Constraining the composition and quantity of organic matter used by abundant marine Thaumarchaeota.

Marine Group I (MGI) Thaumarchaeota were originally described as chemoautotrophic nitrifiers, but molecular and isotopic evidence suggests heterotrophic and/or mixotrophic capabilities. Here, we investigated the quantity and composition of organic matter assimilated by individual, uncultured MGI cells from the Pacific Ocean to constrain their potential for mixotrophy and heterotrophy. We observed that most MGI cells did not assimilate carbon from any organic substrate provided (glucose, pyruvate, oxaloacetate, protein, urea, and amino acids). The minority of MGI cells that did assimilate it did so exclusively from nitrogenous substrates (urea, 15% of MGI and amino acids, 36% of MGI), and only as an auxiliary carbon source (<20% of that subset's total cellular carbon was derived from those substrates). At the population level, MGI assimilation of organic carbon comprised just 0.5%-11% of total biomass carbon. We observed extensive assimilation of inorganic carbon and urea- and amino acid-derived nitrogen (equal to that from ammonium), consistent with metagenomic and metatranscriptomic analyses performed here and previously showing a widespread potential for MGI to perform autotrophy and transport and degrade organic nitrogen. Our results constrain the quantity and composition of organic matter used by MGI and suggest they use it primarily to meet nitrogen demands for anabolism and nitrification.

HT-SIP: a semi-automated stable isotope probing pipeline identifies cross-kingdom interactions in the hyphosphere of arbuscular mycorrhizal fungi.

BACKGROUND: Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing-SIP-remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance-the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling. RESULTS: Our HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to 13C-AMF hyphosphere DNA from a 13CO2 plant labeling study and created metagenome-assembled genomes (MAGs) using high-resolution SIP metagenomics (14 metagenomes per gradient). SIP confirmed the AMF Rhizophagus intraradices and associated MAGs were highly enriched (10-33 atom% 13C), even though the soils' overall enrichment was low (1.8 atom% 13C). We assembled 212 13C-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived 13C were from the phyla Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia-oxidizing archaeon genus Nitrososphaera. CONCLUSIONS: Our semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP-fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat-generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs' phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling. Video Abstract.

Plant-associated fungi support bacterial resilience following water limitation.

Drought disrupts soil microbial activity and many biogeochemical processes. Although plant-associated fungi can support plant performance and nutrient cycling during drought, their effects on nearby drought-exposed soil microbial communities are not well resolved. We used H218O quantitative stable isotope probing (qSIP) and 16S rRNA gene profiling to investigate bacterial community dynamics following water limitation in the hyphospheres of two distinct fungal lineages (Rhizophagus irregularis and Serendipita bescii) grown with the bioenergy model grass Panicum hallii. In uninoculated soil, a history of water limitation resulted in significantly lower bacterial growth potential and growth efficiency, as well as lower diversity in the actively growing bacterial community. In contrast, both fungal lineages had a protective effect on hyphosphere bacterial communities exposed to water limitation: bacterial growth potential, growth efficiency, and the diversity of the actively growing bacterial community were not suppressed by a history of water limitation in soils inoculated with either fungus. Despite their similar effects at the community level, the two fungal lineages did elicit different taxon-specific responses, and bacterial growth potential was greater in R. irregularis compared to S. bescii-inoculated soils. Several of the bacterial taxa that responded positively to fungal inocula belong to lineages that are considered drought susceptible. Overall, H218O qSIP highlighted treatment effects on bacterial community structure that were less pronounced using traditional 16S rRNA gene profiling. Together, these results indicate that fungal-bacterial synergies may support bacterial resilience to moisture limitation.

Bacterial response to spatial gradients of algal-derived nutrients in a porous microplate.

Photosynthetic microalgae are responsible for 50% of the global atmospheric CO2 fixation into organic matter and hold potential as a renewable bioenergy source. Their metabolic interactions with the surrounding microbial community (the algal microbiome) play critical roles in carbon cycling, but due to methodological limitations, it has been challenging to examine how community development is influenced by spatial proximity to their algal host. Here we introduce a copolymer-based porous microplate to co-culture algae and bacteria, where metabolites are constantly exchanged between the microorganisms while maintaining physical separation. In the microplate, we found that the diatom Phaeodactylum tricornutum accumulated to cell abundances ~20 fold higher than under normal batch conditions due to constant replenishment of nutrients through the porous structure. We also demonstrate that algal-associated bacteria, both single isolates and complex communities, responded to inorganic nutrients away from their host as well as organic nutrients originating from the algae in a spatially predictable manner. These experimental findings coupled with a mathematical model suggest that host proximity and algal culture growth phase impact bacterial community development in a taxon-specific manner through organic and inorganic nutrient availability. Our novel system presents a useful tool to investigate universal metabolic interactions between microbes in aquatic ecosystems.

Characterizing Chemoautotrophy and Heterotrophy in Marine Archaea and Bacteria With Single-Cell Multi-isotope NanoSIP.

Characterizing and quantifying in situ metabolisms remains both a central goal and challenge for environmental microbiology. Here, we used a single-cell, multi-isotope approach to investigate the anabolic activity of marine microorganisms, with an emphasis on natural populations of Thaumarchaeota. After incubating coastal Pacific Ocean water with 13C-bicarbonate and 15N-amino acids, we used nanoscale secondary ion mass spectrometry (nanoSIMS) to isotopically screen 1,501 individual cells, and 16S rRNA amplicon sequencing to assess community composition. We established isotopic enrichment thresholds for activity and metabolic classification, and with these determined the percentage of anabolically active cells, the distribution of activity across the whole community, and the metabolic lifestyle-chemoautotrophic or heterotrophic-of each cell. Most cells (>90%) were anabolically active during the incubation, and 4-17% were chemoautotrophic. When we inhibited bacteria with antibiotics, the fraction of chemoautotrophic cells detected via nanoSIMS increased, suggesting archaea dominated chemoautotrophy. With fluorescence in situ hybridization coupled to nanoSIMS (FISH-nanoSIMS), we confirmed that most Thaumarchaeota were living chemoautotrophically, while bacteria were not. FISH-nanoSIMS analysis of cells incubated with dual-labeled (13C,15N-) amino acids revealed that most Thaumarchaeota cells assimilated amino-acid-derived nitrogen but not carbon, while bacteria assimilated both. This indicates that some Thaumarchaeota do not assimilate intact amino acids, suggesting intra-phylum heterogeneity in organic carbon utilization, and potentially their use of amino acids for nitrification. Together, our results demonstrate the utility of multi-isotope nanoSIMS analysis for high-throughput metabolic screening, and shed light on the activity and metabolism of uncultured marine archaea and bacteria.

Methyl Iodide Fumigation of Bacillus anthracis Spores.

Fumigation techniques such as chlorine dioxide, vaporous hydrogen peroxide, and paraformaldehyde previously used to decontaminate items, rooms, and buildings following contamination with Bacillus anthracis spores are often incompatible with materials (e.g., porous surfaces, organics, and metals), causing damage or residue. Alternative fumigation with methyl bromide is subject to U.S. and international restrictions due to its ozone-depleting properties. Methyl iodide, however, does not pose a risk to the ozone layer and has previously been demonstrated as a fumigant for fungi, insects, and nematodes. Until now, methyl iodide has not been evaluated against Bacillus anthracis. Sterne strain Bacillus anthracis spores were subjected to methyl iodide fumigation at room temperature and at 550C. Efficacy was measured on a log-scale with a 6-log reduction in CFUs being considered successful compared to the U.S. Environmental Protection Agency biocide standard. Such efficacies were obtained after just one hour at 55 °C and after 12 hours at room temperature. No detrimental effects were observed on glassware, PTFE O-rings, or stainless steel. This is the first reported efficacy of methyl iodide in the reduction of Bacillus anthracis spore contamination at ambient and elevated temperatures.