Draft Genome Sequence of Raoultella ornithinolytica TNT, a Trinitrotoluene-Denitrating and Plant Growth-Promoting Strain Isolated from Explosive-Contaminated Soil

GENOME ANNOUNCEMENT

Trinitrotoluene (TNT)-contaminated sites pose risks to human and environmental health, making remediation a priority. Detoxification by indigenous bacteria or use of TNT-transforming bacteria in bioaugmented rhizoremediation is preferred to invasive and costly excavation or incineration techniques.

A TNT-detoxifying strain was isolated from TNT-contaminated forest soil at a military site in Belgium. Identified as Raoultella ornithinolytica B6 by partial 16S rRNA gene sequencing and phenotypic profiling, the closest related partial 16S rRNA gene sequence (97.8%) was from strain B6 (GenBank accession no. CP004142) (1).

Here, genomic DNA was isolated using a DNeasy blood and tissue kit (Qiagen, Venlo, the Netherlands), treated with RNase I, and purified by phenol:chloroform extraction. Purified DNA was digested and sequencing adaptors ligated using an Ion Xpress Plus fragment library kit (Life Technologies Inc., Burlington, Ontario, Canada). Adaptor-ligated DNA was size selected (480 bp) on a 2% E-Gel SizeSelect agarose gel, and Agencourt AMPure XP beads (Beckman Coulter, Mississauga, Ontario, Canada) were used for purifications. An Ion library quantitation kit was used prior to amplification and enrichment with an Ion PGM Template OT2 400 kit on an Ion OneTouch 2 system. An Ion Sphere quality control kit was used prior to sequencing with an Ion PGM 400 sequencing kit and a single 316v2 Chip on an Ion Torrent PGM (Life Technologies, Inc., Carlsbad, CA).

In total, 2.49 million reads (mean length, 282 bases) generated 703 Mb of data, of which 938,910 reads were assembled using MIRA version 3.9.9 (2) into 39 contigs, giving a consensus length of 5,652,765 bp at 48× coverage. Mauve (3) was used to order the contigs and compare the genome with the closest related reference genome, that of R. ornithinolytica B6 (accession no. CP004142). The annotation was completed using the PGAP (NCBI) pipeline (4). The genome of R. ornithinolytica TNT consists of a single circular chromosome (55.5% G+C content), which includes 4,935 coding genes, 180 pseudogenes, 34 rRNAs (5S, 16S, 23S), 80 tRNAs, and 7 noncoding RNAs (ncRNAs).

In liquid cultures, strain TNT demonstrated rapid TNT transformation activity in minimal medium (113 mg liter^-1 TNT, 0.1 mg liter^-1 NH₄Cl, and 5 g liter^-1 glucose). Analyses of the draft genome confirmed the presence of genes coding for multiple TNT-transforming enzymes, including nitroreductase A, nitroreductase B, and N-ethylmaleimide (NEM) reductase. The NEM product is similar to NemA in Escherichia coli and to XenB and PetN of Enterobacter cloacae, and it is possibly responsible for nitrite release from TNT (5–9). Strain TNT uses nitrite as the sole N source under aerobic conditions, and genes for a complete nitrite assimilation pathway are present. This strain produces indole, is Voges-Proskauer positive, utilizes glycerol, glucose, fructose, and sucrose as carbon sources, and grows at 20°C. Genes for plant growth-promoting characteristics are present, corroborating results from phenotypic tests: auxin biosynthesis, acetoin production, 1-aminocyclopropane-1-carboxylate deaminase activity, siderophore production, phosphorous solubilization, and chemotaxis. R. ornithinolytica TNT is a promising strain for rhizoremediation applications.