Flagellimonas baculiformis sp. nov. and Flagellimonas crocea sp. nov., isolated from surface seawater of the Pacific Ocean.

Two Gram-negative, non-spore-forming, non-motile, non-flagellated bacteria, designated strains D6T and DH64T, were isolated from surface water of the Pacific Ocean. For strain D6T, growth occurred at 10-40 °C, pH 5.5-9.0 and in the presence of 0-8.0 % NaCl (w/v). For strain DH64T, growth occurred at 10-40 °C, pH 5.5-8.5 and in the presence of 0.5-8.0 % NaCl (w/v). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains D6T and DH64T both belonged to the genera Flagellimonas, with the highest sequence identities to Flagellimonas taeanensis JCM 17757T (98.2 %) and Flagellimonas marinaquae JCM 11811T (98.6 %), respectively. The 16S rRNA gene sequence identity between strains D6T and DH64T was 95.9 %. The average amino acid identity and digital DNA-DNA hybridization values between the two strains and the nearest phylogenetic neighbours were 66.7-93.3 % and 16.1-38.5 %, respectively. The major respiratory quinone of both strains was menaquinone-6. The major polar lipid was phosphatidylethanolamine. The major fatty acids were identified similarly as iso-C15 : 1 G, iso-C15 : 0 and iso-C17 : 0 3-OH. The genomic G+C contents of strains D6T and DH64T were determined to be 45.5 and 42.6 mol%, respectively. The combined genotypic and phenotypic data show that the strains represent two novel species within genera Flagellimonas, for which the names Flagellimonas baculiformis sp. nov. and Flagellimonas crocea sp. nov. are proposed, with type strains D6T (=MCCC M28982T=KCTC 92604T) and DH64T (=MCCC M28986T=KCTC 92975T).

Aerobic Anoxygenic Phototrophic Bacteria in the Marine Environments Revealed by Raman/Fluorescence-Guided Single-Cell Sorting and Targeted Metagenomics.

Aerobic anoxygenic phototrophic bacteria (AAPB) contribute profoundly to the global carbon cycle. However, most AAPB in marine environments are uncultured and at low abundance, hampering the recognition of their functions and molecular mechanisms. In this study, we developed a new culture-independent method to identify and sort AAPB using single-cell Raman/fluorescence spectroscopy. Characteristic Raman and fluorescent bands specific to bacteriochlorophyll a (Bchl a) in AAPB were determined by comparing multiple known AAPB with non-AAPB isolates. Using these spectroscopic biomarkers, AAPB in coastal seawater, pelagic seawater, and hydrothermal sediment samples were screened, sorted, and sequenced. 16S rRNA gene analysis and functional gene annotations of sorted cells revealed novel AAPB members and functional genes, including one species belonging to the genus Sphingomonas, two genera affiliated to classes Betaproteobacteria and Gammaproteobacteria, and function genes bchCDIX, pucC2, and pufL related to Bchl a biosynthesis and photosynthetic reaction center assembly. Metagenome-assembled genomes (MAGs) of sorted cells from pelagic seawater and deep-sea hydrothermal sediment belonged to Erythrobacter sanguineus that was considered as an AAPB and genus Sphingomonas, respectively. Moreover, multiple photosynthesis-related genes were annotated in both MAGs, and comparative genomic analysis revealed several exclusive genes involved in amino acid and inorganic ion metabolism and transport. This study employed a new single-cell spectroscopy method to detect AAPB, not only broadening the taxonomic and genetic contents of AAPB in marine environments but also revealing their genetic mechanisms at the single-genomic level.

Qipengyuania benthica sp. nov. and Qipengyuania profundimaris sp. nov., two novel Erythrobacteraceae members isolated from deep-sea environments.

Two Gram-stain-negative, rod-shaped and non-motile strains, designated as DY56-A-20T and G39T, were isolated from deep-sea sediment of the Pacific Ocean and deep-sea seawater of the Indian Ocean, respectively. Strain DY56-A-20T was found to grow at 15-37 °C (optimum, 28 °C), at pH 6.0-10.0 (optimum, pH 6.5-7.0) and in 0.5-6.0 % (w/v) NaCl (optimum, 1.0-2.0 %), while strain G39T was found to grow at 10-42 °C (optimum, 35-40 °C), at pH 5.5-10.0 (optimum, pH 6.5-7.0) and in 0-12.0 % (w/v) NaCl (optimum, 1.0-2.0 %). The 16S rRNA gene sequence identity analysis indicated that strain DY56-A-20T had the highest sequence identity with Qipengyuania marisflavi KEM-5T (97.6 %), while strain G39T displayed the highest sequence identity with Qipengyuania citrea H150T (98.8 %). The phylogenomic reconstruction indicated that both strains formed independent clades within the genus Qipengyuania. The digital DNA-DNA hybridization and average nucleotide identity values between strains DY56-A-20T/G39T and Qipengyuania/Erythrobacter type strains were 17.8-23.8 % and 70.7-81.1 %, respectively, which are below species delineation thresholds. The genome DNA G+C contents were 65.0 and 63.5 mol% for strains DY56-A-20T and G39T, respectively. The predominant cellular fatty acids (>10 %) of strain DY56-A-20T were C17 : 1 ω6c, summed feature 8 and summed feature 3, and the major cellular fatty acids of strain G39T were C17 : 1 ω6c and summed feature 8. The major polar lipids in both strains were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid and an unidentified polar lipid. The only respiratory quinone present in both strains was ubiquinone-10. Based on those genotypic and phenotypic results, the two strains represent two novel species belonging to the genus Qipengyuania, for which the names Qipengyuania benthica sp. nov. and Qipengyuania profundimaris sp. nov. are proposed. The type strain of Q. benthica is DY56-A-20T (=MCCC M27941T=KCTC 92309T), and the type strain of Q. profundimaris is G39T (=MCCC M30353T=KCTC 8208T).

Genomic insight into strategy, interaction and evolution of nitrifiers in metabolizing key labile-dissolved organic nitrogen in different environmental niches.

Ammonia-oxidizing archaea (AOA) and bacteria (AOB), nitrite-oxidizing bacteria (NOB), and complete ammonia oxidizers (comammox) are responsible for nitrification in nature; however, some groups have been reported to utilize labile-dissolved organic nitrogen (LDON) for satisfying nitrogen demands. To understand the universality of their capacity of LDON metabolism, we collected 70 complete genomes of AOA, AOB, NOB, and comammox from typical environments for exploring their potentials in the metabolism of representative LDON (urea, polyamines, cyanate, taurine, glycine betaine, and methylamine). Genomic analyses showed that urea was the most popular LDON used by nitrifiers. Each group harbored unique urea transporter genes (AOA: dur3 and utp, AOB: utp, and NOB and comammox: urtABCDE and utp) accompanied by urease genes ureABC. The differentiation in the substrate affinity of these transporters implied the divergence of urea utilization efficiency in nitrifiers, potentially driving them into different niches. The cyanate transporter (cynABD and focA/nirC) and degradation (cynS) genes were detected mostly in NOB, indicating their preference for a wide range of nitrogen substrates to satisfy high nitrogen demands. The lack of genes involved in the metabolism of polyamines, taurine, glycine betaine, and methylamines in most of nitrifiers suggested that they were not able to serve as a source of ammonium, only if they were degraded or oxidized extracellularly as previously reported. The phylogenetic analyses assisted with comparisons of GC% and the Codon Adaptation Index between target genes and whole genomes of nitrifiers implied that urea metabolic genes dur3 and ureC in AOA evolved independently from bacteria during the transition from Thaumarchaeota to AOA, while utp in terrestrial AOA was acquired from bacteria via lateral gene transfer (LGT). Cyanate transporter genes cynS and focA/nirC detected only in a terrestrial AOA Candidadus Nitrsosphaera gargensis Ga9.2 could be gained synchronously with Nitrospira of NOB by an ancient LGT. Our results indicated that LDON utilization was a common feature in nitrifiers, but metabolic potentials were different among nitrifiers, possibly being intensely interacted with their niches, survival strategies, and evolutions.

Global analysis of the biosynthetic chemical space of marine prokaryotes.

BACKGROUND: Marine prokaryotes are a rich source of novel bioactive secondary metabolites for drug discovery. Recent genome mining studies have revealed their great potential to bio-synthesize novel secondary metabolites. However, the exact biosynthetic chemical space encoded by the marine prokaryotes has yet to be systematically evaluated. RESULTS: We first investigated the secondary metabolic potential of marine prokaryotes by analyzing the diversity and novelty of the biosynthetic gene clusters (BGCs) in 7541 prokaryotic genomes from cultivated and single cells, along with 26,363 newly assembled medium-to-high-quality genomes from marine environmental samples. To quantitatively evaluate the unexplored biosynthetic chemical space of marine prokaryotes, the clustering thresholds for constructing the biosynthetic gene cluster and molecular networks were optimized to reach a similar level of the chemical similarity between the gene cluster family (GCF)-encoded metabolites and molecular family (MF) scaffolds using the MIBiG database. The global genome mining analysis demonstrated that the predicted 70,011 BGCs were organized into 24,536 mostly new (99.5%) GCFs, while the reported marine prokaryotic natural products were only classified into 778 MFs at the optimized clustering thresholds. The number of MF scaffolds is only 3.2% of the number of GCF-encoded scaffolds, suggesting that at least 96.8% of the secondary metabolic potential in marine prokaryotes is untapped. The unexplored biosynthetic chemical space of marine prokaryotes was illustrated by the 88 potential novel antimicrobial peptides encoded by ribosomally synthesized and post-translationally modified peptide BGCs. Furthermore, a sea-water-derived Aquimarina strain was selected to illustrate the diverse biosynthetic chemical space through untargeted metabolomics and genomics approaches, which identified the potential biosynthetic pathways of a group of novel polyketides and two known compounds (didemnilactone B and macrolactin A 15-ketone). CONCLUSIONS: The present bioinformatics and cheminformatics analyses highlight the promising potential to explore the biosynthetic chemical diversity of marine prokaryotes and provide valuable knowledge for the targeted discovery and biosynthesis of novel marine prokaryotic natural products. Video Abstract.

Metagenome-based analysis of carbon-fixing microorganisms and their carbon-fixing pathways in deep-sea sediments of the southwestern Indian Ocean.

Carbon fixation by chemoautotrophic microorganisms in the dark ocean makes a large contribution to oceanic primary production and the global carbon cycle. In contrast to the Calvin cycle-dominated carbon-fixing pathway in the marine euphotic zone, carbon-fixing pathways and their hosts in deep-sea areas are diverse. In this study, four deep-sea sediment samples close to hydrothermal vents in the southwestern Indian Ocean were collected and processed using metagenomic analysis to investigate carbon fixation potential. Functional annotations revealed that all six carbon-fixing pathways had genes to varied degrees present in the samples. The reductive tricarboxylic acid cycle and Calvin cycle genes occurred in all samples, in contrast to the Wood-Ljungdahl pathway, which previous studies found mainly in the hydrothermal area. The annotations also elucidated the chemoautotrophic microbial members associated with the six carbon-fixing pathways, and the majority of them containing key carbon fixation genes belonged to the phyla Pseudomonadota and Desulfobacterota. The binned metagenome-assembled genomes revealed that key genes for the Calvin cycle and the 3-hydroxypropionate/4-hydroxybutyrate cycle were also found in the order Rhodothermales and the family Hyphomicrobiaceae. By identifying the carbon metabolic pathways and microbial populations in the hydrothermal fields of the southwest Indian Ocean, our study sheds light on complex biogeochemical processes in deep-sea environments and lays the foundation for further in-depth investigations of carbon fixation processes in deep-sea ecosystems.

Microbulbifer zhoushanensis sp. nov., Microbulbifer sediminum sp. nov. and Microbulbifer guangxiensis sp. nov., three marine bacteria isolated from a tidal flat.

Three strains, TT30T, TT37T and L3T, were isolated from tidal flat samples. Cells were Gram-stain-negative, non-motile and rod shaped. Cells of strains TT30T and TT37T were able to grow in a medium containing 1.0-15.0 % (w/v) NaCl (optimum, 3.0 and 4.0 %, respectively), and cells of strain L3T was able to grow in a medium containing 1.0-10.0 % (w/v) NaCl (optimum, 1.0 %). Growth of the three strains was observed at pH 6.0-10.0 and at 10-40 °C. Strains TT30T, TT37T and L3T showed the highest similarity to Microbulbifer hydrolyticus DSM 11525T (97.7 %), M. yueqingensis CGMCC 1.10658T (98.0 %) and M. elongatus DSM 6810T (97.9 %), respectively. Results of phylogenetic analyses indicated that the three isolates represented two distinct lineages within the genus Microbulbifer. The DNA G+C contents of strains TT30T, TT37T and L3T were 61.3, 60.9 and 60.2%, respectively. The average nucleotide identity and in silico DNA-DNA hybridization values among strains TT30T, TT37T and L3T and the reference strains were 84.4-87.4 and 19.6-28.9 %, respectively. Differential phenotypic properties, chemotaxonomic differences, phylogenetic distinctiveness, together with the genomic data, demonstrated that strains TT30T, TT37 T and L3T represent novel species of the genus Microbulbifer, which are named Microbulbifer zhoushanensis sp. nov. (TT30T=KCTC 92167T=MCCC 1K07276T), Microbulbifer sediminum sp. nov. (TT37T=KCTC 92168T=MCCC 1K07277T) and Microbulbifer guangxiensis sp. nov. (L3T=KCTC 92165T=MCCC 1K07278T).

Secondary Metabolic Potential of Kutzneria.

Kutzneria is a rare genus of Actinobacteria that harbors a variety of secondary metabolite gene clusters and produces several interesting types of bioactive secondary metabolites. Recent efforts have partially elucidated the biosynthetic pathways of some of these bioactive natural products, suggesting the diversity and specificity of secondary metabolism within this genus. Here, we summarized the chemical structures, biosynthetic pathways, and key metabolic enzymes of the secondary metabolites isolated from Kutzneria strains. In-depth comparative genomic analysis of all six available high-quality Kutzneria genomes revealed that the majority (77%) of the biosynthetic gene cluster families of Kutzneria were untapped and identified homologues of key metabolic enzymes in the putative gene clusters, including cytochrome P450s, halogenases, and flavin-dependent N-hydroxylases. The present study suggests that Kutzneria exhibits great potential to synthesize novel secondary metabolites, encodes a variety of valuable metabolic enzymes, and also provides valuable information for the targeted discovery and biosynthesis of novel natural products from Kutzneria.

Membranihabitans maritimus sp. nov., a marine bacterium isolated from deep seawater.

A Gram-stain-negative, non-motile, rod-shaped bacterial strain, designated C281T, was isolated from seawater sampled at the Marshallese seamount chain. Results of 16S rRNA gene analysis revealed that strain C281T was most closely related to Membranihabitans marinus CZ-AZ5T with 92.7 % sequence similarity. Phylogenetic analysis indicated that the new isolate represented a novel species by forming a distinctive lineage within the family Saprospiraceae. The DNA G+C content of strain C281T was 38.4 mol%. The genome sizes of strain C281T and the reference strain M. marinus CZ-AZ5T were 5 962 917 and 5 395 999 bp, respectively. The average nucleotide identity and in silico DNA-DNA hybridization values between strains C281T and M. marinus CZ-AZ5T were found to be low (69.3 and 17.6 %, respectively). Different functional genes were found in the genome of strain C281T, such as CZC CBA, polysaccharide utilization loci and linear azol(in)e-containing peptide cluster coding genes. The NaCl range for growth was 0.5-15.0 %. Positive results were obtained for hydrolysis of Tween 60 and urease. MK-7 was the sole respiratory quinone. The major fatty acids were C16 : 1 ω6c and/or C16 : 1 ω7c, iso-C15 : 0 and iso-C15 : 1 F. The major polar lipids of strain C281T were phosphatidylethanolamine, phosphatidylglycerol, two unidentified lipids and five unidentified glycolipids. On the basis of its taxonomic characteristics, the isolate represents a novel species of the genus Membranihabitans, for which the name Membranihabitans maritimus sp. nov. (type strain C281T=KCTC 92171T=MCCC M27001T) is proposed.

Understanding the mechanisms of halotolerance in members of Pontixanthobacter and Allopontixanthobacter by comparative genome analysis.

Halotolerant microorganisms have developed versatile mechanisms for coping with saline stress. With the increasing number of isolated halotolerant strains and their genomes being sequenced, comparative genome analysis would help understand the mechanisms of salt tolerance. Six type strains of Pontixanthobacter and Allopontixanthobacter, two phylogenetically close genera, were isolated from diverse salty environments and showed different NaCl tolerances, from 3 to 10% (w/v). Based on the co-occurrence greater than 0.8 between halotolerance and open reading frame (ORF) among the six strains, possible explanations for halotolerance were discussed regarding osmolyte, membrane permeability, transportation, intracellular signaling, polysaccharide biosynthesis, and SOS response, which provided hypotheses for further investigations. The strategy of analyzing genome-wide co-occurrence between genetic diversity and physiological characteristics sheds light on how microorganisms adapt to the environment.

Metagenomic Features Characterized with Microbial Iron Oxidoreduction and Mineral Interaction in Southwest Indian Ridge.

The Southwest Indian Ridge (SWIR) is one of the typical representatives of deep-sea ultraslow-spreading ridges, and has increasingly become a hot spot of studying subsurface geological activities and deep-sea mining management. However, the understanding of microbial activities is still limited on active hydrothermal vent chimneys in SWIR. In this study, samples from an active black smoker and a diffuse vent located in the Longqi hydrothermal region were collected for deep metagenomic sequencing, which yielded approximately 290 GB clean data and 295 mid-to-high-quality metagenome-assembled genomes (MAGs). Sulfur oxidation conducted by a variety of Gammaproteobacteria, Alphaproteobacteria, and Campylobacterota was presumed to be the major energy source for chemosynthesis in Longqi hydrothermal vents. Diverse iron-related microorganisms were recovered, including iron-oxidizing Zetaproteobacteria, iron-reducing Deferrisoma, and magnetotactic bacterium. Twenty-two bacterial MAGs from 12 uncultured phyla harbored iron oxidase Cyc2 homologs and enzymes for organic carbon degradation, indicated novel chemolithoheterotrophic iron-oxidizing bacteria that affected iron biogeochemistry in hydrothermal vents. Meanwhile, potential interactions between microbial communities and chimney minerals were emphasized as enriched metabolic potential of siderophore transportation, and extracellular electron transfer functioned by multi-heme proteins was discovered. Composition of chimney minerals probably affected microbial iron metabolic potential, as pyrrhotite might provide more available iron for microbial communities. Collectively, this study provides novel insights into microbial activities and potential mineral-microorganism interactions in hydrothermal vents. IMPORTANCE Microbial activities and interactions with minerals and venting fluid in active hydrothermal vents remain unclear in the ultraslow-spreading SWIR (Southwest Indian Ridge). Understanding about how minerals influence microbial metabolism is currently limited given the obstacles in cultivating microorganisms with sulfur or iron oxidoreduction functions. Here, comprehensive descriptions on microbial composition and metabolic profile on 2 hydrothermal vents in SWIR were obtained based on cultivation-free metagenome sequencing. In particular, autotrophic sulfur oxidation supported by minerals was presumed, emphasizing the role of chimney minerals in supporting chemosynthesis. Presence of novel heterotrophic iron-oxidizing bacteria was also indicated, suggesting overlooked biogeochemical pathways directed by microorganisms that connected sulfide mineral dissolution and organic carbon degradation in hydrothermal vents. Our findings offer novel insights into microbial function and biotic interactions on minerals in ultraslow-spreading ridges.

Sphingomicrobium nitratireducens sp. nov., isolated from a tidal flat in Guangxi.

An aerobic, yellow-pigmented and Gram-stain-negative strain, designated as O-35 T, was isolated from a tidal flat sediment collected in Dangjiang Town, the southern China. Colonies of strain O-35 T were circular with 0.5-1.0 mm in diameter, convex and smooth. Cells of strain O-35 T were coccoid-shaped, non-spore forming, non-motile and the strain could reduce nitrate. Growth of strain O-35 T was observed at 15-40 °C (optimum 30 °C), at pH 6.0-9.5 (optimum 7.5-8.0) and in 0.5-5.0% NaCl (optimum 2%, w/v). Strain O-35 T showed 16S rRNA gene sequence identities of 97.3-97.5% with Sphingomicrobium lutaoense CC-TBT-3 T and Sphingomicrobium aestuariivivum AH-M8T, higher than the rest of Sphingomicrobium type strains. Phylogenetic trees based on the 16S rRNA gene and the core-genome sequences demonstrated that strain O-35 T was affiliated within the genus Sphingomicrobium. Overall genome relatedness index calculations revealed that strain O-35 T had < 75.8% of average nucleotide identity and < 19.2% of digital DNA-DNA hybridization values with Sphingomicrobium type strains. The sole isoprenoid quinone was ubiquinone-10. The major fatty acids (> 10%) were summed feature 8, summed feature 3, C16:0 and C18:1 2-OH. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, sphingoglycolipid, two unidentified glycolipids, one unidentified lipid and one unidentified phospholipid. On the basis of the phenotypic, chemotaxonomic and genomic properties, strain O-35 T represents a novel species in the genus Sphingomicrobium, for which the name Sphingomicrobium nitratireducens sp. nov. is proposed. The type strain is O-35 T (= KCTC 92308 T = MCCC 1K07589T).

Pontibrevibacter nitratireducens gen. nov., sp. nov., a member of the family Rhodobacteraceae isolated from seawater of the Indian Ocean and intertidal zone.

Two Gram-stain-negative, facultatively anaerobic, non-motile, rod-shaped bacteria, strains h42T and ALG8, were isolated individually from the Indian Ocean and intertidal zone of Zhoushan, China. The results of 16S rRNA gene sequence analysis showed that the sequence similarity between strains h42T and ALG8 was 99.7 %, and the closest related strains were Monaibacterium marinum C7T (97.77 and 97.62 %) and Pontivivens insulae GYSW-23T (95.31 and 95.45 %). Phylogenetic analysis based on 16S rRNA gene sequences shows that these two novel strains belong to a distinct new lineage of the family Rhodobacteraceae in the order Rhodobacterales. The average nucleotide identity and in silico DNA-DNA hybridization values between the two novel strains and M. marinum C7T and P. insulae GYSW-23T were 72.73-78.15 % and 19.70-20.80 %, respectively. The DNA G+C content of strains h42T and ALG8 was 62.36 % and 62.17 mol %. The major fatty acids (>10 %) in strain h42T were C18 : 0, C19 : 0 cyclo ω8c and summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c), and in strain ALG8 were C19 : 0 cyclo ω8c and summed feature 8 (C18 : 1ω6c and/or C18 : 1 ω7c). The predominant isoprenoid ubiquinone of the two novel strains was Q-10; their major polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, two unidentified glycolipids, an unidentified aminoglycolipid, an unidentified phospholipid and an unidentified lipid. Based on the results of the morphological, physiological, chemotaxonomic and phylogenetic analysis of these two strains, a novel species of a new genus in the family Rhodobacteraceae is proposed, named as Pontibrevibacter nitratireducens gen. nov., sp. nov. The type strain and non-type strain of P. nitratireducens are h42T (=KCTC 72875T=CGMCC 1.17849T=MCCC 1K04735T) and ALG8 (=KCTC 82194=MCCC 1K04733).

Genome sequence of five Zetaproteobacteria metagenome-assembled genomes recovered from hydrothermal vent Longqi, Southwest Indian Ridge.

Southwest Indian Ridge (SWIR) increasingly becomes the hot spot of deep-sea mining and extreme life research. Strategies for future environmental conservation are undoubtedly required which makes investigation of microbial iron metabolisms imperative. Through deep metagenome sequencing, five iron-oxidizing Zetaproteoabacteria metagenome-assembled genomes were recovered from Longqi hydrothermal vent in the SWIR. Phylogenetic analysis revealed that two of the MAGs might represent novel genus in Zetaproteobacteria while other MAGs were related to Mariprofundus. Functional profile suggested that they might be aerobic chemolithoautotrophic species with genes encoding cytochrome c oxidase, iron oxidase cyc2 homologs and carbon fixation CBB pathway. Versatile capabilities of synthesizing diverse amino acids and cofactors were indicated while possession of various metal ion transporters could be vital to heavy metal resistance. Our work has provided more understanding about phylogenetic and functional features of iron-oxidizing Zetaproteobacteria, which might be important to investigate iron biogeochemistry and mineral oxidation in SWIR.

Thiosulfatihalobacter marinus gen. nov. sp. nov., a novel member of the family Roseobacteraceae, isolated from the West Pacific Ocean.

Two strains (GL-11-2T and ZH2-Y79) were isolated from the seawater collected from the West Pacific Ocean and the East China Sea, respectively. Cells were Gram-stain-negative, strictly aerobic, non-motile and rod-shaped. Cells grew in the medium containing 0.5-7.5 % NaCl (w/v, optimum, 1.0-3.0 %), at pH 6.0-8.0 (optimum, pH 6.5-7.0) and at 4-40 °C (optimum, 30 °C). H2S production occurred in marine broth supplemented with sodium thiosulphate. The almost-complete 16S rRNA gene sequences of the two isolates were identical, and exhibited the highest similarity to Pseudoruegeria aquimaris JCM 13603T (97.5 %), followed by Ruegeria conchae TW15T (97.2%), Shimia aestuarii DSM 15283T (97.1 %) and Ruegeria lacuscaerulensis ITI-1157T (97.0 %). Phylogenetic analysis revealed that the isolates were affiliated with the family Roseobacteraceae and represented an independent lineage. The sole isoprenoid quinone was ubiquinone 10. The principal fatty acids were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and cyclo-C19 : 0 ω8c. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and diphosphatidylglycerol. The DNA G+C content was 62.3 mol%. The orthologous average nucleotide identity, in silico DNA-DNA hybridization and average amino acid identity values among the genomes of strain GL-11-2T and the reference strains were 73.2-79.0, 20.3-22.5 and 66.0-80.8 %, respectively. Strains GL-11-2ᵀ and ZH2-Y79 possessed complete metabolic pathways for thiosulphate oxidation, dissimilatory nitrate reduction and denitrification. Phylogenetic distinctiveness, chemotaxonomic differences and phenotypic properties revealed that the isolates represent a novel genus and species of the family Roseobacteraceae, belonging to the class Alphaproteobacteria, for which the name Thiosulfatihalobacter marinus gen. nov., sp. nov. (type strain, GL-11-2T=KCTC 82723T=MCCC M20691T) is proposed.

Mesobacterium pallidum gen. nov., sp. nov., Heliomarina baculiformis gen. nov., sp. nov. and Oricola indica sp. nov., three novel Alphaproteobacteria members isolated from deep-sea water in the southwest Indian ridge.

Three Gram-staining-negative, aerobic and rod-shaped strains, designated as T40-1T, T40-3T and JL-62T, were isolated from the deep-sea water in the southwest Indian ridge. For strain T40-1T, growth occurred at 15-37 °C (optimum, 28 °C), pH 6.0-9.0 (optimum, pH 7.5) and in the presence of 0.5-5.0 % NaCl (w/v; optimum, 2.0 %). Strain T40-3T could grow at 15-40 °C (optimum, 28 °C), with 0.5-11.0 % NaCl (optimum, 2.0 %, w/v) at pH 6.0-9.5 (optimum, 8.0). The temperature, pH and salinity ranges for growth of strain JL-62T were 15-40 °C (optimum, 30 °C), pH 5.5-9.0 (optimum, pH 7.5-8.0) and 0.5-9.0 % NaCl (w/v; optimum, 4.0 %). Ubiquinone-10 was the sole ubiquinone in all strains, the major fatty acids (>20 %) were summed feature 8 (C18 : 1 ω7c / C18 : 1 ω6c). The major polar lipids of strains T40-1T and T40-3T were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Strain JL-62T contained phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and sulfoquinovosyldiacylglycerol as major polar lipids. Phylogenetic trees based on 16S rRNA gene and core-genomic sequences revealed affiliation of strains T40-1Tand T40-3T to the family Roseobacteraceae and formed two independent clades from other Roseobacteraceae genera, and those two strains had average nucleotide identities of 62.0-72.0 % to their phylogenetically related species which fell into to the genus boundary range, indicating that they represent two novel genera. While strain JL-62T represents a novel species in the genus Oricola belonging to the family Phyllobacteriaceae, which was supported by overall genomic relatedness index calculations. The DNA G+C contents of strains T40-1T, T40-3T and JL-62T were 66.5, 60.1 and 62.1 mol %, respectively. Based on the polyphasic taxonomic data, strains T40-1T (=MCCC M24557T=KCTC 82975T) and T40-3T (=MCCC 1K05135T=KCTC 82976T) are classified as representing two novel genera belonging to the family Roseobacteraceae with the names Mesobacterium pallidum gen. nov., sp. nov. and Heliomarina baculiformis gen. nov., sp. nov. are proposed, and strain JL-62T (=MCCC M24579T=KCTC 82974T) is proposed to represent a novel species within the genus Oricola with the name Oricola indica sp. nov. is proposed.

Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93.

Introduction: CPT11 (Irinotecan; 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an important camptothecin-based broad-spectrum anticancer prodrug. The activation of its warhead, SN38 (7-ethyl-10-hydroxycamptothecin), requires hydrolysis by carboxylesterases. NPC (7-ethyl-10-[4-(1-piperidino)-1-amino] carbonyloxycamptothecin) is a metabolic derivative of CPT11 and is difficult to be hydrolyzed by human carboxylesterase. Microbial carboxylesterase with capability on both CPT11 and NPC hydrolysis is rarely reported. A marine microbial carboxylesterase, E93, was identified to hydrolyze both substrates in this study. This enzyme was an appropriate subject for uncovering the catalytic mechanism of carboxylesterases to CPT11 and NPC hydrolysis. Methods: X-ray diffraction method was applied to obtain high-resolution structure of E93. Molecular docking was adopted to analyze the interaction of E93 with p-NP (p-nitrophenyl), CPT11, and NPC substrates. Mutagenesis and enzymatic assay were adopted to verify the binding pattern of substrates. Results: Three core regions (Region A, B, and C) of the catalytic pocket were identified and their functions on substrates specificity were validated via mutagenesis assays. The Region A was involved in the binding with the alcohol group of all tested substrates. The size and hydrophobicity of the region determined the binding affinity. The Region B accommodated the acyl group of p-NP and CPT11 substrates. The polarity of this region determined the catalytic preference to both substrates. The Region C specifically accommodated the acyl group of NPC. The interaction from the acidic residue, E428, contributed to the binding of E93 with NPC. Discussion: The study analyzed both unique and conserved structures of the pocket in E93, for the first time demonstrating the discrepancy of substrate-enzyme interaction between CPT11 and NPC. It also expanded the knowledge about the substrate specificity and potential application of microbial Family VII carboxylesterases.

TRIM21 regulates pyroptotic cell death by promoting Gasdermin D oligomerization.

Gasdermin-D (GSDMD), the executioner of pyroptotic cell death when it is cleaved by inflammatory caspases, plays a crucial role in host defense and the response to danger signals. So far, there are no known mechanisms, other than cleavage, for regulating GSDMD. Here, we show that tripartite motif protein TRIM21 acts as a positive regulator of GSDMD-dependent pyroptosis. TRIM21 interacted with GSDMD via its PRY-SPRY domain, maintaining GSDMD stable expression in resting cells yet inducing the N-terminus of GSDMD (GSDMD-N) aggregation during pyroptosis. TRIM21-deficient cells displayed a reduced cell death in response to NLRP3 or NLRC4 inflammasome activation. Genetic ablation of TRIM21 in mice conferred protection from LPS-induced inflammation and dextran sulfate sodium-induced colitis. Therefore, TRIM21 plays an essential role in GSDMD-mediated pyroptosis and may be a viable target for controlling and treating inflammation-associated diseases.

Luteirhabdus pelagi gen. nov., sp. nov., a novel member of the family Flavobacteriaceae, isolated from the West Pacific Ocean.

A Gram-stain-negative, aerobic, and yellow-pigmented bacterium, designated A3-108T, was isolated from seawater of the West Pacific Ocean. Cells were non-motile and rod-shaped, with carotenoid-type pigments. Strain A3-108T grew at pH 6.0-8.5 (optimum 6.5) and 15-40 °C (optimum 28 °C), in the presence of 0.5-10% (w/v) NaCl (optimum 1.0%). It possessed the ability to produce H2S. Based on the 16S rRNA gene analysis, strain A3-108T exhibited highest similarity with Aureisphaera salina A6D-50T (90.6%). Phylogenetic analysis shown that strain A3-108T affiliated with members of the family Flavobacteriaceae and represented an independent lineage. The principal fatty acids were iso-C15:0, iso-C17:0 3-OH, iso-C15:1 G, and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The sole isoprenoid quinone was MK-6. The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified aminolipid and one unidentified lipid. The ANIb, in silico DDH and AAI values among the genomes of strain A3-108T and three reference strains were 67.3-71.1%, 18.7-22.1%, and 58.8-71.4%, respectively. The G + C content was 41.0%. Distinctness of the phylogenetic position as well as differentiating chemotaxonomic and other phenotypic traits revealed that strain A3-108T represented a novel genus and species of the family Flavobacteriaceae, for which the name Luteirhabdus pelagi gen. nov., sp. nov. is proposed (type strain, A3-108T = CGMCC 1.18821T = KCTC 82563T).