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Standards in Genomic Sciences

Open Access

High quality draft genome sequence of Bacteroides barnesiae type strain BL2T (DSM 18169T) from chicken caecum

  • Mitsuo Sakamoto1Email author,
  • Alla L. Lapidus2, 3,
  • James Han4,
  • Stephan Trong4,
  • Matthew Haynes4,
  • T. B. K. Reddy4,
  • Natalia Mikhailova4,
  • Marcel Huntemann4,
  • Amrita Pati4,
  • Natalia N. Ivanova4,
  • Rüdiger Pukall5,
  • Victor M. Markowitz6,
  • Tanja Woyke4,
  • Hans-Peter Klenk5,
  • Nikos C. Kyrpides4, 7 and
  • Moriya Ohkuma1
Standards in Genomic Sciences201510:48

Received: 29 August 2014

Accepted: 21 July 2015

Published: 2 August 2015


Bacteroides barnesiae Lan et al. 2006 is a species of the genus Bacteroides, which belongs to the family Bacteroidaceae. Strain BL2T is of interest because it was isolated from the gut of a chicken and the growing awareness that the anaerobic microbiota of the caecum is of benefit for the host and may impact poultry farming. The 3,621,509 bp long genome with its 3,059 protein-coding and 97 RNA genes is a part of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG) project.


Strictly anaerobicNon-motileRod-shapedGram-negativeCecumPoultry Bacteroidaceae


Strain BL2T (= DSM 18169 = CCUG 54636 = JCM 13652) is the type strain of Bacteroides barnesiae which belongs to the genus Bacteroides [1]. The species epithet is derived from the name of Ella M. Barnes, a British microbiologist, who has contributed much to our knowledge of intestinal bacteriology and anaerobic bacteriology in general. B. barnesiae strain BL2T was isolated from caecum of a healthy chicken. Four other strains belonging to the same species have been isolated from the same source [1]. The genus Bacteroides represents one of the predominant anaerobic genera found in chicken caecum [24]. Bacteroides species are thought to play a fundamental role in the breakdown of complex molecules (such as polysaccharides) into simpler compounds that are used by the animal host as well as the microorganisms themselves [5, 6], in the utilization of nitrogenous substances and in the biotransformation of bile acids and other steroids [7]. They also play a role as beneficent protectors of the gut against pathogenic microorganisms [8]. Here we present a summary classification and set of features for B. barnesiae strain BL2T, together with the description of the complete genomic sequencing and annotation.

Organism information

Classification and features

A 1301 bp long contig contained the most complete 16S rRNA gene copy in the draft genome. This partial gene differed by 7 nucleotides (0.5 %) from the 16S rRNA reference sequence (AB253726) generated for the original description of B. barnesiae [1]. Such a difference is not unusual when comparing original sequences from the time organisms were initially described with sequences of type strain genomes sequenced in the KMG project [9], a problem that was only partially resolved in the sequencing orphan species initiative (SOS) [10]. A representative 16S rRNA gene sequence of strain BL2T was compared with GenBank using NCBI BLAST. The single most frequent genus found was Bacteroides . The highest-scoring environmental sequences (up to 99.8 % sequence identity), including HQ784912 (‘gastrointestinal specimens clone ELU0102-T240-S-NI_000093’), were all from a study on gastrointestinal specimens linked to inflammatory bowel diseases phenotype in human ileum [11] and indicate that close relatives of strain BL2T and representatives of B. barnesiae are also relevant to human health. Fig. 1 shows the phylogenetic position of B. barnesiae in a 16S rRNA gene sequence-based tree.
Fig. 1

Phylogenetic tree based on the 16S rRNA gene sequences showing the relationship of Bacteroides barnesiae strain BL2T among the genus Bacteroides . The tree was constructed by the neighbor-joining method. Numbers at nodes indicate the percentage bootstrap values of 1000 replicates. Bars, 0.01 substitutions per nucleotide position. Accession numbers are given for each strain

The cells of B. barnesiae are pleomorphic rods (0.5-1.4 × 0.8-10.6 μm) (Fig. 2). The cells are usually arranged singly or in pairs [1]. B. barnesiae is a Gram-negative, non-sporeforming bacterium (Table 1) that is described as non-motile, with only seven genes associated with motility having been found in the genome (see below). The optimum temperature for growth of strain BL2T is 37 °C. B. barnesiae is a strictly anaerobic chemoorganotroph and is able to ferment glucose, lactose, sucrose, maltose, salicin, xylose, cellobiose, mannose and raffinose [1]. The organism hydrolyzes esculin but does not liquefy gelatin, and neither reduces nitrate nor produces indole from tryptophan [1]. B. barnesiae does not utilize mannitol, arabinose, glycerol, melezitose, sorbitol, rhamnose or trehalose [1]. Growth is possible in the presence of bile [1]. Major fermentation products from broth (1 % peptone, 1 % yeast extract, and 1 % glucose each (w/v)) are acetic acid and succinic acid, whereas isovaleric acid is produced in small amounts [1]. B. barnesiae shows activity for α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, α-arabinosidase, N-acetyl-β-glucosaminidase, α-fucosidase, alkaline phosphatase, leucyl glycine arylamidase, alanine arylamidase and glutamyl glutamic acid arylamidase but no activity urease, catalase, arginine dihydrolase, β-galactosidase 6-phosphate, β-glucuronidase, glutamic acid decarboxylase and arginine, proline, phenylalanine, leucine, pyroglutamic acid, tyrosine, glycine, histidine and serine arylamidase [1].
Fig. 2

Light microscope image of strain BL2T

Table 1

Classification and general features of Bacteroides barnesiae strain BL2T in accordance with the MIGS recommendations [33] published by the Genome Standards Consortium [34] and the NamesforLife database [35]




Evidence code


Current classification

Domain Bacteria

TAS [36]


Phylum Bacteroidetes

TAS [37, 38]


Class Bacteroidia

TAS [38, 39]


Order Bacteroidales

TAS [38, 40]


Family Bacteroidaceae

TAS [41, 42]


Genus Bacteroides

TAS [42, 43]


Species Bacteroides barnesiae

TAS [1]


Strain BL2T

TAS [1]


Gram stain


TAS [1]


Cell shape

Pleomorphic rods

TAS [1]




TAS [1]




TAS [1]


Temperature range


TAS [1]


Optimum temperature

37 °C

TAS [1]


pH range; Optimum

Not reported


Carbon source

Mono- and polysaccharides

TAS [1]


Energy metabolism


TAS [1]




TAS [1]



Not reported



Oxygen requirement

Strictly anaerobic

TAS [1]


Biotic relationship


TAS [1]






Biosafety level





Chicken caecum

TAS [1]


Geographic location


TAS [1]


Sample collection time

Not reported




Not reported




Not reported




Not reported




Not reported


Evidence codes - 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 [44]

B. barnesiae strain BL2T contains menaquinones MK-10 (58 %) and MK-11 (34 %) as principal respiratory quinones, small amounts of MK-8, MK-9 and MK-12 (2 % each) are found as minor components [1]. The major fatty acids found were anteiso-C15:0 (32 %), iso-C15:0 (15 %), 3-hydroxy C16:0 (10 %) and C16:0 (10 %). Fatty acids C14:0 (4 %), C15:0 (2 %), C18:1 ω9c (4 %), C18:2 ω6,9c (2 %) and 3-hydroxy iso-C17:0 (7 %) were found in minor amounts [1]. Chemotaxonomic features are in line with known features from other representatives of the genus [1].

Genome sequencing information

Genome project history

The organism was selected for sequencing on the basis of its phylogenetic position [1214]. Sequencing of B. barnesiae strain BL2T is part of Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes project [9] which aims not only to increase the sequencing coverage of key reference microbial genomes [15], but also to generate a large genomic basis for the discovery of genes encoding novel enzymes [16]. The genome project is deposited in the Genomes OnLine Database [17] and the permanent draft genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute using state of the art sequencing technology [18]. A summary of the project information is shown in Table 2.
Table 2

Genome sequencing project information





Finishing quality

Level 2: High-Quality Draft


Libraries used

Illumina Std. shotgun library


Sequencing platforms

Illumina HiSeq 2000


Fold coverage

122.7 ×



Velvet v. 1.1.04; ALLPATHS v. r41043


Gene calling method



Locus Tag



Genbank ID



Genbank Date of Release









Source Material Identifier

DSM 18169


Project relevance

Tree of Life, GEBA-KMG

Growth conditions and genomic DNA preparation

B. barnesiae strain BL2T, DSM 18169, was grown anaerobically in DSMZ medium 429 (Columbia Blood Agar) at 37 °C [19]. DNA was isolated from 0.5-1 g of cell paste using JetFlex genomic DNA purification (GENOMED) following the standard protocol as recommended by the manufacturer with and additional protease K (50 μl; 21 mg/ml) digest for 60 min. at 58 °C followed by addition of 200 μl Protein Precipitation Buffer after protein precipitation and overnight incubation on ice. DNA is available through the DNA Bank Network [20].

Genome sequencing and assembly

The permanent draft genome of B. barnesiae strain BL2T was generated using Illumina technology [18, 21]. An Illumina Standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 11,109,700 reads totaling 1,666.5 Mb. All general aspects of library construction and sequencing performed at the DOE-JGI can be found at [22]. All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts [23]. Following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet [24], (2) 1–3 kb simulated paired end reads were created from Velvet Contigs using wgsim [25], (3) Illumina reads were assembled with simulated read pairs using Allpaths–LG (version r41043) [26]. Parameters for assembly steps were: 1) Velvet (velveth: 63 –shortPaired and velvetg: −very clean yes –export- Filtered yes –min contig lgth 500 –scaffolding no –cov cutoff 10) 2) wgsim (−e 0 –1 100 –2 100 –r 0 –R 0 –X 0) 3) Allpaths–LG (PrepareAllpathsInputs: PHRED 64 = 1 PLOIDY = 1 FRAG COVERAGE = 125 JUMP COVERAGE = 25 LONG JUMP COV = 50, RunAllpathsLG: THREADS = 8 RUN = std shredpairs TARGETS = standard VAPI WARN ONLY = True OVERWRITE = True). The final draft assembly contained 47 contigs in 43 scaffolds. The total size of the genome is 3.6 Mb and the final assembly is based on 443.6 Mb of Illumina data, which provides an average 122.7 × coverage of the genome.

Genome annotation

Genes were identified using Prodigal [27] as part of the DOE-JGI genome annotation pipeline [28, 29], following by a round of manual curation using the JGI GenePRIMP pipeline [30]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information non-redundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro database. These data sources were combined to assert a product description for each predicted protein. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes-Expert Review platform [31].

Genome properties

The assembly of the draft genome sequence consists of 43 scaffolds amounting to 3,621,509 bp, and the G + C content is 46.8 % (Table 3). Of the 3,156 genes predicted, 3,059 were protein-coding genes, and 97 RNAs. The majority of the protein-coding genes (71.7 %) were assigned 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.
Table 3

Genome statistics



% of total

Genome size (bp)



DNA coding region (bp)



DNA G + C content (bp)



DNA scaffolds



Total genes



Protein coding genes



RNA genes



Genes with function prediction



Genes assigned to COGs



Genes with Pfam domains



Genes with signal peptides



Genes with transmembrane helices



CRISPR repeats


Table 4

Number of genes associated with the general COG functional categories



% age





Translation, ribosomal structure and biogenesis




RNA processing and modification








Replication, recombination and repair




Chromatin structure and dynamics




Cell cycle control, cell division, chromosome partitioning




Nuclear structure




Defense mechanisms




Signal transduction mechanisms




Cell wall/membrane/envelope biogenesis




Cell motility








Extracellular structures




Intracellular trafficking, secretion, and vesicular transport




Posttranslational modification, protein turnover, chaperones




Energy production and conversion




Carbohydrate transport and metabolism




Amino acid transport and metabolism




Nucleotide transport and metabolism




Coenzyme transport and metabolism




Lipid transport and metabolism




Inorganic ion transport and metabolism




Secondary metabolites biosynthesis, transport and catabolism




General function prediction only




Function unknown




Not in COGs

Insights from the genome sequence

B. barnesiae strain BL2T, Bacteroides salanitronis strain BL78T and Bacteroides gallinarum strain C35T were isolated from the cecum of the same healthy chicken [1]. The GGDC (Genome-to-Genome Distance Calculator) web server (GGDC 2.0) [32] was used for the estimation of the overall similarity between the three Bacteroides genomes. The comparison of B. barnesiae with B. salanitronis and B. gallinarum revealed that 11.1 % and 5.2 %, respectively, of the average of the genome lengths are covered with HSPs (high-scoring segment pairs). The identity within the HSPs was 83.6 % and 84.6 %, respectively, whereas the identity over the whole genome was 9.3 % and 4.4 %, respectively. The comparison of B. gallinarum with B. salanitronis revealed that 5.4 % of the genome is covered with HSPs, with an identity within in the HSPs of 84.1 % and an identity over the whole genome of 4.6 %. According to these calculations the similarity between B. barnesiae and B. salanitronis is higher than the similarity between B. barnesiae and B. gallinarum as well as the similarity between B. gallinarum and B. salanitronis .

The genome size of B. barnesiae (3.6 Mb) is significantly smaller than those of B. salanitronis (4.3 Mb) and B. gallinarum (4.9 Mb).


B. barnesiae strain BL2T genome consists of a single chromosome of 3.6 Mb predicted to encode 3,156 genes. Strain BL2T has a relatively small genome in comparison to other sequenced Bacteroides species isolated from the same chicken (4.3-4.9 Mb). These differences of genome size may be the results of adaptation in this niche. Further study will be necessary for elucidation of this idea.



One thousand microbial genomes


Joint genome institute


Sequencing orphan species


Genome-to-genome distance calculator



We would like to gratefully acknowledge the help of Iljana Schröder for growing B. barnesiae cultures, and Evelyne-Marie Brambilla for DNA extraction and quality control (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. A.L. was supported in part by Russian Ministry of Science Mega-grant no.11.G34.31.0068 (PI. Dr Stephen J O’Brien).

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Tsukuba, Japan
Theodosius Dobzhansky Center for Genome Bionformatics, St. Petersburg State University, St. Petersburg, Russia
Algorithmic Biology Lab, St. Petersburg Academic University, St. Petersburg, Russia
DOE Joint Genome Institute, Walnut Creek, USA
Leibniz-Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory, Berkeley, USA
Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia


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