Lacticaseibacillus parahuelsenbergensis sp. nov., Lacticaseibacillus styriensis sp. nov. and Lacticaseibacillus zeae subsp. silagei subsp. nov., isolated from different grass and corn silage.

Four rod-shaped, non-motile, non-spore-forming, facultative anaerobic, Gram-stain-positive lactic acid bacteria, designated as EB0058T, SCR0080, LD0937T and SCR0063T, were isolated from different corn and grass silage samples. The isolated strains were characterized using a polyphasic approach and EB0058T and SCR0080 were identified as Lacticaseibacillus zeae by 16S rRNA gene sequence analysis. Based on whole-genome sequence-based characterization, EB0058T and SCR0080 were separated into a distinct clade from Lacticaseibacillus zeae DSM 20178T, together with CECT9104 and UD2202, whose genomic sequences are available from NCBI GenBank. The average nucleotide identity (ANI) values within the new subgroup are 99.9 % and the digital DNA-DNA hybridization (dDDH) values are 99.3-99.9 %, respectively. In contrast, comparison of the new subgroup with publicly available genomic sequences of L. zeae strains, including the type strain DSM 20178T, revealed dDDH values of 70.2-72.5 % and ANI values of 96.2-96.6 %. Based on their chemotaxonomic, phenotypic and phylogenetic characteristics, EB0058T and SCR0080 represent a new subspecies of L. zeae. The name Lacticaseibacillus zeae subsp. silagei subsp. nov. is proposed with the type strain EB0058T (=DSM 116376T=NCIMB 15474T). According to the results of 16S rRNA gene sequencing, LD0937T and SCR0063T are members of the Lacticaseibacillus group. The dDDH value between the isolates LD0937T and SCR0063T was 67.6 %, which is below the species threshold of 70 %, clearly showing that these two isolates belong to different species. For both strains, whole genome-sequencing revealed that the closest relatives within the Lacticaseibacillus group were Lacticaseibacillus huelsenbergensis DSM 115425 (dDDH 66.5 and 65.9 %) and Lacticaseibacillus casei DSM 20011T (dDDH 64.1 and 64.9 %). Based on the genomic, chemotaxonomic and morphological data obtained in this study, two novel species, Lacticaseibacillus parahuelsenbergensis sp. nov. and Lacticaseibacillus styriensis sp. nov. are proposed and the type strains are LD0937T (=DSM 116105T=NCIMB 15471T) and SCR0063T (=DSM 116297T=NCIMB 15473T), respectively.

Streptococcus raffinosi sp. nov., isolated from human breast milk samples.

Novel Gram-positive, catalase-negative, α-haemolytic cocci were isolated from breast milk samples of healthy mothers living in Hanoi, Vietnam. The 16S rRNA gene sequences of these strains varied by 0-2 nucleotide polymorphisms. The 16S rRNA gene sequence of one strain, designated as BME SL 6.1T, showed the highest similarity to those of Streptococcus salivarius NCTC 8618T (99.4 %), Streptococcus vestibularis ATCC 49124T (99.4 %), and Streptococcus thermophilus ATCC 19258T (99.3 %) in the salivarius group. Whole genome sequencing was performed on three selected strains. Phylogeny based on 631 core genes clustered the three strains into the salivarius group, and the strains were clearly distinct from the other species in this group. The average nucleotide identity (ANI) value of strain BME SL 6.1T exhibited the highest identity with S. salivarius NCTC 8618T (88.4 %), followed by S. vestibularis ATCC 49124T (88.3 %) and S. thermophilus ATCC 19258T (87.4 %). The ANI and digital DNA-DNA hybridization values between strain BME SL 6.1T and other species were below the cut-off value (95 and 70 %, respectively), indicating that it represents a novel species of the genus Streptococcus. The strains were able to produce α-galactosidase and acid from raffinose and melibiose. Therefore, we propose to assign the strains to a new species of the genus Streptococcus as Streptococcus raffinosi sp. nov. The type strain is BME SL 6.1T (=VTCC 12812T=NBRC 116368T).

Thioclava litoralis sp. nov., a novel species of alphaproteobacterium, isolated from surface seawater.

A Gram-negative, aerobic, rod-shaped, non-motile bacterium, designated as FTW29T, was isolated from surface seawater sampled in Futian district, Shenzhen, China. Growth of strain FTW29T was observed at 15-42 ℃ (optimum, 28-30 ℃), pH 4.0-9.0 (optimum, pH 5.5-7.5) and in the presence of 0.5-10% NaCl (optimum, 3.0% NaCl). Strain FTW29T showed 95.0-96.8% 16 S rRNA gene sequence similarity to various type strains of the genera Thioclava, Sinirhodobacter, Rhodobacter, Haematobacter and Frigidibacter of the family Paracoccaceae, and its most closely related strains were Thioclava pacifica DSM 10,166T (96.8%) and Thioclava marina 11.10-0-13T (96.7%). The phylogenomic tree constructed on the bac120 gene set showed that strain FTW29T formed a clade with the genus Thioclava, with a bootstrap value of 100%. The evolutionary distance values between FTW29T and type strains of the genus Thioclava were 0.17-0.19, which are below the recommended standard (0.21-0.23) for defining a novel genus in the family Paracoccaceae. In strain FTW29T, the major fatty acids identified were summed feature 8 (C18:1ω7c) and C16:0, and the predominant respiratory quinones were ubiquinone-10 and ubiquinone-9. The composition of polar lipids in strain FTW29T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminolipid, two unidentified glycolipids and an unidentified lipid. The genome of strain FTW29T comprised one circle chromosome and six plasmids, with a G + C content of 61.4%. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain FTW29T and seven type strains of the genus Thioclava were 76.6-78.4%, 53.2-56.4% and 19.3-20.4%, respectively. Altogether, the phenotypic, phylogenetic and chemotaxonomic evidence illustrated in this study suggested that strain FTW29T represents a novel species of the genus Thioclava, with the proposed name Thioclava litoralis sp. nov. The type strain is FTW29T (= KCTC 82,841T = MCCC 1K08523T).

Pleionea litopenaei sp. nov., isolated from the gastric tract of juvenile Pacific white shrimp Litopenaeus vannamei.

A Gram-stain-negative, aerobic, rod-shaped and motile strain HL-JVS1T, was isolated from the gastric tract of a juvenile Pacific white shrimp. Molecular phylogenetic analysis based on 16S rRNA gene sequences of strain HL-JVS1T revealed its affiliation with the genus Pleionea, with close relatives including Pleionea mediterranea MOLA115T (97.5%) and Pleionea sediminis S1-5-21T (96.2%). The complete genome of strain HL-JVS1T consisted of a circular 4.4 Mb chromosome and two circular plasmids (6.6 and 35.0 kb) with a G + C content of 43.1%. The average nucleotide identity and digital DNA-DNA hybridization values between strain HL-JVS1T and the type strains of described Pleionea species were 69.7-70.4% and 18.3-18.6%, respectively. Strain HL-JVS1T grew at 10-40 °C (optimum, 30 °C) in the presence of 0.5 - 9.0% (w/v) sea salts (optimum, 2.0 - 2.5%), and at pH range of 5.5 - 10.0 (optimum, pH 6.5). The major fatty acids (> 10%) were summed feature 9 (iso-C17:1 ω9c and/or C16:0 10-methyl) (23.3%), iso-C16:0 (14.5%), iso-C11:0 3-OH (13.8%) and iso-C15:0 (11.0%). The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid, two unidentified aminolipids, and two unidentified lipids. The respiratory quinone was ubiquinone-8. The comprehensive phylogenetic, phylogenomic, phenotypic and chemotaxonomic results showed that strain HL-JVS1T is distinct from other Pleionea species. Hence, we propose strain HL-JVS1T as a novel species belonging to the genus Pleionea, for which the name Pleionea litopenaei sp. nov. is proposed with HL-JVS1T (= KCCM 90514T = JCM 36490T) as the type strain.

Chitinophaga pollutisoli sp. nov., isolated from contaminated sediment.

A Gram-stain-negative, yellow-pigmented, and facultatively aerobic bacterium, designated strain GPA1T, was isolated from plastic waste landfill soil in the Republic of Korea. The cells were non-motile short rods exhibiting oxidase-negative and catalase-positive activities. Growth was observed at 15-40 °C (optimum, 30 °C), at pH 6.0-9.0 (optimum, pH 7.0-8.0) and in the presence of 0-2.5 % (w/v) NaCl (optimum, 0 %). Menaquinone-7 was the sole respiratory quinone, and iso-C15 : 0, C16 : 1 ω5c, and iso-C17 : 0 3-OH were the major cellular fatty acids (>10 % of the total fatty acids). Phosphatidylethanolamine was identified as a major polar lipid. Phylogenetic analyses based on 16S rRNA gene sequences and 120 concatenated marker protein sequences revealed that strain GPA1T formed a distinct lineage within the genus Chitinophaga. The genome of strain GPA1T was 6078 kb in size with 53.8 mol% G+C content. Strain GPA1T exhibited the highest similarity to Chitinophaga rhizosphaerae T16R-86T, with a 98.6 % 16S rRNA gene sequence similarity, but their average nucleotide identity and digital DNA-DNA hybridization values were 82.5 and 25.9 %, respectively. Based on its phenotypic, chemotaxonomic, and phylogenetic characteristics, strain GPA1T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga pollutisoli sp. nov. is proposed. The type strain is GPA1T (=KACC 23415T=JCM 36644T).

Spartinivicinus poritis sp. nov., a red pigment-producing bacterium isolated from a scleractinian coral Porites lutea.

A Gram-stain-negative, red pigment-producing, aerobic, and rod-shaped bacterial strain (A2-2T) was isolated from a bleached scleractinian coral (Porites lutea). Strain A2-2T grew with 1.0-7.0 % (w/v) NaCl (optimum, 3.0 %), at pH 6.0-11.0 (optimum, pH 8.0), and at 18-41 °C (optimum, 35 °C). Results of phylogenetic analysis based on 16S rRNA gene sequences suggested that strain A2-2T fell within the genus Spartinivicinus and was closely related to Spartinivicinus ruber S2-4-1HT (98.1 % sequence similarity) and Spartinivicinus marinus SM1973T (98.0 % sequence similarity). The predominant cellular fatty acids of strain A2-2T were C16 : 0 (31.0 %), summed feature 3 (29.0 %), summed feature 8 (11.7 %), C12 : 0 3-OH (6.4 %), and C10 : 0 3-OH (5.5 %), while the major respiratory quinone was Q-9. The polar lipids mainly comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and an unidentified phospholipid. The genome size of strain A2-2T was 6.8 Mb, with a G+C content of 40.2 mol%. The DNA-DNA hybridization value was 24.2 % between A2-2T and S. ruber S2-4-1HT and 36.9 % between A2-2T and S. marinus SM1973T, while the average nucleotide identity values were 80.1 and 88.8 %, respectively. Based on these findings, strain A2-2T could be recognized to represent a novel species of the genus Spartinivicinus, for which the name Spartinivicinus poritis sp. nov. is proposed. The type strain is A2-2T (=MCCC 1K08228T=KCTC 8323T).

Saccharopolyspora mangrovi sp. nov., a novel mangrove soil actinobacterium with distinct metabolic potential revealed by comparative genomic analysis.

A novel mangrove soil-derived actinomycete, strain S2-29T, was found to be most closely related to Saccharopolyspora karakumensis 5K548T based on 16 S rRNA sequence (99.24% similarity) and genomic phylogenetic analyses. However, significant divergence in digital DNA-DNA hybridization, average nucleotide identity, and unique biosynthetic gene cluster possession distinguished S2-29T as a distinct Saccharopolyspora species. Pan genome evaluation revealed exceptional genomic flexibility in genus Saccharopolyspora, with > 95% accessory genome content. Strain S2-29T harbored 718 unique genes, largely implicated in energetic metabolisms, indicating different metabolic capacities from its close relatives. Several uncharacterized biosynthetic gene clusters in strain S2-29T highlighted the strain's untapped capacity to produce novel functional compounds with potential biotechnological applications. Designation as novel species Saccharopolyspora mangrovi sp. nov. (type strain S2-29T = JCM 34,548T = CGMCC 4.7716T) was warranted, expanding the known Saccharopolyspora diversity and ecology. The discovery of this mangrove-adapted strain advances understanding of the genus while highlighting an untapped source of chemical diversity.

Polymerase Chain Reaction and Dot-Blot Hybridization for Leptospira Detection in Water Samples.

The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by probe hybridization in the presence of carrier DNA. It is based on the transfer of a known amount of DNA onto an inert solid support, such as a nylon membrane, utilizing the dot-blot apparatus and without electrophoretic separation. Nylon membranes have the advantage of high nucleic acid binding capacity (400 µg/cm2), high strength, and are positively or neutrally charged. The probe used is a highly specific ssDNA fragment of 18 to 20 bases long labeled with digoxigenin (DIG). The probe will conjugate with the Leptospira DNA. Once the probe has hybridized with the target DNA, it is detected by an anti-digoxigenin antibody, allowing its easy detection through its emissions revealed in an X-ray film. The dots with an emission will correspond to the DNA fragments of interest. This method employs the non-isotopic labeling of the probe, which may have a very long half-life. The drawback of this standard immuno-label is a lower sensitivity than isotopic probes. Nevertheless, it is mitigated by coupling polymerase chain reaction (PCR) and dot-blot assays. This approach enables the enrichment of the target sequence and its detection. Additionally, it may be used as a quantitative application when compared against a serial dilution of a well-known standard. A dot-blot application to detect Leptospira from the three main clades in water samples is presented here. This methodology can be applied to large amounts of water once they have been concentrated by centrifugation to provide evidence of the presence of Leptospiral DNA. This is a valuable and satisfactory tool for general screening purposes, and may be used for other non-culturable bacteria that may be present in water, enhancing the comprehension of the ecosystem.

Pedobacter deserti sp. nov., a novel species isolated from desert soil.

An aerobic, Gram-stain-negative bacterium, designated as SYSU D00382T, was sourced from soil of Gurbantunggut Desert, PR China. The strain was short-rod-shaped, oxidase-positive and catalase-negative, with yellow-colored, convex, round, and smooth colonies on TSA plate. Growth and proliferation occurred at 4-37 °C (optimal: 28-30 °C), pH 5.0-8.0 (optimal: pH 6.0-7.0) and NaCl concentration of 0-2.5% (optimal: 0-0.5%). The 16S rRNA gene based phylogenetic assessment showed that SYSU D00382T belonged to the genus Pedobacter, and was most closely related to Pedobacter ginsengisoli Gsoil 104T with similarity of 97.7%. The genomic DNA G+C content of SYSU D00382T was 46.4%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between SYSU D00382T and P. ginsengisoli Gsoil 104T were 75.7% and 17.5%, respectively. The main polar lipid was phosphatidylethanolamine. The major fatty acids (> 5%) were iso-C15:0, iso-C17:0 3-OH, summed features 3 and 9. The sole respiratory quinone identified was MK-7. The phylogeny based on 16S rRNA gene and whole-genome sequences revealed that SYSU D00382T formed a robust lineage with P. ginsengisoli Gsoil 104T. Based on phenotypic, phylogenetic and genotypic data, a novel specie named Pedobacter deserti sp. nov. is proposed. The type strain is SYSU D00382T (= CGMCC 1.18627T = MCCC 1K04972T = KCTC 82279T).

Undibacterium cyanobacteriorum sp. nov., an auxin-producing bacterium isolated from fresh water during cyanobacterial bloom period.

A novel Gram-negative, white-pigmented, and auxin-producing strain, 20NA77.5T, was isolated from fresh water during cyanobacterial bloom period. Pairwise comparison of the 16S rRNA gene sequences showed that strain 20NA77.5T belonged to the genus Undibacterium and exhibited the highest sequence similarity to the type strains of Undibacterium danionis (98.00%), Undibacterium baiyunense (97.93%), Undibacterium macrobrachii (97.92%), and Undibacterium fentianense (97.71%). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain 20NA77.5T and its related type strains were below 79.93 and 23.80%, respectively. The predominant fatty acids (> 10% of the total fatty acids) were C16:0 and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The genomic DNA G + C content of strain 20NA77.5T was found to be 48.61%. Based on the phylogenetic distinctness, chemotaxonomic features, and phenotypic features, strain 20NA77.5T is considered to represent a novel species of the genus Undibacterium, for which the name Undibacterium cyanobacteriorum sp. nov is proposed. The type strain is 20NA77.5T (= KCTC 8005T = LMG 33136T).

Aquibaculum arenosum gen. nov., sp. nov., a novel member of the family Rhodovibrionaceae, isolated from sea sand.

A Gram-negative, non-motile, and creamy-white coloured bacterium, designated CAU 1616T, was isolated from sea sand collected at Ayajin Beach, Goseong-gun, Republic of Korea. The bacterium was found to grow optimally at 37 °C, pH 8.0-8.5, and with 1-5 % (w/v) NaCl. Phylogenetic analyses based on the 16S rRNA gene sequences placed strain CAU 1616T within the order Rhodospirillales. The highest 16S rRNA gene sequence similarity was to Fodinicurvata fenggangensis YIM D812T (94.1 %), Fodinicurvata sediminis YIM D82T (93.7 %), Fodinicurvata halophila BA45ALT (93.6 %) and Algihabitans albus HHTR 118T (92.3 %). Comparing strain CAU 1616T with closely related species (Fodinicurvata fenggangensis YIM D812T and Fodinicurvata sediminis YIM D82T), the average nucleotide identity based on blast+ values were 69.7-69.8 %, the average amino acid identity values were 61.3-61.4 %, and the digital DNA-DNA hybridization values were 18.4-18.5 %. The assembled draft genome of strain CAU 1616T had 29 contigs with an N50 value of 385.8 kbp, a total length of 3 490 371 bp, and a DNA G+C content of 65.1 mol%. The predominant cellular fatty acids were C18 : 1 2-OH, C19 : 0 cyclo ω8c, and summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c). The major respiratory quinone was Q-10. Based on phenotypic, phylogenetic, and chemotaxonomic evidence, strain CAU 1616T represents a novel genus in the family Rhodovibrionaceae, for which the name Aquibaculum arenosum gen. nov., sp. nov. is proposed. The type strain is CAU 1616T (=KCTC 82428T=MCCC 1K06089T).

Maribellus mangrovi sp. nov., an iron-reducing bacterium isolated from mangrove sediment.

A novel facultatively anaerobic and Gram-stain-negative bacterium, designated FJH33T, was isolated from mangrove sediment sampled in Zhangzhou, PR China. Cells of strain FJH33T were rod-shaped or slightly curved-shaped, with widths of 0.3-0.5 µm and lengths of 1.0-3.0 µm. Optimum growth of strain FJH33T occurred in the presence of 3 % NaCl (w/v), at 33 °C and at pH 7.0. Oxidase activity was negative, while catalase activity was positive. Its iron-reducing ability was determined. Based on 16S rRNA gene sequence similarity, strain FJH33T was most closely related to Maribellus luteus XSD2T (95.1 %), followed by Maribellus sediminis Y2-1-60T (95.0 %) and Maribellus maritimus 5E3T (94.9 %). Genome analysis of strains FJH33T and M. luteus XSD2T revealed low genome relatedness, with an average nucleotide identity value of 73.8% and a digital DNA-DNA hybridization value of 19.0%. Phylogenetic trees built from 16S rRNA genes and genome sequences showed that strain FJH33T represents a relatively independent phylogenetic lineage within the genus Maribellus. The major cellular fatty acids (≥10 %) were iso-C15 : 0 and C18 : 1 ω9c. The sole respiratory quinone was MK-7. The polar lipids consisted of phosphatidylethanolamine, diphosphatidylcholine, diphosphatidyglycerol and one unidentified lipid. The DNA G+C content was 41.4 mol%. Based on the integrated results of phylogenetic, physiological, biochemical and chemotaxonomic characterizations, we propose that strain FJH33T represents a novel species of the genus Maribellus, for which the name Maribellus mangrovi sp. nov. is proposed. The type strain is FJH33T (=KCTC 102210T=MCCC 1H01459T).

Noviherbaspirillum album sp. nov., an airborne bacteria isolated from an urban area of Beijing, China.

A Gram-negative, ellipsoidal to short-rod-shaped, motile bacterium was isolated from Beijing's urban air. The isolate exhibited the closest kinship with Noviherbaspirillum aerium 122213-3T, exhibiting 98.4 % 16S rRNA gene sequence similarity. Phylogenetic analyses based on 16S rRNA gene sequences and genomes showed that it clustered closely with N. aerium 122213-3T, thus forming a distinct phylogenetic lineage within the genus Noviherbaspirillum. The average nucleotide identity and digital DNA-DNA hybridization values between strain I16B-00201T and N. aerium 122213-3T were 84.6 and 29.4 %, respectively. The respiratory ubiquinone was ubiquinone 8. The major fatty acids (>10 %) were summed feature 3 (C16:1ω6c/C16:1ω7c, 43.3 %), summed feature 8 (C18:1ω7c/C18:1ω6c, 15.9 %) and C12:0 (11.0 %). The polyamine profile showed putrescine as the predominant compound. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, unknown lipids and unknown phosphatidylaminolipids. The phenotypic, phylogenetic and chemotaxonomic results consistently supported that strain I16B-00201T represented a novel species of the genus Noviherbaspirillum, for which the name Noviherbaspirillum album sp. nov. is proposed, with I16B-00201T (=CPCC 100848T=KCTC 52095T) designated as the type strain. Its DNA G+C content is 59.4 mol%. Pan-genome analysis indicated that some Noviherbaspirillum species possess diverse nitrogen and aromatic compound metabolism pathways, suggesting their potential value in pollutant treatment.

Microbacterium algihabitans sp. nov., Microbacterium phycohabitans sp. nov., and Microbacterium galbum sp. nov., isolated from dried beach seaweeds.

Three actinobacterial strains, KSW2-21T, KSW2-29T and KSW4-17T, were isolated from dried seaweeds collected around Gwakji Beach in Jeju, Republic of Korea. Their taxonomic positions were determined based on genomic, physiological and morphological characteristics. The isolates were Gram-positive, aerobic, non-motile, rod-shaped bacteria characterized by the following chemotaxonomic features: ornithine as the cell wall diamino acid, the N-glycolyl type of murein, MK-11 as the predominant menaquinone, polar lipids including diphosphatidylglycerol, phosphatidylglycerol, two unidentified glycolipids and four unidentified phospholipids, with anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0 as the the major fatty acids. The 16S rRNA gene phylogeny showed that the novel strains formed three distinct sublines within the genus Microbacterium. Strain KSW4-17T formed a tight cluster with the type strain of Microbacterium hydrothermale, while strains KSW2-21T and KSW2-29T occupied distinct positions between the type strains of M. hydrothermale and Microbacterium testaceum. Strains KSW4-17T and KSW2-29T showed 99.9 % rRNA gene sequence similarity to M. hydrothermale CGMCC 1.12512T, while strain KSW2-21T revealed 99.4 % 16S rRNA gene sequence similarity to the type strains of M. hydrothermale and M. testaceum. The genome sizes and genomic G+C contents of the three isolates ranged from 3.44 to 3.74 Mbp and from 70.3 to 70.8 mol%, respectively. The phylogenomic tree based on 92 core gene sequences exhibited similar topologies to the 16S rRNA gene phylogeny. The comparison of overall genomic relatedness indices, such as average nucleotide indentity and digital DNA-DNA hybridization, supported that the isolates represent three new species of the genus Microbacterium. Based on the results obtained here, Microbacterium algihabitans sp. nov. (type strain, KSW2-21T=KACC 23322T=DSM 116381T), Microbacterium phycohabitans sp. nov. (type strain KSW2-29T=KACC 22350T=NBRC 115221T) and Microbacterium galbum sp. nov. (type strain, KSW4-17T=KACC 23323T=DSM 116383T) are proposed.

Phase Separation Modulates the Thermodynamics and Kinetics of RNA Hybridization.

Biomolecular condensates can influence cellular function in a number of ways, including by changing the structural dynamics and conformational equilibria of the molecules partitioned within them. Here we use methyl transverse relaxation optimized spectroscopy (methyl-TROSY) NMR in conjunction with 2'-O-methyl labeling of RNA to characterize the thermodynamics and kinetics of RNA-RNA base pairing in condensates formed by the C-terminal intrinsically disordered region of CAPRIN1, an RNA-binding protein involved in RNA transport, translation, and stability. CAPRIN1 condensates destabilize RNA-RNA base pairing, resulting from a ∼270-fold decrease and a concomitant ∼15-fold increase in the on- and off-rates for duplex formation, respectively. The ∼30-fold slower diffusion of RNA single strands within the condensed phase partially accounts for the reduced on-rate, but the further ∼9-fold reduction likely reflects shedding of CAPRIN1 chains that are interacting with the RNA prior to hybridization. Our study emphasizes the important role of protein solvation in modulating nucleic acid recognition processes inside condensates.

Flavobacterium adhaerens sp. nov. and Flavobacterium maritimum sp. nov., two novel flavobacteria isolated from the Pearl River Estuary.

Two strains, designated as SYSU M80004T and SYSU M80005T, were isolated from water sampled in the Pearl River Estuary, Guangzhou, Guangdong, PR China. The strains were Gram-stain-negative and aerobic. Strain SYSU M80004T could grow at pH 6.0-8.0 (optimum, pH 7.0), 22-30 °C (optimum, 28 °C) and in the presence of 0-1 % NaCl (w/v; optimum 0 %). Strain SYSU M80005T could grow at pH 6.0-8.0 (optimum, pH 7.0), 4-37 °C (optimum, 28 °C) and in the presence of 0-1 % NaCl (w/v; optimum 0%). Both strains contained MK-6 as the predominant menaquinone. C16 : 0 and iso-C15 : 0 were identified as the major fatty acids (>10 %) of strain SYSU M80004T while strain SYSU M80005T contained iso-C15 : 0 and iso-C17 : 0 3-OH as major fatty acids. Phosphatidylethanolamine was present as the major polar lipid in both strains. The average nucleotide identity and digital DNA-DNA hybridization values between these two strains and their closest relatives were 73.5-79.3 % and 19.6-23.2 %, respectively. Phylogenetic analysis based on the 16S rRNA gene and genomic sequences indicated they belonged to the genus Flavobacterium. Therefore, on the basis of phenotypic, physiological, chemotaxonomic and genomic evidence, two novel species, Flavobacterium adhaerens sp. nov. (type strain=SYSU M80004T=CDMCC 1.4522T=KCTC 102268T) and Flavobacterium maritimum sp. nov. (type strain=SYSU M80005T=CGMCC 1.4523T= KCTC 102269T) are proposed.

Planococcus shenhongbingii sp. nov., Planococcus shixiaomingii sp. nov. and Planococcus liqunii sp. nov., isolated from soil of the Qinghai-Tibet Plateau.

Six novel bacterial strains, designated N016T, N017, N022T, N028, N056T, and N064, were isolated from soil sampled on the Qinghai-Tibet Plateau. Cells were aerobic, orange or yellow, globular or rod-shaped, non-motile, non-spore-forming, Gram-stain-positive, catalase-positive and oxidase-negative. All the isolates were salt-tolerant and could grow in the range of 4-42 °C. Results of phylogenomic analyses based on 16S rRNA gene sequences and core genomic genes showed that the three pairs of strains (N016T/N017, N022T/N028, and N056T/N064) were closely related to the members of the genus Planococcus, and clustered with Planococcus ruber, Planococcus glaciei, and Planococcus chinensis. The digital DNA-DNA hybridization and average nucleotide identity values of the six novel strains with other members of the genus Planococcus were within the ranges of 18.7-53 % and 70.58-93.49 %, respectively, all below the respective recommended thresholds of 70.0 % and 95-96 %. The genomic DNA G+C content of the six strains ranged from 43.5 to 46.0 mol%. The major fatty acids of the six strains were anteiso-C15 : 0, iso-C14 : 0, and C16 : 1 ω7c alcohol. The predominant polar lipids of strains N016T, N022T, and N056T were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Menaquinones 7 and 8 were the respiratory quinones. The results of the above analyses indicated that the six strains represent three novel species of the genus Planococcus, for which the names Planococcus shenhongbingii sp. nov. (type strain N016T=GDMCC 1.4062T=JCM 36224T), Planococcus shixiaomingii sp. nov. (type strain N022T=GDMCC 1.4063T=JCM 36225T), and Planococcus liqunii sp. nov. (type strain N056T=GDMCC 1.4064T=JCM 36226T) are proposed.

Comparative genome analysis of the genus Marivirga and proposal of two novel marine species: Marivirga arenosa sp. nov., and Marivirga salinae sp. nov.

BACKGROUND: The phylum Bacteroidota represents a significant proportion of heterotrophic bacteria found in marine ecosystems. Members of the phylum Bacteroidota are actively involved in the degradation of biopolymers such as polysaccharides and proteins. Bacteroidota genomes exhibit a significant enrichment of various enzymes, including carbohydrate-active enzymes (CAZymes), carboxypeptidases, esterases, isomerases, peptidases, phosphatases, and sulfatases. The genus Marivirga, a member of the family Marivirgaceae within the phylum Bacteroidota, comprises six documented species. During a microbial diversity study, three novel Marivirga strains (BKB1-2 T, ABR2-2, and BDSF4-3 T) were isolated from the West Sea, Republic of Korea. RESULTS: To explore the taxonomic status and genomic characteristics of the novel isolates, we employed a polyphasic taxonomic approach, which included phylogenetic, chemotaxonomic and comprehensive genome analysis. The three isolates were Gram-stain-negative, aerobic, rod-shaped, moderately halophilic, and had a gliding motility. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values among the two isolates, BKB1-2 T and BDSF4-3 T, and the six reference strains were 70.5-76.5% for ANI and 18.1-25.7% for dDDH. Interestingly, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the strains harbor genes for a comprehensive pathway for dissimilatory nitrate reduction to ammonium (DNRA), as well as other nitrogen pathways for the reduction of nitrite, nitric oxide, and nitrous oxide. Additionally, the antiSMASH analysis indicated that the strains contained three to eight biosynthetic gene clusters (BGCs) associated with the synthesis of secondary metabolites. Furthermore, the strains carried a high number of CAZyme ranging from 53 to 152, which was also demonstrated by an in vitro analysis of degradation of the polysaccharide cellulose, chitin, laminarin, starch, and xylan. Additionally, all the strains carried genes for the metabolism of heavy metals, and exhibited tolerance to heavy metals, with minimum inhibitory concentrations (MICs) in millimoles (mM) in ranges of Co2+ (3-6), Cu2+ (0.2-0.4), Ni2+ (3-5), Zn2+ (2-4), Mn2+ (20-50), and Hg2+ (0.3). CONCLUSIONS: Based on polyphasic taxonomic approach, the three isolated strains represent two novel species names Marivirga arenosa sp. nov. (BKB1-2 T = KCTC 82989 T = InaCC B1618T), and Marivirga salinae sp. nov. (BDSF4-3 T = KCTC 82973 T = InaCC B1619T).

One-pot electrochemical detection of foodborne pathogen based on in situ nucleic acid amplification and wash-free assay.

A signal amplification electrochemical biosensor chip was developed to integrate loop-mediated isothermal amplification (LAMP) based on in situ nucleic acid amplification and methyl blue (MB) serving as the hybridization redox indicator for sensitive and selective foodborne pathogen detection without a washing step. The electrochemical biosensor chip was designed by a screen-printed carbon electrode modified with gold nanoparticles (Au NPs) and covered with polydimethylsiloxane membrane to form a microcell. The primers of the target were immobilized on the Au NPs by covalent attachment for in situ amplification. The electroactive MB was used as the electrochemical signal reporter and embedded into the double-stranded DNA (dsDNA) amplicons generated by LAMP. Differential pulse voltammetry was introduced to survey the dsDNA hybridization with MB, which differentiates the specifically electrode-unbound and -bound labels without a washing step. Pyrene as the back-filling agent can further improve response signaling by reducing non-specific adsorption. This method is operationally simple, specific, and effective. The biosensor showed a detection linear range of 102-107 CFU mL-1 with the limit of detection of 17.7 CFU mL-1 within 40 min. This method showed promise for on-site testing of foodborne pathogens and could be integrated into an all-in-one device.

Self-Quenched Fluorophore-DNA Labels for Super-Resolution Fluorescence Microscopy.

Protein labeling through transient and repetitive hybridization of short, fluorophore-labeled DNA oligonucleotides has become widely applied in various optical super-resolution microscopy methods. The main advantages are multitarget imaging and molecular quantification. A challenge is the high background signal originating from the presence of unbound fluorophore-DNA labels in solution. Here, we report the self-quenching of fluorophore dimers conjugated to DNA oligonucleotides as a general concept to reduce the fluorescence background. Upon hybridization, the fluorescence signals of both fluorophores are restored. We expand the toolbox of fluorophores suitable for self-quenching and report their spectra and hybridization equilibria. We apply self-quenched fluorophore-DNA labels to stimulated emission depletion microscopy and single-molecule localization microscopy and report improved imaging performances.