Whole-genome sequence data and analysis of type strains ‘Pusillimonas nitritireducens’ and ‘Pusillimonas subterraneus’ isolated from nitrate- and radionuclide-contaminated groundwater in Russia

Two strains, ‘Pusillimonas nitritireducens’ JR1/69-2-13T and ‘Pusillimonas subterraneus’ JR1/69-3-13T, of aerobic, motile, Gram-negative, non-spore-forming, organotrophic, psychrotolerant bacteria were isolated from a sample of nitrate- and radionuclide-contaminated groundwater in Russia. Here we describe the draft genomes of these strains. The sequenced and annotated genome of the strain JR1/69-2-13T contained 4.3 Mbp with 4108 protein-coding genes. The genome of the strain JR1/69-3-13T contained 4.5 Mbp with 4260 protein-coding genes. Genome analysis of both strains provides an insight into the genomic basis of their resistance to nitrate, heavy metals and metalloids. The draft genome sequences of strains ‘Pusillimonas nitritireducens’ JR1/69-2-13T and ‘Pusillimonas subterraneus’ JR1/69-3-13T are available at DDBJ/EMBL/GenBank under the accession nos. https://www.ncbi.nlm.nih.gov/nuccore/PDNV00000000 and https://www.ncbi.nlm.nih.gov/nuccore/PDNW00000000, respectively.


a b s t r a c t
Two strains, 'Pusillimonas nitritireducens' JR1/69-2-13 T and 'Pusillimonas subterraneus' JR1/69-3-13 T , of aerobic, motile, Gramnegative, non-spore-forming, organotrophic, psychrotolerant bacteria were isolated from a sample of nitrate-and radionuclidecontaminated groundwater in Russia. Here we describe the draft genomes of these strains. The sequenced and annotated genome of the strain JR1/69-2-13 T contained 4.3 Mbp with 4108 proteincoding genes. The genome of the strain JR1/69-3-13 T contained 4.5 Mbp with 4260 protein-coding genes. Genome analysis of both strains provides an insight into the genomic basis of their resistance to nitrate, heavy metals and metalloids. The draft genome sequences of strains 'Pusillimonas nitritireducens' JR1/69-2-13 T

Value of the data
Draft genome sequences of 'P. nitritireducens' and 'P. subterraneus' will create an opportunity for comparative taxonomic studies of the closely related Pusillimonas -Candidimonas group.
Draft genomes and identified genes of new strains will provide insights into the molecular mechanisms by which these strains transform nitrate or nitrite to dinitrogen gas and survive in the nitrate-and radionuclides-contaminated environment.
This data set will be useful for the scientific community, working in the area of functional and phylogenetic diversity of microorganisms in environments contaminated with radioactive waste, and for development of biotechnologies for in situ bioremediation of groundwater by nitrate removal.

Data
In the present work, we describe the draft genome sequences and gene prediction of two strains of aerobic organotrophic bacteria, JR1/69-2-13 T and JR1/69-3-13 T (Fig. 1), isolated from nitrate-and radionuclides-contaminated groundwater collected near the industrial reservoir for liquid radioactive waste (Ozyorsk town, South Urals, Russia) [1]. Functional and phylogenetic diversity of the microorganisms inhabiting the subterranean water-bearing horizons associated with the plants processing uranium and other radionuclides remain insufficiently studied [2,3]. Two strains, JR1/69-2-13 T and JR1/69-3-13 T , were chosen for genome sequencing with the goal of determination of their taxonomic position and identification of the genetic determinants providing for their occurrence in the environment contaminated with radioactive waste. Based on the 16S rRNA gene phylogeny data, both strains were found to belong to the domain Bacteria, the phylum Proteobacteria, the class Betaproteobacteria, the order Burkholderiales, the family Alcaligenaceae [4][5][6][7][8]. The 98.7% similarity level between the 16S rRNA gene sequences of these two strains indicated that they could probably belong to different species. The 16S rRNA gene sequences of the strains JR1/69-2-13 T and JR1/69-3-13 T were phylogenetically closely related to those of the species of the genera Pusillimonas and Candidimonas [9][10][11][12][13][14][15]. As was demonstrated in previous studies, differentiation between the genera of the family Alcaligenaceae on the basis of their phenotypic characteristics is not possible [9,10,14,16]. Genomic features of strains Pusillimonas nitritireducens' JR1/69-2-13 T and 'Pusillimonas subterraneus' JR1/69-3-13 T are presented in Table 1. Numerous genes responsible for heavy metal tolerance and detoxification were identified in their genomes. In the JR1/69-3-13 T genome, the denitrification gene  cluster included the genes predicted to encode the enzymes for sequential reduction of nitrate to dinitrogen gas, which was consistent with the complete denitrification phenotype of the strain. In the JR1/69-2-13 T genome, the genes encoding nitrite reduction to N 2 were revealed. In silico DNA-DNA hybridization (dDDH) values against reference genomes of Pusillimonas and Candidimonas strains were within the range of 19.3-28.8, and were below the 70% threshold to differentiate bacterial species [17]. These data and position of strains JR1/69-2-13 T and JR1/69-3-13 T on the tree of 492 concatenated core protein-coding genes (Fig. 2) testify to their belonging to two novel species within the Pusillimonas genus with proposed names 'Pusillimonas nitritireducens' and 'Pusillimonas subterraneus', respectively. The draft genome sequences of strains JR1/69-2-13 T and JR1/69-3-13 T are deposited in DDBJ/EMBL/Genbank under the accession nos. PDNV00000000 and PDNW00000000, respectively. The versions described in this paper are the first versions of the genome of each strain.  (Ozyorsk town, South Urals, Russia) [1,18]. Two strains were purified by successive transfer from the liquid TEG medium containing bacto-trypton (5.0 g L À 1 ), yeast extract (2.5 g L À 1 ), glucose (1.0 g L À 1 ), and distilled water (1 L, pH 7.0) to solid TEG medium with agar-agar (15.0 g L À 1 ). Bacteria were incubated at 22-30°C. Strains JR1/69-2-13 T and JR1/69-3-13 T were deposited in the Russian Collection of Microorganisms as VKM В-3222 T and VKM В-3223 T , respectively.

DNA isolation and sequencing
Strains JR1/69-2-13 T and JR1/69-3-13 T were grown in TEG liquid medium at 30°C for 72 h. The cells were harvested by centrifugation. The cell integrity was accessed by transmission electron microscopy (JEOL JEM-1010, Japan) of the cells negatively stained with 1% phosphotungstic acid ( Fig. 1A and B). DNA of each strain was isolated from the biomass by the phenol-chloroform-based method as described previously [19]. NEBNext Ultra DNA library preparation kit (New England Biolabs, USA) was used to prepare fragment libraries for genome sequencing. Next-generation shotgunsequencing was performed on MiSeq (Illumina Inc., USA) at the Immanuel Kant Baltic Federal University (Kaliningrad, Russian Federation).

Genome annotation
Identification of protein-coding sequences and primary annotation was performed using the NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP) [21]. Additional gene prediction and functional annotation were performed in the Rapid Annotation using Subsystems Technology (RAST) server [22] and Integrated Microbial Genome-Expert Review pipeline [23], respectively. Analysis of metal resistance was performed with the Antibacterial Biocide and Metal Resistance Genes Database (BacMet) [24]. In silico DNA-DNA hybridization (dDDH) was carried out with the online genome-togenome calculator (GGDC 2.0) provided by the DSMZ [17].