Genome characterization of a Stutzerimonas stutzeri strain isolated from oil-contaminated soil in Tatarstan

Oil spills resulting from anthropogenic activities such as extraction, transportation or storage of petroleum products cause serious harm to the biosphere and are potentially hazardous to human health. Bioremediation using microorganisms is a promising technology for cleaning up such contaminated sites, since it is cost-effective and leads to complete mineralization of harmful compounds. The discovery of new local microbial strains capable of effective oil biodegradation provides prospects for the development of a reliable and cost-effective strategy for the reclamation of oil-contaminated soils. This study presents data on the determination and analysis of the complete genome nucleotide sequence of the DIA-8 strain, which was isolated from oil-contaminated soil in the Republic of Tatarstan. Yellow, shiny, convex colonies with irregular edges, ranging from 0.5 to 3 mm in diameter, were observed when the S. stutzeri DIA-8 strain was cultured on dense LB nutrient agar (See Fig. 1). Stutzerimonas stutzeri is a non-fluorescent, denitrifying bacterium widely distributed in the environment. This bacterial species has attracted significant attention due to its unique metabolic properties, including denitrification, nitrogen fixation, and the degradation of aromatic compounds, and its resistance to metals and potential applications in bioremediation. Analysis of this strain’s genome contributes to a better understanding of the genetic basis of oil degradation, the production of secondary metabolites, potential virulence, and other important physiological functions. Furthermore, the findings of this study may serve as the basis for the development of a cost-effective and environmentally friendly method for the remediation of crude oil-contaminated soils. Genomic DNA was extracted using the ExtractDNA Blood & Cells kit (CJSC Eurogen, Russia). Sequencing was performed on a MiSeq genetic analyzer (Illumina, USA) in paired-end mode (2x300 bp). The MiSeq Reagent Kit v3 was used for sequencing. De novo genome assembly was performed using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) v3.6.2 (https://www.bv-brc.org). Digital DNA-DNA hybridization (dDDH) was calculated using the GGDC v3.0 genome distance calculator (http://ggdc.dsmz.de/distcalc2.php). Average Nucleotide Identity (ANI) values were calculated using the JSpeciesWS online service. A phylogenetic tree based on whole-genome data was constructed using the TYGS web server (https://tygs.dsmz.de). Functional genome annotation was performed using the RAST 2.0 server (https://rast.nmpdr.org) with default settings. The PathogenFinder web server (https://cge.food.dtu.dk/services/PathogenFinder/) and the Virulence Factor Database (VFDB) were used for the prediction of bacterial pathogenicity. Plasmid DNA was isolated using a LumiSpin® PLASMID kit (Lumiprob RUS LLC, Russia&EAEU). Plasmid DNA sequencing was performed by the biotechnology company Cloning Facility (Moscow) using Nanopore technology. The assembled genome of S. stutzeri strain DIA-8 consisted of 28 contigs (> 500 bp) with an N50 of 648,363 bp and a total length of 4,496,503 bp. ANI and dDDH results between S. stutzeri DIA-8 and other S. stutzeri species showed genome similarity values ranging from 97.31% to 97.90% for ANI and from 80.60% to 85.60% for dDDH (See Table 1). These values are above the recommended threshold values of 95% (ANI) and 70% (dDDH) for prokaryotic species delineation, respectively. The TYGS results showed that the DIA-8 strain branch clustered within the S. stutzeri species clade. This indicates that the DIA-8 strain belongs to the S. stutzeri species (See Fig. 2). During the mapping of the complete genome nucleotide sequence, we identified several genes encoding proteins responsible for the catabolism of aromatic compounds. According to RAST server annotation, the S. stutzeri DIA-8 genome contains 60 genes involved in aromatic compound metabolism (See Fig. 3), with 31 genes involved in peripheral pathways and 27 genes involved in the central pathways of aromatic compound catabolism. Aromatic ring cleavage enzymes identified in DIA-8 included benzoate-1,2-dioxygenase (EC 1.14.12.10), 1,6-dihydroxycyclohexa-2,4-diene-1-carboxylate dehydrogenase (EC 1.3.1.25), catechol-1,2-dioxygenase (EC 1.13.11.1), muconate cycloisomerase (EC 5.5.1.1), muconolactone Δ-isomerase (EC 5.3.3.4), 3-oxoadipateenol-lactonase (EC 3.1.1.24), 3-oxoadipate-CoA transferase (EC 2.8.3.6), and 3-oxoadipyl-CoA thiolase (EC 2.3.1.174) (See Fig. 4). Bioinformatic analysis of the S. stutzeri DIA-8 genome sequence also revealed genes involved in the synthesis of glycolipid biosurfactants and heavy metal resistance. The genome analysis indicated that this strain has a low pathogenic potential, similar to other nonvirulent environmental isolates of the genus Pseudomonas, which suggests its potential use as a biotechnological product. Sequencing of the isolated plasmid DNA sample revealed that it is a mixture of two plasmids. One plasmid was 15,416 bp in size and contained 20 protein-coding genes and one gene encoding a non-coding RNA molecule (PrrB/RsmZ). The genes on this plasmid, identified through annotation, are responsible for replication and plasmid transfer between bacterial cells. The second plasmid was 2,450 bp in size and contained three genes encoding proteins of unknown function. Based on our genomic analysis, Stutzerimonas stutzeri DIA-8, isolated from oil-contaminated soil in the Republic of Tatarstan, shows promise for the development of bioremediation agents for polluted ecosystems. The strain’s genome contains genes that play a crucial role in the degradation of hydrocarbon compounds. The obtained results will contribute to further research of the physiological properties of the strain and its potential for restoring the productivity of disturbed lands. The complete genome nucleotide sequence has been deposited in the international GenBank database under accession number GCA_037081755.1. The article contains 4 Figures, 1 Table, 36 References. The Authors declare no conflict of interest.

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