Complete genome sequence of Tsukamurella paurometabola type strain (no. 33T)

Tsukamurella paurometabola corrig. (Steinhaus 1941) Collins et al. 1988 is the type species of the genus Tsukamurella, which is the type genus to the family Tsukamurellaceae. The species is not only of interest because of its isolated phylogenetic location, but also because it is a human opportunistic pathogen with some strains of the species reported to cause lung infection, lethal meningitis, and necrotizing tenosynovitis. This is the first completed genome sequence of a member of the genus Tsukamurella and the first genome sequence of a member of the family Tsukamurellaceae. The 4,479,724 bp long genome contains a 99,806 bp long plasmid and a total of 4,335 protein-coding and 56 RNA genes, and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain no. 33 T (= DSM 20162 = ATCC 8368 = JCM 10117) is the type strain of the species Tsukamurella paurometabola, which in turn is the type species of the genus Tsukamurella [1,2]. Currently, there are eleven species within the genus Tsukamurella [1,3], which is named in honor of Michio Tsukamura, a Japanese microbiologist [1]. The species epithet derives from the Greek words paurus meaning little and metabolus meaning changeable, referring to a metabolism that is little changeable [1]. Strain no. 33 T was first isolated from the mycetome and ovaries of Cimex lectularis (bedbug) in a study on the bacterial flora of Hexapoda by Edward A. Stein-haus in 1941 [2]. T. paurometabola was formerly also known as Corynebacterium paurometabolum (basonym) [1,4] as well as under its heterotypic synonym Rhodococcus aurantiacus [5,6], until Collins et al. revised the controversial taxonomic position of the species in 1988 [1] and J. P. Euzéby corrected the species epithet according to the rules of to the International Code of Nomenclature of Bacteria (1990 Revision) [7]. T. paurometabola is known, albeit rarely, to be an opportunistic pathogen for humans, especially in patients with predisposing conditions, such as immunosuppression (leukemia, solid tumors, and HIV infection) [8,9], chronic lung disease (tuberculosis) [9], and most often indwelling foreign bodies (long-term use of indwelling catheters) [10][11][12][13]. Here we present a summary classification and a set of features for T. paurometabola no. 33 T , together with the description of the complete genomic sequencing and annotation.

Classification and features
The phylogenetic neighborhood of T. paurometabola no. 33 T in a 16S rRNA based tree is shown in Figure 1. The sequences of the two identical 16S rRNA gene copies in the genome differ by one nucleotide from the previously published 16S rRNA sequence (AF283280).

Figure 1.
Phylogenetic tree highlighting the position of T. paurometabola relative to the other type strains within the genus Tsukamurella. The tree was inferred from 1,447 aligned characters [14,15] of the 16S rRNA gene sequence under the maximum likelihood criterion [16] and rooted with the members of the closely related genus Dietzia. 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 [17] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [18] are labeled with one asterisk, those registered as 'Complete and Publish.
A representative genomic 16S rRNA sequence of strain no. 33 T was compared using NCBI BLAST 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 [19] and the relative frequencies, of taxa and keywords (reduced to their stem [20]) were determined, weighted by BLAST scores. The most frequently occurring genera were Tsukamurella (34.7%), Mycobacterium (32.5%), Dietzia (20.6%) and Rhodococcus (12.1%) (220 hits in total). Regarding the seven hits to sequences from members of the species, the average identity within HSPs was 99.3%, whereas the average coverage by HSPs was 96.7%. Regarding the 45 hits to sequences from other members of the genus, the average identity within HSPs was 99.2%, whereas the average coverage by HSPs was 96.2%. Among all other species, the one yielding the highest score was Tsukamurella strandjordii, (NR_025113), which corresponded to an identity of 99.5% and a HSP coverage of 100.0%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highestscoring environmental sequence was DQ366095 ('on Oil Degrading Consortium oil polluted soil clone MH1 Pitesti'), which showed an identity of 99.2% and an HSP coverage of 99.0%. The most frequently occurring keywords within the labels of environmental samples which yielded hits were 'skin' (9.6%), 'human' (4.8%), 'microbiom, tempor, topograph' (4.2%), 'sea' (3.8%) and 'sediment' (1.8%) (30 hits in total). Environmental samples which yielded hits of a higher score than the highest scoring species were not found. These environmental labels are in line with the locations reported for the isolation of Tsukamurella strains, such as soil, human sputum, and bed bug [2,21]. The cells of T. paurometabola are straight to slightly curved rods with a size of 0.5-0.8 × 1.0-5 µm and occur singly, in pairs, or in masses [2,21] ( Figure 2). The organism is Gram-positive, weakly acid-fast (some strains are strongly acid-fast), non-sporeforming and non-motile [2,21] (Table  1). The organism contains metachromatic granules [2]. Colonies of T. paurometabola are small (diameter, 0.5-2.0 mm) with convex elevation, have entire edges (sometimes rhizoidal), are dryish but easily emulsified and are white to creamy to orange in color [3.15]. T. paurometabola is strictly aerobic and chemoorganotrophic bacterium [1]. Reaction is positive for catalase and pyrazinamidase [1]. Acid is produced from some sugars [1]. The organism does not produce nitriles from nitrates [2]. Indole is not produced by T. paurometabola [2]. The organism is nonpathogenic for guinea pigs [2]. In general T. paurometabola strains grow in the range 10°C to 35°C. Strain no. 33 T does not grow at 45°C [1]. The strain did not survive heating at 60°C for 15 minutes [1]. Some strains of T. paurometabola produce acid from fructose, galactose, glucose, glycerol, inositol, manitol, mannose, sorbitol, sucrose, and trehalose [1]. Acid is not produced from Larabinose, L-rhamnose, or D-xylose [1]. Some strains of T. paurometabola grow on ethanol, fructose, galactose, glucose, inositol, mannitol, mannose, melizitose, sorbitol, sucrose, trehalose, xylose, n-butanol, isobutanol, 2,3-butylene glycol, propanol, propylene glycol, citrate, fumarate, malate, pyruvate, and succinate [1]. The organism does not grow on adonitol, arabinose, inulin, lactose, raffinose, or rhamnose [1]. Acetamide and nicotinamide are used as sole nitrogen sources but not benzamide [1]. Acetamide, glutamate, glucosamine, monoethanolamine, and serine are used as sole sources of carbon and nitrogen [1]. T. paurometabola is able to degrade Tween 20, Tween 40, Tween 60, and Tween 80, but not adenine, casein, or elastin [1]. Some strains of T. paurometabolum degrade xanthine and tyrosine [1]. The organism produces β-galactosidase and urease, but not arylsulfatase or α-esterase [1]. T. paurometabolum is resistant to ethambutol (5 µg/ml), 5-fluorouracil (20 µg/ml), mitomycin C (10 µg/ml), and picric acid (0.2% w/v) [1]. The organism is susceptible to bleomycin (5 µg/ml) [1].  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 of the Gene Ontology project [31]. If the evidence code is IDA, the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [33], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [34]. The genome project is deposited in the Genome On Line Database [18] and the complete genome sequence is deposited in Gen-Bank. 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. paurometabola no. 33 T , DSM 2016, was grown in medium 535 (Trypticase soy broth medium) [35] at 28°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 modification st/LALMice for cell lysis as described in [24]. DNA is available through the DNA Bank Network [36].

Genome sequencing and assembly
The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [37]. Pyrosequencing reads were assembled using the Newbler assembler (Roche). Large Newbler contigs were broken into 4,920 overlapping fragments of 1,000 bp and entered into assembly as pseudoreads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the parallel phrap assembler [38]. Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones [39]. Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 516 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000.

Genome annotation
Genes were identified using Prodigal [40] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [41]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, Uni-Prot, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and In-terPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review (IMG-ER) platform [42].

Genome properties
The genome consists of a 4,379,918 bp long chromosome and a 99,806 bp long plasmid, both with a G+C content of 68.4% (Figure 3 and Table 3). Of the 4,391 genes predicted, 4,335 were proteincoding genes, and 56 RNAs; 93 pseudogenes were also identified. The majority of the protein-coding genes (68.7%) 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.