Time-shifted expression of acetoclastic and methylotrophic methanogenesis by a single Methanosarcina genomospecies predominates the methanogen dynamics in Philippine rice field soil.

BACKGROUND: The final step in the anaerobic decomposition of biopolymers is methanogenesis. Rice field soils are a major anthropogenic source of methane, with straw commonly used as a fertilizer in rice farming. Here, we aimed to decipher the structural and functional responses of the methanogenic community to rice straw addition during an extended anoxic incubation (120 days) of Philippine paddy soil. The research combined process measurements, quantitative real-time PCR and RT-PCR of particular biomarkers (16S rRNA, mcrA), and meta-omics (environmental genomics and transcriptomics). RESULTS: The analysis methods collectively revealed two major bacterial and methanogenic activity phases: early (days 7 to 21) and late (days 28 to 60) community responses, separated by a significant transient decline in microbial gene and transcript abundances and CH4 production rate. The two methanogenic activity phases corresponded to the greatest rRNA and mRNA abundances of the Methanosarcinaceae but differed in the methanogenic pathways expressed. While three genetically distinct Methanosarcina populations contributed to acetoclastic methanogenesis during the early activity phase, the late activity phase was defined by methylotrophic methanogenesis performed by a single Methanosarcina genomospecies. Closely related to Methanosarcina sp. MSH10X1, mapping of environmental transcripts onto metagenome-assembled genomes (MAGs) and population-specific reference genomes revealed this genomospecies as the key player in acetoclastic and methylotrophic methanogenesis. The anaerobic food web was driven by a complex bacterial community, with Geobacteraceae and Peptococcaceae being putative candidates for a functional interplay with Methanosarcina. Members of the Methanocellaceae were the key players in hydrogenotrophic methanogenesis, while the acetoclastic activity of Methanotrichaceae members was detectable only during the very late community response. CONCLUSIONS: The predominant but time-shifted expression of acetoclastic and methylotrophic methanogenesis by a single Methanosarcina genomospecies represents a novel finding that expands our hitherto knowledge of the methanogenic pathways being highly expressed in paddy soils. Video Abstract.

Metagenomic insights into genetic factors driving bacterial niche differentiation between bulk and rhizosphere soils.

Cellular motility is crucial for effective colonization of the rhizosphere, but it is not yet clear whether bacterial motility is particularly linked to other genetic traits. Here, we applied genome-resolved metagenomics and phylogenomics to investigate the ecological significance of cellular motility for niche differentiation and the links between the genetic makeup of motile bacteria and rhizosphere colonization within a four-decade maize field experiment. Indeed, highly diverse sets of genes encoding cellular motility, including chemotaxis, flagellar assembly and motility proteins, and utilization of polymeric carbon were the important predictors of bacterial niche differentiation between bulk and rhizosphere soils. This is well exemplified by metagenome-assembled genomes encoding high motility capacity (hmc_MAGs). Their collective abundance was, on average, sixfold higher in rhizosphere soil than in bulk soil. All bulk-soil-derived MAGs showed low motility capacities (lmc). The hmc_MAGs were highly enriched in beneficial traits involved in carbohydrate utilization, assimilatory (nasA) and dissimilatory (nirBD) nitrate reduction, inorganic phosphate solubilization (gcd), and organic phosphate mineralization (phoD). Belonging to the families Sphingomonadaceae, Burkholderiaceae and Steroidobacteraceae, the hmc_MAGs showed a ninefold greater enrichment in these traits than proteobacterial lmc_MAGs and a twofold greater enrichment than 264 genomes publicly available for the above three families, thereby substantiating that a specific rhizosphere effect acted on the microbes represented by the hmc_MAGs. The particular link between the genetic capacities for high cellular motility and increased carbohydrate depolymerization as the key determinant for plant-selected rhizosphere colonization was further substantiated by the analysis of public bulk-rhizosphere soil metagenomes retrieved from wheat and cucumber field sites.

Harnessing biological nitrogen fixation in plant leaves.

The importance of biological nitrogen fixation (BNF) in securing food production for the growing world population with minimal environmental cost has been increasingly acknowledged. Leaf surfaces are one of the biggest microbial habitats on Earth, harboring diverse free-living N2-fixers. These microbes inhabit the epiphytic and endophytic phyllosphere and contribute significantly to plant N supply and growth. Here, we summarize the contribution of phyllosphere-BNF to global N cycling, evaluate the diversity of leaf-associated N2-fixers across plant hosts and ecosystems, illustrate the ecological adaptation of N2-fixers to the phyllosphere, and identify the environmental factors driving BNF. Finally, we discuss potential BNF engineering strategies to improve the nitrogen uptake in plant leaves and thus sustainable food production.

Asparagine Uptake: a Cellular Strategy of Methylocystis to Combat Severe Salt Stress.

Methylocystis spp. are known to have a low salt tolerance (≤1.0% NaCl). Therefore, we tested various amino acids and other well-known osmolytes for their potential to act as an osmoprotectant under otherwise growth-inhibiting NaCl conditions. Adjustment of the medium to 10 mM asparagine had the greatest osmoprotective effect under severe salinity (1.50% NaCl), leading to partial growth recovery of strain SC2. The intracellular concentration of asparagine increased to 264 ± 57 mM, with a certain portion hydrolyzed to aspartate (4.20 ± 1.41 mM). In addition to general and oxidative stress responses, the uptake of asparagine specifically induced major proteome rearrangements related to the KEGG level 3 categories of "methane metabolism," "pyruvate metabolism," "amino acid turnover," and "cell division." In particular, various proteins involved in cell division (e.g., ChpT, CtrA, PleC, FtsA, FtsH1) and peptidoglycan synthesis showed a positive expression response. Asparagine-derived 13C-carbon was incorporated into nearly all amino acids. Both the exometabolome and the 13C-labeling pattern suggest that in addition to aspartate, the amino acids glutamate, glycine, serine, and alanine, but also pyruvate and malate, were most crucially involved in the osmoprotective effect of asparagine, with glutamate being a major hub between the central carbon and amino acid pathways. In summary, asparagine induced significant proteome rearrangements, leading to major changes in central metabolic pathway activity and the sizes of free amino acid pools. In consequence, asparagine acted, in part, as a carbon source for the growth recovery of strain SC2 under severe salinity. IMPORTANCE Methylocystis spp. play a major role in reducing methane emissions into the atmosphere from methanogenic wetlands. In addition, they contribute to atmospheric methane oxidation in upland soils. Although these bacteria are typical soil inhabitants, Methylocystis spp. are thought to have limited capacity to acclimate to salt stress. This called for a thorough study into potential osmoprotectants, which revealed asparagine as the most promising candidate. Intriguingly, asparagine was taken up quantitatively and acted, at least in part, as an intracellular carbon source under severe salt stress. The effect of asparagine as an osmoprotectant for Methylocystis spp. is an unexpected finding. It may provide Methylocystis spp. with an ecological advantage in wetlands, where these methanotrophs colonize the roots of submerged vascular plants. Collectively, our study offers a new avenue into research on compounds that may increase the resilience of Methylocystis spp. to environmental change.

Methylocystis sp. Strain SC2 Acclimatizes to Increasing NH4+ Levels by a Precise Rebalancing of Enzymes and Osmolyte Composition.

A high NH4+ load is known to inhibit bacterial methane oxidation. This is due to a competition between CH4 and NH3 for the active site of particulate methane monooxygenase (pMMO), which converts CH4 to CH3OH. Here, we combined global proteomics with amino acid profiling and nitrogen oxides measurements to elucidate the cellular acclimatization response of Methylocystis sp. strain SC2 to high NH4+ levels. Relative to 1 mM NH4+, a high (50 mM and 75 mM) NH4+ load under CH4-replete conditions significantly increased the lag phase duration required for proteome adjustment. The number of differentially regulated proteins was highly significantly correlated with an increasing NH4+ load. The cellular responses to increasing ionic and osmotic stress involved a significant upregulation of stress-responsive proteins, the K+ "salt-in" strategy, the synthesis of compatible solutes (glutamate and proline), and the induction of the glutathione metabolism pathway. A significant increase in the apparent Km value for CH4 oxidation during the growth phase was indicative of increased pMMO-based oxidation of NH3 to toxic hydroxylamine. The detoxifying activity of hydroxlyamine oxidoreductase (HAO) led to a significant accumulation of NO2- and, upon decreasing O2 tension, N2O. Nitric oxide reductase and hybrid cluster proteins (Hcps) were the candidate enzymes for the production of N2O. In summary, strain SC2 has the capacity to precisely rebalance enzymes and osmolyte composition in response to increasing NH4+ exposure, but the need to simultaneously combat both ionic-osmotic stress and the toxic effects of hydroxylamine may be the reason why its acclimatization capacity is limited to 75 mM NH4+. IMPORTANCE In addition to reducing CH4 emissions from wetlands and landfills, the activity of alphaproteobacterial methane oxidizers of the genus Methylocystis contributes to the sink capacity of forest and grassland soils for atmospheric methane. The methane-oxidizing activity of Methylocystis spp. is, however, sensitive to high NH4+ concentrations. This is due to the competition of CH4 and NH3 for the active site of particulate methane monooxygenase, thereby resulting in the production of toxic hydroxylamine with an increasing NH4+ load. An understanding of the physiological and molecular response mechanisms of Methylocystis spp. is therefore of great importance. Here, we combined global proteomics with amino acid profiling and NOx measurements to disentangle the cellular mechanisms underlying the acclimatization of Methylocystis sp. strain SC2 to an increasing NH4+ load.

Genome-Resolved Metagenomics Reveals Distinct Phosphorus Acquisition Strategies between Soil Microbiomes.

Enhancing soil phosphate solubilization is a promising strategy for agricultural sustainability, while little is known about the mechanisms of how microorganisms cope with differing phosphorus availability. Using a combination of genome-resolved metagenomics and amplicon sequencing, we investigated the microbial mechanisms involved in phosphorus cycling under three agricultural treatments in a wheat-maize rotation system and two natural reforestation treatments. Available soil phosphorus was the key factor shaping bacterial and fungal community composition and function across our agricultural and reforestation sites. Membrane-bound quinoprotein glucose dehydrogenase (PQQGDH) and exopolyphosphatases (PPX) governed microbial phosphate solubilization in agroecosystems. In contrast, genes encoding glycerol-3-phosphate transporters (ugpB, ugpC, and ugpQ) displayed a significantly greater abundance in the reforestation soils. The gcd gene encoding PQQGDH was found to be the best determinant for bioavailable soil phosphorus. Metagenome-assembled genomes (MAGs) affiliated with Cyclobacteriaceae and Vicinamibacterales were obtained from agricultural soils. Their MAGs harbored not only gcd but also the pit gene encoding low-affinity phosphate transporters. MAGs obtained from reforestation soils were affiliated with Microtrichales and Burkholderiales. These contain ugp genes but no gcd, and thereby are indicative of a phosphate transporter strategy. Our study demonstrates that knowledge of distinct microbial phosphorus acquisition strategies between agricultural and reforestation soils could help in linking microbial processes with phosphorus cycling. IMPORTANCE The soil microbiome is the key player regulating phosphorus cycling processes. Identifying phosphate-solubilizing bacteria and utilizing them for release of recalcitrant phosphate that is bound to rocks or minerals have implications for improving crop nutrient acquisition and crop productivity. In this study, we combined functional metagenomics and amplicon sequencing to analyze microbial phosphorus cycling processes in natural reforestation and agricultural soils. We found that the phosphorus acquisition strategies significantly differed between these two ecosystems. A microbial phosphorus solubilization strategy dominated in the agricultural soils, while a microbial phosphate transporter strategy was observed in the reforestation soils. We further identified microbial taxa that contributed to enhanced phosphate solubilization in the agroecosystem. These microbes are predicted to be beneficial for the increase in phosphate bioavailability through agricultural practices.

Genome-resolved metagenomics identifies the particular genetic traits of phosphate-solubilizing bacteria in agricultural soil.

Bacteria play a key role in phosphate solubilization, but related genome-centric research on agricultural microbiomes is scarce. Here, we reconstructed 472 metagenome-assembled genomes (MAGs) covering agricultural soils from six long-term field trials across China. A total of 79 MAGs contained gcd encoding quinoprotein glucose dehydrogenase (GCD), which is the key biomarker for phosphate solubilization. Our findings showed that all GCD-MAGs represent potentially novel species, with gcd copy numbers varying from 1 to 10 per genome. Large genome size, a high ratio of glycosyl hydrolase genes, and increased capacity for carbohydrate utilization were specific traits of GCD-MAGs. Notably, the gcd copy number showed a significant and positive correlation with genome size. Generated using a machine learning approach, our findings were validated in a dataset of 692 genotypes covering the 18 bacterial families to which the 79 GCD-MAGs belong. Our results improve the knowledge of both the diversity and the genetic composition of phosphate-solubilizing bacteria. In particular, they reveal a genomic link between phosphate solubilization capacity and increased potential for carbohydrate metabolism, which may accelerate targeted engineering and improve management practices for sustainable agriculture.

Deciphering microbial mechanisms underlying soil organic carbon storage in a wheat-maize rotation system.

A link between microbial life history strategies and soil organic carbon storage in agroecosystems is presumed, but largely unexplored at the gene level. We aimed to elucidate whether and how differential organic material amendments (manure versus peat-vermiculite) affect, relative to sole chemical fertilizer application, the link between microbial life history strategies and soil organic carbon storage in a wheat-maize rotation field experiment. To achieve this goal, we combined bacterial 16S rRNA gene and fungal ITS amplicon sequencing, metagenomics and the assembly of genomes. Fertilizer treatments had a significantly greater effect on microbial community composition than aggregate size, with soil available phosphorus and potassium being the most important community-shaping factors. Limitation in labile carbon was linked to a K-selected oligotrophic life history strategy (Gemmatimonadetes, Acidobacteria) under sole chemical fertilizer application; defined by a significant enrichment of genes involved in resource acquisition, polymer hydrolysis, and competition. By contrast, excess of labile carbon promoted an r-selected copiotrophic life history strategy (Cytophagales, Bacillales, Mortierellomycota) under manure treatment; defined by a significant enrichment of genes involved in cellular growth. A distinct life history strategy was not observed under peat-vermiculite treatment, but rather a mix of both K-selected (Acidobacteria) and r-selected (Actinobacteria, Mortierellomycota) microorganisms. Compared to sole chemical fertilizer application, soil organic carbon storage efficiency was significantly increased by 26.5% and 50.0% under manure and peat-vermiculite treatments, respectively. Taken together, our results highlight the importance of organic material amendments, but in particular a one-time peat-vermiculite application, to promote soil organic carbon storage as a potential management strategy for sustainable agriculture.

Metagenomic insights into nitrogen and phosphorus cycling at the soil aggregate scale driven by organic material amendments.

The soil microbiome, existing as interconnected communities closely associated with soil aggregates, is the key driver in nutrient cycling. However, the underlying genomic information encoding the machinery of the soil microbiome involved in nutrient cycling at the soil aggregate scale is barely known. Here comparative metagenomics and genome binning were applied to investigate microbial functional profiles at the soil aggregate scale under different organic material amendments in a long-term field experiment. Soil samples were sieved into large macroaggregates (>2 mm), macroaggregates (0.25-2 mm) and microaggregates (<0.25 mm). Microbial taxonomic and functional alpha diversity were significantly correlated to soil NO3- and SOC. The highest abundance of nasB, nirK, and amoA genes, which are responsible for denitrification and ammonia oxidizers driving nitrification, was observed in microaggregates. Both manure and peat treatments significantly decreased the abundance of napA and nrfA that encode enzymes involved in dissimilatory nitrate reduction to ammonium (DNRA). As a biomarker for soil inorganic P solubilization, the relative abundance of gcd was significantly increased in macroaggregates and large macroaggregates. Three nearly complete genomes of Nitrososphaeraceae (AOA) and seven bacterial genomes were shown to harbor a series of genes involved in nitrification and P solubilization, respectively. Our study provides comprehensive insights into the microbial genetic potential for DNRA and P-solubilizing activity across different soil aggregate fractions and fertilization regimes.

Seasonality affects function and complexity but not diversity of the rhizosphere microbiome in European temperate grassland.

Knowledge on how grassland microbiota responds on gene expression level to winter-summer change of seasons is poor. Here, we used a combination of quantitative PCR-based assays and metatranscriptomics to assess the impact of seasonality on the rhizospheric microbiota in temperate European grassland. Bacteria dominated, being at least one order of magnitude more abundant than fungi. Despite a fivefold summer increase in bacterial community size, season had nearly no effect on microbiome diversity. It, however, had a marked impact on taxon-specific gene expression, with 668 genes significantly differing in relative transcript abundance between winter and summer samples. Acidobacteria, Bacteroidetes, Planctomycetes, and Proteobacteria showed a greater relative gene expression activity in winter, while mRNA of Actinobacteria and Fungi was, relative to other taxa, significantly enriched in summer. On functional level, mRNA involved in protein turnover (e.g., transcription and translation) and cell maintenance (e.g., chaperones that protect against cell freezing damage such as GroEL and Hsp20) were highly enriched in winter. By contrast, mRNA involved in central carbon and amino acid metabolisms had a greater abundance in summer. Among carbohydrate-active enzymes, transcripts of GH36 family (hemicellulases) were highly enriched in winter, while those encoding GH3 family (cellulases) showed increased abundance in summer. The seasonal differences in plant polymer breakdown were linked to a significantly greater microbial network complexity in winter than in summer. Conceptually, the winter-summer change in microbiome functioning can be well explained by a shift from stress-tolerator to high-yield life history strategy.

Unexpected metabolic versatility among type II methanotrophs in the Alphaproteobacteria.

Aerobic methane-oxidizing bacteria, or methanotrophs, play a crucial role in the global methane cycle. Their methane oxidation activity in various environmental settings has a great mitigation effect on global climate change. Alphaproteobacterial methanotrophs were among the first to be taxonomically characterized, nowadays unified in the Methylocystaceae and Beijerinckiaceae families. Originally thought to have an obligate growth requirement for methane and related one-carbon compounds as a source of carbon and energy, it was later shown that various alphaproteobacterial methanotrophs are facultative, able to grow on multi-carbon compounds such as acetate. Most recently, we expanded our knowledge of the metabolic versatility of alphaproteobacterial methanotrophs. We showed that Methylocystis sp. strain SC2 has the capacity for mixotrophic growth on H2 and CH4. This mini-review will summarize the change in perception from the long-held paradigm of obligate methanotrophy to today's recognition of alphaproteobacterial methanotrophs as having both facultative and mixotrophic capabilities.

Hydrogen utilization by Methylocystis sp. strain SC2 expands the known metabolic versatility of type IIa methanotrophs.

Methane, a non-expensive natural substrate, is used by Methylocystis spp. as a sole source of carbon and energy. Here, we assessed whether Methylocystis sp. strain SC2 is able to also utilize hydrogen as an energy source. The addition of 2% H2 to the culture headspace had the most significant positive effect on the growth yield under CH4 (6%) and O2 (3%) limited conditions. The SC2 biomass yield doubled from 6.41 (±0.52) to 13.82 (±0.69) mg cell dry weight per mmol CH4, while CH4 consumption was significantly reduced. Regardless of H2 addition, CH4 utilization was increasingly redirected from respiration to fermentation-based pathways with decreasing O2/CH4 mixing ratios. Theoretical thermodynamic calculations confirmed that hydrogen utilization under oxygen-limited conditions doubles the maximum biomass yield compared to fully aerobic conditions without H2 addition. Hydrogen utilization was linked to significant changes in the SC2 proteome. In addition to hydrogenase accessory proteins, the production of Group 1d and Group 2b hydrogenases was significantly increased in both short- and long-term incubations. Both long-term incubation with H2 (37 d) and treatments with chemical inhibitors revealed that SC2 growth under hydrogen-utilizing conditions does not require the activity of complex I. Apparently, strain SC2 has the metabolic capacity to channel hydrogen-derived electrons into the quinone pool, which provides a link between hydrogen oxidation and energy production. In summary, H2 may be a promising alternative energy source in biotechnologically oriented methanotroph projects that aim to maximize biomass yield from CH4, such as the production of high-quality feed protein.

Limnoglobus roseus gen. nov., sp. nov., a novel freshwater planctomycete with a giant genome from the family Gemmataceae.

The family Gemmataceae accommodates aerobic, chemoorganotrophic planctomycetes, which inhabit various freshwater ecosystems, wetlands and soils. Here, we describe a novel member of this family, strain PX52T, which was isolated from a boreal eutrophic lake in Northern Russia. This isolate formed pink-pigmented colonies and was represented by spherical cells that occurred singly, in pairs or aggregates and multiplied by budding. Daughter cells were highly motile. PX52T was an obligate aerobic chemoorganotroph, which utilized various sugars and some heteropolysaccharides. Growth occurred at pH 5.0-7.5 (optimum pH 6.5) and at temperatures between 10 and 30 °C (optimum 20-25 °C). The major fatty acids were C18 : 1É·7c, C18 : 0 and ßOH-C16:0; the major intact polar lipid was trimethylornithine, and the quinone was MK-6. The complete genome of PX52T was 9.38 Mb in size and contained nearly 8000 potential protein-coding genes. Among those were genes encoding a wide repertoire of carbohydrate-active enzymes (CAZymes) including 33 glycoside hydrolases (GH) and 87 glycosyltransferases (GT) affiliated with 17 and 12 CAZy families, respectively. DNA G+C content was 65.6 mol%. PX52T displayed only 86.0-89.8 % 16S rRNA gene sequence similarity to taxonomically described Gemmataceae planctomycetes and differed from them by a number of phenotypic characteristics and by fatty acid composition. We, therefore, propose to classify it as representing a novel genus and species, Limnoglobus roseus gen. nov., sp. nov. The type strain is strain PX52T (=KCTC 72397T=VKM B-3275T).

Thriving in Wetlands: Ecophysiology of the Spiral-Shaped Methanotroph Methylospira mobilis as Revealed by the Complete Genome Sequence.

Candidatus Methylospira mobilis is a recently described spiral-shaped, micro-aerobic methanotroph, which inhabits northern freshwater wetlands and sediments. Due to difficulties of cultivation, it could not be obtained in a pure culture for a long time. Here, we report on the successful isolation of strain Shm1, the first axenic culture of this unique methanotroph. The complete genome sequence obtained for strain Shm1 was 4.7 Mb in size and contained over 4800 potential protein-coding genes. The array of genes encoding C1 metabolic capabilities in strain Shm1 was highly similar to that in the closely related non-motile, moderately thermophilic methanotroph Methylococcus capsulatus Bath. The genomes of both methanotrophs encoded both low- and high-affinity oxidases, which allow their survival in a wide range of oxygen concentrations. The repertoire of signal transduction systems encoded in the genome of strain Shm1, however, by far exceeded that in Methylococcus capsulatus Bath but was comparable to those in other motile gammaproteobacterial methanotrophs. The complete set of motility genes, the presence of both the molybdenum-iron and vanadium-iron nitrogenases, as well as a large number of insertion sequences were also among the features, which define environmental adaptation of Methylospira mobilis to water-saturated, micro-oxic, heterogeneous habitats depleted in available nitrogen.

Efficient Tandem LysC/Trypsin Digestion in Detergent Conditions.

All shotgun proteomics experiments rely on efficient proteolysis steps for sensitive peptide/protein identification and quantification. Previous reports suggest that the sequential tandem LysC/trypsin digest yields higher recovery of fully tryptic peptides than single-tryptic proteolysis. Based on the previous studies, it is assumed that the advantageous effect of tandem proteolysis requires a high sample denaturation state for the initial LysC digest. Therefore, to date, all systematic assessments of LysC/trypsin proteolysis are done in chaotropic environments such as urea. Here, sole trypsin is compared with LysC/trypsin and it is shown that tandem digestion can be carried with high efficiency in Mass Spectrometry-compatible detergents, thereby resulting in higher quantitative yields of fully cleaved peptides. It is further demonstrated that higher cleavage efficiency of tandem digests has a positive impact on absolute protein quantification using intensity-based absolute quantification (iBAQ) values. The results of the examination of divergent urea tandem conditions imply that beneficial effects of the initial LysC digest do not depend on the sample denaturation state, but, are mainly caused by different target specificities of LysC and trypsin. The observed detergent compatibility enables tandem digestion schemes to be implemented in efficient cellular solubilization proteomics procedures without the need for buffer exchange to chaotropic environments.

Metatranscriptomics reveals a differential temperature effect on the structural and functional organization of the anaerobic food web in rice field soil.

BACKGROUND: The expected increase in global surface temperature due to climate change may have a tremendous effect on the structure and function of the anaerobic food web in flooded rice field soil. Here, we used the metatranscriptomic analysis of total RNA to gain a system-level understanding of this temperature effect on the methanogenic food web. RESULTS: Mesophilic (30 °C) and thermophilic (45 °C) food web communities had a modular structure. Family-specific rRNA dynamics indicated that each network module represents a particular function within the food webs. Temperature had a differential effect on all the functional activities, including polymer hydrolysis, syntrophic oxidation of key intermediates, and methanogenesis. This was further evidenced by the temporal expression patterns of total bacterial and archaeal mRNA and of transcripts encoding carbohydrate-active enzymes (CAZymes). At 30 °C, various bacterial phyla contributed to polymer hydrolysis, with Firmicutes decreasing and non-Firmicutes (e.g., Bacteroidetes, Ignavibacteriae) increasing with incubation time. At 45 °C, CAZyme expression was solely dominated by the Firmicutes but, depending on polymer and incubation time, varied on family level. The structural and functional community dynamics corresponded well to process measurements (acetate, propionate, methane). At both temperatures, a major change in food web functionality was linked to the transition from the early to late stage. The mesophilic food web was characterized by gradual polymer breakdown that governed acetoclastic methanogenesis (Methanosarcinaceae) and, with polymer hydrolysis becoming the rate-limiting step, syntrophic propionate oxidation (Christensenellaceae, Peptococcaceae). The thermophilic food web had two activity stages characterized first by polymer hydrolysis and followed by syntrophic oxidation of acetate (Thermoanaerobacteraceae, Heliobacteriaceae, clade OPB54). Hydrogenotrophic Methanocellaceae were the syntrophic methanogen partner, but their population structure differed between the temperatures. Thermophilic temperature promoted proliferation of a new Methanocella ecotype. CONCLUSIONS: Temperature had a differential effect on the structural and functional continuum in which the methanogenic food web operates. This temperature-induced change in food web functionality may not only be a near-future scenario for rice paddies but also for natural wetlands in the tropics and subtropics.

Crude-MS Strategy for in-Depth Proteome Analysis of the Methane-Oxidizing Methylocystis sp. strain SC2.

Methylocystis sp. strain SC2 is a representative of the alphaproteobacterial methane oxidizers or type IIa methanotrophs. These microorganisms play a crucial role in methane cycling. Here, we developed an efficient analytical proteomics workflow for strain SC2. It tackles the major challenges related to the high amount of integral membrane proteins that need to be efficiently solubilized and digested for downstream analysis. Each step of the workflow, including cell lysis, protein solubilization and digestion, and MS peptide quantification, was assessed and optimized. Our new crude-lysate-MS approach proved to increase protein quantification accuracy and proteome coverage of strain SC2. It captured 62% of the predicted SC2 proteome, with up to 10-fold increase in membrane-associated proteins relative to less effective conditions. The use of crude cell lysate for downstream analysis showed to be highly efficient for SC2 and other members of the family Methylocystaceae. Using two contrasting nitrogen conditions, we further validated our workflow efficiency by analyzing the SC2 proteome for differentially expressed proteins involved in methane and nitrogen metabolism. Our crude-MS approach may be applied to a variety of proteomic workflows incorporating cell types with challenging solubilization properties. Data are available via ProteomeXchange with identifier PXD009027.

Unusual Genomic Traits Suggest Methylocystis bryophila S285 to Be Well Adapted for Life in Peatlands.

The genus Methylocystis belongs to the class Alphaproteobacteria, the family Methylocystaceae, and encompasses aerobic methanotrophic bacteria with the serine pathway of carbon assimilation. All Methylocystis species are able to fix dinitrogen and several members of this genus are also capable of using acetate or ethanol in the absence of methane, which explains their wide distribution in various habitats. One additional trait that enables their survival in the environment is possession of two methane-oxidizing isozymes, the conventional particulate methane monooxygenase (pMMO) with low-affinity to substrate (pMMO1) and the high-affinity enzyme (pMMO2). Here, we report the finished genome sequence of Methylocystis bryophila S285, a pMMO2-possessing methanotroph from a Sphagnum-dominated wetland, and compare it to the genome of Methylocystis sp. strain SC2, which is the first methanotroph with confirmed high-affinity methane oxidation potential. The complete genome of Methylocystis bryophila S285 consists of a 4.53 Mb chromosome and one plasmid, 175 kb in size. The genome encodes two types of particulate MMO (pMMO1 and pMMO2), soluble MMO and, in addition, contains a pxmABC-like gene cluster similar to that present in some gammaproteobacterial methanotrophs. The full set of genes related to the serine pathway, the tricarboxylic acid cycle as well as the ethylmalonyl-CoA pathway is present. In contrast to most described methanotrophs including Methylocystis sp. strain SC2, two different types of nitrogenases, that is, molybdenum-iron and vanadium-iron types, are encoded in the genome of strain S285. This unique combination of genome-based traits makes Methylocystis bryophila well adapted to the fluctuation of carbon and nitrogen sources in wetlands.

Metatranscriptomics reveals the hydrolytic potential of peat-inhabiting Planctomycetes.

Members of the phylum Planctomycetes are common inhabitants of northern Sphagnum-dominated wetlands. Evidence is accumulating that, in these environments, some planctomycetes may be involved in degrading polymeric organic matter. The experimental data, however, remain scarce due to the low number of characterized representatives of this phylum. In a previous study, we used metatranscriptomics to assess the activity response of peat-inhabiting microorganisms to biopolymers abundantly present in native peat. The community responses to cellulose, xylan, pectin, and chitin availability were analysed relative to unamended controls. Here, we re-analysed these metatranscriptomes and retrieved a total of 1,602,783 rRNA and 35,522 mRNA sequences affiliated with the Planctomycetes. Each of the four polymers induced specific planctomycete responses. These were most pronounced on chitin. The two groups with increased 16S rRNA transcript pools were Gemmata- and Phycisphaera-like planctomycetes. Among uncultivated members of the Planctomycetaceae, two increased transcript pools were detected in pectin-amended samples and belonged to Pirellula-like bacteria. The analysis of taxonomically assigned mRNA reads confirmed the specific response of Gemmata-related planctomycetes to chitin amendment suggesting the presence of chitinolytic capabilities in these bacteria.

Response of Methylocystis sp. Strain SC2 to Salt Stress: Physiology, Global Transcriptome, and Amino Acid Profiles.

Soil microorganisms have to rapidly respond to salt-induced osmotic stress. Type II methanotrophs of the genus Methylocystis are widely distributed in upland soils but are known to have a low salt tolerance. Here, we tested the ability of Methylocystis sp. strain SC2 to adapt to increased salinity. When exposed to 0.75% NaCl, methane oxidation was completely inhibited for 2.25 h and fully recovered within 6 h. Growth was inhibited for 23.5 h and then fully recovered. Its transcriptome was profiled after 0 min (control), 45 min (early response), and 14 h (late response) of stress exposure. Physiological and transcriptomic stress responses corresponded well. Salt stress induced the differential expression of 301 genes, with sigma factor σ32 being a major controller of the transcriptional stress response. The transcript levels of nearly all the genes involved in oxidizing CH4 to CO2 remained unaffected, while gene expression involved in energy-yielding reactions (nuoA-N) recovered concomitantly with methane oxidation from salt stress shock. Glutamate acted as an osmoprotectant. Its accumulation in late stress response corresponded to increased production of glutamate dehydrogenase 1. Chromosomal genes whose products (stress-induced protein, DNA-binding protein from starved cells, and CsbD family protein) are known to confer stress tolerance showed increased expression. On plasmid pBSC2-1, genes encoding type IV secretion system and single-strand DNA-binding protein were upregulated in late response, suggesting stress-induced activation of the plasmid-borne conjugation machinery. Collectively, our results show that Methylocystis sp. strain SC2 is able to adapt to salt stress, but only within a narrow range of salinities.IMPORTANCE Besides the oxic interface of methanogenic environments, Methylocystis spp. are widely distributed in upland soils, where they may contribute to the oxidation of atmospheric methane. However, little is known about their ability to cope with changes in soil salinity. Growth and methane oxidation of Methylocystis sp. strain SC2 were not affected by the presence of 0.5% NaCl, while 1% NaCl completely inhibited its activity. This places strain SC2 into the low-salt-tolerance range reported for other Methylocystis species. Our results show that, albeit in a narrow range, strain SC2 is able to respond and adapt to salinity changes. It possesses various stress response mechanisms, which allow resumption of growth within 24 h when exposed to 0.75% NaCl. Presumably, these mechanisms allow Methylocystis spp., such as strain SC2, to thrive in upland soils and to adapt to certain fluctuations in soil salinity.