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.

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.

A Deep-Sea Bacterium Is Capable of Degrading Polyurethane.

Plastic wastes have been recognized as the most common and durable marine contaminants, which are not only found in the shallow water, but also on the sea floor. However, whether deep-sea microorganisms have evolved the capability of degrading plastic remains elusive. In this study, a deep-sea bacterium Bacillus velezensis GUIA was found to be capable of degrading waterborne polyurethane. Transcriptomic analysis showed that the supplement of waterborne polyurethane upregulated the expression of many genes related to spore germination, indicating that the presence of plastic had effects on the growth of strain GUIA. In addition, the supplement of waterborne polyurethane also evidently upregulated the expressions of many genes encoding lipase, protease, and oxidoreductase. Liquid chromatography-mass spectrometry (LC-MS) results showed that potential enzymes responsible for plastic degradation in strain GUIA were identified as oxidoreductase, protease, and lipase, which was consistent with the transcriptomic analysis. In combination of in vitro expression and degradation assays as well as Fourier transform infrared (FTIR) analysis, we demonstrated that the oxidoreductase Oxr-1 of strain GUIA was the key degradation enzyme toward waterborne polyurethane. Moreover, the oxidoreductase Oxr-1 was also shown to degrade the biodegradable polybutylene adipate terephthalate (PBAT) film indicating its wide application potential. IMPORTANCE The widespread and indiscriminate disposal of plastics inevitably leads to environmental pollution. The secondary pollution by current landfill and incineration methods causes serious damage to the atmosphere, land, and rivers. Therefore, microbial degradation is an ideal way to solve plastic pollution. Recently, the marine environment is becoming a hot spot to screen microorganisms possessing potential plastic degradation capabilities. In this study, a deep-sea Bacillus strain was shown to degrade both waterborne polyurethane and biodegradable PBAT film. The FAD-binding oxidoreductase Oxr-1 was demonstrated to be the key enzyme mediating plastic degradation. Our study not only provided a good candidate for developing bio-products toward plastic degradation but also paved a way to investigate the carbon cycle mediated by plastic degradation in deep-sea microorganisms.

Study of Microbial Sulfur Metabolism in a Near Real-Time Pathway through Confocal Raman Quantitative 3D Imaging.

As microbial sulfur metabolism significantly contributes to the formation and cycling of deep-sea sulfur, studying their sulfur metabolism is important for understanding the deep-sea sulfur cycle. However, conventional methods are limited in near real-time studies of bacterial metabolism. Recently, Raman spectroscopy has been widely used in studies on biological metabolism due to its low-cost, rapid, label-free, and nondestructive features, providing us with new approaches to solve the above limitation. Here, we used the confocal Raman quantitative 3D imaging method to nondestructively detect the growth and metabolism of Erythrobacter flavus 21-3 in the long term and near real time, which possessed a pathway mediating the formation of elemental sulfur in the deep sea, but the dynamic process was unknown. In this study, its dynamic sulfur metabolism was visualized and quantitatively assessed in near real time using 3D imaging and related calculations. Based on 3D imaging, the growth and metabolism of microbial colonies growing under both hyperoxic and hypoxic conditions were quantified by volume calculation and ratio analysis. Additionally, unprecedented details of growth and metabolism were uncovered by this method. Due to this successful application, this method is potentially significant for analyzing the in situ biological processes of microorganisms in the future. IMPORTANCE Microorganisms contribute significantly to the formation of deep-sea elemental sulfur, so studies on their growth and dynamic sulfur metabolism are important to understand the deep-sea sulfur cycle. However, near real-time in situ nondestructive metabolic studies of microorganisms remain a great challenge due to the limitations of existing methods. We thus used an imaging-related workflow by confocal Raman microscopy. More detailed descriptions of the sulfur metabolism of E. flavus 21-3 were disclosed, which perfectly complemented previous research results. Therefore, this method is potentially significant for analyzing the in-situ biological processes of microorganisms in the future. To our knowledge, this is the first label-free and nondestructive in situ technique that can provide temporally persistent 3D visualization and quantitative information about bacteria.

Deep-Sea In Situ Insights into the Formation of Zero-Valent Sulfur Driven by a Bacterial Thiosulfate Oxidation Pathway.

Zero-valent sulfur (ZVS) distributes widely in the deep-sea cold seep, which is an important immediate in the sulfur cycle of cold seep. In our previous work, we described a novel thiosulfate oxidation pathway determined by thiosulfate dehydrogenase (TsdA) and thiosulfohydrolase (SoxB) mediating the conversion of thiosulfate to ZVS in the deep-sea cold seep bacterium Erythrobacter flavus 21-3. However, the occurrence and ecological role of this pathway in the deep-sea cold seep were obscure. Here, we cultured E. flavus 21-3 in the deep-sea cold seep for 10 days and demonstrated its capability of forming ZVS in the in situ field. Based on proteomic, stoichiometric analyses and microscopic observation, we found that this thiosulfate oxidation pathway benefited E. flavus 21-3 to adapt the cold seep conditions. Notably, ~25% metagenomes assembled genomes derived from the shallow sediments of cold seeps contained both tsdA and soxB, where presented abundant sulfur metabolism-related genes and active sulfur cycle. Our results suggested that the thiosulfate oxidation pathway determined by TsdA and SoxB existed across many bacteria inhabiting in the cold seep and frequently used by microbes to take part in the active cold seep biogeochemical sulfur cycle. IMPORTANCE The contribution of microbes to the deep-sea cold seep sulfur cycle has received considerable attention in recent years. In the previous study, we isolated E. flavus 21-3 from deep-sea cold seep sediments and described a novel thiosulfate oxidation pathway in the laboratorial condition. It provided a new clue about the formation of ZVS in the cold seep. However, because of huge differences between laboratory and in situ environment, whether bacteria perform the same thiosulfate oxidation pathway in the deep-sea cold seep should be further confirmed. In this work, we verified that E. flavus 21-3 formed ZVS using this pathway in deep-sea cold seep through in situ cultivation, which confirmed the importance of this thiosulfate oxidation pathway and provided an in situ approach to study the real metabolism of deep-sea microorganisms.

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.

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.

Iocasia fonsfrigidae NS-1 gen. nov., sp. nov., a Novel Deep-Sea Bacterium Possessing Diverse Carbohydrate Metabolic Pathways.

Resolving metabolisms of deep-sea microorganisms is crucial for understanding ocean energy cycling. Here, a strictly anaerobic, Gram-negative strain NS-1 was isolated from the deep-sea cold seep in the South China Sea. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain NS-1 was most closely related to the type strain Halocella cellulosilytica DSM 7362T (with 92.52% similarity). A combination of phylogenetic, genomic, and physiological traits with strain NS-1, was proposed to be representative of a novel genus in the family Halanaerobiaceae, for which Iocasia fonsfrigidae NS-1 was named. It is noteworthy that I. fonsfrigidae NS-1 could metabolize multiple carbohydrates including xylan, alginate, starch, and lignin, and thereby produce diverse fermentation products such as hydrogen, lactate, butyrate, and ethanol. The expressions of the key genes responsible for carbohydrate degradation as well as the production of the above small molecular substrates when strain NS-1 cultured under different conditions, were further analyzed by transcriptomic methods. We thus predicted that part of the ecological role of Iocasia sp. is likely in the fermentation of products from the degradation of diverse carbohydrates to produce hydrogen as well as other small molecules, which are in turn utilized by other members of cold seep microbes.

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.

Threonine dehydratase enhances bacterial cadmium resistance via driving cysteine desulfuration and biomineralization of cadmium sulfide nanocrystals.

Biomineralization is often used by microorganisms to sequester heavy metal ions and provides a potential means for remediating increasing levels of heavy metal pollution. Bacteria have been shown to utilize cysteine for the biomineralization of metal sulfide. Indeed, in the present study, the supplement of L-cysteine was found to significantly improve both cadmium resistance and removal abilities of a deep-sea bacterium Pseudomonas stutzeri 273 through cadmium sulfide (CdS) nanoparticle biomineralization. With a proteomic approach, threonine dehydratase of P. stutzeri 273 (psTD) was proposed to be a key factor enhancing bacterial cadmium resistance through catalyzing L-cysteine desulfuration, H2S generation and CdS nanoparticle biomineralization. Consistently, deletion of the gene encoding psTD in P. stutzeri 273 resulted in the decline of H2S generation, decrease of cadmium resistance, and reduction of cadmium removal ability, confirming the unique function of psTD directing the formation of CdS nanoparticles. Correspondingly, the single-enzyme biomineralization of CdS nanoparticle driven by psTD was further developed, and psTD was shown to act as a capping reagent for the mineralization reaction, which controlling the size and structure of nanocrystals. Our results provide important clues for the construction of engineered bacteria for cadmium bioremediation and widen the synthesis methods of nanomaterials.

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.

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.

A novel bacterial thiosulfate oxidation pathway provides a new clue about the formation of zero-valent sulfur in deep sea.

Zero-valent sulfur (ZVS) has been shown to be a major sulfur intermediate in the deep-sea cold seep of the South China Sea based on our previous work, however, the microbial contribution to the formation of ZVS in cold seep has remained unclear. Here, we describe a novel thiosulfate oxidation pathway discovered in the deep-sea cold seep bacterium Erythrobacter flavus 21-3, which provides a new clue about the formation of ZVS. Electronic microscopy, energy-dispersive, and Raman spectra were used to confirm that E. flavus 21-3 effectively converts thiosulfate to ZVS. We next used a combined proteomic and genetic method to identify thiosulfate dehydrogenase (TsdA) and thiosulfohydrolase (SoxB) playing key roles in the conversion of thiosulfate to ZVS. Stoichiometric results of different sulfur intermediates further clarify the function of TsdA in converting thiosulfate to tetrathionate (-O3S-S-S-SO3-), SoxB in liberating sulfone from tetrathionate to form ZVS and sulfur dioxygenases (SdoA/SdoB) in oxidizing ZVS to sulfite under some conditions. Notably, homologs of TsdA, SoxB, and SdoA/SdoB widely exist across the bacteria including in Erythrobacter species derived from different environments. This strongly indicates that this novel thiosulfate oxidation pathway might be frequently used by microbes and plays an important role in the biogeochemical sulfur cycle in nature.