Genome-resolved metagenomics provides insights into the microbial-mediated sulfur and nitrogen cycling in temperate seagrass meadows

The presence of seagrasses facilitates numerous microbial-mediated biogeochemical cycles, with sulfur- and nitrogen-cycling microorganisms playing crucial roles as regulators. Despite efforts to comprehend the diversity of microbes in seagrass ecosystems, the metabolic functions of these benthic microorganisms in seagrass sediments remain largely unknown. Using metagenomics, we provide insights into the sulfur- and nitrogen-cycling pathways and key metabolic capacities of microorganisms in both Z. japonica-colonized and unvegetated sediments over a seasonal period. Taxonomic analysis of N and S cycling genes revealed that δ- and γ- proteobacteria dominated the benthic sulfate-reducing bacteria, while α- and γ-proteobacteria played a significant role in the sulfur-oxidation processes. The proteobacterial lineages were also major contributors to the benthic nitrogen cycling. However, at a finer taxonomic resolution, microbial participants in different processes were observed to be highly diverse and mainly driven by environmental factors such as temperature and salinity. The gene pools of sulfur and nitrogen cycles in the seagrass sediments were dominated by genes involved in sulfide oxidation (fccA) and hydroxylamine oxidation (hao), respectively. Seagrass colonization elevated the relative abundance of genes responsible for sulfite production (phsC), hydroxylamine oxidation (hao), and nitrogen fixation (nifK), but suppressed sulfur oxidation (soxXYZ) and denitrification (nosZ and nirS). The prevalence of proteobacterial lineages functioned with versatile capabilities in both sulfur and nitrogen cycles in seagrass ecosystems, highlighting tight couplings between these processes, which was further supported by the recovery of 83 metagenome-assembled genomes (MAGs). These findings broaden our understanding of the biogeochemical processes that are mediated by microorganisms in seagrass ecosystems

Spatiotemporal assembly and functional composition of planktonic microeukaryotic communities along productivity gradients in a subtropical lake

Microeukaryotes play crucial roles in the microbial loop of freshwater ecosystems, functioning both as primary producers and bacterivorous consumers. However, understanding the assembly of microeukaryotic communities and their functional composition in freshwater lake ecosystems across diverse environmental gradients remains limited. Here, we utilized amplicon sequencing of 18S rRNA gene and multivariate statistical analyses to examine the spatiotemporal and biogeographical patterns of microeukaryotes in water columns (at depths of 0.5, 5, and 10 m) within a subtropical lake in eastern China, covering a 40 km distance during spring and autumn of 2022. Our results revealed that complex and diverse microeukaryotic communities were dominated by Chlorophyta (mainly Chlorophyceae), Fungi, Alveolata, Stramenopiles, and Cryptophyta lineages. Species richness was higher in autumn than in spring, forming significant hump-shaped relationships with chlorophyll a concentration (Chl-a, an indicator of phytoplankton biomass). Microeukaryotic communities exhibited significant seasonality and distance-decay patterns. By contrast, the effect of vertical depth was negligible. Stochastic processes mainly influenced the assembly of microeukaryotic communities, explaining 63, 67, and 55% of community variation for spring, autumn, and both seasons combined, respectively. Trait-based functional analysis revealed the prevalence of heterotrophic and phototrophic microeukaryotic plankton with a trade-off along N:P ratio, Chl-a, and dissolved oxygen (DO) gradients. Similarly, the mixotrophic proportions were significantly and positively correlated with Chl-a and DO concentrations. Overall, our findings may provide useful insights into the assembly patterns of microeukaryotes in lake ecosystem and how their functions respond to environmental changes

Comparative Transcriptomics Reveals Distinct Gene Expressions of a Model Ciliated Protozoan Feeding on Bacteria-Free Medium, Digestible, and Digestion-Resistant Bacteria

Bacterivory is an important ecological function of protists in natural ecosystems. However, there are diverse bacterial species resistant to protistan digestion, which reduces the carbon flow to higher trophic levels. So far, a molecular biological view of metabolic processes in heterotrophic protists during predation of bacterial preys of different digestibility is still lacking. In this study, we investigated the growth performance a ciliated protozoan Tetrahymena thermophila cultivated in a bacteria-free Super Proteose Peptone (SPP) medium (control), and in the media mixed with either a digestion-resistant bacterial species (DRB) or a digestible strain of E. coli (ECO). We found the protist population grew fastest in the SPP and slowest in the DRB treatment. Fluorescence in situ hybridization confirmed that there were indeed non-digested, viable bacteria in the ciliate cells fed with DRB, but none in other treatments. Comparative analysis of RNA-seq data showed that, relative to the control, 637 and 511 genes in T. thermophila were significantly and differentially expressed in the DRB and ECO treatments, respectively. The protistan expression of lysosomal proteases (especially papain-like cysteine proteinases), GH18 chitinases, and an isocitrate lyase were upregulated in both bacterial treatments. The genes encoding protease, glycosidase and involving glycolysis, TCA and glyoxylate cycles of carbon metabolic processes were higher expressed in the DRB treatment when compared with the ECO. Nevertheless, the genes for glutathione metabolism were more upregulated in the control than those in both bacterial treatments, regardless of the digestibility of the bacteria. The results of this study indicate that not only bacterial food but also digestibility of bacterial taxa modulate multiple metabolic processes in heterotrophic protists, which contribute to a better understanding of protistan bacterivory and bacteria-protists interactions on a molecular basis

Detection of Prokaryotes on the Astomatous Ciliated Protist Kentrophoros flavus (Ciliophora, Karyorelictea) Revealed A Consistently Associated Muribaculaceae-Like Bacterium

The interactions between symbiotic bacterial consortia and their protist hosts in benthic environments have attracted increasing interest in recent years. In the present study, we investigated the diversity of potentially associated bacteria for an astomatous ciliate, Kentrophoros flavus, collected in the intertidal zone of Yantai, China. For the first time, the diversity of the associated bacteria in the species K. flavus was examined using 16S rRNA-based techniques (clone libraries and PacBio sequencing) and the fluorescence in situ hybridization (FISH) technique. The 16S rRNA-based sequencing revealed a higher diversity of associated bacteria in K. flavus than previously expected. In addition to a genus-typical thiotrophic symbiont, the "Candidatus Kentron" stain YE, we provide evidence showing the consistent existence of one Muribaculaceae-like bacterium that was secondarily abundant among the bacterial operational taxonomic units (OTUs). Fluorescence in situ hybridization (FISH) with three specific probes and double-label FISH experiments with "Candidatus Kentron" probes showed that the Muribaculaceae-like bacterium was abundant and merged with the "Candidatus Kentron" stain YE on the cell surface of the host. A phylogenetic analysis of the bacterial 16S rRNA gene showed that the bacterium was a distinct branch in Muribaculaceae, members of which are primarily reported from gut microbiome. The name "Muribaculaceae-like bacterium associated with Kentrophoros flavus" (MLAKF) is proposed for the new bacterium. The higher 16S rRNA diversity in K. flavus and the discovery of MLAKF on the cell surface both suggest a potential bacterial consortium that interacts with the host K. flavus

Environmental Factors and Pollution Stresses Select Bacterial Populations in Association With Protists

International audienceDigestion-resistant bacteria (DRB) refer to the ecological bacterial group that can be ingested, but not digested by protistan grazers, thus forming a specific type of bacteria-protist association. To test the hypothesis that the environment affects the assembly of DRB in protists, a mixotrophic ciliate, Paramecium bursaria, and a heterotrophic ciliate, Euplotes vannus, were reared at different temperatures, light conditions, and concentration gradients of antibiotic oxytetracycline and heavy metals. Community profiling indicated that the composition of DRB in both species varied significantly across the manipulated conditions, except for in P. bursaria under light/dark treatments. Clone library analysis of bacterial 16S rRNA genes showed that DRB were diverse. Pseudomonas became more abundant during the warmer treatment of P. bursaria, whereas the dominance of Pseudoalteromonas weakened and Vibrio became more abundant in E. vannus at a higher temperature. During the treatment of diel light:dark cycles, Aestuariibacter and Alteromonas were selected for in E. vannus but not Pseudoalteromonas, which was highly represented in the all-light and alldark treatments. In contrast, P. bursaria consistently hosted Nevskia, Curvibacter, and Asticcacaulis under all light conditions. There were many bacterial species co-resistant to oxytetracycline and to protistan digestion, in which Sphingomonas, Alteromonas, Aestuariibacter, Puniceicoccaceae (Verrucomicrobia), Pseudomonas, and Sulfitobacter were frequently abundant. Flectobacillus and Aestuariibacter were major lead-resistant bacteria associated with the studied protists. Acinetobacter and Hydrogenophaga were abundant in the P. bursaria treated with a high dose of mercury. Aestuariibacter was found as a dominant group of DRB in E. vannus across all cadmium treatments. In summary, this study demonstrates for the first time that environmental stress selects for bacterial populations associated with protists and that there are diverse bacterial species that not only are resistant to pollution stresses but can also survive protistan predation. This work highlights that bacteria-protists associations need to be taken into account in understanding ecological and environmental issues, such as resilience of bacterial community and function, microbial co-occurrence, and quantity and distribution of antibiotic resistant bacteria and genes

Distinct seasonality of chytrid-dominated benthic fungal communities in the neritic oceans (Bohai Sea and North Yellow Sea)

Benthic fungal diversities in Bohai Sea and North Yellow Sea were investigated using pyrosequencing of 18S rDNA. Overall, Chytridiomycota dominated, followed by Basidiomycota, Ascomycota and Cryptomycota, in terms of alpha diversities and relative abundance. The beta diversity of benthic fungi showed a significant seasonality but no regional differences, accounted for by contrasting relative abundances of Chytridiomycota and Basidiomycota. Significantly seasonal changes in Chytridiomycota and Basidiomycota assemblage structure were also observed, but not for Ascomycota and Cryptomycota. Environmental filtering was more important than water depth and geographic distance in shaping the distribution of benthic fungi in the neritic oceans. The overall fungal beta diversity co-varied with concentration of chlorophyll a, pH, and salinity, distance from land, and water depth. The assemblage structure of benthic Chytridiomycota, Basidiomycota, Ascomycota and Cryptomycota co-varied with different sets of environmental parameters, suggesting their niche differentiations in the coastal sediments. (C) 2017 Elsevier Ltd and British Mycological Society. All rights reserved

Coupling between Ribotypic and Phenotypic Traits of Protists across Life Cycle Stages and Temperatures

Relationships between ribotypic and phenotypic traits of protists across life cycle stages remain largely unknown. Herein, we used single cells of two soil and two marine ciliate species to examine phenotypic and ribotypic traits and their relationships across lag, log, plateau, cystic stages and temperatures. We found that Colpoda inflata and Colpoda steinii demonstrated allometric relationships between 18S ribosomal DNA (rDNA) copy number per cell (CNPC), cell volume (CV), and macronuclear volume across all life cycle stages. Integrating previously reported data of Euplotes vannus and Strombidium sulcatum indicated taxon-dependent rDNA CNPC-CV functions. Ciliate and prokaryote data analysis revealed that the rRNA CNPC followed a unified power-law function only if the rRNA-deficient resting cysts were not considered. Hence, a theoretical framework was proposed to estimate the relative quantity of resting cysts in the protistan populations with total cellular rDNA and rRNA copy numbers. Using rDNA CNPC was a better predictor of growth rate at a given temperature than rRNA CNPC and CV, suggesting replication of redundant rDNA operons as a key factor that slows cell division. Single-cell high-throughput sequencing and analysis after correcting sequencing errors revealed multiple rDNA and rRNA variants per cell. Both encystment and temperature affected the number of rDNA and rRNA variants in several cases. The divergence of rDNA and rRNA sequence in a single cell ranged from 1% to 10% depending on species. These findings have important implications for inferring cell-based biological traits (e.g., species richness, abundance and biomass, activity, and community structure) of protists using molecular approaches. IMPORTANCE Based on phenotypic traits, traditional surveys usually characterize organismal richness, abundance, biomass, and growth potential to describe diversity, organization, and function of protistan populations and communities. The rRNA gene (rDNA) and its transcripts have been widely used as molecular markers in ecological studies of protists. Nevertheless, the manner in which these molecules relate to cellular (organismal) and physiological traits remains poorly understood, which could lead to misinterpretations of protistan diversity and ecology. The current research highlights the dynamic nature of cellular rDNA and rRNA contents, which tightly couple with multiple phenotypic traits in ciliated protists. We demonstrate that quantity of resting cysts and maximum growth rate of a population can be theoretically estimated using ribotypic trait-based models. The intraindividual sequence polymorphisms of rDNA and rRNA can be influenced by encystment and temperature, which should be considered when interpreting species-level diversity and community structure of microbial eukaryotes

Seagrass (Zostera marina) promotes nitrification potential and selects specific ammonia oxidizers in coastal sediments

Purpose Seagrasses accelerate sedimentation, release oxygen and organic matter through their roots, and compete with ammonia oxidizers for ammonia/ammonium in surface sediments and overlying water, all of which can influence benthic aerobic nitrification. To understand the effects of seagrass vegetation on benthic nitrification, the heterogeneity of nitrification activities and functional microbial communities between seagrass-vegetated and adjacent bare sediments was investigated. Materials and methods Surface (0-5 cm) sediments were sampled from a Zostera marina-colonized coastal lagoon in northern China. The potential nitrification rates (PNR) and relative contributions of ammonia-oxidizing bacteria (PNRaob) and ammonia-oxidizing archaea (PNRaoa) were determined based on the sediment slurry incubation with kanamycin inhibition method. The abundances and community compositions of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were determined using qPCR, clone library, and high-throughput sequencing. Results and discussion The total PNR (PNRtotal) in the seagrass-colonized sediments were significant higher than those in the bare sediments (P 0.05) in amoA gene abundance and diversity was observed between the two habitats. Most AOB genotypes were affiliated to Nitrosomonas, of which NL7 was selectively enriched in the vegetated region, while N. cryotolerans prevailed in the bare. Two distinct AOA groups Crenarchaeota 1.1b and Crenarchaeota 1.1a dominated in vegetated and unvegetated sediments, respectively. Dissolved oxygen of overlying water and TOC:TN of sediment significantly influenced AOB community, while AOA community was strongly driven by nitrate and metal iron in sediments. Conclusions Seagrass vegetation substantially enhances nitrification potential and selects specific ammonia oxidizers in coastal sediments

Genome-resolved metagenomics provides insights into the microbial-mediated sulfur and nitrogen cycling in temperate seagrass meadows

The presence of seagrasses facilitates numerous microbial-mediated biogeochemical cycles, with sulfur- and nitrogen-cycling microorganisms playing crucial roles as regulators. Despite efforts to comprehend the diversity of microbes in seagrass ecosystems, the metabolic functions of these benthic microorganisms in seagrass sediments remain largely unknown. Using metagenomics, we provide insights into the sulfur- and nitrogen-cycling pathways and key metabolic capacities of microorganisms in both Z. japonica-colonized and unvegetated sediments over a seasonal period. Taxonomic analysis of N and S cycling genes revealed that delta- and gamma- proteobacteria dominated the benthic sulfate-reducing bacteria, while alpha- and gamma-proteobacteria played a significant role in the sulfur-oxidation processes. The proteobacterial lineages were also major contributors to the benthic nitrogen cycling. However, at a finer taxonomic resolution, microbial participants in different processes were observed to be highly diverse and mainly driven by environmental factors such as temperature and salinity. The gene pools of sulfur and nitrogen cycles in the seagrass sediments were dominated by genes involved in sulfide oxidation (fccA) and hydroxylamine oxidation (hao), respectively. Seagrass colonization elevated the relative abundance of genes responsible for sulfite production (phsC), hydroxylamine oxidation (hao), and nitrogen fixation (nifK), but suppressed sulfur oxidation (soxXYZ) and denitrification (nosZ and nirS). The prevalence of proteobacterial lineages functioned with versatile capabilities in both sulfur and nitrogen cycles in seagrass ecosystems, highlighting tight couplings between these processes, which was further supported by the recovery of 83 metagenome-assembled genomes (MAGs). These findings broaden our understanding of the biogeochemical processes that are mediated by microorganisms in seagrass ecosystems

Dynamics and Distribution of Marine Synechococcus Abundance and Genotypes during Seasonal Hypoxia in a Coastal Marine Ranch

Marine Synechococcus are an ecologically important picocyanobacterial group widely distributed in various oceanic environments. Little is known about the dynamics and distribution of Synechococcus abundance and genotypes during seasonal hypoxia in coastal zones. In this study, an investigation was conducted in a coastal marine ranch along two transects in Muping, Yantai, where hypoxic events (defined here as the dissolved oxygen concentration <3 mg L-1) occurred in the summer of 2015. The hypoxia occurred in the bottom waters from late July and persisted until late August. It was confined at nearshore stations of the two transects, one running across a coastal ranch and the other one outside. During this survey, cell abundance of Synechococcus was determined with flow cytometry, showing great variations ranging from 1 x 10(4) to 3.0 x 10(5) cells mL(-1), and a bloom of Synechococcus occurred when stratification disappeared and hypoxia faded out outside the ranch. Regression analysis indicated that dissolved oxygen, pH, and inorganic nutrients were the most important abiotic factors in explaining the variation in Synechococcus cell abundance. Diverse genotypes (mostly belonged to the sub-clusters 5.1 and 5.2) were detected using clone library sequencing and terminal restriction fragment length polymorphism analysis of the 16S-23S rRNA internal transcribed spacer region. The richness of genotypes was significantly related to salinity, temperature, silicate, and pH, but not dissolved oxygen. Two environmental factors, temperature and salinity, collectively explained 17% of the variation in Synechococcus genotype assemblage. With the changes in population composition in diverse genotypes, the Synechococcus assemblages survived in the coastal hypoxia event and thrived when hypoxia faded out