DNA-DNA Hybridization Research Articles

Pseudocitrobacter cyperus, a novel bacterial species recovered from Cyperus alternifolius in Egypt

BackgroundStrain Cyp38ST was isolated as an endophyte from the plant Cyperus alternifolius, collected along the banks of the River Nile in 2019. Preliminary analysis tentatively identified Cyp38ST as belonging to the genus Pseudocitrobacter.MethodsThe preliminary identification of Cyp38ST was performed using the VITEK®2 identification system, MALDI-TOF-MS, and 16S rRNA gene sequencing. To confirm its taxonomic classification, the draft genome of Cyp38ST was generated using DNBseq, and the genome-based taxonomic evaluation was conducted by calculating the overall genome-relatedness indices (OGRIs) such as Average Nucleotide Identity (ANI), digital DNA-DNA hybridization (dDDH), and the tetra-nucleotide signatures (Tetra). Additionally, the biochemical features, antimicrobial susceptibility profiles, and fatty acid methyl ester content of Cyp38ST were characterized.ResultsVITEK®2 misidentified Cyp38ST as Citrobacter werkmanii, MALDI-TOF-MS identified it as Pseudocitrobacter faecalis. While the 16S rRNA gene showed more than 99.0% similarity to other Pseudocitrobacter species, the calculated OGRIs were lower than the thresholds recommended for species assignment to all currently known Pseudocitrobacter species. Furthermore, the phylogenomic analysis revealed that Cyp38ST forms a distinct species cluster within the genus Pseudocitrobacter. Cyp38ST was predicted as a potential human pathogen and carried a unique ß-lactamase-coding gene.ConclusionHere we present Cyp38ST (= CCASU-2024-73T) as the type strain of a novel species within the genus Pseudocitrobacter to which we propose the name Pseudocitrobacter cyperus sp. nov. We provide a full description of the novel species and present its genome sequence and annotation. The discovery of this novel species highlights the potential of endophytic bacteria associated with unique plant hosts to harbor previously uncharacterized microbial diversity.

A new mesophilic member of the Chloroflexota phylum 'Ca. Сhloroploca septentrionalis' from the meromictic lake Bol'shie Khruslomeny separated from the White Sea.

A new filamentous phototrophic bacterium Khr17 was isolated as an enrichment culture from the brackish polar lake Bol'shie Khruslomeny. The organism was a halotolerant, strictly anaerobic phototroph possessing photosystem II. Sulfide was required for phototrophic growth. The cells of bacterium Khr17 formed nonmotile, wavy trichomes surrounded by a sheath. The cells contained chlorosomes, gas vesicles, and storage granules. The antenna pigments of bacterium Khr17 were bacteriochlorophyll c and β- and γ-carotenes. The genome of Khr17 bacterium carries all the genes responsible for CO2 fixation via the 3-hydroxypropionate pathway. The genes encoding the proteins of the nitrogenase complex were not found. The DNA G+C content was 59.9%. The 16S rRNA gene sequence of isolate Khr17 exhibited 99.4% similarity to related species. The average nucleotide identity and digital DNA-DNA hybridization values for the isolate showed 91.9% and 46.9% similarity, respectively, to other 'Ca. Chloroploca' species. Based on its phenotypic and phylogenetic characteristics, classification of Khr17 as member of a new species, 'Ca. Chloroploca septentrionalis' sp. nov., was proposed. Members of the genus 'Ca. Chloroploca' have previously not been found in Arctic areas and in the plankton of meromictic lakes.

Pseudonocardia spirodelae sp. nov., isolated from duckweed and formal proposal to reclassify Pseudonocardia antarctica as a later heterotypic synonym of Pseudonocardia alni and reclassify Pseudonocardia carboxydivorans as Pseudonocardia alni subsp. carboxydivorans.

A novel Pseudonocardia strain DW16-2T, isolated from duckweed (Spirodela polyrhiza), was taxonomically studied in detail. The analysis based on its 16S rRNA gene sequence revealed that the strain was most closely related to Pseudonocardia carboxydivorans Y8T (98.8%), followed by Pseudonocardia tropica YIM 61452T (98.7%), Pseudonocardia antarctica DVS 5a1T (98.7%) and Pseudonocardia alni DSM 44104T (98.7%). The average nucleotide identity (ANI) based on blast and digital DNA-DNA hybridization (dDDH) relatedness values between strain DW16-2T and their closest type strains were below the threshold values for identifying a novel species. Morphological, physiological and chemotaxonomic features of strain DW16-2T were typical for the genus Pseudonocardia by forming extensively branched substrate mycelium and aerial mycelium that fragmented into rod-shaped spore, with a smooth surface. The whole-cell hydrolysates of strain DW16-2T contained meso-diaminopimelic acid as the diagnostic diamino acid, and the whole-cell sugars were arabinose, galactose, glucose and a trace amount of ribose. The polar lipids contained phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and unidentified phospholipids. The menaquinone (MK) was MK-8(H4). The cellular fatty acids (>5 %) were iso-C16 : 0, iso-C16 : 1 H, summed feature 3: C16 : 1 ω7c/C16 : 1 ω6c; C16 : 1 ω6c/C16 : 1 ω7c, C17 : 1 ω8c and anteiso-C17 : 0. Characterization based on chemotaxonomic, phenotypic, genotypic and phylogenetic evidence demonstrated that strain DW16-2T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia spirodelae sp. nov. (type strain DW16-2T = TBRC 16418T = NBRC 115857T) is proprosed. In addition, the comparison of the whole genome sequences suggested that P. alni and P. antarctica belong to the same species and P. carboxydivorans is a subspecies of P. alni. Therefore, it is proposed that P. antarctica Prabahar et al. 2004 is reclassified as a later heterotypic synonym of P. alni (Evtushenko et al. 1989) Warwick et al. 1994, and P. carboxydivorans Park et al. 2008 is proposed as a subspecies of P. alni (Evtushenko et al. 1989) Warwick et al. 1994.

Genome-based reclassification of Suonthocola fibrivorans as a later heterotypic synonym of Diplocloster agilis.

Both the genera Suonthocola and Diplocloster are members of the family Lachnospiraceae. Their type species, both Suonthocola fibrivorans Sanger_33T and Diplocloster agilis ASD5720T, were isolated from human faeces. A comparison of their 16S rRNA gene sequences revealed 100% similarity, suggesting their close relatedness and the possibility of belonging to the same species. To clarify their taxonomic relationship, genome-based analyses were carried out. Overall genomic relatedness indices analyses indicated that S. fibrivorans Sanger_33T shared average amino acid identity and percentage of conserved proteins prediction values higher than the Lachnospiraceae-specific genus-level thresholds with D. agilis ASD5720T, as well as the other two existing species of the genus Diplocloster. Additionally, S. fibrivorans Sanger_33T formed a distinct branch with D. agilis ASD5720T, clustering with the other two species of the genus Diplocloster into the same clade in both the 16S rRNA gene phylogenetic tree and the core-genome phylogenomic tree. Essentially, both the average nucleotide identity and digital DNA-DNA hybridization prediction values between S. fibrivorans Sanger_33T and D. agilis ASD5720T were above the recommended species boundaries. It is thus clear that S. fibrivorans Sanger_33T and D. agilis ASD5720T constitute the same species. On the basis of the earliest valid publication, priority is given to D. agilis Chaplin et al. 2022. Based on this, the orphan species S. fibrivorans Hitch et al. 2022 is reclassified as a later heterotypic synonym of D. agilis Chaplin et al. 2022, along with the reclassification of the genus Suonthocola Hitch et al. 2022 as a later synonym of Diplocloster Chaplin et al. 2022.

Clostridium sediminicola sp. nov., a crude oil aggregation-forming anaerobic bacterium isolated from marine sediment.

A crude oil aggregation-forming, strictly anaerobic, Gram-stain-positive, spore-forming, rod-shaped, motile and mesophilic bacterium, named strain SH18-2T, was isolated from marine sediment near Sado Island in the Sea of Japan. The temperature, salinity and pH ranges of this strain for the growth were 15-40 °C (optimum 35 °C), 0.5-6.0% NaCl (w/v; optimum 2.0%) and pH 6.0-8.0 (optimum 7.5), respectively. A phylogenetic analysis of the 16S rRNA gene sequence showed that this strain belongs to the genus Clostridium. It was most closely related to Clostridium grantii A-1T (97.02% homology). The draft genome size was 5616089 bp, with a G+C content of 29.7 mol%. The digital DNA-DNA hybridization values determined using the Genome-to-Genome Distance Calculator and the average nucleotide identity between this strain and C. grantii A-1T were 20.30 and 78.24%, respectively. Whole-organism hydrolysates of strain SH18-2T contained meso-diaminopimelic acid; the major fatty acid was C16:0, and the polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, glycolipid and three unidentified polar lipids. From the results of genotypic, phenotypic and chemotaxonomic analyses, SH18-2T (=NBRC 116030T=DSM 115762T) is the type strain and represents a novel species of the genus Clostridium, and the name Clostridium sediminicola sp. nov. is proposed.

Natronorarus salvus gen. nov., sp. nov., Halalkalicoccus ordinarius sp. nov., and Halalkalicoccus salilacus sp. nov., halophilic archaea from a soda lake and two saline lakes, and proposal to classify the genera Halalkalicoccus and Natronorarus into Halalkalicoccaceae fam. nov. in the order Halobacteriales within the class Halobacteria.

Four novel halophilic archaeal strains CGA53T, CG83T, FCH27T, and SEDH24 were isolated from a soda lake and two saline lakes in China, respectively. Strain CGA53T showed the highest 16S rRNA gene similarity (92.6%) to Salinilacihabitans rarus AD-4T, and the other three strains were found to be related to Halalkalicoccus species with similarities of 97.6-98.3%. Metagenomic studies indicated that these four strains are low abundant inhabitants detected in these hypersaline environments, and only one MAG of Chagannuoer Soda Lake (CG) could be assigned to the genus Halalkalicoccus. Their growth occurred at 20-60°C (optima, 42, 37, 37-42, and 35°C), 0.9-5.1M NaCl (optima, 3.9, 2.6, 3.5, and 3M), and 0-1.0M MgCl2 (optima, 0.5, 0.7, and 0.1) and pH5.5-10.5 (optima, 9.0, 7.5, 7.0, and 7.0), respectively. Phylogenetic and phylogenomic analyses revealed that strains CG83T, FCH27T, and SEDH24 cluster with the current species of the genus Halalkalicoccus, and strain CGA53T forms an independent branch separated from this genus. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and average amino acid identity (AAI) values among strains CGA53T, CG83T, FCH27T, SEDH24, and the type species of the current genera within the class Halobacteria were 67.4-81.6%, 16.5-28.6% and 49.7-74.1%, respectively, clearly lower than the cutoff values for species demarcation. Strain CGA53T may represent a novel species of a new genus according to the cutoff value for genus demarcation of 65% AAI. Diverse differential phenotypic characteristics, such as nutrition, biochemical activities, antibiotic sensitivity, and H2S formation, were found among these four strains and Halalkalicoccus species. Genome-based classification supported that strains CGA53T, CG83T, FCH27T, SEDH24, and the current species of Halalkalicoccus represent a novel family of the order Halobacteriales within the class Halobacteria.

Pseudomonas retamae sp. nov., a novel endophytic bacterium with plant growth-promoting potential, isolated from root nodules of Retama monosperma in Northwestern Algeria.

A thorough polyphasic taxonomic study, integrating genome-based taxonomic approaches, was carried out to characterize the RB5T strain isolated from root nodules of Retama monosperma growing on the coastal dunes of Bousfer Beach (Oran, Algeria). The 16S rRNA gene sequence analysis revealed that strain RB5T had the highest similarity to Pseudomonas granadensis LMG27940T (98.94%) and Pseudomonas gozinkensis IzPS32dT (98.73%). Phylogenetic studies, including both 16S rRNA gene sequence and multilocus sequence analysis using 16S rRNA, gyrB and rpoD housekeeping genes, positioned RB5T in a distinct branch alongside its closest relative, P. granadensis LMG27940T. Phylogenomic analysis using the Bac120 marker set and Type (Strain) Genome Server confirmed the unique position of RB5T and its close relationship with P. granadensis LMG27940T. Similarly, genomic comparisons using average nucleotide identity based on blast (ANIb) and digital DNA-DNA hybridization (dDDH) revealed values of 92.85 and 59.3%, respectively, when compared with its closest relative, P. granadensis LMG27940T. Both values fall below the established species delimitation thresholds of 95-96% for ANIb and 70% for dDDH, providing strong genomic evidence that strain RB5T represents a novel species. Further average nucleotide identity comparisons with unclassified Pseudomonas spp. (384 genomes) and metagenomic-derived genomes from the Genome Taxonomy Database (GTDB) showed values between 84.27 and 89.2%, indicating that strain RB5T belongs to a unique evolutionary line. The genome of RB5T, with a size of 6 311 310 bp and a G+C content of 60%, harbours several key genes associated with plant growth-promoting traits, making it a promising candidate for sustainable agriculture. Phenotypically, RB5T strain is an aerobic, rod-shaped, Gram-negative, non-spore-forming bacterium that is motile with a single polar flagellum. It grows under a wide range of temperature (4-42 °C) and pH (5-10) conditions and tolerates up to 6% (w/v) NaCl. The main cellular fatty acid composition of RB5T includes C16:0, C17:0 cyclo and the summed features 3 consisting of C16:1 ω7c/C16:1 ω6c. Based on the phylogenetic, phenotypic, chemotaxonomic and genome comparison analyses, strain RB5T was identified as a novel species of the genus Pseudomonas, for which the name Pseudomonas retamae sp. nov. is proposed. The type strain is RB5T (=DSM 117471T=LMG 33633T=CIP 112482T).

Pseudoalteromonas qingdaonensis sp. nov., isolated from the intestines of Ilyoplax deschampsi.

A Gram-stain-negative, aerobic, motile, catalase-positive, oxidase-positive, short rod-shaped marine bacterium, designated as YIC-827T, was isolated from Qingdao, Shandong Province, China. The results showed that cells of strain YIC-827T could grow optimally at 25-35 °C, pH 6.5-7.5 and 2-7% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that the strain YIC-827T was a member of the genus Pseudoalteromonas. The closest relative to this strain was Pseudoalteromonas ruthenica KMM 300T, with a similarity of 98.39%. The digital DNA-DNA hybridization value between the new isolate and phylogenetically related species is 19.6%. Strain YIC-827T could decompose sodium alginate, casein and esters (Tween 20, Tween 40, Tween 60 and Tween 80), but could not hydrolyse starch, cellulose and DNA. The fatty acid profile of a strain consists of a large number of C16:0, C18:1 ω7c and C16:1 ω7c/C16:1 ω6c. The G+C content of the DNA of this strain was determined to be 48.93%. Based on phenotypic characteristics, phylogenetic analysis and DNA-DNA correlation data, the strain YIC-827 T represents a novel species of the genus Pseudoalteromonas with the name Pseudoalteromonas qingdaonensis sp. nov. The type strain of P. qingdaonensis sp. is strain YIC-827T (=MCCC 1K08807T=CGMCC 1.62085T=KCTC 8212T).

Roseomonas cutis sp. nov., isolated from human epidermis.

Five pink-pigmented bacterial strains, isolated from human skin and classified within the genus Roseomonas, were examined. Among them, four were identified as Roseomonas mucosa, while strain OT10T was deemed to be a potential novel species. Strain OT10T exhibited characteristics, such as Gram-stain-negative, oxidase positive, motile, strictly aerobic and rod shaped. The cells had multiple flagella at one end, arranged in a lophotrichous pattern. The predominant cellular fatty acids in OT10T were C18:1 ω7c/C18:1 ω6c and C18:1 2OH; ubiquinone (Q)-10 was identified as the sole quinone. Major polar lipids included phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine and two aminolipids. The G+C content of the genome was determined to be 72.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence similarities revealed that strain OT10T is closely related to Roseomonas gilardii subsp. gilardii ATCC 49956T (97.7%), Roseomonas gilardii subsp. rosea ATCC BAA-691T (97.7%) and R. mucosa ATCC BAA-692T (97.5%). For the comparative genomic analyses, whole-genome sequencing was also conducted for strain OT10T. Considering the chemotaxonomic, genotypic and phenotypic features, as well as the low average nucleotide identity and digital DNA-DNA hybridization values compared to its closest phylogenomic neighbours, OT10T is proposed to be a novel species named Roseomonas cutis sp. nov., with OT10T designated as the type strain (=KCTC 92087T =JCM 34968T).

Pseudomonas fungipugnans sp. nov., a potently antifungal bacterium isolated from moss.

A novel strain, 681, was isolated from a moss sample taken from the Chrutzelried woods in Canton Zürich, Switzerland. The strain showed potent activity against several fungi and oomycetes. It was affiliated to the Pseudomonas genus by 16S rRNA gene sequence phylogeny. Genome sequencing showed a G+C content of 59.9 mol%. The highest average nucleotide identity was 86.63%, and the highest digital DNA-DNA hybridization value was 32.2% (with Pseudomonas nunensis ln5), considerably below the thresholds for species delineation. Multi-locus phylogeny using 81 concatenated sequences indicated that the strain represented a new species within the Pseudomonas mandelii subgroup. This study details its characterization as a new species. The 681 genome was screened in silico using antiSMASH to reveal candidate secondary metabolite clusters for the strong antifungal activity exhibited by 681. Of the 15 clusters identified, we disrupted the seven best and tested for activity against Fusarium solani. The pyrrolnitrin, rhizoxin, novel PKS-NRPS and acaterin gene clusters contributed significantly to the observed antifungal activity. Phenotypic analyses found that strain 681 cells were aerobic, Gram-negative, motile rods (mean length 2.60 µm and mean width of 0.67 µm) with one to three polar flagella. Optimal growth was at 30 °C, but growth also occurred at 8 °C. The pH range was 6-7, with some growth at pH 8. Robust growth occurred at 0-3% (w/v) NaCl and weak growth at 4% (w/v) NaCl, with the optimum at 1% (w/v) NaCl. Strain 681 was oxidase positive, hydrolysed arginine under anaerobic conditions, showed intense fluorescence on King's medium B and produced a hypersensitive response in tobacco leaves. Following our investigations, we propose the designation Pseudomonas fungipugnans sp. nov. for this new species. The type strain, 681, is available from the DSMZ and LMG/BCCM culture collections under the designations DSM 115721 and LMG 33039, respectively.

Paenibacillus zeirhizosphaerae sp. nov., isolated from surface of the maize (Zea mays) roots in a horticulture field, Hungary.

A novel Gram-stain-positive, rod-shaped, endospore-forming bacterium with peritrichous flagella, designated as P96T was isolated from the surface of maize roots. Strain P96T grew optimally at 28 °C, pH 7.0. The strain contained A1γ meso-Dpm-direct in the cell-wall peptidoglycan. The dominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The genome size of strain P96T was 4.8 Mb, and the G+C content was 50.01%. Phylogenomic analyses based on the whole-genome sequences classified the strain into the genus Paenibacillus. The digital DNA-DNA hybridization and average nucleotide identity relatedness analysis resulted in values below the threshold for prokaryotic species delineation, with the highest values observed for Paenibacillus enshidis KCTC 33519T (29.4 and 85.2%, respectively). Genotypic data together with phenotypic properties supported the classification of strain P96T as representative of a novel species of the genus Paenibacillus, for which the name Paenibacillus zeirhizosphaerae sp. nov. is proposed. The type strain is P96T (=LMG 32802T = NCAIM B 02678T).

Pseudomonas spirodelae, sp. nov., a bacterium isolated from the roots of the aquatic plant Spirodela polyrhiza.

A polyphasic taxonomic study was carried out on strain T5W1T, isolated from the roots of the aquatic plant Spirodela polyrhiza. This isolate is Gram-negative, rod-shaped, motile, aerobic and non-pigmented. Nearly complete 16S rRNA gene sequence homology related the strain to Pseudomonas, with 98.4% similarity to P. guineae, P. peli and P. leptonychotis. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) with the closest phylogenetic neighbour of T5W1T showed differences at the species level, further confirmed by differences in several physiological characteristics. The main fatty acids are summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C18 : 1 ω7c, and C16 : 0. The DNA G+C content is 59.3 mol%. Q-9 is the primary ubiquinone found, and phosphatidylethanolamine is the dominant polar lipid, with lesser amounts of phosphatidylglycerol, diphosphatidylglycerol and phosphatidylserine. Based on the results obtained, this bacterium is assigned to the genus Pseudomonas as a new species with the name Pseudomonas spirodelae sp. nov., type strain T5W1T (=NRRL B-65714T =DSM 118085T).

Arvimicrobium flavum gen. nov., sp. nov., A Novel Genus in the Family Phyllobacteriaceae Isolated From Forest Soil.

During the study of microbial diversity of forest soil in the Republic of Korea, a yellow pigment-producing, Gram-stain-negative, rod-shaped, motile bacterium was isolated and designated as strain 1W2T. This strain grew at temperature of 10-37°C, at pH of 5.0-9.0, and at NaCl concentration of 0-3.0% (w/v). The 16S rRNA gene sequencing and genome sequencing revealed that strain 1W2T is a member of the family Phyllobacteriaceae but exhibits low similarity with known genera, suggesting that this strain is a new genus within the family. This strain showed the closest similarity to the genera Mesorhizobium (96.6-96.9%), Aminobacter (96.4 -96.6%), Aquamicrobium (96.5-96.7%), and Pseudaminobacter (96.6-96.7%). The nearest relative of 1W2T was Mesorhizobium shangrilense CCBAU 65327T with the 16S rRNA gene sequence similarity of 96.9%. The genome size was 5,545,526bp with DNA G + C content of 64.7%. The values of overall genomic relatedness indices between strain 1W2T and the reference members were 20.4-21.3% for digital DNA-DNA hybridization, 74.0-76.6% for average nucleotide identity, and 68.1-61.2% for amino acids identity. Chemotaxonomic profiling revealed that Q-10 was the sole ubiquinone; summed feature 8 (C18:1ω7c and/or C18:1ω6c), iso-C13:0, and C19:0 cyclo ω8c were the predominant fatty acids; and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylcholine, and phosphatidylethanolamine were the major polar lipids. Based on these data, strain 1W2T represents a novel species of a new genus in the family Phyllobacteriaceae. Accordingly, we proposed the name Arvimicrobium flavum gen. nov., sp. nov., with the type strain 1W2T (= KCTC 92441T = NBRC 116019T).

Leifsonia stereocauli sp. nov., Isolated from Stereocaulon tomentosum Fr. Lichen.

A Gram-stain-positive, aerobic, yellow-pigmented, catalase-positive, oxidase-positive, non-motile with no flagella and irregularly rod-shaped, denominated strain YIM 134122T, was isolated from a Stereocaulon tomentosum Fr. lichen gathered on Baima Snow Mountain in Diqing Tibetan Autonomous Prefecture, Yunnan Province, China. The novel strain grew at pH 6.0-8.0 (optimum, pH 7.0-7.2), 4-35℃ (optimum,25-35℃), and with the existence of 0-10% (w/v) NaCl (optimum, 0-2%). Phylogenetic analysis on the basis of 16S rRNA gene sequences indicated that strain YIM 134122T belonged to the genus Leifsonia and presented high levels of 16S rRNA gene sequence similarity with Leifsonia kafniensis JCM 17021T (97.92%) and Leifsonia psychrotolerans CGMCC 1.12166T (97.79%). Based on the draft genome sequence, the G + C content of strain YIM 134122T was found to be 68.41%, and the average nucleotide identity and digital DNA-DNA hybridizations values between strain YIM 134122T and Leifsonia kafniensis JCM 17021T and Leifsonia psychrotolerans CGMCC 1.12166T were 73.78% and 73.75%, and 19.6% and 19.9%, respectively, which were extremely lower than the generally proposed threshold value for species delineation. The predominant polar lipids in strain YIM 134122T consisted of phosphatidylglycerol, diphosphatidylglycerol, three unknown glycolipids, two unknown phospholipids, and one unknown lipid, whereas MK-10, MK-11 and MK-12 were the main menaquinones. The major fatty acids (> 4%) of the strain contained iso-C15:0, anteiso-C15:0, iso-C16:0 and anteiso-C17:0. On the basis of phylogenetic, chemotaxonomic, phenotypic and genotypic evidences, it is concluded that strain YIM 134122T represents a novel species of the genus Leifsonia, for with the name Leifsonia stereocauli sp. nov. is proposed. The type strain is YIM 134122T (= CGMCC 1.62033T = MCCC 1K08590T = KCTC 59219T).

Identification and Bioactivity Analysis of a Novel Bacillus Species, B. maqinnsis sp. nov. Bos-x6-28, Isolated from Feces of the Yak (Bos grunniens).

Background: The identification of novel bacterial species from the intestines of yaks residing on the Qinghai-Tibet Plateau is pivotal in advancing our understanding of host-microbiome interactions and represents a promising avenue for microbial drug discovery. Methods: In this study, we conducted a polyphasic taxonomic analysis and bioactive assays on a Bacillus strain, designated Bos-x6-28, isolated from yak feces. Results: The findings revealed that strain Bos-x6-28 shares a high 16S rRNA gene sequence similarity (98.91%) with B. xiamenensis HYC-10T and B. zhangzhouensis DW5-4T, suggesting close phylogenetic affinity. Physiological and biochemical characterizations demonstrated that Bos-x6-28 could utilize nine carbon sources, including D-galactose, inositol, and fructose, alongside nine nitrogen sources, such as threonine, alanine, and proline. Analysis of biochemical markers indicated that Bos-x6-28's cell wall hydrolysates contained mannose, glucose, and meso-2,6-diaminopimelic acid, while menaquinone-7 (MK-7), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylglycerol (DPG) were found in the cell membrane. The primary cellular fatty acids included C16:0 (28.00%), cyclo-C17:0 (19.97%), C14:0 (8.75%), cyclo-C19:0 (8.52%), iso-C15:0 (5.49%), anteiso-C15:0 (4.61%), and C12:0 (3.15%). Whole-genome sequencing identified a genome size of 3.33 Mbp with 3353 coding genes. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses confirmed Bos-x6-28 as a novel species, hereby named B. maqinnsis Bos-x6-28 (MCCC 1K09379). Further genomic analysis unveiled biosynthetic gene clusters encoding bioactive natural compounds, including β-lactones, sactipeptides, fengycin, and lichenysin analogs. Additionally, in vitro assays demonstrated that this strain exhibits antibacterial and cytotoxic activities. Conclusions: These findings collectively indicate the novel Bacillus species B. maqinnsis Bos-x6-28 as a promising source for novel antibiotic and antitumor agents.

Description of Massilia orientalis sp. nov., Isolated from Rhizosphere Soil of Ageratina adenophora.

A novel bacteria strain, designated YIM B02787T, was isolated from rhizosphere soil of Ageratina adenophora, in Yunnan, southwest China. The strain was aerobic, Gram-stain-negative, rod-shaped and motile with one polar flagellum. Growth occurred at 4-45°C (optimum, 20-30°C) and pH 6.0-10.0 (optimum, 7.0-8.0), and in presence of 0-1% (w/v) NaCl. Phylogenetic analyses based on both 16S rRNA gene and genome sequences data revealed that strain YIM B02787T belongs to the genus Massilia, being closely related to Massilia phosphatilytica KCTC 52513T (98.93% similarity), M. putida KCTC 42761T (98.86%), and M. kyonggiensis JCM 19189T (98.78%). The DNA G+C content was 65.9%. The digital DNA-DNA hybridization and average nucleotide identity values between the isolate strain and aforementioned closely neighbors were low, at 35.8-48.9 and 88.5-92.5%, respectively. Strain YIM B02787T contained Q-8 as the ubiquinone and major fatty acids were summed feature 3 (C16:1ω7c and/or C16:1ω6c, 45.5%) and C16:0 (27.5%). The polar lipid profile consisted of phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, two unidentified phospholipids, two unidentified aminophospholipids, and one unidentified polar lipid. On the basis of its phylogenetic, phenotypic and chemotaxonomic characteristics, strain YIM B02787Trepresents a novel species of the genus Massilia, for which the name Massilia orientalis sp. nov. is proposed. The type strain is YIM B02787T (= NBRC 116628T = CGMCC 1.61539T).

Genomic Characterization of Probiotic Purple Nonsulfur Bacteria Cereibacter sphaeroides Strains S3W10 and SS15: Implications for Enhanced Shrimp Aquaculture.

Cereibacter sphaeroides strains S3W10 and SS15, isolated from shrimp ponds, exhibit potential probiotic benefits for aquaculture. In this study, the genomic features of S3W10 and SS15 were thoroughly characterized to evaluate their probiotic properties and safety for aquaculture use. The genomes of S3W10 and SS15 consist of 130 and 74 contigs, with sizes of 4.6 Mb and 4.4 Mb and GC contents of 69.2%. Average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and phylogenomic analyses confirmed that these strains belong to C. sphaeroides. Genome annotation predicted 4260 coding sequences (CDS) in S3W10 and 4086 CDS in SS15, including genes associated with stress tolerance, nutrient absorption, and antioxidant activity. Notably, genes related to vitamin B12 synthesis, digestive enzyme production, and carotenoid biosynthesis, which support shrimp health, were identified in both genomes. CAZyme analysis identified 116 and 115 carbohydrate-active enzymes in S3W10 and SS15, respectively, supporting adaptation to gastrointestinal environments and the host immune response. Pan-genome analysis across C. sphaeroides strains revealed 7918 gene clusters, highlighting the open pan-genome structure of this species and its high genetic diversity. Further bioinformatic analyses assessing mobile genetic elements, antibiotic-resistance genes, and virulence factors demonstrated the safety of both strains for aquaculture, as no plasmids or virulence genes were identified. The genomic insights in this study provide a deeper understanding of the strains' adaptability and functional potential, aligning with previous in vitro and in vivo studies and highlighting their potential for use in shrimp cultivation.

Segatella asaccharophila sp. nov., an anaerobic pectinophile isolated from a two-phase methane fermentation system.

An obligately anaerobic, Gram-stain-negative, non-spore-forming and non-motile rod (strain LPYR103-PreT) was isolated from a two-phase methane fermentation system. Using 16S rRNA gene sequence-based phylogenetic analysis, strain LPYR103-PreT was classified in the genus Segatella. The 16S rRNA gene sequence similarity, average nucleotide identity and digital DNA-DNA hybridization between strain LPYR103-PreT and its phylogenetically nearest species - Segatella cerevisiae JCM 30867T - were 94.4, 77.9 and 23.4%, respectively. The genome size of strain LPYR103-PreT was 3 326 733 bp, and its genomic DNA G+C content was 44.05%. The most abundant cellular fatty acid was anteiso-C15 : 0. The growth of strain LPYR103-PreT was stimulated by the addition of pectin, d-galacturonate and d-glucuronate; in contrast, the strain exhibited poor growth in the presence of common sugars, such as glucose. Therefore, strain LPYR103-PreT was classified as a pectinophile - a bacterium that shows a preference for pectin and a few related compounds as substrates. Glucose is degraded by type strains of 12 species belonging to the genus Segatella; thus, strain LPYR103-PreT is the first described pectinophile belonging to this genus. Strain LPYR103-PreT produced succinate and acetate as its major metabolic end products. Based on the differences in the phylogenetic, genomic, physiological and chemotaxonomic characteristics of strain LPYR103-PreT and related species, the name Segatella asaccharophila sp. nov. is proposed to accommodate strain LPYR103-PreT (= NRIC 0997T = JCM 37351T=DSM 118531T = KCTC 25923T).

Sphingomonas longa sp. nov., Sphingomonas mollis sp. nov. and Sphingomonas aurea sp. nov.: three novel Sphingomonas species isolated from soil.

Three Gram-negative, aerobic and non-motile bacterial strains, BT552T, BT553T and KR1UV-12T, were isolated from soil samples in Gwangju-si and Gangneung-si, the Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequence showed that strains BT552T, BT553T and KR1UV-12T clustered to a distinct clade within the family Sphingomonadaceae (order Sphingomonadales, class Alphaproteobacteria). The strains exhibited the highest genetic similarity with representatives of the genus Sphingomonas; moreover, strains BT552T and BT553T tightly clustered with Sphingomonas melonis DAPP-PG 224T (98.2 and 98.1 %) and Sphingomonas aquatilis JSS-7T (98.1 and 98.0 %), while strain KR1UV-12T clustered with S. melonis DAPP-PG 224T (97.9%) and Sphingomonas rubra BH3T (97.8%), respectively. The major cellular fatty acids of all three strains were summed feature 8 (C18:1 ω7c/C18:1 ω6c), comprising 44.7, 46.4 and 48.5%. Additionally, their respiratory quinone is Q-10, and polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phospholipids, sphingolipid and phosphatidylcholine. They all grow well at an optimum temperature of 25 °C, at pH 7. The draft genomes of strains BT552T, BT553T and KR1UV-12T measures 4 035 561 bp, 3 941 714 bp and 3 418 792 bp, respectively, comprising 3 804 3648 and 3236 coding sequences and 50, 48 and 45 RNA genes. The average nucleotide identity analysis and digital DNA-DNA hybridization values between BT552T, BT553T and KR1UV-12T and closely related Sphingomonas species range from 72.7 to 80.2% and 19.4 to 24.3%, respectively. Based on phenotypic, genotypic and chemotaxonomic data, these three strains BT552T, BT553T and KR1UV-12T represent three novel bacterial species within the genus Sphingomonas for which the names Sphingomonas longa sp. nov. (type strain BT552T= KCTC 82094T =NBRC 114993T), Sphingomonas mollis sp. nov. (type strain BT553T =KCTC 82095T =NBRC 114994T) and Sphingomonas aurea sp. nov. (type strain KR1UV-12T = KCTC 92959T = TBRC 18506T) are proposed.

Actinomadura monticuli sp. nov., isolated from Darangshi Oreum (a volcanic cone), and the reclassification of Actinomadura glauciflava Lu et al. 2003 as a later heterotypic synonym of Actinomadura luteofluorescens (Shinobu 1962) Preobrazhenskaya et al. 1975 (Approved Lists 1980).

Two mycelium-forming actinobacterial strains, designated as DLS-47 and DLS-62T, were isolated from volcanic ash collected from the surface of a rock on the peak of Darangshi Oreum (a volcanic cone) in Jeju, Republic of Korea, and their taxonomic positions were investigated by a polyphasic approach. Both of the isolates showed growth at 20-42 °C, pH 6.0-9.0 and 0-1% (w/v) NaCl. Furthermore, DLS-47 was found to grow at 45 °C, while strain DLS-62T grew at pH 10.0 and 3% (w/v) NaCl. The 16S rRNA gene-based phylogeny showed that both of the isolates belonged to the genus Actinomadura; strain DLS-47 was most closely related to Actinomadura chokoriensis DSM 45346T (100% sequence identity), while strain DLS-62T formed a tight cluster with Actinomadura bangladeshensis DSM 45347T (99.5% sequence similarity). Morphological and chemotaxonomic characteristics supported the affiliation of the two isolates to the genus Actinomadura. Phylogenomic analysis based on 92 core gene sequences showed that both of the isolates were most closely related to A. chokoriensis DSM 45346T. Strain DLS-47 shared 100% of orthologous average nucleotide identity and digital DNA-DNA hybridization values with A. chokoriensis DSM 45346T, while strain DLS-62T showed orthologous average nucleotide identity ≤89.8% and digital DNA-DNA hybridization values ≤39.4% with strain DLS-47 and members of the genus Actinomadura. The results of phenotypic assays and comparison of overall genomic relatedness indices support the conclusion that strain DLS-47 (= KACC 23347=DSM 116423) is a strain of A. chokoriensis, while strain DLS-62T (= KACC 23345T = DSM 116424T) represents a new species of the genus Actinomadura, for which the name Actinomadura monticuli sp. nov. is proposed. Also, Actinomadura glauciflava Lu et al. 2003 is reclassified as a later heterotypic synonym of Actinomadura luteofluorescens (Shinobu 1962) Preobrazhenskaya et al. 1975 (Approved Lists 1980) based on analysis of overall genomic relatedness indices and phenotypic similarity.