Rice Paddy Nitrospirae Carry and Express Genes Related to Sulfate Respiration: Proposal of the New Genus "Candidatus Sulfobium".

Nitrospirae spp. distantly related to thermophilic, sulfate-reducing Thermodesulfovibrio species are regularly observed in environmental surveys of anoxic marine and freshwater habitats. Here we present a metaproteogenomic analysis of Nitrospirae bacterium Nbg-4 as a representative of this clade. Its genome was assembled from replicated metagenomes of rice paddy soil that was used to grow rice in the presence and absence of gypsum (CaSO4·2H2O). Nbg-4 encoded the full pathway of dissimilatory sulfate reduction and showed expression of this pathway in gypsum-amended anoxic bulk soil as revealed by parallel metaproteomics. In addition, Nbg-4 encoded the full pathway of dissimilatory nitrate reduction to ammonia (DNRA), with expression of its first step being detected in bulk soil without gypsum amendment. The relative abundances of Nbg-4 were similar under both treatments, indicating that Nbg-4 maintained stable populations while shifting its energy metabolism. Whether Nbg-4 is a strict sulfate reducer or can couple sulfur oxidation to DNRA by operating the pathway of dissimilatory sulfate reduction in reverse could not be resolved. Further genome reconstruction revealed the potential to utilize butyrate, formate, H2, or acetate as an electron donor; the Wood-Ljungdahl pathway was expressed under both treatments. Comparison to publicly available Nitrospirae genome bins revealed the pathway for dissimilatory sulfate reduction also in related Nitrospirae recovered from groundwater. Subsequent phylogenomics showed that such microorganisms form a novel genus within the Nitrospirae, with Nbg-4 as a representative species. Based on the widespread occurrence of this novel genus, we propose for Nbg-4 the name "Candidatus Sulfobium mesophilum," gen. nov., sp. nov.IMPORTANCE Rice paddies are indispensable for the food supply but are a major source of the greenhouse gas methane. If it were not counterbalanced by cryptic sulfur cycling, methane emission from rice paddy fields would be even higher. However, the microorganisms involved in this sulfur cycling are little understood. By using an environmental systems biology approach with Italian rice paddy soil, we could retrieve the population genome of a novel member of the phylum Nitrospirae This microorganism encoded the full pathway of dissimilatory sulfate reduction and expressed it in anoxic paddy soil under sulfate-enriched conditions. Phylogenomics and comparison to the results of environmental surveys showed that such microorganisms are actually widespread in freshwater and marine environments. At the same time, they represent an undiscovered genus within the little-explored phylum Nitrospirae Our results will be important for the design of enrichment strategies and postgenomic studies to further understanding of the contribution of these novel Nitrospirae spp. to the global sulfur cycle.

Coproheme decarboxylases - Phylogenetic prediction versus biochemical experiments.

Coproheme decarboxylases (ChdCs) are enzymes responsible for the catalysis of the terminal step in the coproporphyrin-dependent heme biosynthesis pathway. Phylogenetic analyses confirm that the gene encoding for ChdCs is widespread throughout the bacterial world. It is found in monoderm bacteria (Firmicutes, Actinobacteria), diderm bacteria (e. g. Nitrospirae) and also in Archaea. In order to test phylogenetic prediction ChdC representatives from all clades were expressed and examined for their coproheme decarboxylase activity. Based on available biochemical data and phylogenetic analyses a sequence motif (-Y-P-M/F-X-K/R-) is defined for ChdCs. We show for the first time that in diderm bacteria an active coproheme decarboxylase is present and that the archaeal ChdC homolog from Sulfolobus solfataricus is inactive and its physiological role remains elusive. This shows the limitation of phylogenetic prediction of an enzymatic activity, since the identified sequence motif is equally conserved across all previously defined clades.

Effects of variable-sized polyethylene microplastics on soil chemical properties and functions and microbial communities in purple soil.

Microplastic contamination of soil has drawn increased attention due to the ecological harm it poses to the soil ecosystem. However, little is known about how microplastic particle sizes affect soil chemical properties and microbial communities, particularly in purple soil. In this study, a four-week incubation experiment was conducted to evaluate the effect of polyethylene microplastics (PE MPs) with different particle sizes (i.e., 300 and 600 µm) on soil properties, extracellular polymeric substances (EPS), enzyme activities, and microbial communities in purple soil. When compared to 600 µm-PE MPs, 300 µm-PE MPs reduced contents of dissolved organic matter (DOM), EPS, and ß-1,4-N-acetylglucosaminidase (NAG) activity, but increased the cation exchange capacity (CEC). High-throughput 16S rRNA gene sequencing revealed that the 300 µm-PE MPs resulted in an increase in the phylum Nitrospirae, which is associated with microplastic degradation. The data implied that smaller PE MPs improved the growth of polyethylene-degrading bacteria by adsorbing more EPS and DOM, resulting in the degradation of microplastics. Co-occurrence network analysis revealed that smaller PE MPs had lower toxicity to microbial populations than larger PE MPs, increasing the stability of the network. CEC and ß-1,4-glucosidase (BG) were found to be the two major factors affecting the microbial communities by redundancy analysis (RDA). The study highlighted how microplastic particle sizes affect soil bacterial communities and soil functions.

Enhanced fixation of dissolved inorganic carbon by algal-bacterial aerobic granular sludge during treatment of low-organic-content wastewater.

The microalgae-based wastewater treatment technologies are believed to contribute to carbon neutrality. This study investigated the inorganic carbon fixation performance in the algal-bacterial aerobic granular sludge (A-BAGS) process under cultivation at different concentrations of organic carbon (OC) and inorganic carbon (IC). The results indicated that A-BAGS in treating wastewater containing organics of 77 mg-C/L contributed little to the fixation of inorganic carbon, while the highest inorganic carbon removal efficiency of 50 % was achieved at the influent IC of 100 mg/L and OC of 7 mg/L. This high IC condition contributed to enhanced biomass growth rate and enhanced extracellular polymeric substances, while it did not affect the granular stability and nitrification efficiency. The microbial diversity was also largely enhanced. The results demonstrated the great potential of A-BAGS for simultaneous resource recovery in wastewater and inorganic carbon fixation, while operation conditions need to be further optimized.

Combined intensive management of fertilization, tillage, and organic material mulching regulate soil bacterial communities and functional capacities by altering soil potassium and pH in a Moso bamboo forest.

Intensive management is a common practice in agricultural and forestry ecosystems to improve soil quality and crop yield by influencing nutrient supply and soil microbiota; however, the linkage between soil nutrients and bacterial community and functional capacities in intensively managed economic forests has not been well studied. In this study, we investigated the soil properties such as available potassium (AK), available nitrogen (AN), available phosphorus (AP), ammonium (NH 4 + ), nitrate (NO 3 - ), organic matter (OM), total nitrogen (TN), total phosphorus (TP), bacterial diversity and community composition, potential functions of rhizome roots, and soil microbiota across a chronosequence of intensively managed Moso bamboo (Phyllostachys edulis) forests. Our results demonstrated that the combined intensive management (deep tillage, fertilization, and organic material mulching) in this study caused a significant increase in the concentrations of AK, AN, AP, NH 4 + , NO 3 - , OM, TN, and TP (P < 0.05). However, they led to a remarkable decrease in pH (P < 0.05). Such changes lowered the Shannon diversity of the soil and rhizome root microbiota but did not significantly affect the community composition and functional capacity. Soil bacterial community variation was predominantly mediated by soil total potassium (TK) (15.02%), followed by pH (11.29%) and AK (11.13%). We further observed that Nitrospirae accounted for approximately 50% of the variation in soil pH, NO 3 - , NH 4 + , and AK, indicating its importance in soil nutrient cycling, especially nitrogen cycling. Accordingly, we propose that the management-induced changes in soil parameters reshaped the bacterial community structure and keystone bacterial assemblage, leading to the differentiation of microbial functions.

Comparative Genomics on Cultivated and Uncultivated Freshwater and Marine "Candidatus Manganitrophaceae" Species Implies Their Worldwide Reach in Manganese Chemolithoautotrophy.

Chemolithoautotrophic manganese oxidation has long been theorized but only recently demonstrated in a bacterial coculture. The majority member of the coculture, "Candidatus Manganitrophus noduliformans," is a distinct but not yet isolated lineage in the phylum Nitrospirota (Nitrospirae). Here, we established two additional MnCO3-oxidizing cultures using inocula from Santa Barbara (California) and Boetsap (South Africa). Both cultures were dominated by strains of a new species, designated "Candidatus Manganitrophus morganii." The next most abundant members differed in the available cultures, suggesting that while "Ca. Manganitrophus" species have not been isolated in pure culture, they may not require a specific syntrophic relationship with another species. Phylogeny of cultivated "Ca. Manganitrophus" and related metagenome-assembled genomes revealed a coherent taxonomic family, "Candidatus Manganitrophaceae," from both freshwater and marine environments and distributed globally. Comparative genomic analyses support this family being Mn(II)-oxidizing chemolithoautotrophs. Among the 895 shared genes were a subset of those hypothesized for Mn(II) oxidation (Cyc2 and PCC_1) and oxygen reduction (TO_1 and TO_2) that could facilitate Mn(II) lithotrophy. An unusual, plausibly reverse complex 1 containing 2 additional pumping subunits was also shared by the family, as were genes for the reverse tricarboxylic acid carbon fixation cycle, which could enable Mn(II) autotrophy. All members of the family lacked genes for nitrification found in Nitrospira species. The results suggest that "Ca. Manganitrophaceae" share a core set of candidate genes for the newly discovered manganese-dependent chemolithoautotrophic lifestyle and likely have a broad, global distribution. IMPORTANCE Manganese (Mn) is an abundant redox-active metal that cycles in many of Earth's biomes. While diverse bacteria and archaea have been demonstrated to respire Mn(III/IV), only recently have bacteria been implicated in Mn(II) oxidation-dependent growth. Here, two new Mn(II)-oxidizing enrichment cultures originating from two continents and hemispheres were examined. By comparing the community composition of the enrichments and performing phylogenomic analysis on the abundant Nitrospirota therein, new insights are gleaned on cell interactions, taxonomy, and machineries that may underlie Mn(II)-based lithotrophy and autotrophy.

Identification and Genomic Characterization of Two Previously Unknown Magnetotactic Nitrospirae.

Magnetotactic bacteria (MTB) are a group of microbes that biomineralize membrane-bound, nanosized magnetite (Fe3O4), and/or greigite (Fe3S4) crystals in intracellular magnetic organelle magnetosomes. MTB belonging to the Nitrospirae phylum can form up to several hundreds of Fe3O4 magnetosome crystals and dozens of sulfur globules in a single cell. These MTB are widespread in aquatic environments and sometimes account for a significant proportion of microbial biomass near the oxycline, linking these lineages to the key steps of global iron and sulfur cycling. Despite their ecological and biogeochemical importance, our understanding of the diversity and ecophysiology of magnetotactic Nitrospirae is still very limited because this group of MTB remains unculturable. Here, we identify and characterize two previously unknown MTB populations within the Nitrospirae phylum through a combination of 16S rRNA gene-based and genome-resolved metagenomic analyses. These two MTB populations represent distinct morphotypes (rod-shaped and coccoid, designated as XYR, and XYC, respectively), and both form more than 100 bullet-shaped magnetosomal crystals per cell. High-quality draft genomes of XYR and XYC have been reconstructed, and they represent a novel species and a novel genus, respectively, according to their average amino-acid identity values with respect to available genomes. Accordingly, the names Candidatus Magnetobacterium cryptolimnobacter and Candidatus Magnetomicrobium cryptolimnococcus for XYR and XYC, respectively, were proposed. Further comparative genomic analyses of XYR, XYC, and previously reported magnetotactic Nitrospirae reveal the general metabolic potential of this MTB group in distinct microenvironments, including CO2 fixation, dissimilatory sulfate reduction, sulfide oxidation, nitrogen fixation, or denitrification processes. A remarkably conserved magnetosome gene cluster has been identified across Nitrospirae MTB genomes, indicating its putative important adaptive roles in these bacteria. Taken together, the present study provides novel insights into the phylogenomic diversity and ecophysiology of this intriguing, yet poorly understood MTB group.

Microbial succession during the transition from active to inactive stages of deep-sea hydrothermal vent sulfide chimneys.

BACKGROUND: Deep-sea hydrothermal vents are highly productive biodiversity hotspots in the deep ocean supported by chemosynthetic microorganisms. Prominent features of these systems are sulfide chimneys emanating high-temperature hydrothermal fluids. While several studies have investigated the microbial diversity in both active and inactive sulfide chimneys that have been extinct for up to thousands of years, little is known about chimneys that have ceased activity more recently, as well as the microbial succession occurring during the transition from active to inactive chimneys. RESULTS: Genome-resolved metagenomics was applied to an active and a recently extinct (~ 7 years) sulfide chimney from the 9-10° N hydrothermal vent field on the East Pacific Rise. Full-length 16S rRNA gene and a total of 173 high-quality metagenome assembled genomes (MAGs) were retrieved for comparative analysis. In the active chimney (L-vent), sulfide- and/or hydrogen-oxidizing Campylobacteria and Aquificae with the potential for denitrification were identified as the dominant community members and primary producers, fixing carbon through the reductive tricarboxylic acid (rTCA) cycle. In contrast, the microbiome of the recently extinct chimney (M-vent) was largely composed of heterotrophs from various bacterial phyla, including Delta-/Beta-/Alphaproteobacteria and Bacteroidetes. Gammaproteobacteria were identified as the main primary producers, using the oxidation of metal sulfides and/or iron oxidation coupled to nitrate reduction to fix carbon through the Calvin-Benson-Bassham (CBB) cycle. Further analysis revealed a phylogenetically distinct Nitrospirae cluster that has the potential to oxidize sulfide minerals coupled to oxygen and/or nitrite reduction, as well as for sulfate reduction, and that might serve as an indicator for the early stages of chimneys after venting has ceased. CONCLUSIONS: This study sheds light on the composition, metabolic functions, and succession of microbial communities inhabiting deep-sea hydrothermal vent sulfide chimneys. Collectively, microbial succession during the life span of a chimney could be described to proceed from a "fluid-shaped" microbial community in newly formed and actively venting chimneys supported by the oxidation of reductants in the hydrothermal fluid to a "mineral-shaped" community supported by the oxidation of minerals after hydrothermal activity has ceased. Remarkably, the transition appears to occur within the first few years, after which the communities stay stable for thousands of years. Video Abstract.

In situ absorbance measurements: a new means to study respiratory electron transfer in chemolithotrophic microorganisms.

Absorbance measurements on intact chemolithotrophic microorganisms that respire aerobically on soluble iron are described that used a novel integrating cavity absorption meter to eliminate the effects of light scattering on the experimental results. Steady state kinetic measurements on ferric iron production by intact cells revealed that the Michaelis Menten equation described the initial rates of product formation for at least 8 different chemolithotrophic microorganisms in 6 phyla distributed equally among the archaea and the Gram negative and Gram positive eubacteria. Cell-monitored turnover measurements during aerobic respiration on soluble iron by the same 12 intact microorganisms revealed six different patterns of iron-dependent absorbance changes, suggesting that there may be at least six different sets of prosthetic groups and biomolecules that can accomplish aerobic respiration on soluble iron. Detailed kinetic studies revealed that the 3-component iron respiratory chain of Acidithiobacillus ferrooxidans functioned as an ensemble with a single macroscopic rate constant when the iron-reduced proteins were oxidized in the presence of excess molecular oxygen. The principal member of this 3-component system was a cupredoxin called rusticyanin that was present in the periplasm of At. ferrooxidans at an approximate concentration of 350 mg/mL, an observation that provides new insights into the crowded environments in the periplasms of Gram negative eubacteria that conduct electrons across their periplasm. The ability to conduct direct spectrophotometric measurements under noninvasive physiological conditions represents a new and powerful approach to examine the rates and extents of biological events in situ without disrupting the complexity of the live cellular environment.

Floodplain soil and its bacterial composition are strongly affected by depth.

We studied bacterial abundance and community structure of five soil cores using high-throughput sequencing of the 16S rRNA gene. Shifts in the soil bacterial composition were more pronounced within a vertical profile than across the landscape. Soil organic carbon (SOC) and nitrogen (N) concentrations decreased exponentially with soil depth and revealed a buried carbon-rich horizon between 0.8 and 1.3 m across all soil cores. This buried horizon was phylogenetically similar to its surrounding subsoils supporting the idea that the type of carbon, not necessarily the amount of carbon was driving the apparent similarities. In contrast to other studies, Nitrospirae was one of our major phyla with relatively high abundances throughout the soil profile except for the surface soil. Although depth is the major driver shaping soil bacterial community structure, positive correlations with SOC and N concentrations, however, were revealed with the bacterial abundance of Acidobacteria, one of the major, and Gemmatimonadetes, one of the minor phyla in our study. Our study showed that bacterial diversity in soils below 2.0 m can be still as high if not higher than in the above laying subsurface soil suggesting that various bacteria throughout the soil profile influence major biogeochemical processes in floodplain soils.

In vitro assembly of the bacterial actin protein MamK from ' Candidatus Magnetobacterium casensis' in the phylum Nitrospirae.

Magnetotactic bacteria (MTB), a group of phylogenetically diverse organisms that use their unique intracellular magnetosome organelles to swim along the Earth's magnetic field, play important roles in the biogeochemical cycles of iron and sulfur. Previous studies have revealed that the bacterial actin protein MamK plays essential roles in the linear arrangement of magnetosomes in MTB cells belonging to the Proteobacteria phylum. However, the molecular mechanisms of multiple-magnetosome-chain arrangements in MTB remain largely unknown. Here, we report that the MamK filaments from the uncultivated 'Candidatus Magnetobacterium casensis' (Mcas) within the phylum Nitrospirae polymerized in the presence of ATP alone and were stable without obvious ATP hydrolysis-mediated disassembly. MamK in Mcas can convert NTP to NDP and NDP to NMP, showing the highest preference to ATP. Unlike its Magnetospirillum counterparts, which form a single magnetosome chain, or other bacterial actins such as MreB and ParM, the polymerized MamK from Mcas is independent of metal ions and nucleotides except for ATP, and is assembled into well-ordered filamentous bundles consisted of multiple filaments. Our results suggest a dynamically stable assembly of MamK from the uncultivated Nitrospirae MTB that synthesizes multiple magnetosome chains per cell. These findings further improve the current knowledge of biomineralization and organelle biogenesis in prokaryotic systems.

Post-translational modifications are enriched within protein functional groups important to bacterial adaptation within a deep-sea hydrothermal vent environment.

BACKGROUND: Post-translational modification (PTM) of proteins is one important strategy employed by bacteria for environmental adaptation. However, PTM profiles in deep-sea microbes remain largely unexplored. RESULTS: We provide here insight into PTMs in a hydrothermal vent microbial community through integration of metagenomics and metaproteomics. In total, 2919 unique proteins and 1306 unique PTMs were identified, whereas the latter included acetylation, deamination, hydroxylation, methylation, nitrosylation, oxidation, and phosphorylation. These modifications were unevenly distributed among microbial taxonomic and functional categories. A connection between modification types and particular functions was demonstrated. Interestingly, PTMs differed among the orthologous proteins derived from different bacterial groups. Furthermore, proteomic mapping to the draft genome of a Nitrospirae bacterium revealed novel modifications for proteins that participate in energy metabolism, signal transduction, and inorganic ion transport. CONCLUSIONS: Our results suggest that PTMs are enriched in specific functions, which would be important for microbial adaptation to extreme conditions of the hydrothermal vent. PTMs in deep-sea are highly diverse and divergent, and much broader investigations are needed to obtain a better understanding of their functional roles.

Crystal growth of bullet-shaped magnetite in magnetotactic bacteria of the Nitrospirae phylum.

Magnetotactic bacteria (MTB) are known to produce single-domain magnetite or greigite crystals within intracellular membrane organelles and to navigate along the Earth's magnetic field lines. MTB have been suggested as being one of the most ancient biomineralizing metabolisms on the Earth and they represent a fundamental model of intracellular biomineralization. Moreover, the determination of their specific crystallographic signature (e.g. structure and morphology) is essential for palaeoenvironmental and ancient-life studies. Yet, the mechanisms of MTB biomineralization remain poorly understood, although this process has been extensively studied in several cultured MTB strains in the Proteobacteria phylum. Here, we show a comprehensive transmission electron microscopy (TEM) study of magnetic and structural properties down to atomic scales on bullet-shaped magnetites produced by the uncultured strain MYR-1 belonging to the Nitrospirae phylum, a deeply branching phylogenetic MTB group. We observed a multiple-step crystal growth of MYR-1 magnetite: initial isotropic growth forming cubo-octahedral particles (less than approx. 40 nm), subsequent anisotropic growth and a systematic final elongation along [001] direction. During the crystal growth, one major {111} face is well developed and preserved at the larger basal end of the crystal. The basal {111} face appears to be terminated by a tetrahedral-octahedral-mixed iron surface, suggesting dimensional advantages for binding protein(s), which may template the crystallization of magnetite. This study offers new insights for understanding magnetite biomineralization within the Nitrospirae phylum.