Complete genome sequence of Jonesia denitrificans type strain (Prevot 55134T)

Jonesia denitrificans (Prevot 1961) Rocourt et al. 1987 is the type species of the genus Jonesia, and is of phylogenetic interest because of its isolated location in the actinobacterial suborder Micrococcineae. J. denitrificans is characterized by a typical coryneform morphology and is able to form irregular nonsporulating rods showing branched and club-like forms. Coccoid cells occur in older cultures. J. denitrificans is classified as a pathogenic organism for animals (vertebrates). The type strain whose genome is described here was originally isolated from cooked ox blood. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of a member of the genus for which a complete genome sequence is described. The 2,749,646 bp long genome with its 2558 protein-coding and 71 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain Prevot 55134 T (= DSM 20603 = ATCC 14870 = CIP 55.134) is the type strain of the species Jonesia denitrificans, the type species of the genus Jonesia [1]. The isolate originated from cooked ox blood [2]. J. denitrificans was originally placed into the genus Listeria and described as L. denitrificans by Prevot in 1961 [3], even though it differed morphologically from other members of the genus Listeria. Later, extensive analysis based on the determination of the G+C content [4], DNA-DNA hybridization [4], peptidoglycan type [5,6], as well as fatty acids and polar lipid pattern [5,7] confirmed the misclassification of the strain. As a consequence the strain was transferred to the new genus Jonesia by Rocourt et al. 1987 [1]. Five years later Stackebrandt and Prauser assigned Jonesia to the family Cellulomonadaceae despite being aware that Jonesia differed from members of other genera of the family e.g. in its G+C content, peptidoglycan type and its composition of isoprenoid quinones. The rationale to place Jonesia into this family was based on the finding that the 16S rRNA gene sequence analysis showed close relationship to Promicromonospora and Cellulomonas at that time [8]. Following extensive phylogenetic in 1995, studies Jonesia was subsequently excluded from the family Cellulomonadacea and placed in the family Jonesiaceae, within the suborder Micrococcineae [9]. With Jonesia quinghaiensis, an environmental isolate from mud of a soda lake in China, a second species of the genus was described by Schumann et al. in 2004 [10]. Two additional environmental strains closely related to J. denitrificans, with 98% and 99% 16S rRNA gene sequence similarity have been reported. These organisms were isolated from the microbial community of feed batch reactors for composting of household biowaste [11]. Here we present a summary classification and a set of features for J. denitrificans Prevot 55134 T together with the description of the complete genomic sequencing and annotation.   [19,20] of the 16S rRNA sequence under the maximum likelihood criterion [21]. Rooting was done with the type strain of Actinomyces bovis, the type species of Actinomyces, which is the type genus of the order Actinomycetales. 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 [22] are shown in blue, published genomes in bold, e.g. the GEBA genomes Beutenbergia cavernae [23], and Sanguibacter keddieii [24].

Classification and features
J. denitrificans type strain cells are Gram-positive, typically coryneform and characterized by irregular rods, 0.3-0.5 µm in diameter and 2-3 µm in length. Coccoid forms occur in older cultures (Table 1),. No endospores are formed. (Figure 2). The cells are motile by means of peritrichous flagella (absent in Figure 2). Colonies range from 0.5 to 1.5 mm in diameter on BHI agar. The optimum temperature for growth is 30°C [16]. The organism is catalase positive, oxidase negative and utilizes Dcellobiose, D-galactose, D-sorbitol, turanose and acetic acid [10]. Cellulose, starch, DNA and RNA are hydrolyzed. In addition, J. denitrificans produces acid from a large variety of sugars, polysaccharides and other compounds as described by Seeliger and Jones in 1986 [16] and is capable of denitrification [25]. The natural habitat of the organism is not known, however, J. denitrificans is a known pathogen of rats and mice when injected intraperitoneally [16].

Chemotaxonomy
The cell wall of strain Prevot 55134 T contains murein of type A4α, composed of L-Lys-L-Ser-D-Glu only [5,6], type A11.48 according to the DSMZ catalogue of strains (http://www.dsmz.de/ microorganisms/main.php?content_id=35). In addition to the amino sugars muramic acid and glucosamine, galactosamine was detectable in the hydrolysate of the cell walls of J. denitrificans [5]. 12-Methyl-tetradonic acid (ai-C15:0) and hexadonic acid (C16:0) constituted the major cellular fatty acid, and minor amounts of 14-methyl-hexadonic acid (ai-C17:0) and tetradecanoic acid (C14:0) were also detected [6,7]. Diphosphatidylglycerol (DPG) and phosphatidylinositol (PI) were identified by TLC as the polar lipids [6] and menaquinone of the MK-9 type was detected as the major component.  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 the Gene Ontology project [18]. 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.

Genome sequencing information 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. The genome project is deposited in the Genomes OnLine Database [22] and the complete genome sequence 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. . DNA was isolated from 0.5-1 g of cell paste using the JGI CTAB-Protocol with a modified protocol for cell lysis (ALM), according to Wu et al. [27].

Genome sequencing and assembly
The genome was sequenced using only the Sanger platform. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website (http://www. jgi.doe.gov/). All reads were assembled using the phrap assembler. Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones [28]. Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 653 Sanger finishing reads were produced. The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequenced reads provided 9.5x coverage of the genome. The final assembly consists of 35,028 Sanger 454 reads.

Genome annotation
Genes were identified using Prodigal [29] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline (http://geneprimp.jgi-psf.org) [30]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant 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 [31].

Genome properties
The single replicon genome is 2,749,646 bp long with a 58.4% GC content ( Table 3 and Figure 3). Of the 2,629 genes predicted, 2,558 were protein coding genes, and 71 RNAs. In addition, 47 pseudogenes were also identified. The majority of the genes (68.3%) were assigned with a putative function, while the remaining ones are annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.