Mechanisms of nucleic acid degradation and high hydrostatic pressure tolerance of a novel deep-sea wall-less bacterium.

Wall-less bacteria are broadly distributed in diverse habitats. They evolved from a common ancestor within the Firmicutes phylum through reductive evolution. Here, we report the cultivation, characterization, and polyphasic taxonomic analysis of the novel free-living wall-less bacterium, Hujiaoplasma nucleasis zrk29. We demonstrated that strain zrk29 had a strong ability to degrade DNA and RNA both under laboratory conditions and in the deep sea. We found that nucleic acids induced strain zrk29 to release chronic bacteriophages which supported strain zrk29 and other marine bacteria to metabolize nucleic acids without lysing host cells. We also showed that strain zrk29 tolerated high hydrostatic pressure via two pathways: (i) by transporting cations into its cells to increase intracellular osmotic pressure and (ii) by adjusting the unsaturated fatty acid chain content in its cell membrane phospholipids to increase cell membrane fluidity. This study extends our understanding of free-living wall-less bacteria and provides a useful model to explore the unique adaptation mechanisms of deep-sea microbes. IMPORTANCE The unique physiology and survival strategies of the Tenericutes bacterium-a typical wall-less bacterium-have fascinated scientists and the public, especially in extreme deep-sea environments where there is high hydrostatic pressure (HHP) and limited availability of nutrients. Here, we have isolated a novel free-living Tenericutes strain from deep-sea sediment and have found that it metabolizes nucleic acids with the support of chronic bacteriophages. This Tenericutes strain tolerates HHP stress by increasing intracellular osmotic pressure and the unsaturated fatty acid chain content of phospholipids in its cell membrane. Our results provide insights into the unique physiology of deep-sea free-living Tenericutes bacteria and highlight the significant role that chronic bacteriophages play in assisting wall-less bacteria to adapt to harsh conditions.

Zebrafish (Danio rerio) TRß- and TTR-based electrochemical biosensors: Construction and application for the evaluation of thyroid-disrupting activity of bisphenols.

Thyroid-disrupting chemicals (TDCs) have received increasing concerns because of their negative health impacts on both wildlife and humans. This study aimed to develop in vitro screening assays for TDCs based on thyroid hormone receptor ß (TRß) and transthyretin (TTR) proteins. Firstly, the recombinant ligand-binding domain of TRß (TRß-LBD) and TTR proteins of zebrafish were produced by eukaryotic expression system and then used as bio-recognition components to construct electrochemical biosensors. In the biosensors, the supported bilayer lipid membrane (s-BLM) was used as a matrix to immobilize proteins, and gold nanoflowers (AuNFs) were used to improve the sensitivity by increasing electroactive surface area. Under the optimizing conditions, the zfTRß-LBD/AuNFs/s-BLM/GCE biosensor had a detection range of 0.23 nM-1.92 µM and a detection limit of 0.07 nM for triiodothyronine (T3), while the zfTTR/AuNFs/s-BLM/GCE biosensor had a detection range of 0.46 nM-3.84 µM, with a detection limit of 0.13 nM. Based on the constructed biosensors, the order of T3 equivalent concentrations of bisphenols was BPA ≈ BPS > BPF > BPAF ≈ BPAP > BPZ, which was similar to the results of recombinant TRß two-hybrid yeast assay. Furthermore, the reliability of the biosensors was validated by molecular docking, in which BPA and BPS showed higher binding affinity to zfTRß-LBD. Therefore, this study provided a valuable tool for efficiently screening TDCs.

Characterization of a Deep-Sea Actinobacterium Strain Uncovers Its Prominent Capability of Utilizing Taurine and Polyvinyl Alcohol.

Actinobacteria represent a large group of important prokaryotes with great application potentials and widely distribute in diverse natural environments including the ocean. However, compared to their terrestrial cultured members, there are much less available marine Actinobacteria, especially deep-sea counterparts. Here, we cultured a bacterial strain of deep-sea actinobacterium, Marmoricola sp. TYQ2, by using a basal medium supplemented with taurine. Consistently, the growth of strain TYQ2 was significantly promoted by the supplement of taurine. Transcriptomic analysis showed that the expressions of genes encoding proteins associated with taurine metabolization and utilization as well as energy generation were evidently up-regulated when taurine was added. Moreover, strain TYQ2 was demonstrated to degrade polyvinyl alcohol (PVA) with the involvement of the redox cycle of extracellular quinol and quinone and the reduction of iron to ferrous, and strain TYQ2 could utilize the degradation products for energy production, thereby supporting bacterial growth. Overall, our experimental results demonstrate the prominent degradation capabilities of Marmoricola sp. TYQ2 toward the organics taurine and PVA.

A Deep-Sea Bacterium Senses Blue Light via a BLUF-Dependent Pathway.

Light is a ubiquitous energy source and environmental signal that broadly impacts the lifestyle of a large number of photosynthetic/nonphotosynthetic microorganisms living in the euphotic layer. However, the responses of deep-sea microbes to light are largely unknown, even though blue light is proposed to be distributed in the deep ocean. Here, we successfully cultured a novel bacterial species, named Spongiibacter nanhainus CSC3.9, from deep-sea cold seep samples by a blue light induction approach. The growth of strain CSC3.9 was obviously promoted by the illumination of blue light. We next determined BLUF (a typical blue light photoreceptor) was the most essential factor directing light sensing of strain CSC3.9 through a combined proteomic and genetic method. The function of light sensing mediated by BLUF was further confirmed by the in vitro-synthesized protein. Notably, homologs of BLUF widely existed across the marine microorganisms (containing Spongiibacter species) derived from different environments, including cold seeps. This strongly indicates that the distribution of light utilization by the nonphototrophic bacteria living in the ocean is broad and has been substantially underestimated. IMPORTANCE Extensive studies have been conducted to explore the mechanisms of light sensing and utilization by microorganisms that live in the photic zone. Strikingly, accumulated evidence shows that light is distributed in the deep biosphere. However, the existence and process of light sensing and utilization by microbes inhabiting the deep ocean have been seldom reported. In the present study, a novel bacterial strain, Spongiibacter nanhainus CSC3.9, was enriched and purified from a deep-sea cold seep sample through a blue light induction method. Combined with genomic, proteomic, genetic, and biochemical approaches, the mechanism of this novel strain sensing blue light through a BLUF-dependent pathway was detailed. Our study provides a good model to study the mechanisms of light sensing mediated by deep-sea nonphototrophic bacteria.

A deep-sea sulfate-reducing bacterium generates zero-valent sulfur via metabolizing thiosulfate.

Zero-valent sulfur (ZVS) is a crucial intermediate in the sulfur geobiochemical circulation and is widespread in deep-sea cold seeps. Sulfur-oxidizing bacteria are thought to be the major contributors to the formation of ZVS. However, ZVS production mediated by sulfate-reducing bacteria (SRB) has rarely been reported. In this study, we isolated and cultured a typical SRB designated Oceanidesulfovibrio marinus CS1 from deep-sea cold seep sediment in the South China Sea. We show that O. marinus CS1 forms ZVS in the medium supplemented with thiosulfate. Proteomic and protein activity assays revealed that thiosulfate reductase (PhsA) and the sulfide:quinone oxidoreductase (SQR) played key roles in driving ZVS formation in O. marinus CS1. During this process, thiosulfate firstly was reduced by PhsA to form sulfide, then sulfide was oxidized by SQR to produce ZVS. The expressions of PhsA and SQR were significantly upregulated when O. marinus CS1 was cultured in a deep-sea cold seep, strongly indicating that strain CS1 might form ZVS in the deep-sea environment. Notably, homologs of phsA and sqr were widely identified from microbes living in sediments of deep-sea cold seep in the South China Sea by the metagenomic analysis. We thus propose that SRB containing phsA and sqr genes potentially contribute to the formation of ZVS in deep-sea cold seep environments.

Marine bacterial exopolysaccharide EPS11 inhibits migration and invasion of liver cancer cells by directly targeting collagen I.

Many natural polysaccharides have significant anticancer activity with low toxicity, but the complex chemical structures make in-depth studies of the involved mechanisms extremely difficult. The purpose of this study was to investigate the effect of the marine bacterial exopolysaccharide (exopolysaccharide 11 [EPS11]) on liver cancer metastasis to explore the underlying target protein and molecular mechanism. We found that EPS11 significantly suppressed cell adhesion, migration, and invasion in liver cancer cells. Proteomic analysis showed that EPS11 induced downregulation of proteins related to the extracellular matrix-receptor interaction signaling pathway. In addition, the direct pharmacological target of EPS11 was identified as collagen I using cellular thermal shift assays. Surface plasmon resonance and pull-down assays further confirmed the specific binding of EPS11 to collagen I. Moreover, EPS11 was shown to inhibit tumor metastasis by directly modulating collagen I activity via the ß1-integrin-mediated signaling pathway. Collectively, our study demonstrated for the first time that collagen I could be a direct pharmacological target of polysaccharide drugs. Moreover, directly targeting collagen I may be a promising strategy for finding novel carbohydrate-based drugs.

Growth promotion of a deep-sea bacterium by sensing infrared light through a bacteriophytochrome photoreceptor.

Photoreceptors are found in all kingdoms of life and bacteriophytochromes (Bphps) are the most abundant photo-sensing receptors in bacteria. Interestingly, BphPs have been linked to some bacterial physiological responses, yet most of the biological processes they regulate are still elusive, especially in non-photosynthetic bacteria. Here, we show that a bacteriophytochrome (CmoBphp) from a deep-sea bacterium Croceicoccus marinus OT19 perceives infrared light (wavelength at 940 nm) and transduces photo-sensing signals to a downstream intracellular transduction cascade for better growth. We discover that the infrared light-mediated growth promotion of C. marinus OT19 is attributed partly to the enhancement of pyruvate and propanoate metabolism. Further study suggests that CmoBphp plays a crucial role in integrating infrared light with intracellular signalling to control the bacterial growth and metabolism. This is the first report that deep-sea non-photosynthetic bacteria can sense infrared light to control growth through a bacteriophytochrome photoreceptor, thus providing new understandings towards light energy utilization by microorganisms.

Characterization of the first cultured free-living representative of Candidatus Izemoplasma uncovers its unique biology.

Candidatus Izemoplasma, an intermediate in the reductive evolution from Firmicutes to Mollicutes, was proposed to represent a novel class of free-living wall-less bacteria within the phylum Tenericutes. Unfortunately, the paucity of pure cultures has limited further insights into their physiological and metabolic features as well as ecological roles. Here, we report the first successful isolation of an Izemoplasma representative from the deep-sea methane seep, strain zrk13, using a DNA degradation-driven method given Izemoplasma's prominent DNA-degradation potentials. We further present a detailed description of the physiological, genomic and metabolic traits of the novel strain, which allows for the first time the reconstruction of the metabolic potential and lifestyle of a member of the tentatively defined Candidatus Izemoplasma. On the basis of the description of strain zrk13, the novel species and genus Xianfuyuplasma coldseepsis is proposed. Using a combined biochemical and transcriptomic method, we further show the supplement of organic matter, thiosulfate or bacterial genomic DNA could evidently promote the growth of strain zrk13. In particular, strain zrk13 could degrade and utilize the extracellular DNA for growth in both laboraterial and deep-sea conditions. Moreover, the predicted genes determining DNA-degradation broadly distribute in the genomes of other Izemoplasma members. Given that extracellular DNA is a particularly crucial phosphorus as well as nitrogen and carbon source for microorganisms in the seafloor, Izemoplasma bacteria are thought to be important contributors to the biogeochemical cycling in the deep ocean.

Marine Bacterial Polysaccharide EPS11 Inhibits Cancer Cell Growth via Blocking Cell Adhesion and Stimulating Anoikis.

Tumor cells that acquire metastatic potential have developed resistance to anoikis, a cell death process, after detachment from their primary site to the second organ. In this study, we investigated the molecular mechanisms of a novel marine bacterial polysaccharide EPS11 which exerts its cytotoxic effects through affecting cancer cell adhesion and anoikis. Firstly, we found that EPS11 could significantly affect cell proliferation and block cell adhesion in A549 cells. We further demonstrated that the expression of several cell adhesion associated proteins is downregulated and the filiform structures of cancer cells are destroyed after EPS11 treatment. Interestingly, the destruction of filiform structures in A549 cells by EPS11 is in a dose-dependent manner, and the inhibitory tendency is very consistent with that observed in the cell adhesion assay, which confirms that filiform structures play important roles in modulating cell adhesion. Moreover, we showed that EPS11 induces apoptosis of A549 cells through stimulating ßIII-tubulin associated anoikis: (i) EPS11 inhibits the expression of ßIII-tubulin in both transcription and translation levels; and (ii) EPS11 treatment dramatically decreases the phosphorylation of protein kinase B (PKB or AKT), a critical downstream effector of ßIII-tubulin. Importantly, EPS11 evidently inhibits the growth of A549-derived tumor xenografts in vivo. Thus, our results suggest that EPS11 may be a potential candidate for human non-small cell lung carcinoma treatment via blocking filiform structure mediated adhesion and stimulating ßIII-tubulin associated anoikis.