Deep-sea in situ and laboratory multi-omics provide insights into the sulfur assimilation of a deep-sea Chloroflexota bacterium.

Chloroflexota bacteria are abundant and globally distributed in various deep-sea ecosystems. It has been reported based on metagenomics data that two deep-sea Chloroflexota lineages (the SAR202 group and Dehalococcoidia class) have the potential to drive sulfur cycling. However, the absence of cultured Chloroflexota representatives is a significant bottleneck toward understanding their contribution to the deep-sea sulfur cycling. In this study, we find that Phototrophicus methaneseepsis ZRK33 isolated from deep-sea sediment has a heterotrophic lifestyle and can assimilate sulfate and thiosulfate. Using combined physiological, genomic, proteomic, and in situ transcriptomic methods, we find that strain ZRK33 can perform assimilatory sulfate reduction in both laboratory and deep-sea conditions. Metabolism of sulfate or thiosulfate by strain ZRK33 significantly promotes the transport and degradation of various macromolecules and thereby stimulates the energy production. In addition, metagenomic results show that genes associated with assimilatory and dissimilatory sulfate reduction are ubiquitously distributed in the metagenome-assembled genomes of Chloroflexota members derived from deep-sea sediments. Metatranscriptomic results also show that the expression levels of related genes are upregulated, strongly suggesting that Chloroflexota bacteria may play undocumented roles in deep-sea sulfur cycling. IMPORTANCE: The cycling of sulfur is one of Earth's major biogeochemical processes and is closely related to the energy metabolism of microorganisms living in the deep-sea cold seep and hydrothermal vents. To date, some of the members of Chloroflexota are proposed to play a previously unrecognized role in sulfur cycling. However, the sulfur metabolic characteristics of deep-sea Chloroflexota bacteria have never been reported, and remain to be verified in cultured deep-sea representatives. Here, we show that the deep-sea Chloroflexota bacterium ZRK33 can perform sulfate assimilation in both laboratory and deep-sea conditions, which expands our knowledge of the sulfur metabolic potential of deep-sea Chloroflexota bacteria. We also show that the genes associated with assimilatory and dissimilatory sulfate reduction ubiquitously distribute in the deep-sea Chloroflexota members, providing hints to the roles of Chloroflexota bacteria in deep-sea sulfur biogeochemical cycling.

Physiological and metabolic insights into the first cultured anaerobic representative of deep-sea Planctomycetes bacteria.

Planctomycetes bacteria are ubiquitously distributed across various biospheres and play key roles in global element cycles. However, few deep-sea Planctomycetes members have been cultivated, limiting our understanding of Planctomycetes in the deep biosphere. Here, we have successfully cultured a novel strain of Planctomycetes (strain ZRK32) from a deep-sea cold seep sediment. Our genomic, physiological, and phylogenetic analyses indicate that strain ZRK32 is a novel species, which we propose be named: Poriferisphaera heterotrophicis. We show that strain ZRK32 replicates using a budding mode of division. Based on the combined results from growth assays and transcriptomic analyses, we found that rich nutrients, or supplementation with NO3- or NH4+ promoted the growth of strain ZRK32 by facilitating energy production through the tricarboxylic acid cycle and the Embden-Meyerhof-Parnas glycolysis pathway. Moreover, supplementation with NO3- or NH4+ induced strain ZRK32 to release a bacteriophage in a chronic manner, without host cell lysis. This bacteriophage then enabled strain ZRK32, and another marine bacterium that we studied, to metabolize nitrogen through the function of auxiliary metabolic genes. Overall, these findings expand our understanding of deep-sea Planctomycetes bacteria, while highlighting their ability to metabolize nitrogen when reprogrammed by chronic viruses.

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.

In situ Raman quantitative monitoring of methanogenesis: Culture experiments of a deep-sea cold seep methanogenic archaeon.

Gas production from several metabolic pathways is a necessary process that accompanies the growth and central metabolism of some microorganisms. However, accurate and rapid nondestructive detection of gas production is still challenging. To this end, gas chromatography (GC) is primarily used, which requires sampling and sample preparation. Furthermore, GC is expensive and difficult to operate. Several researchers working on microbial gases are looking forward to a new method to accurately capture the gas trends within a closed system in real-time. In this study, we developed a precise quantitative analysis for headspace gas in Hungate tubes using Raman spectroscopy. This method requires only a controlled focus on the gas portion inside Hungate tubes, enabling nondestructive, real-time, continuous monitoring without the need for sampling. The peak area ratio was selected to establish a calibration curve with nine different CH4-N2 gaseous mixtures and a linear relationship was observed between the peak area ratio of methane to nitrogen and their molar ratios (A(CH4)/A(N2) = 6.0739 × n(CH4)/n(N2)). The results of in situ quantitative analysis using Raman spectroscopy showed good agreement with those of GC in the continuous monitoring of culture experiments of a deep-sea cold seep methanogenic archaeon. This method significantly improves the detection efficiency and shows great potential for in situ quantitative gas detection in microbiology. It can be a powerful complementary tool to GC.

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.

Characterization of the First Cultured Representative of "Candidatus Thermofonsia" Clade 2 within Chloroflexi Reveals Its Phototrophic Lifestyle.

"Candidatus Thermofonsia" represents a novel class within the phylum Chloroflexi. Metagenomic analysis reveals "Ca. Thermofonsia" harbors phototrophs outside the classically phototrophic Chloroflexia class. Unfortunately, the paucity of pure cultures limits further insights into their potential phototrophy. Here, we report the successful isolation of a "Ca. Thermofonsia" representative (Phototrophicus methaneseepsis ZRK33) from a deep-sea cold seep. Using combined physiological, genomic, and transcriptomic methods, we further show the long-wavelength light (e.g., red and infrared light) could promote the growth of strain ZRK33 and upregulate the expression of genes associated with phototrophy. In particular, strain ZRK33 has a typical phototrophic lifestyle under both laboratory and deep-sea conditions. Strain ZRK33 also possesses the ability to fix inorganic carbon through the 3-hydroxypropionate bicycle in both laboratory and deep-sea in situ environments, and the combined autotrophic, phototrophic, and heterotrophic capabilities endow strain ZRK33 with a photomixotrophic lifestyle. Notably, the predicted genes associated with phototrophy broadly exist in the metagenomes of 27 deep-sea Chloroflexi members, strongly suggesting diverse phototrophic Chloroflexi members are distributed in various unexplored deep biospheres. IMPORTANCE The deep ocean microbiota represents the unexplored majority of global ocean waters. The phylum Chloroflexi is abundant and broadly distributed in various deep-sea ecosystems. It was reported that some members of "Candidatus Thermofonsia" clade 2 might possess phototrophs; however, the absence of cultured representatives is a significant bottleneck toward understanding their phototrophic characteristics. In the present study, we successfully isolated a representative of the novel class "Ca. Thermofonsia" from a deep-sea cold seep by using an enrichment medium constantly supplemented with rifampicin, allowing researchers to isolate more Chloroflexi members in the future. Importantly, outside the classically phototrophic Chloroflexia class, we discover a novel phototrophic clade within the phylum Chloroflexi and demonstrate the existence of phototrophic lifestyles in the deep sea. Thus, this study expands the range of phototrophic Chloroflexi and provides a good model to study the mechanism of phototrophy performed in the deep biosphere.

Isolation, Identification and Characterization of Two Kinds of Deep-Sea Bacterial Lipopeptides Against Foodborne Pathogens.

Under multiple stresses of deep sea, many microorganisms have evolved potentials to produce different metabolites to cope with the stresses they face. In this study, we isolated a bacterial strain Bacillus sp. YJ17 from the deep-sea cold seep. Compared with commercial food preservative nisin, it showed broad and strong antibacterial activities against foodborne pathogens, including multiple resistant bacteria Pseudomonas aeruginosa PAO1 and methicillin-resistant Staphylococcus aureus (MRSA). The active agents were purified by reversed-phase high performance liquid chromatography (RP-HPLC). Analysis of high-energy collision induced dissociation mass spectrometry (HCD-MS) showed that the two active agents belong to family of fengycin and surfactin, and based on results of tandem mass spectrometry (HCD-MS/MS), the amino acid sequence of purified fengycin and surfactin might be Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile and Glu-Leu/Ile-Leu/Ile-Leu/Ile-Val-Asp-Leu/Ile, respectively. Since the purified fengycin and surfactin exhibited strong inhibition against P. aeruginosa PAO1 and MRSA respectively, the inhibition mechanisms of fengycin against P. aeruginosa PAO1 and surfactin against MRSA were investigated by electron microscopy. After treatment with purified fengycin, the morphology of P. aeruginosa PAO1 became abnormal and aggregated together, and obvious cytoplasmic leakage was observed. After treatment with purified surfactin, the MRSA cells clustered together, and cell surface became rough and jagged. Further study showed that reactive oxygen species (ROS) accumulation and cell membrane damage occurred in P. aeruginosa PAO1 and MRSA after treated with fengycin and surfactin, respectively. Furthermore, typical ROS scavenging enzymes catalase (CAT) and superoxide dismutase (SOD) were also significantly reduced in P. aeruginosa PAO1 and MRSA after treated with fengycin and surfactin, respectively. Therefore, the inhibition mechanisms of fengycin against P. aeruginosa PAO1 and surfactin against MRSA are closely related with accumulation of ROS, which might be due to the decreased activity of CAT and SOD after treated with fengycin and surfactin, respectively. Overall, our study provides good candidates from the deep-sea environment to deal with foodborne pathogens, especially multidrug-resistant bacteria.

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.

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.

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 Two Unique Cold-Active Lipases Derived from a Novel Deep-Sea Cold Seep Bacterium.

The deep ocean microbiota has unexplored potential to provide enzymes with unique characteristics. In order to obtain cold-active lipases, bacterial strains isolated from the sediment of the deep-sea cold seep were screened, and a novel strain gcc21 exhibited a high lipase catalytic activity, even at the low temperature of 4 °C. The strain gcc21 was identified and proposed to represent a new species of Pseudomonas according to its physiological, biochemical, and genomic characteristics; it was named Pseudomonas marinensis. Two novel encoding genes for cold-active lipases (Lipase 1 and Lipase 2) were identified in the genome of strain gcc21. Genes encoding Lipase 1 and Lipase 2 were respectively cloned and overexpressed in E. coli cells, and corresponding lipases were further purified and characterized. Both Lipase 1 and Lipase 2 showed an optimal catalytic temperature at 4 °C, which is much lower than those of most reported cold-active lipases, but the activity and stability of Lipase 2 were much higher than those of Lipase 1 under different tested pHs and temperatures. In addition, Lipase 2 was more stable than Lipase 1 when treated with different metal ions, detergents, potential inhibitors, and organic solvents. In a combination of mutation and activity assays, catalytic triads of Ser, Asp, and His in Lipase 1 and Lipase 2 were demonstrated to be essential for maintaining enzyme activity. Phylogenetic analysis showed that both Lipase 1 and Lipase 2 belonged to lipase family III. Overall, our results indicate that deep-sea cold seep is a rich source for novel bacterial species that produce potentially unique cold-active enzymes.

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.

Pseudodesulfovibrio cashew sp. Nov., a Novel Deep-Sea Sulfate-Reducing Bacterium, Linking Heavy Metal Resistance and Sulfur Cycle.

Sulfur cycling is primarily driven by sulfate reduction mediated by sulfate-reducing bacteria (SRB) in marine sediments. The dissimilatory sulfate reduction drives the production of enormous quantities of reduced sulfide and thereby the formation of highly insoluble metal sulfides in marine sediments. Here, a novel sulfate-reducing bacterium designated Pseudodesulfovibrio cashew SRB007 was isolated and purified from the deep-sea cold seep and proposed to represent a novel species in the genus of Pseudodesulfovibrio. A detailed description of the phenotypic traits, phylogenetic status and central metabolisms of strain SRB007 allowed the reconstruction of the metabolic potential and lifestyle of a novel member of deep-sea SRB. Notably, P. cashew SRB007 showed a strong ability to resist and remove different heavy metal ions including Co2+, Ni2+, Cd2+ and Hg2+. The dissimilatory sulfate reduction was demonstrated to contribute to the prominent removal capability of P. cashew SRB007 against different heavy metals via the formation of insoluble metal sulfides.

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.

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.

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).

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.

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.

Genetic and Physiological Adaptations of Marine Bacterium Pseudomonas stutzeri 273 to Mercury Stress.

Mercury-mediated toxicity remains one of the greatest barriers against microbial survival, even though bacterial resistance to mercury compounds can occur. However, the genetic and physiological adaptations of bacteria to mercury stress still remains unclear. Here, we show that the marine bacterium Pseudomonas stutzeri 273 is resistant to 50 µM Hg2+ and removes up to 94% Hg2+ from culture. Using gene homologous recombination and complementation, we show that genes encoding Hg2+-transport proteins MerT, MerP, the mercuric reductase MerA and the regulatory protein MerD are essential for bacterial mercuric resistance when challenged with Hg2+. Further, mercury stress inhibits flagellar development, motility, chemotaxis and biofilm formation of P. stutzeri 273, which are verified by transcriptomic and physiological analyses. Surprisingly, we discover that MerF, a previously reported Hg2+-transporter, determines flagellar development, motility and biofilm formation in P. stutzeri 273 by genetic and physiological analyses. Our results strongly indicate that MerF plays an integral role in P. stutzeri 273 to develop physiological responses to mercury stress. Notably, MerF homologs are also prevalent in different human pathogens. Using this unique target may provide novel strategies to control these pathogenic bacteria, given the role of MerF in flagella and biofilm formation. In summary, our data provide an original report on MerF in bacterial physiological development and suggest that the mer in marine bacteria has evolved through progressive, sequential recruitment of novel functions over time.