Blue light promotes zero-valent sulfur production in a deep-sea bacterium.

Increasing evidence has shown that light exists in a diverse range of deep-sea environments. We unexpectedly found that blue light is necessary to produce excess zero-valent sulfur (ZVS) in Erythrobacter flavus 21-3, a bacterium that has been recently isolated from a deep-sea cold seep. E. flavus 21-3 is able to convert thiosulfate to ZVS using a novel thiosulfate oxidation pathway comprising a thiosulfate dehydrogenase (TsdA) and a thiosulfohydrolase (SoxB). Using proteomic, bacterial two-hybrid and heterologous expression assays, we found that the light-oxygen-voltage histidine kinase LOV-1477 responds to blue light and activates the diguanylate cyclase DGC-2902 to produce c-di-GMP. Subsequently, the PilZ domain-containing protein mPilZ-1753 binds to c-di-GMP and activates TsdA through direct interaction. Finally, Raman spectroscopy and gene knockout results verified that TsdA and two SoxB homologs cooperate to regulate ZVS production. As ZVS is an energy source for E. flavus 21-3, we propose that deep-sea blue light provides E. flavus 21-3 with a selective advantage in the cold seep, suggesting a previously unappreciated relationship between light-sensing pathways and sulfur metabolism in a deep-sea microorganism.

Antibacterial insights into alternariol and its derivative alternariol monomethyl ether produced by a marine fungus.

Alternaria alternata FB1 is a marine fungus identified as a candidate for plastic degradation in our previous study. This fungus has been recently shown to produce secondary metabolites with significant antimicrobial activity against various pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and the notorious aquaculture pathogen Vibrio anguillarum. The antibacterial compounds were purified and identified as alternariol (AOH) and its derivative, alternariol monomethyl ether (AME). We found that AOH and AME primarily inhibited pathogenic bacteria (MRSA or V. anguillarum) by disordering cell division and some other key physiological and biochemical processes. We further demonstrated that AOH could effectively inhibit the unwinding activity of MRSA topoisomerases, which are closely related to cell division and are the potential action target of AOH. The antibacterial activities of AOH and AME were verified by using zebrafish as the in vivo model. Notably, AOH and AME did not significantly affect the viability of normal human liver cells at concentrations that effectively inhibited MRSA or V. anguillarum. Finally, we developed the genetic operation system of A. alternata FB1 and blocked the biosynthesis of AME by knocking out omtI (encoding an O-methyl transferase), which facilitated A. alternata FB1 to only produce AOH. The development of this system in the marine fungus will accelerate the discovery of novel natural products and further bioactivity study.IMPORTANCEMore and more scientific reports indicate that alternariol (AOH) and its derivative alternariol monomethyl ether (AME) exhibit antibacterial activities. However, limited exploration of their detailed antibacterial mechanisms has been performed. In the present study, the antibacterial mechanisms of AOH and AME produced by the marine fungus Alternaria alternata FB1 were disclosed in vitro and in vivo. Given their low toxicity on the normal human liver cell line under the concentrations exhibiting significant antibacterial activity against different pathogens, AOH and AME are proposed to be good candidates for developing promising antibiotics against methicillin-resistant Staphylococcus aureus and Vibrio anguillarum. We also succeeded in blocking the biosynthesis of AME, which facilitated us to easily obtain pure AOH. Moreover, based on our previous results, A. alternata FB1 was shown to enable polyethylene degradation.

Volatile Organic Compounds Produced by a Deep-Sea Bacterium Efficiently Inhibit the Growth of Pseudomonas aeruginosa PAO1.

The deep-sea bacterium Spongiibacter nanhainus CSC3.9 has significant inhibitory effects on agricultural pathogenic fungi and human pathogenic bacteria, especially Pseudomonas aeruginosa, the notorious multidrug-resistant pathogen affecting human public health. We demonstrate that the corresponding antibacterial agents against P. aeruginosa PAO1 are volatile organic compounds (VOCs, namely VOC-3.9). Our findings show that VOC-3.9 leads to the abnormal cell division of P. aeruginosa PAO1 by disordering the expression of several essential division proteins associated with septal peptidoglycan synthesis. VOC-3.9 hinders the biofilm formation process and promotes the biofilm dispersion process of P. aeruginosa PAO1 by affecting its quorum sensing systems. VOC-3.9 also weakens the iron uptake capability of P. aeruginosa PAO1, leading to reduced enzymatic activity associated with key metabolic processes, such as reactive oxygen species (ROS) scavenging. Overall, our study paves the way to developing antimicrobial compounds against drug-resistant bacteria by using volatile organic compounds.

The novel amylase function of the carboxyl terminal domain of Amy63.

α-amylase plays a crucial role in regulating metabolism and health by hydrolyzing of starch and glycogen. Despite comprehensive studies of this classic enzyme spanning over a century, the function of its carboxyl terminal domain (CTD) with a conserved eight ß-strands is still not fully understood. Amy63, identified from a marine bacterium, was reported as a novel multifunctional enzyme with amylase, agarase and carrageenase activities. In this study, the crystal structure of Amy63 was determined at 1.8 Å resolution, revealing high conservation with some other amylases. Interestingly, the independent amylase activity of the carboxyl terminal domain of Amy63 (Amy63_CTD) was newly discovered by the plate-based assay and mass spectrometry. To date, the Amy63_CTD alone could be regarded as the smallest amylase subunit. Moreover, the significant amylase activity of Amy63_CTD was measured over a wide range of temperature and pH, with optimal activity at 60 °C and pH 7.5. The Small-angle X-ray scattering (SAXS) data showed that the high-order oligomeric assembly gradually formed with increasing concentration of Amy63_CTD, implying the novel catalytic mechanism as revealed by the assembly structure. Therefore, the discovery of the novel independent amylase activity of Amy63_CTD suggests a possible missing step or a new perspective in the complex catalytic process of Amy63 and other related α-amylases. This work may shed light on the design of nanozymes to process marine polysaccharides efficiently.

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.

MerF is a novel regulator of deep-sea Pseudomonas stutzeri flagellum biogenesis and motility.

MerF, a proposed bacterial mercury transporter, was surprisingly found to play key roles in the flagellum biogenesis and motility but not mercuric resistance of the deep-sea bacterium Pseudomonas stutzeri 273 in our previous study. However, the mechanism behind this interesting discovery has not been elucidated. Here, we firstly applied the combined transcriptomic and proteomic analysis to the P. stutzeri 273 wild type and merF deletion mutant. The results showed that expressions of extracellular flagellar components and FliS, a key factor controlling the biogenesis of extracellular flagellar filament, were significantly downregulated in the merF deletion mutant. In combination of genetic and biochemical methods, MerF was further demonstrated to regulate the expression of fliS via directly binding to its promoter, which is consistent with the discovery that MerF is essential for bacterial flagellum biogenesis and motility. Importantly, the expression of merF and fliS could be simultaneously upregulated by different heavy metals and MerF homologues exist in both bacterial and archaeal domains. To the best of our knowledge, this is the first report linking the heavy metal transporter and the flagellum biogenesis and motility in microorganisms, which provides a good model to investigate the unexplored adaptation strategies of deep-sea microbes against harsh conditions.

Formation of cadmium sulfide nanoparticles mediates cadmium resistance and light utilization of the deep-sea bacterium Idiomarina sp. OT37-5b.

Heavy metal is one of the major factors threatening the survival of microorganisms. Here, a deep-sea bacterium designated Idiomarina sp. OT37-5b possessing strong cadmium (Cd) tolerance was isolated from a typical hydrothermal vent. Both the Cd-resistance and removal efficiency of Idiomarina sp. OT37-5b were significantly promoted by the supplement of cysteine and meanwhile large amount of CdS nanoparticles were observed. Production of H2 S from cysteine catalysed by methionine gamma-lyase was further demonstrated to contribute to the formation of CdS nanoparticles. Proteomic results showed the addition of cysteine effectively enhanced the efflux of Cd, improved the activities of reactive oxygen species scavenging enzymes, and thereby boosted the nitrogen reduction and energy production of Idiomarina sp. OT37-5b. Notably, the existence of CdS nanoparticles obviously promoted the growth of Idiomarina sp. OT37-5b when exposed to light, indicating this bacterium might grab light energy through CdS nanoparticles. Proteomic analysis revealed the expression levels of essential components for light utilization including electron transport, cytochrome complex and F-type ATPase were significantly up-regulated, which strongly suggested the formation of CdS nanoparticles promoted light utilization and energy production. Our results provide a good model to investigate the uncovered mechanisms of self-photosensitization of nonphotosynthetic bacteria for light-to-chemical production in the deep biosphere.

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.

Calcium protects bacteria against cadmium stress via reducing nitric oxide production and increasing iron acquisition.

Cadmium (Cd) is a common toxic heavy metal in the environment, and bacteria have evolved different strategies against Cd-toxicity. Here, we found that marine bacterium Bacillus sp. 98 could significantly alleviate Cd-toxicity by recruiting calcium (Ca) for reducing excessive intracellular nitric oxide (NO) and enhancing iron acquisition. To investigate the underlying mechanisms, mass spectrometry-based proteomic analysis was applied to Bacillus sp. 98 after treated with Cd supplemented with or without Ca. Compared with bacterial cells treated with Cd only, the proteomic results showed that the expression level of NO synthase was markedly down-regulated, while the expression levels of NO dioxygenase, which is responsible for converting NO to nitrate, and proteins associated with iron uptake were profoundly enhanced when Ca was supplemented. Consistently, bacterial intracellular NO amount was dramatically increased after Bacillus sp. 98 was treated with Cd, and reversed to a normal level when Ca or iron was supplemented. Notably, Ca also protected bacteria against stresses from other heavy metals including Cu, Cr, Mn, Ni and Zn, and this self-protection strategy was adopted as well in zebrafish, which encourages us to develop Ca-associated products against heavy metals toxicity in the future.

Maribellus comscasis sp. nov., a novel deep-sea Bacteroidetes bacterium, possessing a prominent capability of degrading cellulose.

Bacteroidetes are thought to be specialized for the degradation of algae-derived ocean polysaccharides. Here, we show that Bacteroidetes are the predominant phylum in deep-sea sediments and possess more genes associated with polysaccharides degradation than other bacteria. We have isolated a novel Bacteroidetes species from the deep-sea sediments by using a special polysaccharide containing medium, Maribellus comscasis WC007, which possesses 82 putative polysaccharide utilization loci (PULs) containing 374 glycoside hydrolases and 82 SusC/D pairs (Sus indicates starch utilization system; SusC represents the actual TonB-dependent transporter, and SusD is an associated substrate-binding outer membrane lipoprotein) together with 58 sigma/antisigma factors. Through an in-depth analysis of these PULs, strain WC007 can efficiently degrade numerous different polysaccharides including cellulose, pectin, fucoidan, mannan, xylan and starch, which are verified by growth assays. Notably, we find that cellulose has the most significant growth-promoting effect on M. comscasis WC007. And based on scanning electron microscope observation, transcriptomics and metabolomics, we further report on the underlying mechanisms of cellulose degradation and utilization, as well as potential contributions to the carbon cycle. Overall, our results suggest that Bacteroidetes may play key roles in the carbon cycle, likely due to their high abundance and prominent polysaccharide degradation capabilities.

Metagenomic Insights into the Metabolic and Ecological Functions of Abundant Deep-Sea Hydrothermal Vent DPANN Archaea.

Due to their unique metabolism and important ecological roles, deep-sea hydrothermal archaea have attracted great scientific interest. Among these archaea, DPANN superphylum archaea are widely distributed in hydrothermal vent environments. However, DPANN metabolism and ecology remain largely unknown. In this study, we assembled 20 DPANN genomes among 43 reconstructed genomes obtained from deep-sea hydrothermal vent sediments. Phylogenetic analysis suggests 6 phyla, comprised of Aenigmarchaeota, Diapherotrites, Nanoarchaeota, Pacearchaeota, Woesearchaeota, and a new candidate phylum we have designated Kexuearchaeota These are included in the 20 DPANN archaeal members, indicating their broad diversity in this special environment. Analyses of their metabolism reveal deficiencies due to their reduced genome size, including gluconeogenesis and de novo nucleotide and amino acid biosynthesis. However, DPANN archaea possess alternate strategies to address these deficiencies. DPANN archaea also have the potential to assimilate nitrogen and sulfur compounds, indicating an important ecological role in the hydrothermal vent system.IMPORTANCE DPANN archaea show high distribution in the hydrothermal system, although they display small genome size and some incomplete biological processes. Exploring their metabolism is helpful to understand how such small forms of life adapt to this unique environment and what ecological roles they play. In this study, we obtained 20 high-quality metagenome-assembled genomes (MAGs) corresponding to 6 phyla of the DPANN group (Aenigmarchaeota, Diapherotrites, Nanoarchaeota, Pacearchaeota, Woesearchaeota, and a new candidate phylum designated Kexuearchaeota). Further metagenomic analyses provided insights on the metabolism and ecological functions of DPANN archaea to adapt to deep-sea hydrothermal environments. Our study contributes to a deeper understanding of their special lifestyles and should provide clues to cultivate this important archaeal group in the future.

EPS364, a Novel Deep-Sea Bacterial Exopolysaccharide, Inhibits Liver Cancer Cell Growth and Adhesion.

The prognosis of liver cancer was inferior among tumors. New medicine treatments are urgently needed. In this study, a novel exopolysaccharide EPS364 was purified from Vibrio alginolyticus 364, which was isolated from a deep-sea cold seep of the South China Sea. Further research showed that EPS364 consisted of mannose, glucosamine, gluconic acid, galactosamine and arabinose with a molar ratio of 5:9:3.4:0.5:0.8. The relative molecular weight of EPS364 was 14.8 kDa. Our results further revealed that EPS364 was a ß-linked and phosphorylated polysaccharide. Notably, EPS364 exhibited a significant antitumor activity, with inducing apoptosis, dissipation of the mitochondrial membrane potential (MMP) and generation of reactive oxygen species (ROS) in Huh7.5 liver cancer cells. Proteomic and quantitative real-time PCR analyses indicated that EPS364 inhibited cancer cell growth and adhesion via targeting the FGF19-FGFR4 signaling pathway. These findings suggest that EPS364 is a promising antitumor agent for pharmacotherapy.

The cyclic lipopeptides suppress the motility of Vibrio alginolyticus via targeting the Na+ -driven flagellar motor component MotX.

In our previous study, we found that pumilacidin-like cyclic lipopeptides (CLPs) derived from marine bacterium Bacillus sp. strain 176 significantly suppressed the mobile capability and virulence of Vibrio alginolyticus. Here, to further disclose the mechanism of CLPs inhibiting the motility of V. alginolyticus, we first applied transcriptomic analysis to V. alginolyticus treated with or without CLPs. The transcriptomic results showed that the expression of several important components of the Na+ -driven flagellar motor closely related to bacterial motility were markedly suppressed, suggesting that the structure and function of Na+ -driven flagellar motor might be disabled by CLPs. The transcriptomic data were further analysed by the protein-protein interaction network, and the results supported that MotX, one of the essential components of Na+ -driven flagellar motor was most likely the action target of CLPs. In combination of gene knockout, electrophoretic mobility shift assay and immunoblotting techniques, CLPs were demonstrated to affect the rotation of flagella of Vibrio alginolyticus via direct interacting with the Na+ -driven flagellar motor component MotX, which eventually inhibited the bacterial motility. Interestingly, homologues of MotX were found broadly distributed and highly conserved in different pathogenic species, which extends the application range of CLPs as an antibacterial drug targeting bacterial motility in many pathogens.

Structural and Functional Insights into Iturin W, a Novel Lipopeptide Produced by the Deep-Sea Bacterium Bacillus sp. Strain wsm-1.

In the present study, a deep-sea bacterial strain designated Bacillus sp. strain wsm-1 was screened and found to exhibit strong antifungal activity against many plant-pathogenic fungi, and corresponding antifungal agents were thereby purified and determined by tandem mass spectrometry to be two cyclic lipopeptide homologs. These homologs, which were different from any previously reported lipopeptides, were identified to possess identical amino acid sequences of ß-amino fatty acid-Asn-Ser-Asn-Pro-Tyr-Asn-Gln and deduced as two novel lipopeptides designated C14 iturin W and C15 iturin W. Electron microscopy observation indicated that both iturin W homologs caused obvious morphological changes and serious disruption of plasma membrane toward fungal cells, while C15 iturin W exhibited more serious cell damages than C14 iturin W did, which was well consistent with the results of the antifungal activity assays. To improve the yield and antifungal activity of iturin W, the effects of different carbon and nitrogen sources and amino acids on production of C14 iturin W and C15 iturin W were investigated. The results indicated that supplements of most of the detected carbon and nitrogen sources could increase the yield of C14 iturin W, but inhibit the yield of C15 iturin W, while supplements of tryptone and most of the detected amino acids could increase the yield of both C14 iturin W and C15 iturin W.IMPORTANCE Plant disease caused by pathogenic fungi is one of the most devastating diseases, which affects the food safety of the whole world to a great extent. Biological control of plant diseases by microbial natural products is more desirable than traditional chemical control. In this study, we discovered a novel lipopeptide, iturin W, with promising prospects in biological control of plant diseases. Moreover, the effects of different carbon and nitrogen sources and amino acids on production of C14 iturin W and C15 iturin W would provide a reasonable basis for the optimization of the fermentation process of lipopeptides. Notably, the structure of iturin W was different from that of any previously reported lipopeptide, suggesting that deep-sea microorganisms might produce many novel natural products and have significant potential in the development of biological products in the future.

Sorafenib kills liver cancer cells by disrupting SCD1-mediated synthesis of monounsaturated fatty acids via the ATP-AMPK-mTOR-SREBP1 signaling pathway.

Sorafenib is a multikinase inhibitor that is effective in treating advanced liver cancer. Although its mechanism of action through several established cancer-related protein kinase targets is well-characterized, sorafenib induces variable responses among human tumors, and the cause for this variation is yet unknown. To investigate the underlying mechanisms, we applied mass spectrometry-based proteomic analysis to Huh7.5 human liver cancer cells and found that sorafenib significantly affected the expression of the key lipogenic enzymes, especially stearoyl coenzyme A desaturase 1 (SCD1), in these cells. Given that SCD1 catalyzes the most crucial and rate-limiting step in the synthesis of monounsaturated fatty acids (FAs), we performed a lipidomic analysis, which showed a dramatically altered lipid profile in sorafenib-treated cells. Detection and analysis of free FAs showed that the levels of monounsaturated FAs, including oleate, were significantly decreased in those cells treated by sorafenib. Addition of oleate protected liver cancer cells from sorafenib-induced death and alleviated the abnormalities of mitochondrial morphology and function caused by the drug. Treatment with sorafenib suppressed ATP production, resulting in AMPK activation via phosphorylation. Further secondary effects included reduction of the levels of sterol regulatory element-binding protein 1 (SREBP1) and the phosphorylation of mammalian target of rapamycin (mTOR) in liver cancer cells. These effects were partly abolished in the presence of compound C (an AMPK inhibitor) and ATP and adenosine, and SREBP1c overexpression also could be resistant to the effects of sorafenib, suggesting that the sorafenib-induced reduction in cell viability was mediated by the ATP-AMPK-mTOR-SREBP1 signaling pathway. Taken together, our results suggest that sorafenib's anticancer activity in liver cancer cells is based on the inhibition of ATP production, SCD1 expression, and monounsaturated FA synthesis. In addition, the decreased monounsaturated FA synthesis further triggered the more serious reduction of ATP production in sorafenib-treated cells. To our knowledge, this is the first evidence that sorafenib disrupts lipogenesis and triggers liver cancer cell death by targeting SCD1 through the ATP-AMPK-mTOR-SREBP1 pathway.-Liu, G., Kuang, S., Cao, R., Wang, J., Peng, Q., Sun, C. Sorafenib kills liver cancer cells by disrupting SCD1-mediated synthesis of monounsaturated fatty acids via the ATP-AMPK-mTOR- SREBP1 signaling pathway.

Sphingosinithalassobacter tenebrarum sp. nov., isolated from a deep-sea cold seep.

A Gram-stain-negative, facultatively anaerobic, yellow-pigmented, non-motile, rod-shaped bacterium, designated zrk23T, was isolated from a deep-sea cold seep. The strain was characterized by a polyphasic approach to clarify its taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequences placed zrk23T within the genus Sphingosinithalassobacter and showed the highest similarity to Sphingosinithalassobacter portus FM6T (97.93 %). Growth occurs at temperatures from 16 to 45 °C (optimum, 30 °C), at pH values between pH 6.0 and 8.5 (optimum, pH 7.0) and in 0-5.0 % (w/v) NaCl (optimum, 1.5 %). The major fatty acids were C16 : 0, C14 : 0 2-OH and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The major isoprenoid quinone was ubiquinone-10. Predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol, one unidentified phosphoglycolipid, three unidentified glycolipids and three unidentified phospholipids. The G+C content of the genomic DNA was 64.69 %. The average nucleotide identity values between zrk23T and the most closely related available genome, of Sphingosinithalassobacter portus FM6T, was 82.21 %, indicating that zrk23T was clearly distinguished from S. portus. The analysis of genome sequence of zrk23T revealed that there were many genes associated with degradation of aromatic compounds existing in the genome of zrk23T. As a result of the combination of the results of phylogenetic analysis and phenotypic and chemotaxonomic data, zrk23T was considered to represent a novel species of the genus Sphingosinithalassobacter, for which the name Sphingosinithalassobacter tenebrarum sp. nov. is proposed. The type strain is zrk23T (=KCTC 72896T=MCCC 1K04416T).

Proteoglycan from Bacillus sp. BS11 Inhibits the Inflammatory Response by Suppressing the MAPK and NF-κB Pathways in Lipopolysaccharide-Induced RAW264.7 Macrophages.

Inflammation is involved in the pathogenesis of many debilitating diseases. Proteoglycan isolated from marine Bacillus sp. BS11 (EPS11) was shown to have anticancer activity, but its anti-inflammatory potential remains elusive. In the present study, the anti-inflammatory effects and mechanism of EPS11 were evaluated using a lipopolysaccharide (LPS)-induced RAW264.7 macrophage model. Biochemical characterization showed that the total sugar content and protein content of EPS11 were 49.5% and 30.2% respectively. EPS11 was composed of mannose, glucosamine, galactosamine, glucose, galactose, rhamnose, and glucuronic acid. Its molecular weight was determined to be 3.06 × 105 Da. The protein determination of EPS11 was also performed. EPS11 displayed a strong anti-inflammatory effect on LPS-stimulated RAW264.7 macrophages in vitro, which significantly suppressed inflammatory cytokines and mediators (such as NO, TNF-α, IL-6 and IL-1ß, and COX-2). Western blot analysis indicated that EPS11 could downregulate the expression of many key proteins in mitogen-activated protein kinases (MAPKs) and transcription factor nuclear factor-κB (NF-κB) signaling pathways. In particular, EPS11 almost completely inhibited the expression of NF-κB P65, which indicated that EPS11 acted primarily on the NF-κB pathways. These findings offer new insights into the molecular mechanism underlying the anti-inflammatory effect of EPS11.

Characterization of Antifungal Lipopeptide Biosurfactants Produced by Marine Bacterium Bacillus sp. CS30.

This study was initiated to screen for marine bacterial agents to biocontrol Magnaporthe grisea, a serious fungal pathogen of cereal crops. A bacterial strain, isolated from the cold seep in deep sea, exhibited strong growth inhibition against M. grisea, and the strain was identified and designated as Bacillus sp. CS30. The corresponding antifungal agents were purified by acidic precipitation, sequential methanol extraction, Sephadex LH-20 chromatography, and reversed phase high-performance liquid chromatography (RP-HPLC), and two antifungal peaks were obtained at the final purification step. After analysis by mass spectrometry (MS) and tandem MS, two purified antifungal agents were deduced to belong to the surfactin family, and designated as surfactin CS30-1 and surfactin CS30-2. Further investigation showed that although the antifungal activity of surfactin CS30-1 is higher than that of surfactin CS30-2, both of them induced the increased generation of reactive oxygen species (ROS) and caused serious damage to the cell wall and cytoplasm, thus leading to the cell death of M. grisea. Our results also show the differences of the antifungal activity and antifungal mechanism of the different surfactin homologs surfactin CS30-1 and surfactin CS30-2, and highlight them as potential promising agents to biocontrol plant diseases caused by M. grisea.

Marine Bacterial Polysaccharide EPS11 Inhibits Cancer Cell Growth and Metastasis via Blocking Cell Adhesion and Attenuating Filiform Structure Formation.

Our previous results suggested that EPS11, a novel marine bacterial polysaccharide, might be a potential drug candidate for human non-small cell lung carcinoma treatment. In this study, we further investigate the anticancer mechanisms against liver cancer and the anti-metastatic effects in vivo of EPS11. Firstly, we found that EPS11 exerts cytotoxic effects via blocking cell adhesion and destroying filiform structure formation in Huh7.5 cells. Moreover, mass spectrometry-based proteomic analysis of EPS11-treated Huh7.5 cells revealed that expression of many adhesion-related proteins was significantly changed. It is noteworthy that the expression of CD99, a key factor related to cell adhesion, migration and cell death, is remarkably down-regulated after EPS11 treatment. Importantly, over-expression of CD99 partly rescues cell death rate, and improves cell adhesion and migration ability in Huh7.5 treated by EPS11. Thus, we propose that CD99 is a potential action target of EPS11, inhibiting cancer cell proliferation, adhesion and migration. Notably, administration of EPS11 simultaneously with tumor induction evidently reduces tumor nodule formation in the lungs, which strongly indicates that EPS11 has anti-metastatic effects in vivo. Taken together, our results suggest that EPS11 inhibits liver cancer cell growth via blocking cell adhesion and attenuating filiform structure formation, and has potential as an anti-cancer drug, targeting metastasis of cancer cells, in the future.

Fengycins, Cyclic Lipopeptides from Marine Bacillus subtilis Strains, Kill the Plant-Pathogenic Fungus Magnaporthe grisea by Inducing Reactive Oxygen Species Production and Chromatin Condensation.

Rice blast caused by the phytopathogen Magnaporthe grisea poses a serious threat to global food security and is difficult to control. Bacillus species have been extensively explored for the biological control of many fungal diseases. In the present study, the marine bacterium Bacillus subtilis BS155 showed a strong antifungal activity against M. grisea The active metabolites were isolated and identified as cyclic lipopeptides (CLPs) of the fengycin family, named fengycin BS155, by the combination of high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Analyses using scanning and transmission electron microscopy revealed that fengycin BS155 caused morphological changes in the plasma membrane and cell wall of M. grisea hyphae. Using comparative proteomic and biochemical assays, fengycin BS155 was demonstrated to reduce the mitochondrial membrane potential (MMP), induce bursts of reactive oxygen species (ROS), and downregulate the expression level of ROS-scavenging enzymes. Simultaneously, fengycin BS155 caused chromatin condensation in fungal hyphal cells, which led to the upregulation of DNA repair-related protein expression and the cleavage of poly(ADP-ribose) polymerase (PARP). Altogether, our results indicate that fengycin BS155 acts by inducing membrane damage and dysfunction of organelles, disrupting MMP, oxidative stress, and chromatin condensation, resulting in M. grisea hyphal cell death. Therefore, fengycin BS155 and its parent bacterium are very promising candidates for the biological control of M. grisea and the associated rice blast and should be further investigated as such.IMPORTANCE Rice (Oryza sativa L.) is the most important crop and a primary food source for more than half of the world's population. Notably, scientists in China have developed several types of rice that can be grown in seawater, avoiding the use of precious freshwater resources and potentially creating enough food for 200 million people. The plant-affecting fungus Magnaporthe grisea is the causal agent of rice blast disease, and biological rather than chemical control of this threatening disease is highly desirable. In this work, we discovered fengycin BS155, a cyclic lipopeptide material produced by the marine bacterium Bacillus subtilis BS155, which showed strong activity against M. grisea Our results elucidate the mechanism of fengycin BS155-mediated M. grisea growth inhibition and highlight the potential of B. subtilis BS155 as a biocontrol agent against M. grisea in rice cultivation under both fresh- and saltwater conditions.