRNA based tree. All four 16S rRNA gene copies in the genome of strain VPI 2845 T are identical, but differ by two nucleotides from the previously published 16S rRNA sequence (U41350) generated from ATCC 27872.

Figure 1.
Phylogenetic tree highlighting the position of C. ochracea VP 2845 T relative to the other type strains of species within the genus Capnocytophaga and to selected type strains of species belonging to other genera within the Flavobacteriaceae. The tree was inferred from 1,405 aligned characters [13,14] of the 16S rRNA gene sequence under the maximum likelihood criterion [15] and rooted with Joostella and Galbibacter. 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 [16] are shown in blue, published genomes in bold.
C. ochracea is Gram-negative, has no flagellae and is motile by gliding (Table 1, Figure 2). Cells are pigmented and the name 'ochracea' is derived from the yellow color exhibited by harvested cell mass [6]. It is a catalase-and oxidase-negative species. C. ochracea is usually susceptible to a number of antibiotics, however, resistance is increasing in this species [23,24]. Furthermore, C. ochracea is known to possess an immunosuppressive factor [25]. All strains of C. ochracea are capa-ble of fermenting glucose, sucrose, maltose and mannose, whereas most strains ferment amygdalin, fructose, galactose, lactose and raffinose [20]. The optimal growth temperature is 37°C. Nitrate is reduced to nitrite, and dextran, glycogen, starch and aesculin are hydrolysed by most strains. Indole is not produced. Acetic and succinic acid are the main metabolic end products of fermentation [6].

Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position, and is part of the Genomic Encyclopedia of Bacteria and Archaea project. The genome project is deposited in the Genomes OnLine Database [10] and the complete genome sequence in GenBank (CP001632). Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in

Genome sequencing and assembly
The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequenc-ing performed at the JGI can be found at the JGI website. 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 2,919 overlapping fragments of 1,000 bp 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). Possi-ble mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones [33]. Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 226 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 35.1× coverage of the genome. 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 the Gene Ontology project [22]. If the evidence code is IDA, then the property was directly observed for a living isolate by one of the authors or an expert mentioned in the acknowledgements.

Genome annotation
Genes were identified using Prodigal [34] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [35]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, Uni-Prot, TIGRFam, Pfam, PRIAM, KEGG, COG, and In-terPro databases. Additional gene prediction analysis and functional annotation were performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform [36].

Genome properties
The genome is 2,612,925 bp long and comprises one circular chromosome with a 39.6% GC content ( Table 3). Of the 2,252 genes predicted, 2,193 were protein coding genes, and 59 RNAs; 22 pseudogenes were also identified. Genes assigned with putative functions comprised 61.7% of the genome, while the remaining genes 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 Figure 3 and Table   4.