Complete genome sequence of Coriobacterium glomerans type strain (PW2(T)) 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 PW2(T) 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 G enomic E ncyclopedia of Bacteria and Archaea project.

Introduction

Strain PW2T (= DSM 20642 = ATCC 49209 = JCM 10262) is the type strain of [1]. The absence of sequence data in the original description excluded determination of the phylogenetic position of the genus , but taxonomic evidence also excluded an affiliation with , or . The 16S rRNA gene sequence [2] revealed that and [3] are phylogenetic neighbors. Based upon phylogenetic position within the class and a unique set of 16S rRNA gene signature nucleotides both genera were placed in the family , order Coriobacterales, subclass [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 2nd edition of Bergey’s Manual the class was elevated to phylum rank [6] and subsequently the subclass was elevated to class rank [7]. The suborder rank ‘ has been introduced by Garrity and collaborators [8]. is a phylogenetic neighbor of [9] and both genera form one of four sister clades of . 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 [11] revealed that they are actually members of .

Here we present a summary classification and a set of features for PW2T 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 PW2T 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 (61.9%) and (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 (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 is rarely found in environmental samples.

Figure 1 shows the phylogenetic neighborhood of PW2T 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.

Figure 1

Phylogenetic tree highlighting the position of relative to the type strains of the other species within the family . The tree was inferred from 1,401 aligned characters [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 ([23-27], see FP929047 for ).

Table 1

Classification and general features of PW2T according to the MIGS recommendations [28].

MIGS ID	Property	Term	Evidence code
	Current classification	Domain Bacteria	TAS [29]
		Phylum Actinobacteria	TAS [30]
		Class Actinobacteria	TAS [4]
		Subclass Coriobacteridae	TAS [4,31]
		Order Coriobacteriales	TAS [4,31]
		Suborder Coriobacterineae	TAS [32]
		Family Coriobacteriaceae	TAS [4,31]
		Genus Coriobacterium	TAS [1]
		Species Coriobacterium glomerans	TAS [1]
MIGS-7	Subspecific genetic lineage (strain)	PW2T	TAS [1]
MIGS-12	Reference for biomaterial	Haas and König 1988	TAS [1]
	Gram stain	positive	TAS [1]
	Cell shape	rod-shaped	TAS [1]
	Motility	non-motile	TAS [1]
	Sporulation	non-sporulating	TAS [1]
	Temperature range	mesophile	TAS [1]
	Optimum temperature	30°C	TAS [1]
	Salinity	not reported
MIGS-22	Relationship to oxygen	obligate anaerobe	TAS [1]
	Carbon source	not reported
	Energy metabolism	chemoorganotroph	TAS [1]
MIGS-6	Habitat	host, intestinal tract	TAS [1]
MIGS-6.2	pH	not reported
MIGS-15	Biotic relationship	unknown
MIGS-14	Known pathogenicity	none	TAS [1]
MIGS-16	Specific host	Pyrrhocoris apterus L.	TAS [1]
MIGS-18	Health status of Host	unknown
	Biosafety level	1	TAS [33]
MIGS-19	Trophic level	unknown
MIGS-23.1	Isolation	intestinal tract of the red soldier bug	TAS [1]
MIGS-4	Geographic location	Bavaria, Germany	TAS [1]
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 [34].

Morphology and physiology

Cells of PW2T 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 N2-CO2 (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. CO2 and H2 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 N2-CO2 (80:20) as the gas phase, but lacking H2, the formation of ethanol occurs only at a lower concentration. PW2T 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].

Figure 2

Scanning electron micrograph of PW2T

Chemotaxonomy

The peptidoglycan of stain PW2T 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.

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 enomic ncyclopedia of and 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.

Table 2

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

strain PW2T, DSM 20642, was grown anaerobically in an atmosphere of N2-CO2 (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). The initial Newbler assembly consisting of 14 contigs in one scaffold was converted into a phrap [43] 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 [44] 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 [43] 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 [42], Dupfinisher [45], 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 [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.

Table 3

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

Figure 3

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).

Table 4

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