Cultivation of Diverse Novel Marine Bacteria from Deep Ocean Sediment Using Spent Culture Supernatant of Ca. Bathyarchaeia Enrichment.

Most microorganisms resist pure cultivation under conventional laboratory conditions. One of the primary issues for this un-culturability is the absence of biologically produced growth-promoting factors in traditionally defined growth media. However, whether cultivating microbes by providing spent culture supernatant of pivotal microbes in the growth medium can be an effective approach to overcome this limitation is still an under-explored area of research. Here, we used the spent culture medium (SCM) method to isolate previously uncultivated marine bacteria and compared the efficiency of this method with the traditional cultivation (TC) method. In the SCM method, Ca. Bathyarchaeia-enriched supernatant (10%) was used along with recalcitrant organic substrates such as lignin, humic acid, and organic carbon mixture. Ca. Bathyarchaeia, a ubiquitous class of archaea, have the capacity to produce metabolites, making their spent culture supernatant a key source to recover new bacterial stains. Both cultivation methods resulted in the recovery of bacterial species from the phyla Pseudomonadota, Bacteroidota, Actinomycetota, and Bacillota. However, our SCM approach also led to the recovery of species from rarely cultivated groups, such as Planctomycetota, Deinococcota, and Balneolota. In terms of the isolation of new taxa, the SCM method resulted in the cultivation of 80 potential new strains, including one at the family, 16 at the genus, and 63 at the species level, with a novelty ratio of ~ 35% (80/219). In contrast, the TC method allowed the isolation of ~ 10% (19/171) novel strains at species level only. These findings suggest that the SCM approach improved the cultivation of novel and diverse bacteria.

Expanded Archaeal Genomes Shed New Light on the Evolution of Isoprenoid Biosynthesis.

Isoprenoids and their derivatives, essential for all cellular life on Earth, are particularly crucial in archaeal membrane lipids, suggesting that their biosynthesis pathways have ancient origins and play pivotal roles in the evolution of early life. Despite all eukaryotes, archaea, and a few bacterial lineages being known to exclusively use the mevalonate (MVA) pathway to synthesize isoprenoids, the origin and evolutionary trajectory of the MVA pathway remain controversial. Here, we conducted a thorough comparison and phylogenetic analysis of key enzymes across the four types of MVA pathway, with the particular inclusion of metagenome assembled genomes (MAGs) from uncultivated archaea. Our findings support an archaeal origin of the MVA pathway, likely postdating the divergence of Bacteria and Archaea from the Last Universal Common Ancestor (LUCA), thus implying the LUCA's enzymatic inability for isoprenoid biosynthesis. Notably, the Asgard archaea are implicated in playing central roles in the evolution of the MVA pathway, serving not only as putative ancestors of the eukaryote- and Thermoplasma-type routes, but also as crucial mediators in the gene transfer to eukaryotes, possibly during eukaryogenesis. Overall, this study advances our understanding of the origin and evolutionary history of the MVA pathway, providing unique insights into the lipid divide and the evolution of early life.

Description of the first cultured representative of "Candidatus Synoicihabitans" genus, isolated from deep-sea sediment of South China Sea.

In this study we describe the first cultured representative of Candidatus Synoicihabitans genus, a novel strain designated as LMO-M01T, isolated from deep-sea sediment of South China Sea. This bacterium is a facultative aerobe, Gram-negative, non-motile, and has a globular-shaped morphology, with light greenish, small, and circular colonies. Analysis of the 16S rRNA gene sequences of strain LMO-M01T showed less than 93% similarity to its closest cultured members. Furthermore, employing advanced phylogenomic methods such as comparative genome analysis, average nucleotide identity (ANI), average amino acids identity (AAI), and digital DNA-DNA hybridization (dDDH), placed this novel species within the candidatus genus Synoicihabitans of the family Opitutaceae, Phylum Verrucomicrobiota. The genomic analysis of strain LMO-M01T revealed 175 genes, encoding putative carbohydrate-active enzymes. This suggests its metabolic potential to degrade and utilize complex polysaccharides, indicating a significant role in carbon cycling and nutrient turnover in deep-sea sediment. In addition, the strain's physiological capacity to utilize diverse biopolymers such as lignin, xylan, starch, and agar as sole carbon source opens up possibilities for sustainable energy production and environmental remediation. Moreover, the genome sequence of this newly isolated strain has been identified across diverse ecosystems, including marine sediment, fresh water, coral, soil, plants, and activated sludge highlighting its ecological significance and adaptability to various environments. The recovery of strain LMO-M01T holds promise for taxonomical, ecological and biotechnological applications. Based on the polyphasic data, we propose that this ecologically important strain LMO-M01T represents a novel genus (previously Candidatus) within the family Opitutaceae of phylum Verrucomicrobiota, for which the name Synoicihabitans lomoniglobus gen. nov., sp. nov. was proposed. The type of strain is LMO-M01T (= CGMCC 1.61593T = KCTC 92913T).

Plastoquinone synthesis inhibition by tetrabromo biphenyldiol as a widespread algicidal mechanism of marine bacteria.

Algae and bacteria have complex and intimate interactions in the ocean. Besides mutualism, bacteria have evolved a variety of molecular-based anti-algal strategies. However, limited by the unknown mechanism of synthesis and action of these molecules, these strategies and their global prevalence remain unknown. Here we identify a novel strategy through which a marine representative of the Gammaproteobacteria produced 3,3',5,5'-tetrabromo-2,2'-biphenyldiol (4-BP), that kills or inhibits diverse phytoplankton by inhibiting plastoquinone synthesis and its effect cascades to many other key metabolic processes of the algae. Through comparative genomic analysis between the 4-BP-producing bacterium and its algicidally inactive mutant, combined with gene function verification, we identified the gene cluster responsible for 4-BP synthesis, which contains genes encoding chorismate lyase, flavin-dependent halogenase and cytochrome P450. We demonstrated that in near in situ simulated algal blooming seawater, even low concentrations of 4-BP can cause changes in overall phytoplankton community structure with a decline in dinoflagellates and diatoms. Further analyses of the gene sequences from the Tara Oceans expeditions and 2750 whole genome sequences confirmed the ubiquitous presence of 4-BP synthetic genes in diverse bacterial members in the global ocean, suggesting that it is a bacterial tool potentially widely used in global oceans to mediate bacteria-algae antagonistic relationships.

Expanding Asgard members in the domain of Archaea sheds new light on the origin of eukaryotes.

The hypothesis that eukaryotes originated from within the domain Archaea has been strongly supported by recent phylogenomic analyses placing Heimdallarchaeota-Wukongarchaeota branch from the Asgard superphylum as the closest known archaeal sister-group to eukaryotes. However, our understanding is still limited in terms of the relationship between eukaryotes and archaea, as well as the evolution and ecological functions of the Asgard archaea. Here, we describe three previously unknown phylum-level Asgard archaeal lineages, tentatively named Sigyn-, Freyr- and Njordarchaeota. Additional members in Wukongarchaeota and Baldrarchaeota from distinct environments are also reported here, further expanding their ecological roles and metabolic capacities. Comprehensive phylogenomic analyses further supported the origin of eukaryotes within Asgard archaea and a new lineage Njordarchaeota was supposed as the known closest branch with the eukaryotic nuclear host lineage. Metabolic reconstruction suggests that Njordarchaeota may have a heterotrophic lifestyle with capability of peptides and amino acids utilization, while Sigynarchaeota and Freyrarchaeota also have the potentials to fix inorganic carbon via the Wood-Ljungdahl pathway and degrade organic matters. Additionally, the Ack/Pta pathway for homoacetogenesis and de novo anaerobic cobalamin biosynthesis pathway were found in Freyrarchaeota and Wukongrarchaeota, respectively. Some previously unidentified eukaryotic signature proteins for intracellular membrane trafficking system, and the homologue of mu/sigma subunit of adaptor protein complex, were identified in Freyrarchaeota. This study expands the Asgard superphylum, sheds new light on the evolution of eukaryotes and improves our understanding of ecological functions of the Asgard archaea.

The late Archaean to early Proterozoic origin and evolution of anaerobic methane-oxidizing archaea.

Microorganisms, called anaerobic methane-oxidizing archaea (ANME), can reduce a large amount of greenhouse gas methane and therefore have the potential to cool the Earth. We collected nearly all ANMEs genomes in public databases and performed a comprehensive comparative genomic analysis and molecular dating. Our results show that ANMEs originated in the late Archaean to early Proterozoic eon. During this period of time, our planet Earth was experiencing the Great Oxygenation Event and Huronian Glaciation, a dramatic drop in the Earth's surface temperature. This suggests that the emergence of ANMEs may contribute to the reduction of methane at that time, which is an unappreciated potential cause that led to the Huronian Glaciation.

A methylotrophic origin of methanogenesis and early divergence of anaerobic multicarbon alkane metabolism.

Methanogens are considered as one of the earliest life forms on Earth, and together with anaerobic methane-oxidizing archaea, they have crucial effects on climate stability. However, the origin and evolution of anaerobic alkane metabolism in the domain Archaea remain controversial. Here, we present evidence that methylotrophic methanogenesis was the ancestral form of this metabolism. Carbon dioxide-reducing methanogenesis developed later through the evolution of tetrahydromethanopterin S-methyltransferase, which linked methanogenesis to the Wood-Ljungdahl pathway for energy conservation. Anaerobic multicarbon alkane metabolisms in Archaea also originated early, with genes coding for the activation of short-chain or even long-chain alkanes likely evolving from an ethane-metabolizing ancestor. These genes were likely horizontally transferred to multiple archaeal clades including Candidatus (Ca) Bathyarchaeia, Ca. Lokiarchaeia, Ca. Hadarchaeia, and the methanogenic Ca. Methanoliparia.

A methylotrophic origin of methanogenesis and early divergence of anaerobic multicarbon alkane metabolism.

Methanogens are considered as one of the earliest life forms on Earth, and together with anaerobic methane-oxidizing archaea, they have crucial effects on climate stability. Yet, the origin and evolution of anaerobic alkane metabolism in the domain Archaea remain controversial. Here, we show that methanogenesis was already present in the common ancestor of Euryarchaeota, TACK archaea, and Asgard archaea likely in the late Hadean or early Archean eon and that the ancestral methanogen was dependent on methylated compounds and hydrogen. Carbon dioxide-reducing methanogenesis developed later through the evolution of tetrahydromethanopterin S-methyltransferase, which linked methanogenesis to the Wood-Ljungdahl pathway for energy conservation. Multicarbon alkane metabolisms in Archaea also originated early, with genes coding for the activation of short- or even long-chain alkanes likely evolving from an ethane-metabolizing ancestor. These genes were likely horizontally transferred to multiple archaeal clades including Candidatus (Ca) Bathyarchaeota, Ca. Helarchaeota, Ca Hadesarchaeota, and the methanogenic Ca. Methanoliparia.

Insight Into the Pico- and Nano-Phytoplankton Communities in the Deepest Biosphere, the Mariana Trench.

As photoautotrophs, phytoplankton are generally present in the euphotic zone of the ocean, however, recently healthy phytoplankton cells were found to be also ubiquitous in the dark deep sea, i.e., at water depths between 2000 and 4000 m. The distributions of phytoplankton communities in much deeper waters, such as the hadal zone, are unclear. In this study, the vertical distribution of the pico- and nano-phytoplankton (PN) communities from the surface to 8320 m, including the epipelagic, mesopelagic, bathypelagic, and hadal zones, were investigated via both 18S and p23S rRNA gene analysis in the Challenger Deep of the Mariana Trench. The results showed that Dinoflagellata, Chrysophyceae, Haptophyta, Chlorophyta, Prochloraceae, Pseudanabaenaceae, Synechococcaceae, and Eustigmatophyceae, etc., were the predominant PN in the Mariana Trench. Redundancy analyses revealed that depth, followed by temperature, was the most important environmental factors correlated with vertical distribution of PN community. In the hadal zone, the PN community structure was considerably different from those in the shallower zones. Some PN communities, e.g., Eustigmatophyceae and Chrysophyceae, which have the heterotrophic characteristics, were sparse in shallower waters, while they were identified with high relative abundance (94.1% and 20.1%, respectively) at the depth of 8320 m. However, the dinoflagellates and Prochloraceae Prochlorococcus were detected throughout the entire water column. We proposed that vertical sinking, heterotrophic metabolism, and/or the transition to resting stage of phytoplankton might contribute to the presence of phytoplankton in the hadal zone. This study provided insight into the PN community in the Mariana Trench, implied the significance of phytoplankton in exporting organic matters from the euphotic to the hadal zone, and also hinted the possible existence of some undetermined energy metabolism (e.g., heterotrophy) of phytoplankton making themselves adapt and survive in the hadal environment.

Salinisphaera aquimarina sp. nov., isolated from seawater.

A Gram-stain-negative, aerobic, rod-shaped, motile bacterium with a subpolar flagellum, designated strain CCMM005T, was isolated from offshore seawater at Qingdao, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CCMM005T belonged to the genus Salinisphaera and exhibited highest 16S rRNA gene sequence similarity to Salinisphaera dokdonensis CL-ES53T (96.9 %). It showed lower sequence similarities (94.9-96.4 %) with all other representatives of the genus Salinisphaera. Optimal growth occurred in the presence of 4 % (w/v) NaCl, at 30 °C and at pH 7.0. The polar lipids of strain CCMM005T consisted of phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, one unidentified phosphoglycolipid and one unidentified phospholipid. The predominant isoprenoid quinone was Q-8. The major fatty acids were C19 : 0cyclo ω8c, C18 : 0 and C18 : 1ω7c. The DNA G+C content of strain CCMM005T was 65.3 mol%. On the basis of data from this polyphasic study, strain CCMM005T is considered to represent a novel species of the genus Salinisphaera, for which the name Salinisphaera aquimarina sp. nov. is proposed. The type strain is CCMM005T (=MCCC 1K03246T=KCTC 52640T).