A Putatively New Family of Alphaproteobacterial Chloromethane Degraders from a Deciduous Forest Soil Revealed by Stable Isotope Probing and Metagenomics

Background: Chloromethane (CH3 Cl) is the most abundant halogenated organic compound in the atmosphere and substantially responsible for the destruction of the stratospheric ozone layer. Since anthropogenic CH 3 Cl sources have become negligible with the application of the Montreal Protocol (1987), natural sources, such as vegetation and soils, have increased proportionally in the global budget. CH3 Cl-degrading methylotrophs occurring in soils might be an important and overlooked sink.Results & Conclusions: The objective of our study was to link the biotic CH3 Cl sink with the identity of active microorganisms and their biochemical pathways for CH3 Cl degradation in a deciduous forest soil. When tested in laboratory microcosms, biological CH3 Cl consumption occurred in leaf litter, senescent leaves, and organic and mineral soil horizons. Highest consumption rates, around 2 mmol CH3 Cl g -1 dry weight h -1 , were measured in organic soil and senescent leaves, suggesting that top soil layers are active (micro-)biological CH 3 Cl degradation compartments of forest ecosystems. The DNA of these [13C]-CH3 Cl-degrading microbial communities was labelled using stable isotope probing (SIP), and the corresponding taxa and their metabolic pathways studied using high-throughput metagenomics sequencing analysis. [ 13C]-labelled Metagenome-Assembled Genome closely related to the family Beijerinckiaceae may represent a new methylotroph family of Alphaproteobacteria, which is found in metagenome databases of forest soils samples worldwide. Gene markers of the only known pathway for aerobic CH3 Cl degradation, via the methyltransferase system encoded by the CH3 Cl utilisation genes (cmu), were undetected in the DNA-SIP metagenome data, suggesting that biological CH3 Cl sink in this deciduous forest soil operates by a cmu-independent metabolism.

Carbon assimilating fungi from surface ocean to subseafloor revealed by coupled phylogenetic and stable isotope analysis

Fungi are ubiquitous in the ocean and hypothesized to be important members of marine ecosystems, but their roles in the marine carbon cycle are poorly understood. Here, we use 13C DNA stable isotope probing coupled with phylogenetic analyses to investigate carbon assimilation within diverse communities of planktonic and benthic fungi in the Benguela Upwelling System (Namibia). Across the redox stratified water column and in the underlying sediments, assimilation of 13C-labeled carbon from diatom extracellular polymeric substances (13C-dEPS) by fungi correlated with the expression of fungal genes encoding carbohydrate-active enzymes. Phylogenetic analysis of genes from 13C-labeled metagenomes revealed saprotrophic lineages related to the facultative yeast Malassezia were the main fungal foragers of pelagic dEPS. In contrast, fungi living in the underlying sulfidic sediments assimilated more 13C-labeled carbon from chemosynthetic bacteria compared to dEPS. This coincided with a unique seafloor fungal community and dissolved organic matter composition compared to the water column, and a 100-fold increased fungal abundance within the subseafloor sulfide-nitrate transition zone. The subseafloor fungi feeding on 13C-labeled chemolithoautotrophs under anoxic conditions were affiliated with Chytridiomycota and Mucoromycota that encode cellulolytic and proteolytic enzymes, revealing polysaccharide and protein-degrading fungi that can anaerobically decompose chemosynthetic necromass. These subseafloor fungi, therefore, appear to be specialized in organic matter that is produced in the sediments. Our findings reveal that the phylogenetic diversity of fungi across redox stratified marine ecosystems translates into functionally relevant mechanisms helping to structure carbon flow from primary producers in marine microbiomes from the surface ocean to the subseafloor.

Active communities and growth of soil microorganisms are framed by mean annual precipitation in three California annual grasslands

Earth system models project altered precipitation regimes across much of the globe. In California, the winter wet season is predicted to extend into spring, and the summer dry period to lengthen. How altered precipitation will affect soil carbon (C) persistence is a key knowledge gap. However, we do not have a mechanistic understanding of how altered soil moisture regimes will affect microbial population dynamics. Using quantitative stable isotope probing (qSIP), we compared total and active soil microbial communities across three California annual grassland ecosystems that span a rainfall gradient and have developed upon similar parent material. We also assessed multiple edaphic variables, including available C and the radiocarbon (14C) age of soil C. Samples were assayed in the wet season, when we expected environmental conditions would be most similar across sites. We hypothesized that the long-term legacy of soil water limitation would be reflected in lower community growth capacity at the driest site. We also predicted that actively growing communities would be more compositionally similar across the gradient than the total background microbiome. Across the three sites, edaphic parameters such as pH roughly sorted with mean annual precipitation, and soil carbon age increased with precipitation. Bacterial growth rates increased from the driest site to the intermediate site, and rates were comparable between the intermediate and wettest sites. These differences were persistent across major phyla, including the Actinobacteria, Bacteroidetes, and Proteobacteria. Taxonomic identity was a strong predictor of growth, such that the growth rates of a taxon at one site predicted its growth rates at the others. We think this fact, that taxa that grew quickly at one site tended to grow quickly at the others, is likely a consequence of genetically determined physiological traits, and is consistent with the idea that evolutionary history influences growth rate.

Grazing weakens competitive interactions between active methanotrophs and nitrifiers modulating greenhouse-gas emissions in grassland soils

Grassland soils serve as a biological sink and source of the potent greenhouse gases (GHG) methane (CH4) and nitrous oxide (N2O). The underlying mechanisms responsible for those GHG emissions, specifically, the relationships between methane- and ammonia-oxidizing microorganisms in grazed grassland soils are still poorly understood. Here, we characterized the effects of grazing on in situ GHG emissions and elucidated the putative relations between the active microbes involving in methane oxidation and nitrification activity in grassland soils. Grazing significantly decreases CH4 emissions while it increases N2O emissions basing on 14-month in situ measurement. DNA-based stable isotope probing (SIP) incubation experiment shows that grazing decreases both methane oxidation and nitrification processes and decreases the diversity of active methanotrophs and nitrifiers, and subsequently weakens the putative competition between active methanotrophs and nitrifiers in grassland soils. These results constitute a major advance in our understanding of putative relationships between methane- and ammonia-oxidizing microorganisms and subsequent effects on nitrification and methane oxidation, which contribute to a better prediction and modeling of future balance of GHG emissions and active microbial communities in grazed grassland ecosystems.

Calculation and Interpretation of Substrate Assimilation Rates in Microbial Cells Based on Isotopic Composition Data Obtained by nanoSIMS

Stable isotope probing (SIP) combined with nano-scale secondary ion mass spectrometry (nanoSIMS) is a powerful approach to quantify assimilation rates of elements such as C and N into individual microbial cells. Here, we use mathematical modeling to investigate how the derived rate estimates depend on the model used to describe substrate assimilation by a cell during a SIP incubation. We show that the most commonly used model, which is based on the simplifying assumptions of linearly increasing biomass of individual cells over time and no cell division, can yield underestimated assimilation rates when compared to rates derived from a model that accounts for cell division. This difference occurs because the isotopic labeling of a dividing cell increases more rapidly over time compared to a non-dividing cell and becomes more pronounced as the labeling increases above a threshold value that depends on the cell cycle stage of the measured cell. Based on the modeling results, we present formulae for estimating assimilation rates in cells and discuss their underlying assumptions, conditions of applicability, and implications for the interpretation of intercellular variability in assimilation rates derived from nanoSIMS data, including the impacts of storage inclusion metabolism. We offer the formulae as a Matlab script to facilitate rapid data evaluation by nanoSIMS users.

Multisubstrate DNA stable isotope probing reveals guild structure of bacteria that mediate soil carbon cycling

Soil microorganisms determine the fate of soil organic matter (SOM), and their activities compose a major component of the global carbon (C) cycle. We employed a multisubstrate, DNA-stable isotope probing experiment to track bacterial assimilation of C derived from distinct sources that varied in bioavailability. This approach allowed us to measure microbial contributions to SOM processing by measuring the C assimilation dynamics of diverse microorganisms as they interacted within soil. We identified and tracked 1,286 bacterial taxa that assimilated 13C in an agricultural soil over a period of 48 d. Overall 13C-assimilation dynamics of bacterial taxa, defined by the source and timing of the 13C they assimilated, exhibited low phylogenetic conservation. We identified bacterial guilds composed of taxa that had similar 13C assimilation dynamics. We show that C-source bioavailability explained significant variation in both C mineralization dynamics and guild structure, and that the growth dynamics of bacterial guilds differed significantly in response to C addition. We also demonstrate that the guild structure explains significant variation in the biogeographical distribution of bacteria at continental and global scales. These results suggest that an understanding of in situ growth dynamics is essential for understanding microbial contributions to soil C cycling. We interpret these findings in the context of bacterial life history strategies and their relationship to terrestrial C cycling.

Raman Microspectroscopy Goes Viral: Infection Dynamics in the Cosmopolitan Microalga, Emiliania huxleyi

Emiliania huxleyi is a cosmopolitan member of the marine phytoplankton. This species’ capacities for carbon sequestration and sulfur mobilization make it a key player in oceanic biogeochemical cycles that influence climate on a planetary scale. Seasonal E. huxleyi blooms are abruptly terminated by viral epidemics caused by a clade of large DNA viruses collectively known as coccolithoviruses (EhVs). EhVs thereby mediate a significant part of material and energy fluxes associated with E. huxleyi population dynamics. In this study, we use spontaneous Raman microspectroscopy to perform label-free and non-invasive measurements of the macromolecular composition of individual virions and E. huxleyi host cells. Our novel autofluorescence suppression protocol enabled spectroscopic visualization of evolving macromolecular redistributions in individual E. huxleyi cells at different stages of EhV infection. Material transfer from E. huxleyi hosts to single EhV-163 virions was confirmed by combining stable isotope probing (SIP) experiments with Raman microspectroscopy. Inheritance of the host cells’ 13C-enriched isotopic signature was quantified based on red shifts of Raman peaks characteristic of phenylalanine’s phenyl ring. Two-dimensional Raman mapping of EhV-infected E. huxleyi cells revealed that the compact region producing an intense Raman DNA signal (i.e., the nucleus) in healthy E. huxleyi cells becomes diffuse during the first hours of infection. Raman DNA emissions integrated throughout individual cells decreased during the infection cycle. Our observations are consistent with EhV-163 degrading the host’s nuclear DNA, scavenging released nucleotides for its own genome replication, and shedding newly-produced virions prior to host lysis via budding.