Genome Analysis and Potential Ecological Functions of Members of the Genus Ensifer from Subsurface Environments and Description of Ensifer oleiphilus sp. nov.

The current work deals with genomic analysis, possible ecological functions, and biotechnological potential of two bacterial strains, HO-A22T and SHC 2-14, isolated from unique subsurface environments, the Cheremukhovskoe oil field (Tatarstan, Russia) and nitrate- and radionuclide-contaminated groundwater (Tomsk region, Russia), respectively. New isolates were characterized using polyphasic taxonomy approaches and genomic analysis. The genomes of the strains HO-A22T and SHC 2-14 contain the genes involved in nitrate reduction, hydrocarbon degradation, extracellular polysaccharide synthesis, and heavy metal detoxification, confirming the potential for their application in various environmental biotechnologies. Genomic data were confirmed by cultivation studies. Both strains were found to be neutrophilic, chemoorganotrophic, facultatively anaerobic bacteria, growing at 15-33 °C and 0-1.6% NaCl (w/v). The 16S rRNA gene sequences of the strains were similar to those of the type strains of the genus Ensifer (99.0-100.0%). Nevertheless, genomic characteristics of strain HO-A22T were below the thresholds for species delineation: the calculated average nucleotide identity (ANI) values were 83.7-92.4% (<95%), and digital DNA-DNA hybridization (dDDH) values were within the range of 25.4-45.9% (<70%), which supported our conclusion that HO-A22T (=VKM B-3646T = KCTC 92427T) represented a novel species of the genus Ensifer, with the proposed name Ensifer oleiphilus sp. nov. Strain SHC 2-14 was assigned to the species 'Ensifer canadensis', which has not been validly published. This study expanded the knowledge about the phenotypic diversity among members of the genus Ensifer and its potential for the biotechnologies of oil recovery and radionuclide pollution treatment.

Correction: Semenova et al. Physiological and Genomic Characterization of Actinotalea subterranea sp. nov. from Oil-Degrading Methanogenic Enrichment and Reclassification of the Family Actinotaleaceae. Microorganisms 2022, 10, 378.

The authors wish to make the following correction to this paper [...].

Physiological and Genomic Characterization of Actinotalea subterranea sp. nov. from Oil-Degrading Methanogenic Enrichment and Reclassification of the Family Actinotaleaceae.

The goal of the present work was to determine the diversity of prokaryotes involved in anaerobic oil degradation in oil fields. The composition of the anaerobic oil-degrading methanogenic enrichment obtained from an oil reservoir was determined by 16S rRNA-based survey, and the facultatively anaerobic chemoorganotrophic bacterial strain HO-Ch2T was isolated and studied using polyphasic taxonomy approach and genome sequencing. The strain HO-Ch2T grew optimally at 28 °C, pH 8.0, and 1-2% (w/v) NaCl. The 16S rRNA gene sequence of the strain HO-Ch2T had 98.8% similarity with the sequence of Actinotalea ferrariae CF5-4T. The genomic DNA G + C content of strain HO-Ch2T was 73.4%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the genome of strain HO-Ch2T and Actinotalea genomes were 79.8-82.0% and 20.5-22.2%, respectively, i.e., below the thresholds for species delineation. Based on the phylogenomic, phenotypic, and chemotaxonomic characterization, we propose strain HO-Ch2T (= VKM Ac-2850T = KCTC 49656T) as the type strain of a new species within the genus Actinotalea, with the name Actinotalea subterranea sp. nov. Based on the phylogenomic analysis of 187 genomes of Actinobacteria we propose the taxonomic revision of the genera Actinotalea and Pseudactinotalea and of the family Actinotaleaceae. We also propose the reclassification of Cellulomonas carbonis as Actinotalea carbonis comb. nov., Cellulomonas bogoriensis as Actinotalea bogoriensis comb. nov., Actinotalea caeni as Pseudactinotalea caeni comb. nov., and the transfer of the genus Pseudactinotalea to the family Ruaniaceae of the order Ruaniales.

Soehngenia longivitae sp. nov., a Fermenting Bacterium Isolated from a Petroleum Reservoir in Azerbaijan, and Emended Description of the Genus Soehngenia.

A methanogenic enrichment growing on a medium with methanol was obtained from a petroleum reservoir (Republic of Azerbaijan) and stored for 33 years without transfers to fresh medium. High-throughput sequencing of the V4 region of the 16S rRNA gene revealed members of the genera Desulfovibrio, Soehngenia, Thermovirga, Petrimonas, Methanosarcina, and Methanomethylovorans. A novel gram-positive, rod-shaped, anaerobic fermentative bacterium, strain 1933PT, was isolated from this enrichment and characterized. The strain grew at 13-55 °C (optimum 35 °C), with 0-3.0% (w/v) NaCl (optimum 0-2.0%) and in the pH range of 6.7-8.0 (optimum pH 7.0). The 16S rRNA gene sequence similarity, the average nucleotide identity (ANI) and in silico DNA-DNA hybridization (dDDH) values between strain 1933PT and the type strain of the most closely related species Soehngenia saccharolytica DSM 12858T were 98.5%, 70.5%, and 22.6%, respectively, and were below the threshold accepted for species demarcation. Genome-based phylogenomic analysis and physiological and biochemical characterization of the strain 1933PT (VKM B-3382T = KCTC 15984T) confirmed its affiliation to a novel species of the genus Soehngenia, for which the name Soehngenia longivitae sp. nov. is proposed. Genome analysis suggests that the new strain has potential in the degradation of proteinaceous components.

Sphaerochaeta halotolerans sp. nov., a novel spherical halotolerant spirochete from a Russian heavy oil reservoir, emended description of the genus Sphaerochaeta, reclassification of Sphaerochaeta coccoides to a new genus Parasphaerochaeta gen. nov. as Parasphaerochaeta coccoides comb. nov. and proposal of Sphaerochaetaceae fam. nov.

Anaerobic, fermentative, halotolerant bacteria, strains 4-11T and 585, were isolated from production water of two low-temperature petroleum reservoirs (Russia) and were characterized by using a polyphasic approach. Cells of the strains were spherical, non-motile and 0.30-2.5 µm in diameter. Strain 4-11T grew optimally at 35 °C, pH 6.0 and 1.0-2.0% (w/v) NaCl. Both strains grew chemoorganotrophically with mono-, di- and trisaccharides. The major cellular fatty acids of both strains were C14:0, C16:0, C16:1 ω9 and C18:0 3-OH. Major polar lipids were glycolipids and phospholipids. The 16S rRNA gene sequences of the strains 4-11T and 585 had 99.9% similarity and were most closely related to the sequence of Sphaerochaeta associata GLS2T (96.9, and 97.0% similarity, respectively). The G+C content of the genomic DNA of strains 4-11T and 585 were 46.8 and 46.9%, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between the genomes of strain 4-11T and S. associata GLS2T were 73.0 and 16.9%, respectively. Results of phylogenomic metrics analysis of the genomes and 120 core proteins of strains 4-11T and 585 and their physiological and biochemical characteristics confirmed that the strains represented a novel species of the genus Sphaerochaeta, for which the name Sphaerochaeta halotolerans sp. nov. is proposed, with the type strain 4-11T (=VKM B-3269T=KCTC 15833T). Based on the results of phylogenetic analysis, Sphaerochaeta coccoides was reclassified as member of a new genus Parasphaerochaeta gen. nov., Parasphaerochaeta coccoides comb. nov. The genera Sphaerochaeta and Parasphaerochaeta form a separate clade, for which a novel family, Sphaerochaetaceae fam. nov., is proposed.

Geobacillus proteiniphilus sp. nov., a thermophilic bacterium isolated from a high-temperature heavy oil reservoir in China.

A rod-shaped, spore-forming, thermophilic, chemoorganotrophic, aerobic or facultatively anaerobic bacterial strain, 1017T, was isolated from production water sampled at the Dagang oilfield (PR China), and was characterized by using a polyphasic approach. The strain is capable of anaerobic glucose fermentation. Nitrate is reduced to nitrite. Optimal growth was observed at 60-65 °C, at pH between pH 7.0 and 7.5, and with 1-2 % (w/v) NaCl. The major cellular fatty acids were iso-C17 : 0, anteiso-C17 : 0, iso-C15 : 0, iso-C16 : 0 and C16 : 0. The predominant polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analysis based on the 16S rRNA, gyrB and parE gene sequences indicated that the isolate belonged to the genus Geobacillus and was most closely related to Geobacillus thermoleovorans KCTC 3570T (99.5, 96.1 and 97.9 % sequence similarity, respectively). Genome sequencing revealed a genome size of 3.57495 Mb and a DNA G+C content of 51.8 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between the genomes of strain 1017T and G. thermoleovorans KCTC 3570T were 95.9 and 64.9 %, respectively. Results of phylogenomic metrics analysis of the genome and 1172 core genes of strain 1017T and its physiological and biochemical characteristics confirmed that strain 1017T represented a novel species of the genus Geobacillus, for which the name Geobacillusproteiniphilus sp. nov. is proposed. The type strain is 1017T (=VKM B-3132T=KCTC 33986T).

Draft Genome Sequence of a Sulfate-Reducing Bacterium, "Desulfofundulus salinum" 435T, Isolated from a High-Temperature Gas Field in Russia.

The draft genome sequence of the thermophilic sulfate-reducing bacterium "Desulfofundulus salinum" strain 435T, isolated from condensate water of the Igrim high-temperature gas field (Western Siberia, Russia), is presented here. The genome is annotated to elucidate the taxonomic position of strain 435T.

Diversity of Metabolically Active Bacteria in Water-Flooded High-Temperature Heavy Oil Reservoir.

The goal of this work was to study the overall genomic diversity of microorganisms of the Dagang high-temperature oilfield (PRC) and to characterize the metabolically active fraction of these populations. At this water-flooded oilfield, the microbial community of formation water from the near-bottom zone of an injection well where the most active microbial processes of oil degradation occur was investigated using molecular, cultural, radiotracer, and physicochemical techniques. The samples of microbial DNA and RNA from back-flushed water were used to obtain the clone libraries for the 16S rRNA gene and cDNA of 16S rRNA, respectively. The DNA-derived clone libraries were found to contain bacterial and archaeal 16S rRNA genes and the alkB genes encoding alkane monooxygenases similar to those encoded by alkB-geo1 and alkB-geo6 of geobacilli. The 16S rRNA genes of methanogens (Methanomethylovorans, Methanoculleus, Methanolinea, Methanothrix, and Methanocalculus) were predominant in the DNA-derived library of Archaea cloned sequences; among the bacterial sequences, the 16S rRNA genes of members of the genus Geobacillus were the most numerous. The RNA-derived library contained only bacterial cDNA of the 16S rRNA sequences belonging to metabolically active aerobic organotrophic bacteria (Tepidimonas, Pseudomonas, Acinetobacter), as well as of denitrifying (Azoarcus, Tepidiphilus, Calditerrivibrio), fermenting (Bellilinea), iron-reducing (Geobacter), and sulfate- and sulfur-reducing bacteria (Desulfomicrobium, Desulfuromonas). The presence of the microorganisms of the main functional groups revealed by molecular techniques was confirmed by the results of cultural, radioisotope, and geochemical research. Functioning of the mesophilic and thermophilic branches was shown for the microbial food chain of the near-bottom zone of the injection well, which included the microorganisms of the carbon, sulfur, iron, and nitrogen cycles.

Genome sequencing and annotation of Geobacillus sp. 1017, a hydrocarbon-oxidizing thermophilic bacterium isolated from a heavy oil reservoir (China).

The draft genome sequence of Geobacillus sp. strain 1017, a thermophilic aerobic oil-oxidizing bacterium isolated from formation water of the Dagang high-temperature oilfield, China, is presented here. The genome comprised 3.6 Mbp, with the G + C content of 51.74%. The strain had a number of genes responsible for numerous metabolic and transport systems, exopolysaccharide biosynthesis, and decomposition of sugars and aromatic compounds, as well as the genes related to resistance to metals and metalloids. The genome sequence is available at DDBJ/EMBL/GenBank under the accession no MQMG00000000. This genome is annotated for elucidation of the genomic and phenotypic diversity of new thermophilic alkane-oxidizing bacteria of the genus Geobacillus.

Draft Genome Sequence of Aeribacillus pallidus Strain 8m3, a Thermophilic Hydrocarbon-Oxidizing Bacterium Isolated from the Dagang Oil Field (China).

The draft genome sequence of Aeribacillus pallidus strain 8m3, a thermophilic aerobic oil-oxidizing bacterium isolated from production water from the Dagang high-temperature oil field, China, is presented here. The genome is annotated to provide insights into the genomic and phenotypic diversity of the genus Aeribacillus.