Introduction
============

Strain PW2^T^ (= DSM 20642 = ATCC 49209 = JCM 10262) is the type strain of *[Coriobacterium glomerans](http://dx.doi.org/10.1601/nm.5738)* \[[@r1]\]. The absence of sequence data in the original description excluded determination of the phylogenetic position of the genus *[Coriobacterium](http://dx.doi.org/10.1601/nm.5737)*, but taxonomic evidence also excluded an affiliation with *[Bifidobacterium](http://dx.doi.org/10.1601/nm.7677)*, *[Eubacterium](http://dx.doi.org/10.1601/nm.4228)* or *[Lachnospira](http://dx.doi.org/10.1601/nm.4119)*. The 16S rRNA gene sequence \[[@r2]\] revealed that *[Coriobacterium](http://dx.doi.org/10.1601/nm.5737)* and *[Atopobium](http://dx.doi.org/10.1601/nm.5739)* \[[@r3]\] are phylogenetic neighbors. Based upon phylogenetic position within the class *[Actinobacteria](http://dx.doi.org/10.1601/nm.5712)* and a unique set of 16S rRNA gene signature nucleotides both genera were placed in the family *[Coriobacteriaceae](http://dx.doi.org/10.1601/nm.5736)*, order *Coriobacterales*, subclass *[Coriobacteridae](http://dx.doi.org/10.1601/nm.5733)* \[[@r4]\]. The family was expanded by the description of several new genera which at the time of writing encompasses 13 genera and 29 species \[[@r5]\]. In the 2^nd^ edition of Bergey's Manual the class *[Actinobacteria](http://dx.doi.org/10.1601/nm.5712)* was elevated to phylum rank \[[@r6]\] and subsequently the subclass *[Coriobacteridae](http://dx.doi.org/10.1601/nm.5733)* was elevated to class rank \[[@r7]\]. The suborder rank '*[Coriobacterineae](http://dx.doi.org/10.1601/nm.5735)'* has been introduced by Garrity and collaborators \[[@r8]\]. *[Coriobacterium](http://dx.doi.org/10.1601/nm.5737)* is a phylogenetic neighbor of *[Collinsella](http://dx.doi.org/10.1601/nm.5745)* \[[@r9]\] and both genera form one of four sister clades of *[Coriobacteriaceae](http://dx.doi.org/10.1601/nm.5736)*. 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 \[[@r10]\]. 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](http://dx.doi.org/10.1601/nm.5737)* \[[@r11]\] revealed that they are actually members of *[Collinsella](http://dx.doi.org/10.1601/nm.5745)*.

Here we present a summary classification and a set of features for *[C. glomerans](http://dx.doi.org/10.1601/nm.5738)* 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](http://dx.doi.org/10.1601/nm.5738)* PW2^T^ was compared using NCBI BLAST \[[@r12],[@r13]\] 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 \[[@r14]\] and the relative frequencies of taxa and keywords (reduced to their stem \[[@r15]\]) were determined, weighted by BLAST scores. The most frequently occurring genera were *[Collinsella](http://dx.doi.org/10.1601/nm.5745)* (61.9%) and *[Coriobacterium](http://dx.doi.org/10.1601/nm.5737)* (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](http://dx.doi.org/10.1601/nm.20207)* (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](http://dx.doi.org/10.1601/nm.5738)* is rarely found in environmental samples.

[Figure 1](#f1){ref-type="fig"} shows the phylogenetic neighborhood of *[C. glomerans](http://dx.doi.org/10.1601/nm.5738)* 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.

![Phylogenetic tree highlighting the position of *[C. glomerans](http://dx.doi.org/10.1601/nm.5738)* relative to the type strains of the other species within the family *[Coriobacteriaceae](http://dx.doi.org/10.1601/nm.5736)*. The tree was inferred from 1,401 aligned characters \[[@r16],[@r17]\] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion \[[@r18]\]. Rooting was done initially using the midpoint method \[[@r19]\] and then checked for its agreement with the current classification ([Table 1](#t1){ref-type="table"}). 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 \[[@r20]\] (left) and from 1,000 maximum-parsimony bootstrap replicates \[[@r21]\] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD \[[@r22]\] are labeled with one asterisk, those also listed as \'Complete and Published\' with two asterisks (\[[@r23]-[@r27]\], see FP929047 for *[Gordonibacter pamelaeae](http://dx.doi.org/10.1601/nm.14334)*).](sigs.3507020-f1){#f1}

###### Classification and general features of *[C. glomerans](http://dx.doi.org/10.1601/nm.5738)* PW2^T^ according to the MIGS recommendations \[[@r28]\].

  MIGS ID        Property                                   Term                                                                          Evidence code
  ----------- ----------------------------------------- ----------------------------------------------------------------------------- --------------------------
                 Current classification                     Domain *[Bacteria](http://dx.doi.org/10.1601/nm.419)*                         TAS \[[@r29]\]
                                                            Phylum *[Actinobacteria](http://dx.doi.org/10.1601/nm.5712)*                  TAS \[[@r30]\]
                                                            Class *[Actinobacteria](http://dx.doi.org/10.1601/nm.5712)*                   TAS \[[@r4]\]
                                                            Subclass *[Coriobacteridae](http://dx.doi.org/10.1601/nm.5733)*               TAS \[[@r4],[@r31]\]
                                                            Order *[Coriobacteriales](http://dx.doi.org/10.1601/nm.5734)*                 TAS \[[@r4],[@r31]\]
                                                            Suborder *[Coriobacterineae](http://dx.doi.org/10.1601/nm.5735)*              TAS \[[@r32]\]
                                                            Family *[Coriobacteriaceae](http://dx.doi.org/10.1601/nm.5736)*               TAS \[[@r4],[@r31]\]
                                                            Genus *[Coriobacterium](http://dx.doi.org/10.1601/nm.5737)*                   TAS \[[@r1]\]
                                                            Species *[Coriobacterium glomerans](http://dx.doi.org/10.1601/nm.5738)*       TAS \[[@r1]\]
  MIGS-7         Subspecific genetic lineage (strain)       PW2^T^                                                                        TAS \[[@r1]\]
  MIGS-12        Reference for biomaterial                  Haas and König 1988                                                           TAS \[[@r1]\]
                 Gram stain                                 positive                                                                      TAS \[[@r1]\]
                 Cell shape                                 rod-shaped                                                                    TAS \[[@r1]\]
                 Motility                                   non-motile                                                                    TAS \[[@r1]\]
                 Sporulation                                non-sporulating                                                               TAS \[[@r1]\]
                 Temperature range                          mesophile                                                                     TAS \[[@r1]\]
                 Optimum temperature                        30°C                                                                          TAS \[[@r1]\]
                 Salinity                                   not reported                                                              
  MIGS-22        Relationship to oxygen                     obligate anaerobe                                                             TAS \[[@r1]\]
                 Carbon source                              not reported                                                              
                 Energy metabolism                          chemoorganotroph                                                              TAS \[[@r1]\]
  MIGS-6         Habitat                                    host, intestinal tract                                                        TAS \[[@r1]\]
  MIGS-6.2       pH                                         not reported                                                              
  MIGS-15        Biotic relationship                        unknown                                                                   
  MIGS-14        Known pathogenicity                        none                                                                          TAS \[[@r1]\]
  MIGS-16        Specific host                              *Pyrrhocoris apterus* L.                                                      TAS \[[@r1]\]
  MIGS-18        Health status of Host                      unknown                                                                   
                 Biosafety level                            1                                                                             TAS \[[@r33]\]
  MIGS-19        Trophic level                              unknown                                                                   
  MIGS-23.1      Isolation                                  intestinal tract of the red soldier bug                                       TAS \[[@r1]\]
  MIGS-4         Geographic location                        Bavaria, Germany                                                              TAS \[[@r1]\]
  MIGS-5         Time of sample collection                  December 1981                                                                 NAS
  MIGS-4.1       Latitude                                   not reported                                                              
  MIGS-4.2       Longitude                                  not reported                                                              
  MIGS-4.3       Depth                                      not reported                                                              
  MIGS-4.4       Altitude                                   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). Evidence codes are from the Gene Ontology project \[[@r34]\].

Morphology and physiology
-------------------------

Cells of *[C. glomerans](http://dx.doi.org/10.1601/nm.5738)* PW2^T^ stain Gram-positive which is consistent with an electron-dense cell wall, 40 nm wide \[[@r35]\]. The cells are pear-shaped to irregularly shaped rods with length that varies from 0.44 to 1.80 µm ([Figure 2](#f2){ref-type="fig"}). 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 \[[@r1]\] 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](http://dx.doi.org/10.1601/nm.5738)* 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 \[[@r1]\].

![Scanning electron micrograph of *[C. glomerans](http://dx.doi.org/10.1601/nm.5738)* PW2^T^](sigs.3507020-f2){#f2}

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 \[[@r36]\]; A11.31 according to \[[@r37]\]). 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.

Genome sequencing and annotation
================================

Genome project history
----------------------

This organism was selected for sequencing on the basis of its phylogenetic position \[[@r38]\], and is part of the ***G****enomic* ***E****ncyclopedia of* ***[Bacteria](http://dx.doi.org/10.1601/nm.419)****and* ***[Archaea](http://dx.doi.org/10.1601/nm.1)*** project \[[@r39]\]. The genome project is deposited in the Genomes OnLine Database \[[@r22]\] 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 \[[@r40]\]. A summary of the project information is shown in [Table 2](#t2){ref-type="table"}.

###### Genome sequencing project information

  -----------------------------------------------------------------------------------------------------------------
  MIGS ID        Property                        Term
  ----------- ------------------------------- ---------------------------------------------------------------------
  MIGS-31        Finishing quality               Finished

  MIGS-28        Libraries used                  Three genomic libraries: one 454 pyrosequence standard library,\
                                                 one 454 PE library (6.7 kb insert size), one Illumina library

  MIGS-29        Sequencing platforms            Illumina GAii, 454 GS FLX Titanium

  MIGS-31.2      Sequencing coverage             35.8 × Illumina; 68.3 × pyrosequence

  MIGS-30        Assemblers                      Newbler version 2.3, Velvet, Phrap version SPS - 4.24

  MIGS-32        Gene calling method             Prodigal 1.4, GenePRIMP

                 INSDC ID                        CP002628

                 GenBank Date of Release         October 11, 2011

                 GOLD ID                         Gc01723

                 NCBI project ID                 42699

                 Database: IMG-GEBA              2503538010

  MIGS-13        Source material identifier      DSM 20642

                 Project relevance               Tree of Life, GEBA
  -----------------------------------------------------------------------------------------------------------------

Growth conditions and DNA isolation
-----------------------------------

*[C. glomerans](http://dx.doi.org/10.1601/nm.5738)* 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 \[[@r39]\]. DNA is available through the DNA Bank Network \[[@r41]\].

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 \[[@r42]\]. Pyrosequencing reads were assembled using the Newbler assembler (Roche). The initial Newbler assembly consisting of 14 contigs in one scaffold was converted into a phrap \[[@r43]\] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (75.8 Mb) was assembled with Velvet \[[@r44]\] 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 128.4 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 \[[@r43]\] 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 \[[@r42]\], Dupfinisher \[[@r45]\], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 40 additional reactions 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 \[[@r46]\]. 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 \[[@r47]\] as part of the DOE-JGI genome annotation pipeline \[[@r48]\], followed by a round of manual curation using the JGI GenePRIMP pipeline \[[@r49]\]. 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 \[[@r50]\].

Genome properties
=================

The genome statistics are provided in [Table 3](#t3){ref-type="table"} and [Figure 3](#f3){ref-type="fig"}. 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](#t4){ref-type="table"}.

###### Genome Statistics

  **MIGS ID**      **Attribute**                          **Number**      **% of Total**
  ------------- ------------------------------------- ---------------- -------------------
                   Genome size (bp)                       2,115,681       100.00
                   DNA coding region (bp)                 1,879,452       88.83
                   DNA G+C content (bp)                   1,277,733       60.39
  MIGS-9           Number of replicons                    1            
  MIGS-10          Extrachromosomal elements              0            
                   Total genes                            1,858           100.00
                   RNA genes                              54              2.91
                   rRNA operons                           2            
                   tRNA genes                             45              2.42
                   Protein-coding genes                   1,804           97.09
                   Pseudo genes                           36              1.94
                   Genes with function prediction         1,378           74.17
                   Genes in paralog clusters              828             44.56
                   Genes assigned to COGs                 1,500           80.73
                   Genes assigned Pfam domains            1,551           83.48
                   Genes with signal peptides             314             16.90
                   Genes with transmembrane helices       484             26.05
                   CRISPR repeats                         2            

![Graphical map of the chromosome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew (purple/olive).](sigs.3507020-f3){#f3}

###### Number of genes associated with the general COG functional categories

  Code      Value      % age       Description
  ------ ---------- ---------- ----------------------------------------------------------------------
  J         134        8.2         Translation, ribosomal structure and biogenesis
  A         \...       \...        RNA processing and modification
  K         158        9.6         Transcription
  L         75         4.6         Replication, recombination and repair
  B         1          0.1         Chromatin structure and dynamics
  D         19         1.2         Cell cycle control, cell division, chromosome partitioning
  Y         \...       \...        Nuclear structure
  V         43         2.6         Defense mechanisms
  T         62         3.8         Signal transduction mechanisms
  M         98         6.0         Cell wall/membrane biogenesis
  N         \...       \...        Cell motility
  Z         \...       \...        Cytoskeleton
  W         \...       \...        Extracellular structures
  U         17         1.0         Intracellular trafficking and secretion, and vesicular transport
  O         42         2.6         Posttranslational modification, protein turnover, chaperones
  C         63         3.8         Energy production and conversion
  G         317        19.3        Carbohydrate transport and metabolism
  E         105        6.4         Amino acid transport and metabolism
  F         52         3.2         Nucleotide transport and metabolism
  H         59         3.6         Coenzyme transport and metabolism
  I         34         2.1         Lipid transport and metabolism
  P         53         3.2         Inorganic ion transport and metabolism
  Q         11         0.7         Secondary metabolites biosynthesis, transport and catabolism
  R         178        10.8        General function prediction only
  S         122        7.4         Function unknown
  \-        358        19.3        Not in COGs

We would like to gratefully acknowledge the help of Gabriele Gehrich-Schröter for growing *[C. glomerans](http://dx.doi.org/10.1601/nm.5738)* cultures, and Susanne Schneider 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, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, UT-Battelle and Oak Ridge National Laboratory under contract DE-AC05-00OR22725.
