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.

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).

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.

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.

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.

Microbial community dynamics during the early stages of plant polymer breakdown in paddy soil.

We used paddy soil slurries amended with rice straw to identify the microbial populations involved in the methanogenic breakdown of plant polymers. Rice straw greatly stimulated microbial activity over the 28-day incubation period. On day 7, the transient peak concentration of acetate (24 mM) coincided with the onset of increased methane production. Microbial 16S rRNA transcript numbers increased by one to two orders of magnitude, but not the 16S rRNA gene copy numbers. Using metatranscriptomic rRNA, Clostridiaceae, Lachnospiraceae, Ruminococcaceae, Veillonellaceae and Pseudomonadaceae were identified to be the most abundant and the most dynamic bacterial groups. Changes in methanogen rRNA and mRNA abundances corresponded well with methanogenic activity. Acetate determined the abundance ratio between Methanosarcinaceae and Methanosaetaceae. Methanocellaceae dominated hydrogenotrophic methanogenesis. Transcript levels of mRNA families involved in plant polymer breakdown increased slightly with time. Glycosyl hydrolase (GH) transcripts involved in cellulose and chitin breakdown were predominantly expressed by the Firmicutes, whereas those involved in hemicellulose breakdown exhibited more diverse taxonomic sources, including Acidobacteria, Bacteriodetes and Chloroflexi. Taken together, we observed strong population dynamics and the expression of taxonomically diverse GH families, suggesting that not only Firmicutes, but also less abundant groups play a major functional role in the decomposition of rice straw.

Identification of microbial populations driving biopolymer degradation in acidic peatlands by metatranscriptomic analysis.

Northern peatlands play a crucial role in the global carbon balance, serving as a persistent sink for atmospheric CO2 and a global carbon store. Their most extensive type, Sphagnum-dominated acidic peatlands, is inhabited by microorganisms with poorly understood degradation capabilities. Here, we applied a combination of barcoded pyrosequencing of SSU rRNA genes and Illumina RNA-Seq of total RNA (metatranscriptomics) to identify microbial populations and enzymes involved in degrading the major components of Sphagnum-derived litter and exoskeletons of peat-inhabiting arthropods: cellulose, xylan, pectin and chitin. Biopolymer addition to peat induced a threefold to fivefold increase in bacterial cell numbers. Functional community profiles of assembled mRNA differed between experimental treatments. In particular, pectin and xylan triggered increased transcript abundance of genes involved in energy metabolism and central carbon metabolism, such as glycolysis and TCA cycle. Concurrently, the substrate-induced activity of bacteria on these two biopolymers stimulated grazing of peat-inhabiting protozoa. Alveolata (ciliates) was the most responsive protozoa group as confirmed by analysis of both SSU rRNA genes and SSU rRNA. A stimulation of alphaproteobacterial methanotrophs on pectin was consistently shown by rRNA and mRNA data. Most likely, their significant enrichment was due to the utilization of methanol released during the degradation of pectin. Analysis of SSU rRNA and total mRNA revealed a specific response of Acidobacteria and Actinobacteria to chitin and pectin, respectively. Relatives of Telmatobacter bradus were most responsive among the Acidobacteria, while the actinobacterial response was primarily affiliated with Frankiales and Propionibacteriales. The expression of a wide repertoire of carbohydrate-active enzymes (CAZymes) corresponded well to the detection of a highly diverse peat-inhabiting microbial community, which is dominated by yet uncultivated bacteria.

Methylovulum psychrotolerans sp. nov., a cold-adapted methanotroph from low-temperature terrestrial environments, and emended description of the genus Methylovulum.

Two isolates of aerobic methanotrophic bacteria, strains Sph1T and Sph2, were obtained from cold methane seeps in a floodplain of the river Mukhrinskaya, Irtysh basin, West Siberia. Another morphologically and phenotypically similar methanotroph, strain OZ2, was isolated from a sediment of a subarctic freshwater lake, Archangelsk region, northern Russia. Cells of these three strains were Gram-stain-negative, light-pink-pigmented, non-motile, encapsulated, large cocci that contained an intracytoplasmic membrane system typical of type I methanotrophs. They possessed a particulate methane monooxygenase enzyme and utilized only methane and methanol. Strains Sph1T, Sph2 and OZ2 were able to grow at a pH range of 4.0-8.9 (optimum at pH 6.0-7.0) and at temperatures between 2 and 36 °C. Although their temperature optimum was at 20-25 °C, these methanotrophs grew well at lower temperatures, down to 4 °C. The major cellular fatty acids were C16 : 1ω5c, C16 : 1ω6c, C16 : 1ω7c, C16 : 1ω8c, C16 : 0 and C14 : 0; the DNA G+C content was 51.4-51.9 mol%. Strains Sph1T, Sph2 and OZ2 displayed nearly identical (99.1-99.7 % similarity) 16S rRNA gene sequences and belonged to the family Methylococcaceae of the class Gammaproteobacteria. The most closely related organism was Methylovulum miyakonense HT12T (96.0-96.5 % 16S rRNA gene sequence similarity and 90 % pmoA sequence similarity). The novel isolates, however, differed from Methylovulum miyakonense HT12T by cell morphology, pigmentation, absence of soluble methane monooxygenase, more active growth at low temperatures, growth over a broader pH range and higher DNA G+C content. On the basis of these differences, we propose a novel species, Methylovulum psychrotolerans sp. nov., to accommodate these methanotrophs. Strain Sph1T (=LMG 29227T=VKM B-3018T) is the type strain.

DAFGA: diversity analysis of functional gene amplicons.

SUMMARY: Diversity analysis of functional marker genes provides physiological insights into microbial guilds that perform an ecologically relevant process. However, it is challenging to group functional gene sequences to valid taxonomic units, primarily because of differences in the evolutionary rates of individual genes and possible horizontal gene transfer events. We developed a python script package named DAFGA, which estimates the evolutionary rate of a particular functional gene in a standardized manner by relating its sequence divergence to that of the 16S rRNA gene. As a result, DAFGA provides gene-specific parameter sets for operational taxonomic unit clustering and taxonomic assignment at desired rank, and it can be implemented into the diversity measurements offered by QIIME. AVAILABILITY AND IMPLEMENTATION: DAFGA is freely available with a manual and test data from https://github.com/outbig/DAFGA.

Ammonium induces differential expression of methane and nitrogen metabolism-related genes in Methylocystis sp. strain SC2.

Nitrogen source and concentration are major determinants of methanotrophic activity, but their effect on global gene expression is poorly studied. Methylocystis sp. strain SC2 produces two isozymes of particulate methane monooxygenase. These are encoded by pmoCAB1 (low-affinity pMMO1) and pmoCAB2 (high-affinity pMMO2). We used RNA-Seq to identify strain SC2 genes that respond to standard (10 mM) and high (30 mM) NH4(+) concentrations in the medium, compared with 10 mM NO3(-). While the expression of pmoCAB1 was unaffected, pmoCAB2 was significantly downregulated (log2 fold changes of -5.0 to -6.0). Among nitrogen metabolism-related processes, genes involved in hydroxylamine detoxification (haoAB) were highly upregulated, while those for assimilatory nitrate/nitrite reduction, high-affinity ammonium uptake and nitrogen regulatory protein PII were downregulated. Differential expression of pmoCAB2 and haoAB was independently validated by end-point reverse transcription polymerase chain reaction. Methane oxidation by SC2 cells exposed to 30 mM NH4(+) was inhibited at ≤ 400 ppmv CH4 , where pMMO2 but not pMMO1 is functional. When transferred back to standard nitrogen concentration, methane oxidation capability and pmoCAB2 expression were restored. Given that Methylocystis contributes to atmospheric methane oxidation in upland soils, differential expression of pmoCAB2 explains, at least to some extent, the strong inhibitory effect of ammonium fertilizers on this activity.

Gammaproteobacterial methanotrophs dominate cold methane seeps in floodplains of West Siberian rivers.

A complex system of muddy fluid-discharging and methane (CH4)-releasing seeps was discovered in a valley of the river Mukhrinskaya, one of the small rivers of the Irtysh Basin, West Siberia. CH4 flux from most (90%) of these gas ebullition sites did not exceed 1.45 g CH4 h(-1), while some seeps emitted up to 5.54 g CH4 h(-1). The δ(13)C value of methane released from these seeps varied between -71.1 and -71.3‰, suggesting its biogenic origin. Although the seeps were characterized by low in situ temperatures (3.5 to 5°C), relatively high rates of methane oxidation (15.5 to 15.9 nmol CH4 ml(-1) day(-1)) were measured in mud samples. Fluorescence in situ hybridization detected 10(7) methanotrophic bacteria (MB) per g of mud (dry weight), which accounted for up to 20.5% of total bacterial cell counts. Most (95.8 to 99.3%) methanotroph cells were type I (gammaproteobacterial) MB. The diversity of methanotrophs in this habitat was further assessed by pyrosequencing of pmoA genes, encoding particulate methane monooxygenase. A total of 53,828 pmoA gene sequences of seep-inhabiting methanotrophs were retrieved and analyzed. Nearly all of these sequences affiliated with type I MB, including the Methylobacter-Methylovulum-Methylosoma group, lake cluster 2, and several as-yet-uncharacterized methanotroph clades. Apparently, microbial communities attenuating methane fluxes from these local but strong CH4 sources in floodplains of high-latitude rivers have a large proportion of potentially novel, psychrotolerant methanotrophs, thereby providing a challenge for future isolation studies.

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.

Deciphering the functional and structural complexity of the Solar Lake flat mat microbial benthic communities.

The Solar Lake in Taba, Egypt, encompasses one of the few modern-day microbial mats' systems metabolically analogous to Precambrian stromatolites. Solar Lake benthic communities and their adaptation to the Lake's unique limnological cycle have not been described for over two decades. In this study, we revisit the flat mat and describe the summer's shallow water versus exposed microbial community; the latter occurs in response to the seasonal partial receding of water. We employed metagenomic NovaSeq-6000 shotgun sequencing and 16S rRNA, mcrA, and dsrB quantitative PCR. A total of 292 medium-to-high-quality metagenome-assembled genomes (MAGs) were reconstructed. At the structural level, Candidatus Aenigmatarchaeota, Micrarchaeota, and Omnitrophota MAGs were exclusively detected in the shallow-water mats, whereas Halobacteria and Myxococcota MAGs were specific to the exposed microbial mat. Functionally, genes involved in reactive oxygen species (ROS) detoxification and osmotic pressure were more abundant in the exposed than in the shallow-water microbial mats, whereas genes involved in sulfate reduction/oxidation and nitrogen fixation were ubiquitously detected. Genes involved in the utilization of methylated amines for methane production were predominant when compared with genes associated with alternative methanogenesis pathways. Solar Lake methanogen MAGs belonged to Methanosarcinia, Bathyarchaeia, Candidatus Methanofastidiosales, and Archaeoglobales. The latter had the genetic capacity for anaerobic methane oxidation. Moreover, Coleofasciculus chthonoplastes, previously reported to dominate the winter shallow-water flat mat, had a substantial presence in the summer. These findings reveal the taxonomic and biochemical microbial zonation of the exposed and shallow-water Solar Lake flat mat benthic community and their capacity to ecologically adapt to the summer water recession. IMPORTANCE: Fifty-five years ago, the extremophilic "Solar Lake" was discovered on the Red Sea shores, garnering microbiologists' interest worldwide from the 1970s to 1990s. Nevertheless, research on the lake paused at the turn of the millennium. In our study, we revisited the Solar Lake benthic community using a genome-centric approach and described the distinct microbial communities in the exposed versus shallow-water mat unveiling microbial zonation in the benthic communities surrounding the Solar Lake. Our findings highlighted the unique structural and functional adaptations employed by these microbial mat communities. Moreover, we report new methanogens and phototrophs, including an intriguing methanogen from the Archaeoglobales family. We describe how the Solar Lake's flat mat microbial community adapts to stressors like oxygen intrusion and drought due to summer water level changes, which provides insights into the genomic strategies of microbial communities to cope with altered and extreme environmental conditions.

Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia.

Aerobic methanotrophic bacteria consume methane as it diffuses away from methanogenic zones of soil and sediment. They act as a biofilter to reduce methane emissions to the atmosphere, and they are therefore targets in strategies to combat global climate change. No cultured methanotroph grows optimally below pH 5, but some environments with active methane cycles are very acidic. Here we describe an extremely acidophilic methanotroph that grows optimally at pH 2.0-2.5. Unlike the known methanotrophs, it does not belong to the phylum Proteobacteria but rather to the Verrucomicrobia, a widespread and diverse bacterial phylum that primarily comprises uncultivated species with unknown genotypes. Analysis of its draft genome detected genes encoding particulate methane monooxygenase that were homologous to genes found in methanotrophic proteobacteria. However, known genetic modules for methanol and formaldehyde oxidation were incomplete or missing, suggesting that the bacterium uses some novel methylotrophic pathways. Phylogenetic analysis of its three pmoA genes (encoding a subunit of particulate methane monooxygenase) placed them into a distinct cluster from proteobacterial homologues. This indicates an ancient divergence of Verrucomicrobia and Proteobacteria methanotrophs rather than a recent horizontal gene transfer of methanotrophic ability. The findings show that methanotrophy in the Bacteria is more taxonomically, ecologically and genetically diverse than previously thought, and that previous studies have failed to assess the full diversity of methanotrophs in acidic environments.

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.

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.

Complete genome sequence of Methylocystis sp. strain SC2, an aerobic methanotroph with high-affinity methane oxidation potential.

Methylocystis sp. strain SC2 is an aerobic type II methanotroph isolated from a highly polluted aquifer in Germany. A specific trait of the SC2 strain is the expression of two isozymes of particulate methane monooxygenase with different methane oxidation kinetics. Here we report the complete genome sequence of this methanotroph that contains not only a circular chromosome but also two large plasmids.