Screening and Molecular Mechanisms of Novel ACE-Inhibitory Peptides from Gracilariopsis lemaneiformis.

Candidate peptides with novel angiotensin-I-converting enzyme (ACE) inhibitor activity were obtained from hydrolysates of Gracilariopsis lemaneiformis by virtual screening method. Our results showed that G. lemaneiformis peptides (GLP) could significantly lower blood pressure in spontaneously hypertensive rats (SHR). At least 101 peptide sequences of GLP were identified by LC-MS/MS analysis and subjected to virtual screening. A total of 20 peptides with the highest docking score were selected and chemically synthesized in order to verify their ACE-inhibitory activities. Among them, SFYYGK, RLVPVPY, and YIGNNPAKG showed good effects with IC50 values of 6.45 ± 0.22, 9.18 ± 0.42, and 11.23 ± 0.23 µmoL/L, respectively. Molecular docking studies revealed that three peptides interacted with the active center of ACE by hydrogen bonding, hydrophobic interactions, and electrostatic forces. These peptides could form stable complexes with ACE. Furthermore, SFYYGK, RLVPVPY, and YIGNNPAKG significantly reduced systolic blood pressure (SBP) in SHR. YIGNNPAKG exhibited the highest antihypertensive effect, with the largest decrease in SBP (approximately 23 mmHg). In conclusion, SFYYGK, RLVPVPY, and YIGNNPAKG can function as potent therapeutic candidates for hypertension treatment.

Characterization of cold-tolerant trehalose-6-phosphate synthase from the deep-sea bacterium Microbacterium sediminis YLB-01.

A gene encoding the enzyme trehalose-6-phosphate synthase (TPS), which is part of the TPS trehalose synthesis pathway, was cloned from the deep-sea psychrotolerant bacterium Microbacterium sediminis YLB-01 and expressed in Escherichia coli BL21. The exogenously expressed TPS exhibited highest similarity (80.93% identity) to Microbacterium sp. TPS. The purified recombinant TPS was cold-tolerant, with low thermostability. The optimum temperature for TPS activity was 40°C, and the enzyme retained 72.6% of its maximal activity at 4°C. The optimum pH was 7.5. TPS activity was cation-dependent, with Mg2+, Co2+, or Ba2+ being essential for maximum activity. The kinetic constants of the recombinant TPS reaction rates confirmed that it was cold-tolerant. Molecular dynamics analysis showed that TPS was more flexible (0.8741Å) at 4°C than 1GZ5, its homolog in the mesophilic bacterium E. coli, and superposition of the 3D enzyme structures supported this.

Anti-Inflammatory Effects of Fucoxanthinol in LPS-Induced RAW264.7 Cells through the NAAA-PEA Pathway.

Palmitoylethanolamide (PEA) is an endogenous lipid mediator with powerful anti-inflammatory and analgesic functions. PEA can be hydrolyzed by a lysosomal enzyme N-acylethanolamine acid amidase (NAAA), which is highly expressed in macrophages and other immune cells. The pharmacological inhibition of NAAA activity is a potential therapeutic strategy for inflammation-related diseases. Fucoxanthinol (FXOH) is a marine carotenoid from brown seaweeds with various beneficial effects. However, the anti-inflammatory effects and mechanism of action of FXOH in lipopolysaccharide (LPS)-stimulated macrophages remain unclear. This study aimed to explore the role of FXOH in the NAAA-PEA pathway and the anti-inflammatory effects based on this mechanism. In vitro results showed that FXOH can directly bind to the active site of NAAA protein and specifically inhibit the activity of NAAA enzyme. In an LPS-induced inflammatory model in macrophages, FXOH pretreatment significantly reversed the LPS-induced downregulation of PEA levels. FXOH also substantially attenuated the mRNA expression of inflammatory factors, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), and markedly reduced the production of TNF-α, IL-6, IL-1ß, and nitric oxide (NO). Moreover, the inhibitory effect of FXOH on NO induction was significantly abolished by the peroxisome proliferator-activated receptor α (PPAR-α) inhibitor GW6471. All these findings demonstrated that FXOH can prevent LPS-induced inflammation in macrophages, and its mechanisms may be associated with the regulation of the NAAA-PEA-PPAR-α pathway.

Aphidicolin Chemistry of the Deep-Sea-Derived Fungus Botryotinia fuckeliana MCCC 3A00494.

Aphidicolin, a potent DNA polymerase α inhibitor, has been explored in clinical trials for the treatment of cancer. So far, about 300 modified aphidicolins have been discovered. However, none have shown a stronger effect. Herein, we report 71 new (aphidicolins A1-A71, 1-71) and eight known (72-79) aphidicolin congeners from Botryotinia fuckeliana MCCC 3A00494, a fungus isolated from the western Pacific Ocean (-5572 m). The structures of 1-71 were determined through extensive spectroscopic analysis, X-ray crystallography, chemical derivatization, modified Mosher's method, and the ECD exciton chirality method. Compounds 54-57 and 58-64 are novel 6/6/5/6/5 pentacyclic aphidicolins featuring tetrahydrofuran and dihydrofuran rings, respectively, while compounds 65-71 are rare noraphidicolins. Aphidicolin A8 (8) significantly induced apoptosis in T24 (IC50 = 2.5 µM) and HL-60 (IC50 = 6.1 µM) cancer cells by causing DNA damage. By docking its structure to the human DNA polymerase α binding pocket, 8 was found to form tight intermolecular contacts, elaborating aphidicolin A8 as a potently cytotoxic lead compound.

Biosynthetic Functional Gene Analysis of Bis-Indole Metabolites from 25D7, a Clone Derived from a Deep-Sea Sediment Metagenomic Library.

This work investigated the metabolites and their biosynthetic functional hydroxylase genes of the deep-sea sediment metagenomic clone 25D7. 5-Bromoindole was added to the 25D7 clone derived Escherichia coli fermentation broth. The new-generated metabolites and their biosynthetic byproducts were located through LC-MS, in which the isotope peaks of brominated products emerged. Two new brominated bis-indole metabolites, 5-bromometagenediindole B (1), and 5-bromometagenediindole C (2) were separated under the guidance of LC-MS. Their structures were elucidated on the basis of 1D and 2D NMR spectra (COSY, HSQC, and HMBC). The biosynthetic functional genes of the two new compounds were revealed through LC-MS and transposon mutagenesis analysis. 5-Bromometagenediindole B (1) also demonstrated moderately cytotoxic activity against MCF7, B16, CNE2, Bel7402, and HT1080 tumor cell lines in vitro.

Macrolactins from Marine-Derived Bacillus subtilis B5 Bacteria as Inhibitors of Inducible Nitric Oxide and Cytokines Expression.

In order to find new natural products with anti-inflammatory activity, chemical investigation of a 3000-meter deep-sea sediment derived bacteria Bacillus subtilis B5 was carried out. A new macrolactin derivative was isolated and identified as 7,13-epoxyl-macrolactin A (1). Owing to the existence of the epoxy ring, 1 exhibited a significant inhibitory effect on the expression of inducible nitric oxide and cytokines, compared with previously isolated known macrolactins (2-5). Real-time Polymerase Chain Reaction (PCR) analysis showed that the new compound significantly inhibited the mRNA expressions of inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Reverse transcription-PCR analysis demonstrated that the new compound reduced the mRNA expression level of IL-1ß in a concentration-dependent manner.

Shewanella mangrovi sp. nov., an acetaldehyde-degrading bacterium isolated from mangrove sediment.

A taxonomic study was carried out on strain YQH10T, which was isolated from mangrove sediment collected from Zhangzhou, China during the screening of acetaldehyde-degrading bacteria. Cells of strain YQH10T were Gram-stain-negative rods and pale brown-pigmented. Growth was observed at salinities from 0 to 11% and at temperatures from 4 to 42 °C. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YQH10T is affiliated to the genus Shewanella, showing the highest similarity with Shewanella haliotis DW01T (95.7%) and other species of the genus Shewanella (91.4-95.6 %). The principal fatty acids were iso-C15 : 0 and C17 : 1ω8c. The major respiratory quinone was Q-8. The polar lipids comprised phosphatidylethanolamine and phosphatidylglycerol. The genomic DNA had a G+C content of 48.3 mol%. Strain YQH10T can completely degrade 0.02% (w/v) acetaldehyde on 2216E at 28 °C within 48 h. Based on these phenotypic and genotypic data, strain YQH10T represents a novel species of the genus Shewanella, for which the name Shewanella mangrovi sp. nov. is proposed. The type strain is YQH10T ( = MCCC 1A00830T = JCM 30121T).

Two new cytotoxic indole alkaloids from a deep-sea sediment derived metagenomic clone.

Two new indole alkaloids, metagenetriindole A (1) and metagenebiindole A (2), were identified from deep-sea sediment metagenomic clone derived Escherichia coli fermentation broth. The structures of new compounds were elucidated by spectroscopic methods. The two new indole alkaloids demonstrated moderately cytotoxic activity against CNE2, Bel7402 and HT1080 cancer cell lines in vitro.

Microbacterium sediminis sp. nov., a psychrotolerant, thermotolerant, halotolerant and alkalitolerant actinomycete isolated from deep-sea sediment.

An aerobic, rod-shaped, Gram-positive, oxidase- and catalase-positive bacterial isolate, strain YLB-01(T), was characterized using phenotypic and molecular taxonomic methods. 16S rRNA gene sequence analysis revealed that the isolate belonged to the genus Microbacterium and represented an evolutionary lineage that was distinct from recognized species of the genus Microbacterium. The isolate showed <97 % 16S rRNA gene sequence similarity with respect to the type strains of all of the members of the genus Microbacterium with validly published names. Cell-wall hydrolysate from the isolate contained the amino acids ornithine, alanine, glycine homoserine and glutamic acid, and the cell-wall sugars consisted of ribose, glucose and galactose. The main respiratory quinones were MK-8, 9, 10 and 11. The major cellular fatty acids were anteiso-C(15:0) (38.5 %), anteiso-C(17:0) (23.1 %) and C(16:0) (18.9 %). The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, an unidentified phospholipid and two unidentified glycolipids. The DNA G+C content of strain YLB-01(T) was 71 mol %. On the basis of the morphological, physiological and chemotaxonomic data and the results of comparative 16S rRNA gene sequence analysis, this isolate represents a novel species of the genus Microbacterium, for which the name Microbacterium sediminis sp. nov. is proposed. The type strain is YLB-01(T) (= DSM 23767(T) = CCTCC AB2010363(T) = MCCC 1A06153(T)).

Revealing the role of tunable amino acid residues in elastin-like polypeptides (ELPs)-mediated biomimetic silicification.

Elastin-like polypeptides (ELPs) are attractive materials for the green preparation of silica nanoparticles via biomimetic silicification. However, the critical factors affecting the ELP-mediated silicification remain unclear. Herein, the role of tunable amino acid residues of ELPs in silicification was studied using three ELPs (ELPs[V9F-40], ELPs[KV8F-40], and ELPs[K5V4F-40]) and their fusion proteins (ELPs[V9F-40]-SpyCatcher, ELPs[KV8F-40]-SpyCatcher, and ELPs[K5V4F-40]-SpyCatcher) with different contents of lysine residues. Bioinformatics methods were employed for the first time to reveal the key physicochemical parameters correlated with silicification. The specific activity of ELPs was increased with the promotion of lysine content with a high correlation coefficient (R = 0.899). Furthermore, exogenous acidic protein SpyCatcher would hinder the interactions between the silica precursors and ELPs, leading to the significantly decrease in specific activity. The isoelectric point (pI) of ELPs presented the highest correlation to silicification with a coefficient of 0.963. The charges of the ELPs [K5V4F-40] at different pH were calculated based on the sequence or structure. Interestingly, the excellent correlation between charges based on structure and specific activity was obtained. Collectively, the novel methods developed here may pave a new way for rational design of ELPs or other peptides for efficient and green preparation of silica nanomaterials for biomedicine, biocatalysis, and biosensor.

A versatile tag for simple preparation of cutinase towards enhanced biodegradation of polyethylene terephthalate.

Enzymatic degradation of polyethylene terephthalate (PET) suffered from challenges such as complex and costly enzyme preparation, difficult access to PET substrates, poor reusability of free enzymes and sometimes MHET inhibitions. Herein, we propose an "all-in-one" strategy to address these issues with a well-designed elastin-like polypeptides (ELPs) tag. The preparation of the ELPs-tagged cutinase (ET-C) was efficient and easy to scale up by centrifugation, with an activity recovery of 57.55 % and a yield of 160 mg/L. Besides, the activity of the ET-C was 1.3 and 1.66-fold higher in degrading PET micro- and macro-plastics compared to wild-type cutinase. The self-immobilized cutinase (ET-C@SiO2) obtained by the ELPs-mediated biosilicification exhibited high loading capacity, activity, and thermostability and maintained 77.65 % of the original activity after 10 reuses. Interestingly, the product of the ET-C was TPA, whereas the wild-type was TPA and MHET. This is a simple way to release the intermediates inhibition compared with the existing methods. Our results demonstrated the feasibility of the versatile ELPs tag, which will pave an alternative economic way for scalable PET biodegradation.

Ramulus Cinnamomi essential oil exerts an anti-inflammatory effect on RAW264.7 cells through N-acylethanolamine acid amidase inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Ramulus Cinnamomi, the dried twig of Cinnamomum cassia (L.) J.Presl., is a traditional Chinese medicine (TCM) with anti-inflammatory effects. The medicinal functions of Ramulus Cinnamomi essential oil (RCEO) have been confirmed, although the potential mechanisms by which RCEO exerts its anti-inflammatory effects have not been fully elucidated. AIM OF THE STUDY: To investigate whether N-acylethanolamine acid amidase (NAAA) mediates the anti-inflammatory effects of RCEO. MATERIALS AND METHODS: RCEO was extracted by steam distillation of Ramulus Cinnamomi, and NAAA activity was detected using HEK293 cells overexpressing NAAA. N-Palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), both of which are NAAA endogenous substrates, were detected by liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The anti-inflammatory effects of RCEO were analyzed in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and the cell viability was measured with a Cell Counting Kit-8 (CCK-8) kit. The nitric oxide (NO) in the cell supernatant was measured using the Griess method. The level of tumor necrosis factor-α (TNF-α) in the RAW264.7 cell supernatant was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The chemical composition of RCEO was assessed by gas chromatography-mass spectroscopy (GC-MS). The molecular docking study for (E)-cinnamaldehyde and NAAA was performed by using Discovery Studio 2019 software (DS2019). RESULTS: We established a cell model for evaluating NAAA activity, and we found that RCEO inhibited the NAAA activity with an IC50 of 5.64 ± 0.62 µg/mL. RCEO significantly elevated PEA and OEA levels in NAAA-overexpressing HEK293 cells, suggesting that RCEO might prevent the degradation of cellular PEA and OEA by inhibiting the NAAA activity in NAAA-overexpressing HEK293 cells. In addition, RCEO also decreased NO and TNF-α cytokines in lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, the GC-MS assay revealed that more than 93 components were identified in RCEO, of which (E)-cinnamaldehyde accounted for 64.88%. Further experiments showed that (E)-cinnamaldehyde and O-methoxycinnamaldehyde inhibited NAAA activity with an IC50 of 3.21 ± 0.03 and 9.62 ± 0.30 µg/mL, respectively, which may represent key components of RCEO that inhibit NAAA activity. Meanwhile, docking assays revealed that (E)-cinnamaldehyde occupies the catalytic cavity of NAAA and engages in a hydrogen bond interaction with the TRP181 and hydrophobic-related interactions with LEU152 of human NAAA. CONCLUSIONS: RCEO showed anti-inflammatory effects by inhibiting NAAA activity and elevating cellular PEA and OEA levels in NAAA-overexpressing HEK293 cells. (E)-cinnamaldehyde and O-methoxycinnamaldehyde, two components in RCEO, were identified as the main contributors of the anti-inflammatory effects of RCEO by modulating cellular PEA levels through NAAA inhibition.

Two new compounds from marine-derived fungus Penicillium sp. F11.

Two new compounds, penicillone A (1) and penicillactam (2), were isolated together with 17 known compounds from a marine-derived fungus Penicillium sp. F11. The structures of the new compounds as well as a firstly literatural reported known compound (3) were assigned by spectroscopic methods including 1D/2D NMR and MS analysis techniques. Their cytotoxicities against HT1080, Cne2, and Bel7402 cell lines were also evaluated.

Genomic analysis of Shewanella eurypsychrophilus YLB-09 reveals backgrounds related to its deep sea environment adaptation.

Shewanella eurypsychrophilus YLB-09 is a psychrophilic and piezotolerant bacterium that was isolated from 2699 m deep sea sediments of the Southwest Indian Ocean. The complete genome sequence of the strain Shewanella eurypsychrophilus YLB-09 was analyzed. The genome of Shewanella eurypsychrophilus YLB-09 contained one single circular chromosome 6,225,487 base pairs with a 43.6 mol% G + C content of 52 ribosomal RNA genes and 5124 protein-coding genes. YLB-09 has the largest number of genes related to energy production and conversion among 22 available complete genomes of Shewanella genus. Meanwhile， a large quantity of genes encoding flagellum/fimbrial-related proteins and two major secondary metabolic gene clusters were found in YLB-09. These data could provide insights into the mechanism of this strain in adapting to deep sea extreme environments.

Engineering and screening of novel ß-1,3-xylanases with desired hydrolysate type by optimized ancestor sequence reconstruction and data mining.

Engineering of hydrolases to shift their hydrolysate types has not been attempted so far, though computer-assisted enzyme design has been successful. A novel integrative strategy for engineering and screening the ß-1,3-xylanase with desired hydrolysate types was proposed, with the purpose to solve problems that the separation and preparation of ß-1,3-xylo-oligosaccharides was in high cost yet in low yield as monosaccharides existed in the hydrolysates. By classifying the hydrolysate types and coding them into numerical values, two robust mathematical models with five selected attributes from molecular docking were established based on LogitBoost and partial least squares regression with overall accuracy of 83.3% and 100%, respectively. Then, they were adopted for efficient screening the potential mutagenesis library of ß-1,3-xylanases that only product oligosaccharides. The virtually designed AncXyl10 was selected and experimentally verified to produce only ß-1,3-xylobiose (60.38%) and ß-1,3-xylotriose (39.62%), which facilitated the preparation of oligosaccharides with high purity. The underlying mechanism of AncXyl10 may associated with the gap processing and ancestral amino acid substitution in the process of ancestral sequence reconstruction. Since many carbohydrate-active enzymes have highly conserved active sites, the strategy and their biomolecular basis will shield a new light for engineering carbohydrates hydrolase to produce specific oligosaccharides.

A Novel Inovirus Reprograms Metabolism and Motility of Marine Alteromonas.

Members from the Inoviridae family with striking features are widespread, highly diverse, and ecologically pervasive across multiple hosts and environments. However, a small number of inoviruses have been isolated and studied. Here, a filamentous phage infecting Alteromonas abrolhosensis, designated ϕAFP1, was isolated from the South China Sea and represented a novel genus of Inoviridae. ϕAFP1 consisted of a single-stranded DNA genome (5986 bp), encoding eight putative ORFs. Comparative analyses revealed ϕAFP1 could be regarded as genetic mosaics having homologous sequences with Ralstonia and Stenotrophomonas phages. The temporal transcriptome analysis of A. abrolhosensis to ϕAFP1 infection revealed that 7.78% of the host genes were differentially expressed. The genes involved in translation processes, ribosome pathways, and degradation of multiple amino acid pathways at the plateau period were upregulated, while host material catabolic and bacterial motility-related genes were downregulated, indicating that ϕAFP1 might hijack the energy of the host for the synthesis of phage proteins. ϕAFP1 exerted step-by-step control on host genes through the appropriate level of utilizing host resources. Our study provided novel information for a better understanding of filamentous phage characteristics and phage-host interactions. IMPORTANCE Alteromonas is widely distributed and plays a vital role in biogeochemical in marine environments. However, little information about Alteromonas phages is available. Here, we isolated and characterized the biological characteristics and genome sequence of a novel inovirus infecting Alteromonas abrolhosensis, designated ϕAFP1, representing a novel viral genus of Inoviridae. We then presented a comprehensive view of the ϕAFP1 phage-Alteromonas abrolhosensis interactions, elucidating reprogramed host metabolism and motility. Our study provided novel information for better comprehension of filamentous phage characteristics and phage-host interactions.

A novel indole alkaloid from deep-sea sediment metagenomic clone-derived Escherichia coli fermentation broth.

To explore secondary metabolites in deep-sea sediment metagenomic clone-derived Escherichia coli fermentation broth, different kinds of chromatography methods were used in the isolation procedures, while the structures of the isolated compounds were assigned based on the MS analysis and their (1)H and (13)C NMR spectra including 2D NMR techniques such as COSY, HMQC, and HMBC experiments. As a result, a novel compound was isolated and characterized as N-{1-[4-(acetylamino)phenyl]-3-hydroxy-1-(1H-indol-3-yl)propan-2-yl}-2,2-dichloroacetamide (1). In addition, eight known compounds were also obtained. Fatty acid amide hydrolase and monoacylglycerol lipase were used to screen analgesic activity, and the new compound showed analgesic activity to some extent in pharmacological test.

Highly thermostable and promiscuous ß-1,3-xylanasen designed by optimized ancestral sequence reconstruction.

The ancestor of ß-1,3-xylanases (AncXyl09) were reconstructed by the optimized ancestral sequences reconstruction strategy to solve the poor catalytic performances of existing ß-1,3-xylanases. The results showed that the half-life at 50 °C was 65.08 h, indicating good thermostability. The large number of hydrogen bonds and the disulfide bonds were the major attributes related with the thermal stability of Anxyl09. Interestingly, AncXyl09 could hydrolyze lichen besides the original substrate of ß-1, 3-xylan, which is the first reported ß-1,3-xylanase with substrate promiscuity. Moreover, the hydrolytic products are mainly disaccharides, the content of ß-1,3-xylobiose and lichoridiose more than 70% as determined by high performance liquid chromatography (HPLC), which could significantly facilitate the separation and purification of oligosaccharides. The successful design of AncXyl09 was the representative of the semi-rationally engineered ß-1, 3-xylanase, which will shield a new light on the ß-1,3-xylanase engineering, active oligosaccharide preparation and marine algae resource utilization.

Genomic analysis of Microbacterium sediminis YLB-01T reveals backgrounds related to its deep-sea environment adaptation.

Microbacterium sediminis YLB-01T, a piezotolerant and psychrotolerant actinomycete, was isolated from deep-sea sediment of the South-West Indian Ocean and could be a good model for understanding the adaptation of extremophiles to the benthic piezosphere. Here, we report the analysis of the complete genome sequence of strain YLB-01T. The genome sequence consists of a single circular chromosome comprising 2,792,195 bp and a linear plasmid comprising 127,669 bp with G + C content of 71.76 and 68.49 mol%, respectively. In this regard, strain YLB-01T possesses the smallest genome size but the highest G + C content among the genus Microbacterium sequenced to date. As the first complete genome sequence of the genus Microbacterium isolated from deep-sea environment, the strain YLB-01T genome is unique or enriched in genes involved in xenobiotics biodegradation and metabolism, compatible solutes, and transposases, some of which might be related to bacterial enhancement of ecological fitness in the deep sea.

Characterization of two novel psychrophilic and piezotolerant strains, Shewanella psychropiezotolerans sp. nov. and Shewanella eurypsychrophilus sp. nov, adapted to an extreme deep-sea environment.

Three marine bacterial strains designated YLB-06T, YLB-08T and YLB-09 were isolated under high hydrostatic pressure from deep-sea sediment samples collected from the Southwest Indian Ocean. They were Gram-stain-negative, oxidase- and catalase-positive, facultative anaerobic and motile. In addition, the strains were capable of growing at 0-20 °C (optimum 4-10 °C) and 0.1-40 MPa (optimum 0.1 MPa), were psychrophiles and piezotolerant, and could use trimethylamine N-oxide (TMAO), DMSO, elemental sulfur and insoluble Fe (III) as terminal electron acceptors during anaerobic growth. Strain YLB-06T could also use nitrate, and strains YLB-08T and YLB-09 could use nitrite as a terminal electron acceptor. Phylogenetic tree analyses based on 16S rRNA gene sequences and 400 optimized universal marker sequences indicated that the strains belonged to the genus Shewanella. The 16S rRNA gene highest similarity, together with the estimated ANI and DDH values for these strains with their related type strains, were below the respective thresholds for species differentiation. The ANI and DDH values between YLB-08T and YLB-09 were 99.9% and 91.8%, respectively, implying that they should belong to the same genospecies. The YLB-06T genome had duplicated genes, and multiple movement modalities, attachment modalities, biofilm synthesis systems, intercellular interactions and a strong antioxidant system, which were all beneficial for survival in an extreme deep-sea environment. The G + C contents of strains YLB-06T, YLB-08T and YLB-09 were 45.1, 43.5 and 43.6 mol%, respectively. Based on polyphasic taxonomic properties, two novel psychropiezotolerant species are proposed, Shewanella psychropiezotolerans sp. nov. with YLB-06T (=MCCC 1A12715T = KCTC 62907T) and S. eurypsychrophilus sp. nov with YLB-08T (=MCCC 1A12718T = KCTC 62909T) as type strains.