Complete genome sequence of Bacteroides helcogenes type strain (P 36-108T)

Bacteroides helcogenes Benno et al. 1983 is of interest because of its isolated phylogenetic location and, although it has been found in pig feces and is known to be pathogenic for pigs, occurrence of this bacterium is rare and it does not cause significant damage in intensive animal husbandry. The genome of B. helcogenes P 36-108T is already the fifth completed and published type strain genome from the genus Bacteroides in the family Bacteroidaceae. The 3,998,906 bp long genome with its 3,353 protein-coding and 83 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain P 36-108 T (= DSM 20613 = ATCC 35417 = JCM 6297) is the type strain of Bacteroides helcogenes, one of currently 39 species in the genus Bacteroides [1,2]. The species epithet of B. helcogenes is derived from the Greek noun helkos meaning 'abscess' and the Greek verb gennaio meaning 'produce', referring to the pathogenic, probably intestinal, abscess-producing properties of the species [2]. B. helcogenes strain P36-108 T was isolated from a pig abscess in Japan, and described by Benno et al. in 1983 [2]. Nine further isolates of B. helcogenes have been obtained from pig abscesses whereas two other isolates origi-nated from pig feces. Here we present a summary classification and a set of features for B. helcogenes P 36-108 T , together with the description of the complete genomic sequencing and annotation.

Classification and features
A representative genomic 16S rRNA sequence of B. helcogenes was compared using NCBI BLAST under default values (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database [3] and the relative frequen-Standards in Genomic Sciences cies, weighted by BLAST scores, of taxa and keywords (reduced to their stem [4]) were determined. The single most frequent genus was Bacteroides (100%) (33 hits in total). Regarding the 21 hits to sequences from other members of the genus, the average identity within HSPs was 92.7%, whereas the average coverage by HSPs was 84.5%. Among all other species, the one yielding the highest score was Bacteroides ovatus, which corresponded to an identity of 93.4% and a HSP coverage of 86.6%. The highest-scoring environmental sequence was AM275453 ('fecal microbiota irritable bowel syndrome patients differs significantly from that of healthy subjects'), which showed an identity of 95.5% and a HSP coverage of 84.3%. The most frequently occurring keywords within the labels of environmental samples which yielded hits were 'human' (11.0%), 'fecal' (9.5%), 'microbiota' (8.8%), 'sequenc' (5.4%) and 'gut' (5.4%) (217 hits in total). The most frequently occurring keywords within the labels of environmental samples which yielded hits of a higher score than the highest scoring species were 'fecal/human' (13.3%), 'feedlot' (5.2%), 'bowel, faecal, healthi, irrit, microbiota, patient, significantli, subject, syndrom' (2.7%) and 'beef, cattl, coli, escherichia, feedbunk, habitat, marc, materi, neg, pen, primari, secondari, stec, surfac, synecolog, top, west' (2.6%) (6 hits in total). Most of these keywords are in accordance with the isolation sites of the different isolates and strongly suggest that B. helcogenes, like many other species of the genus Bacteroides, is associated with the intestinal tract of the host in the case of B. helcogenes, this host is the pig [2]. Figure 1 shows the phylogenetic neighborhood of B. helcogenes P 36-108 T in a 16S rRNA based tree. The sequences of the five 16S rRNA gene copies in the genome differ from each other by up to 20 nucleotides, and differ by up to 13 nucleotides from the previously published 16S rRNA sequence (AB200227).

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [27], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [28]. The genome project is deposited in the Genomes OnLine Database [10] and the complete genome sequence is deposited 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. helcogenes relative to those type strains within the genus that appeared within a monophyletic Bacteroides main clade in preliminary analyses. Note that several of the Bacteroides type strain 16S rRNA sequences (from B. cellulosolvens, B. galacturonicus, B. pectinophilus, B. vulgatus) did not cluster together with this clade (data not shown, but see [5]) and were omitted from the main phylogenetic inference analysis. The same holds for the sequence from Anaerorhabdus furcosa (GU585668; also Bacteroidaceae). Other Bacteroides species lacked a sufficiently long 16S rRNA sequence and also had to be omitted (B. coagulans, B. xylanolyticus). The tree was inferred from 1,414 aligned characters [6,7] of the 16S rRNA gene sequence under the maximum likelihood criterion [8] and rooted with the type strain of the family 'Prevotellaceae'. 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 [9] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [10] are shown in blue, published genomes [11] and Prevotella melaninogenica released Genbank accession CP002122 in bold.  Altitude not reported NAS 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 of the Gene Ontology project [25]. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.

Growth conditions and DNA isolation
B. helcogenes P 36-108 T , DSM 20613, was grown anaerobically in medium 104 (PYG Medium) [29] at 37°C. DNA was isolated from 0.5-1 g of cell paste using MasterPure Gram-positive DNA purification kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer, with modification st/DL for cell lysis as described in Wu et al. [28]. DNA is available through the DNA Bank Network [30,31].

Genome sequencing and assembly
The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [32]. Pyrosequencing reads were assembled using the Newbler assembler version 2. shatter libraries were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [36]. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 93 × coverage of the genome. The final assembly contained 500,148 pyrosequence and 6,257,254 Illumina reads.

Genome annotation
Genes were identified using Prodigal [37] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [38]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, Uni-Prot, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and In-terPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review (IMG-ER) platform [39].

Genome properties
The genome consists of a 3,998,906 bp long chromosome with a GC content of 44.7% (Figure 3 and Table 3). Of the 3,436 genes predicted, 3,353 were protein-coding genes, and 83 RNAs; 109 pseudogenes were also identified. The majority of the protein-coding genes (64.5%) were assigned with a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.