Complete genome sequence of Coriobacterium glomerans type strain (PW2T) from the midgut of Pyrrhocoris apterus L. (red soldier bug)

Coriobacterium glomerans Haas and König 1988, is the only species of the genus Coriobacterium, family Coriobacteriaceae, order Coriobacteriales, phylum Actinobacteria. The bacterium thrives as an endosymbiont of pyrrhocorid bugs, i.e. the red fire bug Pyrrhocoris apterus L. The rationale for sequencing the genome of strain PW2T is its endosymbiotic life style which is rare among members of Actinobacteria. Here we describe the features of this symbiont, together with the complete genome sequence and its annotation. This is the first complete genome sequence of a member of the genus Coriobacterium and the sixth member of the order Coriobacteriales for which complete genome sequences are now available. The 2,115,681 bp long single replicon genome with its 1,804 protein-coding and 54 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain PW2 T (= DSM 20642 = ATCC 49209 = JCM 10262) is the type strain of Coriobacterium glomerans [1]. The absence of sequence data in the original description excluded determination of the phylogenetic position of the genus Coriobacterium, but taxonomic evidence also excluded an affiliation with Bifidobacterium, Eubacterium or Lachnospira. The 16S rRNA gene sequence [2] revealed that Coriobacterium and Atopobium [3] are phylogenetic neighbors. Based upon phylogenetic position within the class Actinobacteria and a unique set of 16S rRNA gene signature nucleotides both genera were placed in the family Coriobacteriaceae, order Coriobacterales, subclass Coriobacteridae [4]. The family was expanded by the description of several new genera which at the time of writing encompasses 13 genera and 29 species [5]. In the 2 nd edition of Bergey's Manual the class Actinobacteria was elevated to phylum rank [6] and subsequently the subclass Coriobacteridae was elevated to class rank [7]. The suborder rank 'Coriobacterineae' has been introduced by Garrity and collaborators [8].
Coriobacterium is a phylogenetic neighbor of Collinsella [9] and both genera form one of four sister clades of Coriobacteriaceae. Besides the type strain a few other closely related strains (e.g. accession numbers FJ554833, FJ554832, FJ554836, FJ554835) were isolated from Pyrrhocoris apterus L. and a related pyrrhocorid host. Their localization in the midgut, the rectum and feces of the red firebug and the vertical transmission route via application of the symbiont to the surface of the eggs was determined via PCR amplification and FISH hybridization. Horizontal transmission also occurred via symbiont-containing material [10]. BLAST re-analysis of 16S rRNA gene sequences of other strains and clones (e.g. accession numbers AJ131149, AJ131150, AJ245921) reported to be members of Coriobacterium [11] revealed that they are actually members of Collinsella.
Here we present a summary classification and a set of features for C. glomerans PW2 T together with the description of the complete genomic sequencing and annotation.

Features of the organism 16S rRNA gene sequence analysis
A representative genomic 16S rRNA gene sequence of C. glomerans PW2 T was compared using NCBI BLAST [12,13] under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database [14] and the relative frequencies of taxa and keywords (reduced to their stem [15]) were determined, weighted by BLAST scores. The most frequently occurring genera were Collinsella (61.9%) and Coriobacterium (38.1%) (29 hits in total). Regarding the five hits to sequences from members of the species, the average identity within HSPs was 97.8%, whereas the average coverage by HSPs was 93.4%. Among all other species, the one yielding the highest score was Collinsella tanakaei (AB490807), which corresponded to an identity of 93.4% and an HSP coverage of 99.4%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was EF399657 (Greengenes short name 'human fecal clone SJTU E 01 75'), which showed an identity of 93.5% and an HSP coverage of 98.4%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were 'human' (20.6%), 'fecal' (19.8%), 'fece' (10.6%), 'lion' (4.7%) and 'intestin' (1.9%) (221 hits in total). Environmental samples which yielded hits of a higher score than the highest scoring species were not found, indicating that C. glomerans is rarely found in environmental samples. Figure 1 shows the phylogenetic neighborhood of C. glomerans PW2 T in a 16S rRNA based tree. The sequences of the two identical 16S rRNA gene copies in the genome differ by six nucleotides from the previously published 16S rRNA sequence (X79048), which contains three ambiguous base calls.

Morphology and physiology
Cells of C. glomerans PW2 T stain Gram-positive which is consistent with an electron-dense cell wall, 40 nm wide [35]. The cells are pear-shaped to irregularly shaped rods with length that varies from 0.44 to 1.80 µm ( Figure 2). Spherical involution forms are common. When attached to the epithelia of the intestines, the bacteria form chains which may reach more than 150 µm in length. Flagella are absent. Colonies, grown anaerobically in an atmosphere of N 2 -CO 2 (80:20) on blood agar (Columbia agar base, BBL), supplemented Schaedler agar (BBL), and TPY agar at 25 and 30°C are about 0.6 mm in diameter and consist of long filamentous chains, bent into hairpins, curls, and loops. A flocculent, wooly sediment with a clear supernatant is formed in fluid media. The bacteria are strictly anaerobic. In TPY medium glucose is fermented to acetic acid, L-lactic acid, and ethanol in a molar ratio of 1.16:1.00:0.95. CO 2 and H 2 also are produced [1] but D-lactic acid, formic acid, volatile short-chain alcohols, or other volatile fatty acids are not formed. Under more stringent anaerobic conditions with N 2 -CO 2 (80:20) as the gas phase, but lacking H 2 , the formation of ethanol occurs only at a lower concentration. C. glomerans PW2 T ferments glucose, L-arabinose, D-xylose, D-ribose, mannose, sucrose, maltose, cellobiose, mannitol, and salicin but not lactose, melibiose, raffinose, inulin, starch, and inositol [1].

Chemotaxonomy
The peptidoglycan of stain PW2 T contains lysine as the diagnostic amino acid in position 3 of the peptide subunit with the interpeptide bridge containing aspartic acid (Lys-Asp type; A4α according to [36]; A11.31 according to [37]). Information on major cell wall sugars, fatty acids, menaquinones and polar lipids is not available. The mol% G+C of DNA was reported to be about 61, and is here confirmed by the genome sequence.  [16,17] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [18]. Rooting was done initially using the midpoint method [19] and then checked for its agreement with the current classification ( Table 1). The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 300 ML bootstrap replicates [20] (left) and from 1,000 maximum-parsimony bootstrap replicates [21] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [22] are labeled with one asterisk, those also listed as 'Complete and Published' with two asterisks ( [23][24][25][26][27], see FP929047 for Gordonibacter pamelaeae).  Phylum Actinobacteria TAS [30] Class 'Actinobacteria' TAS [4] Subclass Coriobacteridae TAS [4,31] Current classification Order Coriobacteriales TAS [4,31] Suborder 'Coriobacterineae' TAS [32] Family Coriobacteriaceae TAS [4,31] Genus

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [38], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [39]. The genome project is deposited in the Genomes OnLine Database [22] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI) using state of the art sequencing technology [40]. A summary of the project information is shown in Table 2.

Growth conditions and DNA isolation
C. glomerans strain PW2 T , DSM 20642, was grown anaerobically in an atmosphere of N 2 -CO 2 (80: 20) in DSMZ medium 104 (modified PYG medium) at 30°C. DNA was isolated from 1-1.5 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/LALM for cell lysis as described in Wu et al. 2009 [39]. DNA is available through the DNA Bank Network [41].

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 [42]. Pyrosequencing reads were assembled using the Newbler assembler (Roche Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [46]. 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 104.1 × coverage of the genome. The final assembly contained 456,305 pyrosequence and 2,106,317 Illumina reads.

Genome annotation
Genes were identified using Prodigal [47] as part of the DOE-JGI genome annotation pipeline [48], followed by a round of manual curation using the JGI GenePRIMP pipeline [49]. 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 [50].

Genome properties
The genome statistics are provided in Table 3 and Figure 3. The genome consists of one chromosome with a total length of 2,115,681 bp and a G+C content of 60.4%. Of the 1,858 genes predicted, 1,804 were protein-coding genes, and 54 RNAs; 36 pseudogenes were also identified. The majority of the protein-coding genes (74.2%) 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.