Complete genome sequence of Sebaldella termitidis type strain (NCTC 11300T)

Sebaldella termitidis (Sebald 1962) Collins and Shah 1986, is the only species in the genus Sebaldella within the fusobacterial family ‘Leptotrichiaceae’. The sole and type strain of the species was first isolated about 50 years ago from intestinal content of Mediterranean termites. The species is of interest for its very isolated phylogenetic position within the phylum Fusobacteria in the tree of life, with no other species sharing more than 90% 16S rRNA sequence similarity. The 4,486,650 bp long genome with its 4,210 protein-coding and 54 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain NCTC 11300 T (= ATCC 33386 TM = NCTC 11300) is the type strain of the species Sebaldella termitidis [1]. The strain was first isolated from posterior intestinal content of Reticulitermes lucifugus (Mediterranean termites) by the French microbiologist Madeleine Sebald [1,2], and was initially classified as Bacteroides termitidis [3]. The unusually low G+C content, as well as biochemical features which did not correspond to those known for the other members of the genus Bacteroides [4], and the subsequently described novel 16S rRNA sequences [5] made the position of B. termitidis within the genus Bacteroides appear controversial, and guided Collins and Shah in 1986 to reclassify B. termitidis as the type strain of the novel genus Sebaldella [1]. Here we present a summary classification and a set of features for S. termitidis NCTC 11300 T , together with the description of the complete genomic sequencing and annotation.

Classification and features
NCTC 11300 T represents an isolated species, with no other cultivated strain known in the literature belonging to the species. An uncultured clone with identical 16S rRNA sequence was identified in a mesophilic anaerobic digester that treats municipal wastewater sludge in Clos de Hilde, France [6], and another uncultured clone, PCD-1 (96.1% 16S rRNA sequence identity), was reported from the digestive tract of the ground beetle Poecilus chalcites [7]. The closest related type strains are those of the genus Leptotrichia, which share 85.9 to 89.96% 16S rRNA sequence similarity [8]. Neither environmental screenings nor metagenomic surveys provided any 16S rRNA sequence with significant sequence similarity to NCTC 11300 T , indicating that members of the species S. termitidis and the genus Sebaldella are not very frequent in the environment (status February 2010). Figure 1 shows the phylogenetic neighborhood of S. termitidis NCTC 11300 T in a 16S rRNA based tree. The sequences of the four identical copies of the 16S rRNA gene in the genome do not differ from the previously published 16S rRNA sequence generated from ATCC 3386 (M58678), which is missing two nucleotides and contains 30 ambiguous base calls. Figure 1. Phylogenetic tree highlighting the position of S. termitidis NCTC 11300 T relative to the other type strains within the family 'Leptotrichiaceae'. The tree was inferred from 1,422 aligned characters [9,10] of the 16S rRNA gene sequence under the maximum likelihood criterion [11] and rooted in accordance with the current taxonomy. 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 [12] are shown in blue, published genomes in bold, e.g. the recently published GEBA genomes from Leptotrichia buccalis [13], and Streptobacillus moniliformis [14].
Cells of strain NCTC 11300 T are Gram-negative, obligately anaerobic, nonmotile, nonsporeforming rods of 0.3 to 0.5 x 2 to 12 μm with central swellings ( Figure 2 and Table 1) [1]. Cells occur singly, in pairs, as well as in filaments [1]. Colonies on surface are transparent to opaque, circular measuring 1-2 mm in diameter, whereas colonies in deep agar are non pigmented and lenticular [1]. The major end products of the glucose metabolism by strain NCTC 11300 T are acetic and lactic acids (with some formic acid) as opposed to succinic and acetic acids dominating in members of the genus Bacteroides [1]. Enzymes of the hexosemonophosphate-shunt are missing, while present in members of the genus Bacteroides [1,4]. A list of additional sugars and alcohols used or not-used for fermentation is provided by Collins and Shah [1].  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 of the Gene Ontology project [21]. If the evidence code is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.

Chemotaxonomy
The cell wall structure of strain NCTC 11300 T has not yet been reported. Nonhydroxylated and 3hydroxyated fatty acids were present [1]. The major long chain fatty acids are saturated and monounsaturated straight chain acids: C16:0 (37%) and C18:1 (41%), with methyl branched acids being absent [1], as opposed to straight-chain saturated, anteiso-and iso-methyl branched-chain acids in members of the genus Bacteroides, which are missing the monounsaturated acids [1]. Menaquinones were not detected, as opposed to members of the genus Bacteroides [1].

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 [22]. The genome project is deposited in the Genome OnLine Database [12] 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.

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 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 4,966 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 qscores. A hybrid 454/Sanger assembly was made using the parallel phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher [24] 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. A total of 796 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. Illumina reads were used to improve the final consensus quality using an inhouse developed tool (the Polisher, unpublished).
The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequence types provided 39.5× coverage of the genome. The final assembly contains 45,934 Sanger and 760,187 pyrosequence reads.

Genome annotation
Genes were identified using Prodigal [25] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [26]. 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 manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform [27].

Genome properties
The genome consists of a 4,418,842 bp long chromosome, and two plasmids with 54,160 bp and 13,648 bp length, respectively, with a 33.4% GC content (Table 3 and Figure 3). Of the 4,264 genes predicted, 4,210 were protein-coding genes, and 54 RNAs; 59 pseudogenes were identified. The majority of the protein-coding genes (60.4%) were assigned with a putative function while those remaining were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.