Genome sequence of the moderately thermophilic, amino-acid-degrading and sulfur-reducing bacterium Thermovirga lienii type strain (Cas60314T)

Thermovirga lienii Dahle and Birkeland 2006 is a member of the genus Thermovirga in the genomically moderately well characterized phylum 'Synergistetes'. Members of this relatively recently proposed phylum ‘Synergistetes’ are of interest because of their isolated phylogenetic position and their diverse habitats, e.g. from humans to oil wells. The genome of T. lienii Cas60314T is the fifth genome sequence (third completed) from this phylum to be published. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,999,646 bp long genome (including one plasmid) with its 1,914 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain Cas60314 T (= DSM 17291 = ATCC BA-1197) is the type strain of the species Thermovirga lienii [1] of the monospecific genus Thermovirga [1]. The strain was originally isolated from 68°C hot oil-well production water from an oil reservoir in the North Sea (Norway) [1]. The genus name Thermovirga was derived from the Greek word for thermê, heat, and the Latin word virga, rod, meaning the hot rod [1]; the species epithet was derived of Lien, in honor of the Norwegian microbiologist Professor Torleiv Lien, for his important contribution in the study of anaerobes from petroleum reservoirs [1]. Whether or not strain Cas60314 T occurs naturally in the oil reservoir is not clear, but is likely because the oil well was not injected with seawater (which eliminates a common source of contamination) [1]. Here we present a summary classification and a set of features for T. lienii Cas60314 T , together with the description of the genomic sequencing and annotation.

Classification and features
A representative genomic 16S rRNA sequence of T. lienii Cas60314 T was compared using NCBI BLAST [2,3] 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 [4] and the relative frequencies of taxa and keywords (reduced to their stem [5]) were determined, weighted by BLAST scores. The most frequently occurring genera were Dethiosulfovibrio (45.6%), Thermanaerovibrio (30.2%), Aminobacterium (12.5%) and Thermovirga (11.7%) (16 hits in total). Regarding the single hit to sequences from members of the species, the average identity within HSPs was 100.0%, whereas the average coverage by HSPs was 92.2%. Among all other species, the one yielding the highest score was Thermanaerovibrio velox (FR733707), which corresponded to an identity of 90.1% and an HSP coverage of 85.5%. (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 HM041945 ('aggregate-forming and crude-oil-adhering microbial biodegradedtemperature petroleum reservoir produced fluid Niiboli oilfield clone NRB28'), which showed an identity of 99.7% and an HSP coverage of 98.3%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were 'digest' (10.9%), 'anaerob' (7.4%), 'sludg' (5.8%), 'mesophil' (5.6%) and 'wastewat' (5.6%) (234 hits in total). The most frequently occurring keywords within the labels of those environmental samples which yielded hits of a higher score than the highest scoring species were 'microbi' (6.3%), 'temperatur' (4.4%), 'oil' (3.6%), 'water' (3.6%) and 'anaerob' (3.2%) (43 hits in total). Although these keywords are not in conflict with the habitat from which strain Cas60314 T was isolated, they do not reveal new insights into the largely unknown natural habitat for members of the species. Figure 1 shows the phylogenetic neighborhood of T. lienii in a 16S rRNA based tree. The sequences of the three 16S rRNA gene copies in the genome differ from each other by up to three nucleotides, and differ by up to three nucleotides from the previously published 16S rRNA sequence (DQ071273). Cells of T. lienii Cas60314 T are Gram-negative staining, motile (only in the early exponential growth phase), straight rods of 0.4 to 0.8 μm diameter and 2 to 3 μm length ( Figure 2) [1]. Cells appeared to be singly, or in chains of 2 to 5 cells, but can also form aggregates of several hundred cells [1]. The growth range of strain Cas60314 T spans from 37-68°C, with an optimum at 58°C, and pH 6.2-8.5, with an optimum at 6.5-7 [1]. Strain Cas60314 T grow best in medium containing 2-3% NaCl [1]. Physiological characteristics such as growth substrates and products formed are described in great detail by Dahle and Birkeland [1]; the organism has a fermentative metabolism and utilizes amino acids, proteinaceous substrates and selected organic acids, but no sugars, fatty acids or alcohols [1]. Strain Cas60314 T reduces cysteine and elemental sulfur to sulfide, but not thiosulfate [1]. The strain could not grow in unreduced medium under an N 2 /air (20:1, v/v) gas phase, but was able to grow with 100% H 2 in the headspace, with peptone as substrate [1].

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [22], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [23]. The genome project is deposited in the Genomes On Line 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.

Growth conditions and DNA isolation
T. lienii strain Cas60314 T , DSM 17291, was grown anaerobically in DSMZ medium 383 (Desulfobacterium medium) [24] at 55°C. DNA was isolated from 0.5-1 g of cell paste using MasterPure Gram-positive DNA purification kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer, with the following modification for cell lysis: 1µl lysozyme and 5 µl mutanolysin added to the standard lysis solution for 40 min at 37°C; after the MPC-step the lysis solution was incubated for one hour on ice. DNA is available through the DNA Bank Network [25].  [6,7] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [8]. Rooting was done initially using the midpoint method [9] 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 1,000 ML bootstrap replicates [10] (left) and from 1,000 maximum parsimony bootstrap replicates [11] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [12] are labeled with one asterisk, those also listed as 'Complete and Published' with two asterisks [13,14]. Dethiosulfovibrio peptidovorans [15] and Aminomonas paucivorans [16] are without a second asterisk, because these publications were based on 'non-complete' permanent draft sequences.  Phylum "Synergistetes" TAS [19] Class Synergistia TAS [19] Order Synergistales TAS [19] Family Synergistaceae TAS [19] Genus 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 the Gene Ontology project [21]. If the evidence code is IDA, then the property was directly observed for a living isolate by one of the authors or an expert mentioned in the acknowledgements. Standards in Genomic Sciences

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 [26]. Pyrosequencing reads were assembled using the Newbler assembler (Roche). The initial Newbler assembly consisting of 127 contigs in one scaffold was converted into a phrap [27] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (379.0 Mb) was assembled with Velvet [28] 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 156.8 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 [27] 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 [26], Dupfinisher [29], 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 811 additional reactions and 23 shatter libraries 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 [30]. The error rate of the completed genome sequence is less than 1 in 100,000.

Genome annotation
Genes were identified using Prodigal [31] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [32].
The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [33], RNAMMer [34], Rfam [35], TMHMM [36], and signalP [37].

Genome properties
The genome consists of a 1,967,774 bp long chromosome and a 31,872 bp long circular plasmid with a 47.1% G+C content (Table 3 and Figure 3). Of the 1,973 genes predicted, 1,914 were proteincoding genes, and 59 RNAs; 38 pseudogenes were also identified. The majority of the protein-coding genes (79.0%) 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.