Uncultured Microorganisms Research Articles

Assessing Normandy Soil Microbial Diversity for Antibacterial Activities Using Traditional Culture and iChip Methods

Uncultured microorganisms represent a promising and untapped source of antibacterial compounds, crucial in the fight against the significant threat of antimicrobial resistance (AMR). In this study, both traditional and isolation chip (iChip) cultivation techniques were employed to enhance the recovery of known and unknown microorganisms from soils located in Normandy, France. The isolates obtained were identified using 16S rDNA or ITS regions analysis and MALDI-TOF mass spectrometry and were screened for antibacterial activity. A total of 386 isolates, belonging to 6 microbial phyla and distributed across 65 genera, were recovered using both methods. In total, 11 isolates are potentially new bacterial species, and 34 were associated with 22 species described recently. The iChip method yielded a higher diversity of microorganisms (47 genera) than the traditional method (38 genera) and was particularly effective in enriching Actinomycetota. Antibacterial screening against target bacteria showed that 85 isolates (22%) exhibited antibacterial activity. The Streptomyces, Pseudomonas, and Bacillaceae taxa accounted for most antibacterial-producing bacteria with some presenting promising undescribed characteristics. Other active isolates were affiliated with less-known antibacterial producers such as Arthrobacter, Chryseobacterium, Delftia, Ensifer, Flavobacterium, Rahnella, and Stenotrophomonas, among others. These results highlight the potential of our microbial collection as a source of new antibacterial natural products.

Bacterial Diversity, Metabolic Profiling, and Application Potential of Antarctic Soil Metagenomes.

Antarctica has attracted increasing interest in understanding its microbial communities, metabolic potential, and as a source of microbial hydrolytic enzymes with industrial applications, for which advances in next-generation sequencing technologies have greatly facilitated the study of unculturable microorganisms. In this work, soils from seven sub-Antarctic islands and Union Glacier were studied using a whole-genome shotgun metagenomic approach. The main findings were that the microbial community at all sites was predominantly composed of the bacterial phyla Actinobacteria and Cyanobacteria, and the families Streptomycetaceae and Pseudonocardiaceae. Regarding the xenobiotic biodegradation and metabolism pathway, genes associated with benzoate, chloroalkane, chloroalkene, and styrene degradation were predominant. In addition, putative genes encoding industrial enzymes with predicted structural properties associated with improved activity at low temperatures were found, with catalases and malto-oligosyltrehalose trehalohydrolase being the most abundant. Overall, our results show similarities between soils from different Antarctic sites with respect to more abundant bacteria and metabolic pathways, especially at higher classification levels, regardless of their geographic location. Furthermore, our results strengthen the potential of Antarctic soils as a source of industrially relevant enzymes.

Microbial Community Structure in the Taklimakan Desert: The Importance of Nutrient Levels in Medium and Culture Methods.

Although the Taklimakan Desert lacks the necessary nutrients and conditions to support an extensive ecosystem, it is a treasure trove of extremophile resources with special structures and functions. We analyzed the bacterial communities using oligotrophic medium and velvet cloth replicate combined with an extended culture duration. We isolated numerous uncultured microorganisms and rare microorganisms belonging to genera not often isolated or recently described, such as Aliihoeflea, Halodurantibacterium, and Indioceanicola. A total of 669 strains were isolated from the soil of the Taklimakan Desert, which were classified into 5 phyla, 7 classes, 25 orders, 42 families, 83 genera, and 379 species. Among them, 148 strains were potential new species. Our data show that even when working with samples from extreme environments, simple approaches are still useful for cultivating stubborn microbes. Through comparing the isolation effects of different nutrient levels on microbial diversity and abundance, the results show that reducing the nutrient level of the medium was more conducive to improving the culturability of microorganisms in low-nutrient environments, while the high-nutrient medium was more suitable for the isolation of dominant fast-growing strains. This study helps to better reflect the diversity of microbial resources and lays a foundation for the further research and utilization of soil microbial resources in the Taklimakan Desert.

Planktonic Microbial Communities of Thermokarst Lakes of Central Yakutia Demonstrate a High Diversity of Uncultivated Prokaryotes with Uncharacterized Functions

Although thermokarst alas lakes of Central Yakutia are of great climatic and economic importance, there is currently virtually no information on microbial communities and microbial processes in these lakes. This paper characterizes the hydrochemical features and presents a primary analysis of the diversity of planktonic microbial communities in three alas lakes of Central Yakutia — Tyungulyu, Taby, and Kharyyalakh. It was shown that in terms of the water physicochemical composition, the studied lakes were quite typical for this region; they had increased alkalinity and trophicity, but differed from each other in microbiological indicators. Chemoheterotrophic prokaryotes predominated in the studied planktonic communities, but a significant proportion of the 16S rRNA gene sequences were most similar to uncultured microorganisms whose functional potential is still unknown.

Nitrogen fixation in the widely distributed marine γ-proteobacterial diazotroph Candidatus Thalassolituus haligoni.

The high diversity and global distribution of heterotrophic bacterial diazotrophs (HBDs) in the ocean has recently become apparent. However, understanding the role these largely uncultured microorganisms play in marine N2 fixation poses a challenge due to their undefined growth requirements and the complex regulation of the nitrogenase enzyme. We isolated and characterized Candidatus Thalassolituus haligoni, a member of a widely distributed clade of HBD belonging to the Oceanospirillales. Analysis of its nifH gene via amplicon sequencing revealed the extensive distribution of Cand. T. haligoni across the Pacific, Atlantic, and Arctic Oceans. Pangenome analysis indicates that the isolate shares >99% identity with an uncultured metagenome-assembled genome called Arc-Gamma-03, recently recovered from the Arctic Ocean. Through combined genomic, proteomic, and physiological approaches, we confirmed that the isolate fixes N2 gas. However, the mechanisms governing nitrogenase regulation in Cand. T. haligoni remain unclear. We propose Cand. T. haligoni as a globally distributed, cultured HBD model species within this understudied clade of Oceanospirillales.

Exploring the secrets of marine microorganisms: Unveiling secondary metabolites through metagenomics.

Marine microorganisms are increasingly recognized as primary producers of marine secondary metabolites, drawing growing research interest. Many of these organisms are unculturable, posing challenges for study. Metagenomic techniques enable research on these unculturable microorganisms, identifying various biosynthetic gene clusters (BGCs) related to marine microbial secondary metabolites, thereby unveiling their secrets. This review comprehensively analyses metagenomic methods used in discovering marine microbial secondary metabolites, highlighting tools commonly employed in BGC identification, and discussing the potential and challenges in this field. It emphasizes the key role of metagenomics in unveiling secondary metabolites, particularly in marine sponges and tunicates. The review also explores current limitations in studying these metabolites through metagenomics, noting how long-read sequencing technologies and the evolution of computational biology tools offer more possibilities for BGC discovery. Furthermore, the development of synthetic biology allows experimental validation of computationally identified BGCs, showcasing the vast potential of metagenomics in mining marine microbial secondary metabolites.

A Comparison of the Microbial Populations in a Culture-Dependent and a Culture-Independent Analysis of Industrial Water Samples

Culture-dependent and culture-independent microbiological methods are two approaches used to study microbial community composition. Culture-dependent methods have been the standard method used for many years but have limited utility with unculturable microorganisms. Culture-independent methods, including molecular techniques, enable direct analysis of microbial DNA without requiring cultivation. Both culture-dependent and -independent methods have roles in advancing our understanding of microbiology, and a combination of these approaches often yields a comprehensive depiction of the microbial diversity within a dynamic system. Bacterial activity reaction tests (BARTs) are a common culture-dependent test used to identify bacteria growing in industrial water samples. In this study, next-generation sequencing (NGS) was used to identify the taxa growing in BARTs and compared with the BART reaction patterns. Additionally, several water samples were analyzed by both BART and NGS analysis to determine whether the bacteria found in the water were also present in the BARTs. The results showed overall agreement between NGS and BARTs, though, in some cases, the most abundant taxa found in the water samples differed from those in the BARTs. This highlights the need for further study into the microbial community dynamics of culture-dependent tests to determine whether they are representative of the original sample.

Microbial diversity and metabolic pathways linked to benzene degradation in petrochemical-polluted groundwater

The rapid advance in shotgun metagenome sequencing has enabled us to identify uncultivated functional microorganisms in polluted environments. While aerobic petrochemical-degrading pathways have been extensively studied, the anaerobic mechanisms remain less explored. Here, we conducted a study at a petrochemical-polluted groundwater site in Henan Province, Central China. A total of twelve groundwater monitoring wells were installed to collect groundwater samples. Benzene appeared to be the predominant pollutant, detected in 10 out of 12 samples, with concentrations ranging from 1.4 μg/L to 5,280 μg/L. Due to the low aquifer permeability, pollutant migration occurred slowly, resulting in relatively low benzene concentrations downstream within the heavily polluted area. Deep metagenome sequencing revealed Proteobacteria as the dominant phylum, accounting for over 63 % of total abundances. Microbial α-diversity was low in heavily polluted samples, with community compositions substantially differing from those in lightly polluted samples. dmpK encoding the phenol/toluene 2-monooxygenase was detected across all samples, while the dioxygenase bedC1 was not detected, suggesting that aerobic benzene degradation might occur through monooxygenation. Sequence assembly and binning yielded 350 high-quality metagenome-assembled genomes (MAGs), with 30 MAGs harboring functional genes associated with aerobic or anaerobic benzene degradation. About 80 % of MAGs harboring functional genes associated with anaerobic benzene degradation remained taxonomically unclassified at the genus level, suggesting that our current database coverage of anaerobic benzene-degrading microorganisms is very limited. Furthermore, two genes integral to anaerobic benzene metabolism, i.e, benzoyl-CoA reductase (bamB) and glutaryl-CoA dehydrogenase (acd), were not annotated by metagenome functional analyses but were identified within the MAGs, signifying the importance of integrating both contig-based and MAG-based approaches. Together, our efforts of functional annotation and metagenome binning generate a robust blueprint of microbial functional potentials in petrochemical-polluted groundwater, which is crucial for designing proficient bioremediation strategies.

BASALT refines binning from metagenomic data and increases resolution of genome-resolved metagenomic analysis

Metagenomic binning is an essential technique for genome-resolved characterization of uncultured microorganisms in various ecosystems but hampered by the low efficiency of binning tools in adequately recovering metagenome-assembled genomes (MAGs). Here, we introduce BASALT (Binning Across a Series of Assemblies Toolkit) for binning and refinement of short- and long-read sequencing data. BASALT employs multiple binners with multiple thresholds to produce initial bins, then utilizes neural networks to identify core sequences to remove redundant bins and refine non-redundant bins. Using the same assemblies generated from Critical Assessment of Metagenome Interpretation (CAMI) datasets, BASALT produces up to twice as many MAGs as VAMB, DASTool, or metaWRAP. Processing assemblies from a lake sediment dataset, BASALT produces ~30% more MAGs than metaWRAP, including 21 unique class-level prokaryotic lineages. Functional annotations reveal that BASALT can retrieve 47.6% more non-redundant opening-reading frames than metaWRAP. These results highlight the robust handling of metagenomic sequencing data of BASALT.

Mechanisms of extracellular electron transfer in anaerobic methanotrophic archaea

Anaerobic methanotrophic (ANME) archaea are environmentally important, uncultivated microorganisms that oxidize the potent greenhouse gas methane. During methane oxidation, ANME archaea engage in extracellular electron transfer (EET) with other microbes, metal oxides, and electrodes through unclear mechanisms. Here, we cultivate ANME-2d archaea (‘Ca. Methanoperedens’) in bioelectrochemical systems and observe strong methane-dependent current (91–93% of total current) associated with high enrichment of ‘Ca. Methanoperedens’ on the anode (up to 82% of the community), as determined by metagenomics and transmission electron microscopy. Electrochemical and metatranscriptomic analyses suggest that the EET mechanism is similar at various electrode potentials, with the possible involvement of an uncharacterized short-range electron transport protein complex and OmcZ nanowires.

Recovery of 1887 metagenome-assembled genomes from the South China Sea

The South China Sea (SCS) is a marginal sea characterized by strong land-sea biogeochemical interactions. SCS has a distinctive landscape with a multitude of seamounts in its basin. Seamounts create “seamount effects” that influence the diversity and distribution of planktonic microorganisms in the surrounding oligotrophic waters. Although the vertical distribution and community structure of marine microorganisms have been explored in certain regions of the global ocean, there is a lack of comprehensive microbial genomic surveys for uncultured microorganisms in SCS, particularly in the seamount regions. Here, we employed a metagenomic approach to study the uncultured microbial communities sampled from the Xianbei seamount region to the North Coast waters of SCS. A total of 1887 non-redundant prokaryotic metagenome-assembled genomes (MAGs) were reconstructed, of which, 153 MAGs were classified as high-quality MAGs based on the MIMAG standards. The community structure and genomic information provided by this dataset could be used to analyze microbial distribution and metabolism in the SCS.

Genomic and physiological properties of Anoxybacterium hadale gen. nov. sp. nov. reveal the important role of dissolved organic sulfur in microbial metabolism in hadal ecosystems.

Hadal zones account for the deepest 45% of the oceanic depth range and play an important role in ocean biogeochemical cycles. As the least-explored aquatic habitat on earth, hadal ecosystems contain a vast diversity of so far uncultured microorganisms that cannot be grown on conventional laboratory culture media. Therefore, it has been difficult to gain a true understanding of the detailed metabolic characteristics and ecological functions of those difficult-to-culture microorganisms in hadal environments. In this study, a novel anaerobic bacterial strain, MT110T, was isolated from a hadal sediment-water interface sample of the Mariana Trench at 10,890 m. The level of 16S rRNA gene sequence similarity and percentage of conserved proteins between strain MT110T and the closest relatives, Anaerovorax odorimutans DSM 5092T (94.9 and 46.6%) and Aminipila butyrica DSM 103574T (94.4 and 46.7%), indicated that strain MT110T exhibits sufficient molecular differences for genus-level delineation. Phylogenetic analyses based on both 16S rRNA gene and genome sequences showed that strain MT110T formed an independent monophyletic branch within the family Anaerovoracaceae. The combined evidence showed that strain MT110T represents a novel species of a novel genus, proposed as Anoxybacterium hadale gen. nov. sp. nov. (type strain MT110T = KCTC 15922T = MCCC 1K04061T), which represents a previously uncultured lineage of the class Clostridia. Physiologically, no tested organic matter could be used as sole carbon source by strain MT110T. Genomic analysis showed that MT110T had the potential capacity of utilizing various carbon sources, but the pathways of sulfur reduction were largely incomplete. Our experiments further revealed that cysteine is one of the essential nutrients for the survival of strain MT110T, and cannot be replaced by sulfite, leucine, or taurine. This result suggests that organic sulfur compounds might play an important role in metabolism and growth of the family Anaerovoracaceae and could be one of the key factors affecting the cultivation of the uncultured microbes. Our study brings a new perspective to the role of dissolved organic sulfur in hadal ecosystems and also provides valuable information for optimizing the conditions of isolating related microbial taxa from the hadal environment.

Metagenomic Insights into Ecophysiology of Zetaproteobacteria and Gammaproteobacteria in Shallow Zones within Deep-sea Massive Sulfide Deposits.

Deep-sea massive sulfide deposits serve as energy sources for chemosynthetic ecosystems in dark, cold environments even after hydrothermal activity ceases. However, the vertical distribution of microbial communities within sulfide deposits along their depth from the seafloor as well as their ecological roles remain unclear. We herein conducted a culture-independent metagenomic ana-lysis of a core sample of massive sulfide deposits collected in a hydrothermally inactive field of the Southern Mariana Trough, Western Pacific, by drilling (sample depth: 0.52‍ ‍m below the seafloor). Based on the gene context of the metagenome-assembled genomes (MAGs) obtained, we showed the metabolic potential of as-yet-uncultivated microorganisms, particularly those unique to the shallow zone rich in iron hydroxides. Some members of Gammaproteobacteria have potential for the oxidation of reduced sulfur species (such as sulfide and thiosulfate) to sulfate coupled to nitrate reduction to ammonia and carbon fixation via the Calvin-Benson-Bassham (CBB) cycle, as the primary producers. The Zetaproteobacteria member has potential for iron oxidation coupled with microaerobic respiration. A comparative ana-lysis with previously reported metagenomes from deeper zones (~2‍ ‍m below the seafloor) of massive sulfide deposits revealed a difference in the relative abundance of each putative primary producer between the shallow and deep zones. Our results expand knowledge on the ecological potential of uncultivated microorganisms in deep-sea massive sulfide deposits and provide insights into the vertical distribution patterns of chemosynthetic ecosystems.

ACHIEVEMENTS AND PERSPECTIVES OF DEVELOPMENT IN SOIL MICROBIOLOGY AT MOSCOW UNIVERSITY

The article summarizes the results of recent research by the staff of Soil Biology Department Faculty of Soil Science of Lomonosov Moscow State University in the field of assessing the genetic potential of microbial communities of soils and their application in the development of fundamental soil and environmental technologies. Promising areas of further work related to the use of the microbial potential of soils for the purpose of bioremediation territories from ecotoxicants, the development of technologies for selfpurification of soils based on the stimulation of natural communities of microorganisms, as well as the use of microbial cultures for biodegradation of petroleum products, pesticides and synthetic polymers. Another important direction is related to the development of scientific basis for the indication of biological objects in the environment and space objects. Within the framework of this direction, genomic analysis of uncultivated microorganisms from the Arctic, Antarctic and other extreme habitats is carried out, and the knowledge gained apply as a model of alien life. Another relevant direction for the Department of Soil Biology is the development of agrobiotechnologies based on the management of the natural soil microbiome, the creation of microbial preparationsstimulators of plant growth and development, microbiological ways to increase the proportion of biological nitrogen in plant nutrition, application of microbial plant endosymbionts and bioinsecticides. An equally important aspect is the search of producers of biologically active substances, such as phytohormones, antibiotics, enzymes, probiotics, hydrolytics of natural and artificial polymers. The considered areas of research in the field of soil biology are important for improving land management, environmental protection and the development of environmental technologies.

The dogma of the sterile uterus revisited: does microbial seeding occur during fetal life in humans and animals?

Opposing conclusions have been drawn regarding the presence of viable bacteria in the healthy pregnant uterus. Current evidence in humans and animals suggests that fetomaternal tissues present only traces of bacteria whose viability is still to be proven. The debate about the pioneer colonization of the fetus is still open, being the 'in utero colonization' hypothesis versus the 'sterile womb paradigm' the two opposing sides. The seed in this field of research sprouted in human medicine in the last decade and became a central topic in other mammals as well. We aimed to review the literature on bacterial colonization of the healthy placenta, amniotic fluid, and meconium as representatives of the fetal environment. What emerges is that confirming the colonization of fetomaternal tissues by viable bacteria is challenging in humans as well as in animals. Contamination represents the major risk in this type of research as it can be related to different parts of the study design. Sampling at natural parturition or postpartum introduces risk for colonization by the vaginal microbiome of the mother or from the environment. Culture does not reveal the presence of unculturable microorganisms, and sequencing does not allow confirming bacterial viability, while also introducing the variability associated with the data analysis. Therefore, on the basis of the present review, we provide some guidelines on the best practices when performing this type of studies. What emerges from the current literature in humans and animals is that fetomaternal tissues are characterized by a very low biomass, that the viability of bacteria eventually present is still to be confirmed, while massive colonization happens at birth, priming the individual, regardless of the species.

In Situ Discrimination and Cultivation of Active Degraders in Soils by Genome-Directed Cultivation Assisted by SIP-Raman-Activated Cell Sorting.

The identification and in situ cultivation of functional yet uncultivable microorganisms are important to confirm inferences regarding their ecological functions. Here, we developed a new method that couples Raman-activated cell sorting (RACS), stable-isotope probing (SIP), and genome-directed cultivation (GDC)─namely, RACS-SIP-GDC─to identify, sort, and cultivate the active toluene degraders from a complex microbial community in petroleum-contaminated soil. Using SIP, we successfully identified the active toluene degrader Pigmentiphaga, the single cells of which were subsequently sorted and isolated by RACS. We further successfully assembled the genome of Pigmentiphaga based on the metagenomic sequencing of 13C-DNA and genomic sequencing of sorted cells, which was confirmed by gyrB gene comparison and average nucleotide identity determination. Additionally, the genotypes and phenotypes of this degrader were directly linked at the single-cell level, and its complete toluene metabolic pathways in petroleum-contaminated soil were reconstructed. Based on its unique metabolic properties uncovered by genome sequencing, we modified the traditional cultivation medium with antibiotics, amino acids, carbon sources, and growth factors (e.g., vitamins and metals), achieving the successful cultivation of RACS-sorted active degrader Pigmentiphaga sp. Our results implied that RACS-SIP-GDC is a state-of-the-art approach for the precise identification, targeted isolation, and cultivation of functional microbes from complex communities in natural habitats. RACS-SIP-GDC can be used to explore specific and targeted organic-pollution-degrading microorganisms at the single-cell level and provide new insights into their biodegradation mechanisms.

Microbial ecosystem assessment and hydrogen oxidation potential of newly discovered vent systems from the Central and South-East Indian Ridge.

In order to expand the knowledge of microbial ecosystems from deep-sea hydrothermal vent systems located on the Central and South-East Indian Ridge, we sampled hydrothermal fluids, massive sulfides, ambient water and sediments of six distinct vent fields. Most of these vent sites were only recently discovered in the course of the German exploration program for massive sulfide deposits and no previous studies of the respective microbial communities exist. Apart from typically vent-associated chemosynthetic members of the orders Campylobacterales, Mariprofundales, and Thiomicrospirales, high numbers of uncultured and unspecified Bacteria were identified via 16S rRNA gene analyses in hydrothermal fluid and massive sulfide samples. The sampled sediments however, were characterized by an overall lack of chemosynthetic Bacteria and the presence of high proportions of low abundant bacterial groups. The archaeal communities were generally less diverse and mostly dominated by members of Nitrosopumilales and Woesearchaeales, partly exhibiting high proportions of unassigned Archaea. Correlations with environmental parameters were primarily observed for sediment communities and for microbial species (associated with the nitrogen cycle) in samples from a recently identified vent field, which was geochemically distinct from all other sampled sites. Enrichment cultures of diffuse fluids demonstrated a great potential for hydrogen oxidation coupled to the reduction of various electron-acceptors with high abundances of Hydrogenovibrio and Sulfurimonas species. Overall, given the large number of currently uncultured and unspecified microorganisms identified in the vent communities, their respective metabolic traits, ecosystem functions and mediated biogeochemical processes have still to be resolved for estimating consequences of potential environmental disturbances by future mining activities.

Metagenomics offers insights into the rhizospheric bacterial diversity of mushrooms from a tropical forest and temperate forest of India

Bacteria promote mushroom growth by providing growth factors and vitamins, preventing pathogen growth, and enhancing spore distribution. Soil bacteria involved in the nitrogen cycle showed association with the abundance of ectomycorrhizal fungi. Uncultured microorganisms comprise a significant proportion of complex soil ecosystem. Nanopore sequencing Technologies used a genome-resolved metagenomics approach to evaluate the rhizospheric bacterial diversity of these mushrooms from the tropical forests of West Bengal (India) and the temperate forests of West Kameng districts in Arunachal Pradesh, India. The ectomycorrhizosphere soil of tropical forest has higher abundance of nitrogen fixing bacteria such as Bradyrhizobium diazoefficiens, B. erythrophlei, and B. elkanii whereas the ectomycorrhizosphere soil of temperate forest has higher abundance of Candidatus Solibacter usitatus, Rhodoplanes sp. Z2-YC6860, Candidatus Koribacter versatilis, Bacillus cereus, Granulicella tundricola, G. mallensis, Bradyrhizobium icense, Bradyrhizobium sp. SK17, Acidobacterium capsulatum, Bacillus weihaiensis, Terriglobus saanensis, Planococcus sp. MB-3u-03, Bradyrhizobium sp. CCGE-LA001, and Bradyrhizobium japonicum These microorganisms have deep impact in the growth and development of ectomycorrhizal partners (trees and mushrooms) and be called as mycorrhiza helper bacteria (MHB).

Metagenomics and Its Tools and Softwares

Background: Throughout all of nature, microorganisms can be found thriving in a variety of environmental circumstances. Most marine life is either unculturable or challenging to culture using conventional techniques. Methods: Through the analysis of genomic data obtained directly from an environmental sample, metagenomics enables the study of all microorganisms, regardless of whether they can be cultured or not. This allows for the identification of the species present as well as the extraction of knowledge about the functionality of microbial communities in their natural habitat. Result: This article summarizes the state-of-the-art metagenomic approaches used and their associated softwares and tools. In order to comprehend and utilise unculturable microorganisms, metagenomics provides access to the enormous diversity of the microbial world and has significantly advanced both academic communities and industrial settings.

Cultivation and genomic characterization of novel and ubiquitous marine nitrite-oxidizing bacteria from the Nitrospirales

Nitrospirales, including the genus Nitrospira, are environmentally widespread chemolithoautotrophic nitrite-oxidizing bacteria. These mostly uncultured microorganisms gain energy through nitrite oxidation, fix CO2, and thus play vital roles in nitrogen and carbon cycling. Over the last decade, our understanding of their physiology has advanced through several new discoveries, such as alternative energy metabolisms and complete ammonia oxidizers (comammox Nitrospira). These findings mainly resulted from studies of terrestrial species, whereas less attention has been given to marine Nitrospirales. In this study, we cultured three new marine Nitrospirales enrichments and one isolate. Three of these four NOB represent new Nitrospira species while the fourth represents a novel genus. This fourth organism, tentatively named “Ca. Nitronereus thalassa”, represents the first cultured member of a Nitrospirales lineage that encompasses both free-living and sponge-associated nitrite oxidizers, is highly abundant in the environment, and shows distinct habitat distribution patterns compared to the marine Nitrospira species. Partially explaining this, “Ca. Nitronereus thalassa” harbors a unique combination of genes involved in carbon fixation and respiration, suggesting differential adaptations to fluctuating oxygen concentrations. Furthermore, “Ca. Nitronereus thalassa” appears to have a more narrow substrate range compared to many other marine nitrite oxidizers, as it lacks the genomic potential to utilize formate, cyanate, and urea. Lastly, we show that the presumed marine Nitrospirales lineages are not restricted to oceanic and saline environments, as previously assumed.