Benchmarking deep learning methods for predicting CRISPR/Cas9 sgRNA on- and off-target activities.

In silico design of single guide RNA (sgRNA) plays a critical role in clustered regularly interspaced, short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. Continuous efforts are aimed at improving sgRNA design with efficient on-target activity and reduced off-target mutations. In the last 5 years, an increasing number of deep learning-based methods have achieved breakthrough performance in predicting sgRNA on- and off-target activities. Nevertheless, it is worthwhile to systematically evaluate these methods for their predictive abilities. In this review, we conducted a systematic survey on the progress in prediction of on- and off-target editing. We investigated the performances of 10 mainstream deep learning-based on-target predictors using nine public datasets with different sample sizes. We found that in most scenarios, these methods showed superior predictive power on large- and medium-scale datasets than on small-scale datasets. In addition, we performed unbiased experiments to provide in-depth comparison of eight representative approaches for off-target prediction on 12 publicly available datasets with various imbalanced ratios of positive/negative samples. Most methods showed excellent performance on balanced datasets but have much room for improvement on moderate- and severe-imbalanced datasets. This study provides comprehensive perspectives on CRISPR/Cas9 sgRNA on- and off-target activity prediction and improvement for method development.

CrnnCrispr: An Interpretable Deep Learning Method for CRISPR/Cas9 sgRNA On-Target Activity Prediction.

CRISPR/Cas9 is a powerful genome-editing tool in biology, but its wide applications are challenged by a lack of knowledge governing single-guide RNA (sgRNA) activity. Several deep-learning-based methods have been developed for the prediction of on-target activity. However, there is still room for improvement. Here, we proposed a hybrid neural network named CrnnCrispr, which integrates a convolutional neural network and a recurrent neural network for on-target activity prediction. We performed unbiased experiments with four mainstream methods on nine public datasets with varying sample sizes. Additionally, we incorporated a transfer learning strategy to boost the prediction power on small-scale datasets. Our results showed that CrnnCrispr outperformed existing methods in terms of accuracy and generalizability. Finally, we applied a visualization approach to investigate the generalizable nucleotide-position-dependent patterns of sgRNAs for on-target activity, which shows potential in terms of model interpretability and further helps in understanding the principles of sgRNA design.

Expression of miRNA-1-3p and its target gene in hair follicle cycle development of Liaoning Cashmere goat.

MicroRNA exerts an important regulatory role in almost all the biological process, including hair follicle development in Liaoning Cashmere goat. In order to improve the Cashmere performance of goat, the regulatory role of microRNA in hair follicle cycle has drawn hotspot attention. However, the molecular mechanisms of miRNA-1-3p involved in hair follicle development are poorly understood. In this study, we found that miRNA-1-3p was less expressed in anagen stage of hair follicle cycle of Cashmere goat than that in telogen stage by using RT-qPCR and immunoblotting analysis, in contrast to the expression pattern of FGF14. The Dual-Luciferase reporter assay was employed to verify the relationship between miRNA-1-3p and FGF14. The results showed that miRNA-1-3p specifically binds to the 3'UTR of FGF14 mRNA, and FGF14 is the target gene of miR-1-3p. In conclusion, this study shows that miRNA-1-3p may regulate hair follicle development in Liaoning Cashmere goats by targeting FGF14.

Description of two novel anaerobic members in the family Clostridiaceae, Anaeromonas gelatinilytica gen.nov., sp. nov., and Anaeromonas frigoriresistens sp. nov., isolated from saline lake sediment.

Cells of members of the family Clostridiaceae, phylum Firmicutes, are generally obligate anaerobic rods. Strains D2Q-14T and D2Q-11T were isolated from sediment of the saline lake Manisi in the Xinjiang Uygur Autonomous Region, PR China. In this study, we employed a polyphasic approach and whole genome analysis of the two isolates. Cells of both isolates were Gram-stain-positive rods that were motile by means of flagella and could utilize sulphate, thiosulphate, elemental sulphur and nitrate as electron acceptors. Phylogenetic analyses based on 16S rRNA gene and whole genome sequences indicated that strains D2Q-14T and D2Q-11T constituted a coherent cluster affiliated to the family Clostridiaceae. In addition, genome analysis revealed that strain D2Q-14Tharboured one nonribosomal peptide synthetase gene cluster, making up 1.4 % of the entire genome. The genome-based analysis, including average nucleotide identity, average amino acid identity and in silico DNA-DNA hybridization, biochemical, phenotypic and chemotaxonomic characterization, indicated that strains D2Q-14T and D2Q-11T represented two novel species of a novel genus in the family Clostridiaceae, for which we propose the names Anaeromonas gelatinilytica gen. nov., sp. nov. and Anaeromonas frigoriresistens sp. nov., with the type strains D2Q-14T (=KCTC 15986T=MCCC 1K04634T) and D2Q-11T (=KCTC 15985T=MCCC 1K04391T), respectively.

Halorubrum salipaludis sp. nov., isolated from the saline-alkaline soil.

A novel extremely halophilic archaeon, designated WN019T, was isolated from the natural saline-alkali wetland soil of Binhai new district, Tianjin, China. Cells of WN019T were aerobic, motile, and pleomorphic rod-shaped, 0.5-0.8 µm in width and 2.0-2.5 µm in length, and the growth occurred optimally at 33-37 °C, pH 7.5-8.0, and in the presence of 15.0-20.0% (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequence comparison showed that the isolate belonged to the genus Halorubrum and exhibited moderate sequence similarity of 97.8% to Halorubrum saccharovorum JCM 8865T. The major respiratory quinones of strain WN019T were MK-8 and MK-8 (H2), and the major polar lipids were glycolipid (GL), phospholipid (PL), phosphatidylglycerol-sulphate (PGS), phosphatidylglycerol (PG) and phosphatidylglycerol-phosphate-methyl ester (Me-PGP). The DNA G + C content of the strain was 67.4 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) value based on whole genome sequences of strain WN019T and Halorubrum saccharovorum JCM 8865T were 87.5% and 35.4%, respectively. Phenotypic, chemotaxonomic, phylogenetic, and genomic analyses suggested that strain WN019T represents a novel species of the genus Halorubrum, for which the name Halorubrum salipaludis sp. nov. is proposed. The type strain is WN019T (= KCTC 4269T = ACCC 19977T).

Bacillus salipaludis sp. nov., isolated from saline-alkaline soil.

A Gram-staining-positive, endospore-forming, aerobic strain, designed WN066T, was isolated from saline-alkali wetland soil of Tianjin, China. Phylogenetic analysis based on 16S rRNA gene sequence indicated WN066T was a member of the genus of Bacillus, and most closely related to Bacillus drentensis DSM 15600T (98.9%), Bacillus cucumis CCM 8651T (98.8%), Bacillus bataviensis DSM 15601T (98.7%) and Bacillus niacini DSM 2923T (98.7%). However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (DDH) values between strain WN066T and the most closely related species were less than the previously proposed cutoff values for differentiating species within the genus, suggesting that this strain represented a novel Bacillus species. The strain grew at 19-42 °C (optimally 33-37 °C) in the presence of 3-20% (w/v) NaCl (optimally 8-12%(w/v)), and at pH 6.5-11.0 (optimally 7.5-8.5). The major cellular fatty acids were iso-C15:0 (24.8%) and anteiso-C15:0 (38.9%). The predominant polar lipids consisted of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). The size of the draft genome was 6,213,503 bp in size and had a G + C content of 38.6 mol %. The peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. On basis of phenotypic, phylogenetic, chemotaxonomic and genomic features, strain WN066T represented a novel species within the genus Bacillus, for which the name B. salipaludis sp. nov. is proposed. The type strain is WN066T (= KCTC 33953T = ACCC 60085T).

Halomonas salipaludis sp. nov., isolated from a saline-alkali wetland soil.

Strain WRN001T, a Gram-staining-negative, strictly aerobic, non-motile bacterium was isolated from the natural saline-alkali wetland soil of Binhai new district, Tianjin, China (38°46' N, 117°13' E). Cells of strain WRN001T were 0.3-0.5 µm in width and 1.5-2.5 µm in length, and the growth occurred optimally at 33-37 °C, pH 7.5-8.0, and in the presence of 8-10% (w/v) NaCl. Based on 16S rRNA gene sequence analysis, the isolate could be affiliated to the genus Halomonas, and the highest 16S rRNA gene sequence similarity of strain WRN001T to its closest relative Halomonas qiaohouensis DSM 26770 T was 97.5%. The size of the genome as presented here was 5,475,884 bp with a G + C content of 63.8 mol %. The major respiratory quinone of strainWRN001T was Q-9, and the dominant fatty acids were summed feature 8, summed feature 3, C10:0, C12:0, C12:0 3-OH, C16:0, and C17:0 cyclo. The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phophatidylcholine (PC), two phospholipids (PL), aminolipid (AL), and three unidentified lipids (L). These data combined with the low digital DDH values between strain WRN001T and the close relative, Halomonas alkalitolerans CGMCC 1.9129 T (42.2%) and based on comparisons with currently available genomes, the highest average nucleotide identity (ANIm) value was 91.4% to Halomonas alkalitolerans CGMCC 1.9129 T (GenBank accession No. GCA_001971685.1). Therefore, we propose a novel species in the genus Halomonas to accommodate this novel isolate: Halomonas salipaludis sp. nov. (type strain WRN001T = KCTC 52853 T = ACCC 19974 T).

Pontibacter flavimaris sp. nov., of the family Hymenobacteraceae, isolated from marine sediment.

A Gram-stain-negative, ovoid or rod-shaped, non-flagellated, motile-by-gliding and aerobic bacteria, designated S10-8T, was isolated from marine sediment of the Yellow Sea. Colonies of strain S10-8T had a pink-red pigmentation and its cells were approximately 0.5-0.8 µm×1.0-2.5 µm in size. Growth occurred at 10-45 °C (optimally at 33-37 °C), in the presence of 0-12.0 % NaCl (optimally at 2.0-5.0 %, w/v) and at pH 5.0-8.5 (optimally at pH 7.0-7.5). Phylogenetic analysis of the 16S rRNA gene indicated that strain S10-8T is a member of the genus Pontibacter within the family Hymenobacteraceae, and the 16S rRNA gene sequence similarity of strain S10-8T to its closest relative Pontibacter actiniarum KCTC 12367T was 96.9 %. Strain S10-8T contained MK-7 as the predominant menaquinone and summed feature 4 (iso-C17:1 I and/or anteiso-C17:1 B) and iso-C15:0 as the major fatty acids. The major polar lipids were phosphatidylethanolamine, an unidentified aminophospholipid and an unidentified lipid. The size of the draft genome was 4 623 791 bp and the G+C content was 53.5 mol%. There were low DNA-DNA hybridization values (<48.3±5.2 %) and average nucleotide identity values (<86.5 %) between strain S10-8T and the most closely related recognized Pontibacter species. Therefore, we propose a novel species in the genus Pontibacter to accommodate the novel isolate: Pontibacter flavimaris sp. nov. (type strain S10-8T=KCTC 42769T=ACCC 19859T).

Salimicrobium humidisoli sp. nov., Isolated from Saline-Alkaline Soil.

A Gram-staining-positive, aerobic, non-endospore-forming, coccus-shaped, non-flagellated, and non-motile bacterium, designated WN024T, was isolated from the natural saline-alkali wetland soil of Binhai new district, Tianjin, China. Cells of strain WN024T were catalase- and oxidase-positive. The isolate was able to grow between 20 and 45 °C (optimal at 33-37 °C), pH 6.5-11.0 (optimal 7.5-8.0), and in the presence of 5.0-25.0% NaCl (optimal at 10.0-15.0%, w/v). The isolate could be affiliated to the genus Salimicrobium and the highest 16S rRNA gene sequence similarity of strain WN024T to its closest relative Salimicrobium salexigens DSM 22782T was 97.9%. The size of the genome was 2,622,223 bp in size with a G + C content of 47.1%. The sole respiratory quinone of strain WN024T was MK-7, the predominant fatty acids were iso-C15:0, anteiso-C15:0 and anteiso-C17:0. The major polar lipids were phosphatidylglycerol (PG), glycolipid (GL), phospholipid (PL) and diphosphatidylglycerol (DPG). The cell-wall diamino acid was meso-diaminopimelic acid. The DNA-DNA hybridization values between strain WN024T and the closest relative S. salexigens DSM 22782T was 47.3 ± 2.3%. The highest average nucleotide identity (ANI) value was 92.3% to S. salexigens DSM 22782T (GenBank Accession No. GCA_900156705.1). Therefore, we propose a novel species in the genus Salimicrobium to accommodate this novel isolate, named Salimicrobium humidisoli sp. nov. The type strain is WN024T (= ACCC 19979T = KCTC 33897T).

Halovibrio salipaludis sp. nov., Isolated from Saline-Alkaline Soil.

A Gram-staining-negative, halophilic, aerobic, oval-shaped or vibrio-shaped, motile by a polar flagellum strain, designated YL5-2T, was isolated from natural saline-alkaline wetland soil of Binhai new district, Tianjin, China. Strain YL5-2T grew optimally at 35 °C, pH 7.5-8.0, and in the presence of 10-25% (w/v) NaCl on MA medium. Phylogenetic analyses based on 16S rRNA gene sequences showed that the isolate belonged to the genus Halovibrio and exhibited high sequence similarity of 97.7% to Halovibrio variabilis DSM 3050T. The sole respiratory ubiquinone of strain YL5-2T is Q-9, and the dominant fatty acids were C18:1ω9c, C16:0, C19:0 cycloω8c, and Summed Feature 8. The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), and lipid (L). The DNA G+C content of the strain was 62.1 mol%. The average nucleotide identity (ANI) based on whole genome sequences of strain YL5-2T and Halovibrio variabilis DSM 3050T was 93.85%, and the dDDH value between these two strains was determined to be 52.0%. Phenotypic, chemotaxonomic, phylogenetic, and genomic analyses suggested that strain YL5-2T represent a novel species of the genus Halovibrio, for which the name Halovibrio salipaludis sp. nov. is proposed. The type strain is YL5-2T (=KCTC 52852T=ACCC 19971T).

Halomonas humidisoli Sp. Nov., Isolated From Saline-Alkaline Soil.

A Gram staining-negative, halophilic, aerobic, non-motile bacteria, designated strain WN018T, were isolated from the natural saline-alkali wetland soil of Binhai new district, Tianjin, China (38°46'N, 117°13'E). Cells of strain WN018T were short rod-shaped, 0.3-0.4 µm wide and 0.5-1.9 µm long, and growth occurred optimally at 30-33 °C, pH 7.5-8.0, and in the presence of 4-8% (w/v) NaCl. Based on 16S rRNA gene sequence analysis, the isolates could be affiliated to the genus Halomonas, exhibiting highest sequence similarity of 97.50% to Halomonas venusta DSM 4743T. The DNA G+C content of the strain was 63.8%. The distinct phylogenetic position and phenotypic traits distinguished the novel isolate from its nearest neighbors. The major respiratory quinone of strain WN018T was Q-9 (91.0%) and Q-8 (9.0%), and the dominant fatty acids were C16:0, C14:0, C10:0, C12:0 3-OH. The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), three phospholipids (PL), aminolipid (AL), and two unidentified lipids (L). The average nucleotide identity (ANI) based on whole-genome sequences of strain WN018T and Halomonas hydrothermalis DSM 15725T was 93.02%, and the dDDH value between these two strains was determined to be 49.7%. Therefore, we propose a novel species in the genus Halomonas to accommodate the novel isolate: Halomonas humidisoli sp. nov. (type strain WN018T = ACCC 19975T = KCTC 52854T).

Bacillus fungorum sp. nov., a bacterium isolated from spent mushroom substrate.

A facultatively anaerobic, Gram-stain-positive, spore-forming Bacillus strain, 17-SMS-01T, isolated from spent mushroom substrate in the Fangshan District, Beijing, PR China, was initially identified as a Bacillus cereus group species based on 16S rRNA gene sequences. Strain 17-SMS-01T had the highest sequence similarities to Bacillus wiedmannii FSL W8-0169T (99.9 %), Bacillus albus N35-10-2T (99.9 %), Bacillus luti TD41T (99.9 %) and Bacillus proteolyticus TD42T (99.9 %). However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (DDH) values between strain 17-SMS-01T and the most closely related species were less than the previously proposed cut-off values of 96 % (ANI) and 70 % (DDH) for differentiating species within the genus, suggesting that this strain represents a novel Bacillus group species. The fatty acid profile of strain 17-SMS-01T, which showed a predominance of iso-C15 : 0 and anteiso-C15 : 0, supported the allocation of the strain to the genus Bacillus. The predominant menaquinone was MK-7 (100%). The major polar lipids were diphosphatidylglycerol, phosphatidyl ethanolamine, phosphatidyl glycerol, an unidentified aminophospholiped and unidentified lipids. The DNA G+C content of the novel strain was 35.0 mol%. The results of physiological and biochemical tests also allowed the phenotypic differentiation of strain 17-SMS-01T from the most closely related recognized species. On the basis of the phylogenetic and phenotypic evidence, strain 17-SMS-01T represents a novel Bacillus species, for which the name Bacillus fungorum sp. nov. is proposed. Type strain of the novel species is 17-SMS-01T (=MCCC 1K03483T=KCTC 33949T).

Quorum sensing signal autoinducer-2 promotes root colonization of Bacillus velezensis SQR9 by affecting biofilm formation and motility.

Root colonization of beneficial rhizobacteria is critical for their beneficial effects. Quorum sensing (QS) has been reported to affect the colonization of many plant pathogens. However, how QS signals regulate root colonization of beneficial rhizobacteria is unclear. In this study, the QS signal AI-2 synthetase-encoding gene luxS was completely deleted from the genome of the plant beneficial rhizobacterium Bacillus velezensis SQR9, and bioluminescence experiments showed that AI-2 production was blocked. Deletion of luxS reduced biofilm formation, motility, and root colonization of B. velezensis SQR9, while addition of exogenous AI-2 to the mutant restored this phenomenon. These results indicated that AI-2 positively affects the root colonization of B. velezensis SQR9. This study provided new insights for enhancing the colonization of beneficial rhizobacteria. KEY POINTS: • LuxS participated in the synthesis of the quorum sensing signal AI-2 in B. velezensis. • AI-2 enhanced motility, biofilm formation, and root colonization of B. velezensis. • AI-2 stimulated the production of γ-polyglutamic acid by B. velezensis.

Anaerophilus nitritogenes gen. nov., sp. nov., isolated from salt lake sediment in Xinjiang Province, China.

An obligately anaerobic, nitrate-reducing bacterial strain (MJB2T) was isolated from sediments of saline in Xinjiang province of China. Cells were Gram-stain-positive rods and motile by means of flagella and formed endospores. The novel strain MJB2T was able to grow at 15-37 °C (optimum 28-30 °C), pH 5.8-9.4 (optimum 7.8) and with 1.0-7.0% NaCl (optimum 5.0-6.0%, w/v). Sulfate, sulfite, thiosulfate, elemental sulfur, nitrite and Fe(III) were not used as terminal electron acceptors. Oxidase and catalase reactions were positive. H2S was producted from L-cystine. Complex substrates such as beef extract, peptone and yeast extract can be used as sole energy sources. The DNA G+C content was 29.4 mol%. The major cellular fatty acids (> 10%) were C14:0, C16:1 cis 7 and C16:1 cis 9. The main polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, three unidentified amino lipids, one unidentified amino glycolipid, two unidentified glycolipid, one unidentified aminophospholipid and two unidentified lipids. No respiratory quinones were detected. According to phylogenetic analysis based on 16S rRNA gene sequences, strain MJB2T was affiliated to the family Clostridiaceae (order Clostridiales) with highest 16S rRNA gene sequence similarity of 95.3% to Crassaminicella profunda Ra1766HT. Strain MJB2T exhibited 74.9% ANI values to C. profunda Ra1766HT. In silico DNA-DNA relatedness value between strain MJB2T and C. profunda Ra1766HT was 19.5%. The distinct biochemical, chemotaxonomic and phylogenetic differences from the previously described taxa supported that strain MJB2T represents a novel species of a new genus, for which the name Anaerophilus nitritogenes gen. nov., sp. nov. is proposed. The type strain is MJB2T (=KCTC 15800T=MCCC 1K03631T).

Aliifodinibius salipaludis sp. nov., Isolated from Saline-Alkaline Soil.

A Gram-staining-negative, oblong or rod-shaped, non-flagellated and facultatively anaerobic bacterium, designated WN023T, was isolated from natural saline-alkali wetland soil of Binhai new district, Tianjin, China. The colonies of strain WN023T on marine agar 2216 (MA) have a brown, circular, wet pigmentation, and its cells are approximately 1-1.5 µm × 2.0-2.5 µm in size. Growth occurred at 15-45 °C, but optimally at 33-37 °C, in the presence of 5.0-25.0% (w/v) NaCl, but optimally at 10.0-15.0% (w/v) NaCl, and at a pH 6.5-11.0, but optimally at 7.5-8.0. Phylogenetic analysis of the 16S rRNA gene indicates that strain WN023T is a member of the genus Aliifodinibius within the family Balneolaceae, and the 16S rRNA gene sequence similarity of strain WN023T with its close relative Aliifodinibius halophilus KCTC 42497T, Aliifodinibius salicampi KACC 19060T, Aliifodinibius roseus KCTC 23442T, Aliifodinibius sediminis ACCC 10714T, and Fodinibius salinus ACCC 10716T was 97.3%, 94.5%, 93.9%, 93.71%, and 92.7%, respectively. Strain WN023T contained menaquinone-7 (MK-7) as the predominant menaquinone, and iso C15:0, Sum in feature 3 (C16:1ω6c and/or C16:1ω7c), and iso C17:1ω9c as the major fatty acids. The major polar lipids were glycolipid (GL), aminolipid (AL), phospholipid (PL), phosphatidylcholine (PC), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), and lipid (L). The size of the draft genome as presented here was 3,583,276 bp in size, and the G+C content is 42.2 mol%. These combined physiological, biochemical, phylogenetic, and genotypic data supported placement of strain WN023T in the genus Aliifodinibius and indicated that it was distinct from all other members in the genus Aliifodinibius. This was also confirmed by the low DNA-DNA hybridization values (38.4%) between strain WN023T and the most closely related recognized Aliifodinibius species-A. halophilus KCTC 42497T. Therefore, we propose a novel species in the genus Aliifodinibius to accommodate the novel isolate: Aliifodinibius salipaludis sp. nov. (type strain WN023T = KCTC 52855T = ACCC 19978T).

A genomic island in a plant beneficial rhizobacterium encodes novel antimicrobial fatty acids and a self-protection shield to enhance its competition.

Rhizobacteria devote a relatively large percentage of their genomes to encode bioactive natural products that are important for competition in the rhizosphere. In this study, a plant beneficial rhizobacterium Bacillus velezensis SQR9 was discovered to produce novel antibacterial fatty acids, Bacillunoic acids, which are encoded on a genomic island (GI). This GI contains a hybrid type I fatty acid synthase (FAS)-polyketide synthase (PKS) system and an ABC transporter. The FAS was predicted to synthesize a primer that was transferred to the PKS to synthesize Bacillunoic acids. The synthesized Bacillunoic acids inhibit the growth of diverse bacteria, with the strongest activity against closely related Bacillus strains, the ABC transporter exported the toxic Bacillunoic acids upon their induction for protecting the producing strain. The inhibition of other Bacillus strains by Bacillunoic acids extended the antimicrobial spectrum of SQR9 and enhanced its competition with closely related root-associated bacteria. So, through the obtaining of this GI by horizontal gene transfer, strain SQR9 not only acquired a competitive weapon but also acquired a self-protecting shield, which increased its competition with other rhizobacteria.

Alkalilacustris brevis gen. nov., sp. nov., isolated from a soda lake.

A Gram-stain-negative, aerobic, non-pigmented and short-rod-shaped bacterium, designated 34079T, was isolated from a water sample of a soda lake in Jilin, a province of China. Strain 34079T grew at 10-50 °C (optimum, 35 °C), pH 7-10 (optimum, pH 8.0-8.5). NaCl was required for growth at the concentration range 1-10.0 % (w/v), with an optimum at 2.5-4 % (w/v). Chemotaxonomic analysis indicated that the sole respiratory quinone was Q-10. The predominant cellular fatty acids (>5 %) were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c) and C16 : 0. The major polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, three unidentified amino lipids, one unidentified amino phosphoglycolipid, one phosphoglycolipid, one unidentified glycolipid, three unidentified phospholipids and two unidentified lipids. The DNA G+C content was 65.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 34079T formed a distinct lineage in the clade of the family 'Rhodobacteraceae' with the highest sequence similarity of 96.1 % to Pararhodobacter aggregans, followed by Rhodobaca bogoriensis DSM 18756T (95.7 %) and Roseibaca ekhonensis DSM 11469T (94.7 %). The distinct biochemical, chemotaxonomic and phylogenetic differences from the previously described taxa supported that strain 34079T represents a novel species of a new genus, for which the name Alkalilacustris brevis gen. nov., sp. nov. is proposed. The type strain is 34079T (=KCTC 62428T=MCCC 1K03493T).

Meridianimarinicoccus roseus gen. nov., sp. nov., a novel genus of the family Rhodobacteraceae isolated from seawater.

A Gram-stain negative, pink-pigmented, strictly aerobic, non-motile and coccoid-shaped bacterial strain, designated TG-679T, was isolated from a deep-sea water sample collected from the South China Sea. Cells of strain TG-679T were catalase- and oxidase-positive, lacked bacteriochlorophyll a and carotenoid. Strain TG-679T was found to grow at 10-40 °C (optimum, 28 °C), pH 5.5-10.0 (optimum, pH 7.5) and with 0.5-2.0 % (w/v) NaCl (optimum, 1.5 %). Chemotaxonomic analysis of strain TG-679T indicated that the sole respiratory quinone was Q-10, the predominant cellular fatty acid was C18 : 1ω7c, and the major polar lipids consisted of phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, phosphatidylcholine, an unidentified phospholipid and two unidentified glycolipids. The genomic DNA G+C content of strain TG-679T was 65.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain TG-679T constituted a separated branch in the family Rhodobacteraceae. Differential phenotypic properties, together with phylogenetic distinctiveness, demonstrated that strain TG-679T is clearly distinct from any validly published genus. Based on polyphasic taxonomic characterization, strain TG-679T is considered to represent a novel species of a novel genus, for which the name Meridianimarinicoccus roseus gen. nov., sp. nov. is proposed. The type strain of the species is TG-679T (=KCTC 62454T=MCCC 1K03496T).

Microbulbifer flavimaris sp. nov., a halophilic Gammaproteobacteria isolated from marine sediment of the Yellow Sea, China.

A Gram-stain-negative, aerobic, non-flagellated, non-motile bacterium, designated strain WRN-8T, was isolated from marine sediment of the Yellow Sea, China (36° 5' 33'' N, 121° 20' 37'' E). Colonies of strain WRN-8T were 0.2-0.3 µm wide, 2.1-2.8 µm long, catalase-positive and oxidase-positive. Colonies on marine agar solid media were circular, wet, smooth, light yellow and approximately 1.3 mm in diameter. Growth occurred optimally at 33-37 °C, pH 7.0-7.5 and in the presence of 2-4 % NaCl (w/v). Phylogenetic analysis of the 16S rRNA gene indicated that strain WRN-8T is a member of the genus Microbulbifer within the family Microbulbiferaceae, and the closest described neighbour in terms of 16S rRNA gene sequence identity is Microbulbifer aestuariivivens KCTC 52569T (98.1 %). The major respiratory quinone of strain WRN-8T is Q-8, its predominant fatty acids are iso-C15 : 0, iso-C17 : 0, C16 : 0, iso-C11 : 03-OH and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), and its major polar lipids are phosphatidylethanolamine, phosphatidylglycerol, glycolipid, an unidentified phospholipid and an unidentified lipid. The draft genome obtained in this study was 3 643 020 bp, and the G+C content was 59.2 mol%. DNA-DNA hybridization (<46.3 %) and average nucleotide identity (<86.7 %) values between strain WRN-8T and the closest-related recognized Microbulbifer species confirmed the novelty of this new species. Therefore, we propose a novel species in the genus Microbulbifer to accommodate the novel isolate: Microbulbifer flavimaris sp. nov. (type strain WRN-8T=KCTC 42989T=ACCC 19926T).

Mining the deep Red-Sea brine pool microbial community for anticancer therapeutics.

BACKGROUND: Microbial species in the brine pools of the Red Sea and the brine pool-seawater interfaces are exposed to high temperature, high salinity, low oxygen levels and high concentrations of heavy metals. As adaptations to these harsh conditions require a large suite of secondary metabolites, these microbes have a huge potential as a source of novel anticancer molecules. METHODS: A total of 60 ethyl-acetate extracts of newly isolated strains from extreme environments of the Red-Sea were isolated and tested against several human cancer cell lines for potential cytotoxic and apoptotic activities. RESULTS: Isolates from the Erba brine-pool accounted for 50% of active bacterial extracts capable of inducing 30% or greater inhibition of cell growth. Among the 60 extracts screened, seven showed selectivity towards triple negative BT20 cells compared to normal fibroblasts. CONCLUSION: In this study, we identified several extracts able to induce caspase-dependent apoptosis in various cancer cell lines. Further investigations and isolation of the active compounds of these Red Sea brine pool microbes may offer a chemotherapeutic potential for cancers with limited treatment options.