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Complete genome sequence of Bacteroides helcogenes type strain (P 36–108T)
Standards in Genomic Sciences volume 4, pages45–53(2011)
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.
Strain P 36–108T (= 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 . B. helcogenes strain P36-108T was isolated from a pig abscess in Japan, and described by Benno et al. in 1983 . Nine further isolates of B. helcogenes have been obtained from pig abscesses whereas two other isolates originated from pig feces. Here we present a summary classification and a set of features for B. helcogenes P 36–108T, 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  and the relative frequencies, weighted by BLAST scores, of taxa and keywords (reduced to their stem ) 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 .
Figure 1 shows the phylogenetic neighborhood of B. helcogenes P 36–108T 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).
The cells of B. helcogenes generally have the shape of short rods (0.5–0.6 εm × 0.8–4.0 µm) which occur singly or in pairs (Figure 2). B. helcogenes is a Gram-negative, non-pigmented and non spore-forming bacterium (Table 1). The organism is originally described as nonmotile and only five genes associated with motility have been found in the genome (see below). The organism grows well at 37°C but does not grow at 4°C or at 45°C . B. helcogenes is strictly anaerobic, chemoorganotrophic and is able to ferment glucose, mannose, fructose, galactose, sucrose, maltose, cellobiose, lactose, xylose, melibiose, raffinose, starch, glycogen, salicin, amygdalin, and xylan . The organism hydrolyzes esculin and starch but does not digest casein, liquify gelatin, reduce nitrate nor produce indole from tryptophan . B. helcogenes does not utilize arabinose, ramnose, ribose, trehalose, inulin, glycerol, mannitol, sorbitol, inositol, adonitol, erythritol or gum Arabic . It does not require hemin for growth but does require the presence of CO2; it does not show hemolysis. Growth is not enhanced by the addition of 20% bile . Major fermentation products from PYFG broth (peptone yeast extract Fildes glucose broth ) are acetic acid and succinic acid; propionic and isobutyric acid are produced in small amounts . B. helcogenes is phosphatase, DNase, β-glucuronidase, and glutamic acid decarboxylase active and urease, catalase, lecithinase and lipase inactive . The organism produces ammonium and chondroitin sulfatase . B. helcogenes can grow in the presence of kanamycin (1mg/ml), vancomycin (10 µg/ml), colistin (10 µg/ml), erythromycin (60 µg/ml) or polymyxin B (10 µg/ml) but not in the presence of cepharothin (10 µg/ml) or Brilliant green (0.001%) .
Little chemotaxonomic information is available for strain P 36–108T. Thus far, only the fatty acid composition has been elucidated. The major fatty acids found (>10%) were anteiso-C15:0, C15:0 and iso-C15:0.3-OH. Also, iso-C15:0, C16:0, and cis C18:1 were detected in a proportion ranging between 5% to 10% of the total fatty acids (unpublished data).
Genome sequencing and annotation
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  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.
Growth conditions and DNA isolation
B. helcogenes P 36–108T, DSM 20613, was grown anaerobically in medium 104 (PYG Medium)  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. . 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 . Pyrosequencing reads were assembled using the Newbler assembler version 2.3-PreRelease-10-21-2009-gcc-4.1.2-threads (Roche). The initial Newbler assembly consisting of 48 contigs in two scaffolds was converted into a phrap assembly by  making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (225.3 Mb) was assembled with Velvet  and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 146.7 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package  was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution , Dupfinisher , or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F.Chang, unpublished). A total of 160 additional reactions and 4 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 . 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.
Genes were identified using Prodigal  as part of the Oak Ridge National Laboratory genome annotation pipeline, 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, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform .
The genome consists of a 3,998,906 bp long chromosome with a GC content of 44.7% (Table 3 and Figure 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.
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We would like to gratefully acknowledge the help of Sabine Welnitz (DSMZ) for growing B. helcogenes cultures. 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, UT-Battelle and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-2.
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Pati, A., Gronow, S., Zeytun, A. et al. Complete genome sequence of Bacteroides helcogenes type strain (P 36–108T). Stand in Genomic Sci 4, 45–53 (2011). https://doi.org/10.4056/sigs.1513795
- strictly anaerobic
- pig abscess
- animal pathogen