Time-series metaproteogenomics of a high-CO2 aquifer reveals active viruses with fluctuating abundances and broad host ranges.

Ecosystems subject to mantle degassing are of particular interest for understanding global biogeochemistry, as their microbiomes are shaped by prolonged exposure to high CO2 and have recently been suggested to be highly active. While the genetic diversity of bacteria and archaea in these deep biosphere systems have been studied extensively, little is known about how viruses impact these microbial communities. Here, we show that the viral community in a high-CO2 cold-water geyser (Wallender Born, Germany) undergoes substantial fluctuations over a period of 12 days, although the corresponding prokaryotic community remains stable, indicating a newly observed "infect to keep in check" strategy that maintains prokaryotic community structure. We characterized the viral community using metagenomics and metaproteomics, revealing 8 654 viral operational taxonomic units (vOTUs). CRISPR spacer-to-protospacer matching linked 278 vOTUs to 32 hosts, with many vOTUs sharing hosts from different families. High levels of viral structural proteins present in the metaproteome (several structurally annotated based on AlphaFold models) indicate active virion production at the time of sampling. Viral genomes expressed many proteins involved in DNA metabolism and manipulation, and encoded for auxiliary metabolic genes, which likely bolster phosphate and sulfur metabolism of their hosts. The active viral community encodes genes to facilitate acquisition and transformation of host nutrients, and appears to consist of many nutrient-demanding members, based on abundant virion proteins. These findings indicate viruses are inextricably linked to the biogeochemical cycling in this high-CO2 environment and substantially contribute to prokaryotic community stability in the deep biosphere hotspots.

Complete Genome Sequence of Citrobacter braakii GW-Imi-1b1, Isolated from Hospital Wastewater in Greifswald, Germany.

The imipenem-resistant Citrobacter braakii strain GW-Imi-1b1 was isolated from a hospital wastewater sample in Greifswald, Germany. The genome comprises one chromosome (5.09 Mb), one prophage (41.9 kb), and 13 plasmids (2 to 140.9 kb). The genome harbors 5,322 coding sequences, shows a high potential for genomic mobility, and includes genes encoding proteins for multiple drug resistances.

Influenza A H1N1 Induced Disturbance of the Respiratory and Fecal Microbiome of German Landrace Pigs - a Multi-Omics Characterization.

Seasonal influenza outbreaks represent a large burden for the health care system as well as the economy. While the role of the microbiome has been elucidated in the context of various diseases, the impact of respiratory viral infections on the human microbiome is largely unknown. In this study, swine was used as an animal model to characterize the temporal dynamics of the respiratory and gastrointestinal microbiome in response to an influenza A virus (IAV) infection. A multi-omics approach was applied on fecal samples to identify alterations in microbiome composition and function during IAV infection. We observed significantly altered microbial richness and diversity in the gastrointestinal microbiome after IAV infection. In particular, increased abundances of Prevotellaceae were detected, while Clostridiaceae and Lachnospiraceae decreased. Moreover, our metaproteomics data indicated that the functional composition of the microbiome was heavily affected by the influenza infection. For instance, we identified decreased amounts of flagellin, correlating with reduced abundances of Lachnospiraceae and Clostridiaceae, possibly indicating involvement of a direct immune response toward flagellated Clostridia during IAV infection. Furthermore, enzymes involved in short-chain fatty acid (SCFA) synthesis were identified in higher abundances, while metabolome analyses revealed rather stable concentrations of SCFAs. In addition, 16S rRNA gene sequencing was used to characterize effects on the composition and natural development of the upper respiratory tract microbiome. Our results showed that IAV infection resulted in significant changes in the abundance of Moraxellaceae and Pasteurellaceae in the upper respiratory tract. Surprisingly, temporal development of the respiratory microbiome structure was not affected. IMPORTANCE Here, we used swine as a biomedical model to elucidate the impact of influenza A H1N1 infection on structure and function of the respiratory and gastrointestinal tract microbiome by employing a multi-omics analytical approach. To our knowledge, this is the first study to investigate the temporal development of the porcine microbiome and to provide insights into the functional capacity of the gastrointestinal microbiome during influenza A virus infection.

Can nutrient uptake by Carex counteract eutrophication in fen peatlands?

Ground- and surface-water-fed peatlands (i.e., fens) of temperate Europe face high anthropogenic nutrient loads from atmospheric deposition, agricultural catchment areas, and from peat decomposition, if drained. As a result, nitrogen loads may exceed a fen's natural nutrient removal capacity, leading to increased eutrophication of adjacent water bodies. Therefore, it is important to address possible means to decrease a fen's nutrient load, including nutrient uptake by fen plants. To assess how much fen plants can contribute to nutrient removal by uptake, nutrient stocks of above- and below-ground biomass need to be quantified. Therefore, we investigated nitrogen, phosphorous, and potassium uptake capacities of sedges (Carex species), which are common dominants in fen plant communities. We grew specimens of five Carex species with varying preferences in nutrient availability under controlled, different nutrient levels. We show that Carex above-ground biomass harvest can remove up to one third of a system's total nitrogen even at high loads of about 40 g nitrogen m-2. Species-specific differences in biomass production, rather than preferences in nutrient availability under natural conditions, were drivers of standing nutrient stocks: Highly productive species, i.e., C. acutiformis and C. rostrata, had highest nutrient standing stocks across all nutrient levels. Amounts of nutrients stored in shoots increased almost linearly with increasing nutrient levels, whereas below-ground nutrient stocks species-specifically increased, saturated, or decreased, with increasing nutrient levels. As a rough estimate, depending on the species, 6-16 cycles of annual above-ground harvest would suffice to decrease nitrogen concentrations from the highest to the lowest level used in this study. Overall, our results indicate that Carex biomass harvest can be an efficient means to counteract anthropogenic nitrogen eutrophication in fens.

Transcriptional response of key metabolic and stress response genes of a nuculanid bivalve, Lembulus bicuspidatus from an oxygen minimum zone exposed to hypoxia-reoxygenation.

Benthic animals inhabiting the edges of marine oxygen minimum zones (OMZ) are exposed to unpredictable large fluctuations of oxygen levels. Sessile organisms including bivalves must depend on physiological adaptations to withstand these conditions. However, as habitats are rather inaccessible, physiological adaptations of the OMZ margin inhabitants to oxygen fluctuations are not well understood. We therefore investigated the transcriptional responses of selected key genes involved in energy metabolism and stress protection in a dominant benthic species of the northern edge of the Namibian OMZ, the nuculanid clam Lembulus bicuspidatus,. We exposed clams to normoxia (~5.8 ml O2 l-1), severe hypoxia (36 h at ~0.01 ml O2 l-1) and post-hypoxic recovery (24 h of normoxia following 36 h of severe hypoxia). Using newly identified gene sequences, we determined the transcriptional responses to hypoxia and reoxygenation of the mitochondrial aerobic energy metabolism (pyruvate dehydrogenase E1 complex, cytochrome c oxidase, citrate synthase, and adenine nucleotide translocator), anaerobic glycolysis (hexokinase (HK), phosphoenolpyruvate carboxykinase (PEPCK), phosphofructokinase, and aldolase), mitochondrial antioxidants (glutaredoxin, peroxiredoxin, and uncoupling protein UCP2) and stress protection mechanisms (a molecular chaperone HSP70 and a mitochondrial quality control protein MIEAP) in the gills and the labial palps of L. bicuspidatus. Exposure to severe hypoxia transcriptionally stimulated anaerobic glycolysis (including HK and PEPCK), antioxidant protection (UCP2), and quality control mechanisms (HSP70 and MIEAP) in the gills of L. bicuspidatus. Unlike UCP2, mRNA levels of the thiol-dependent mitochondrial antioxidants were not affected by hypoxia-reoxygenation stress. Transcript levels of marker genes for aerobic energy metabolism were not responsive to oxygen fluctuations in L. bicuspidatus. Our findings highlight the probable importance of anaerobic succinate production (via PEPCK) and mitochondrial and proteome quality control mechanisms in responses to oxygen fluctuations of the OMZ bivalve L.bicuspidatus. The reaction of L.bicuspidatus to oxygen fluctuations implies parallels to that of other hypoxia-tolerant bivalves, such as intertidal species.

Bacterial symbiont subpopulations have different roles in a deep-sea symbiosis.

The hydrothermal vent tubeworm Riftia pachyptila hosts a single 16S rRNA phylotype of intracellular sulfur-oxidizing symbionts, which vary considerably in cell morphology and exhibit a remarkable degree of physiological diversity and redundancy, even in the same host. To elucidate whether multiple metabolic routes are employed in the same cells or rather in distinct symbiont subpopulations, we enriched symbionts according to cell size by density gradient centrifugation. Metaproteomic analysis, microscopy, and flow cytometry strongly suggest that Riftia symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: While small symbionts actively divide and may establish cellular symbiont-host interaction, large symbionts apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Moreover, in large symbionts, carbon fixation and biomass production seem to be metabolic priorities. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.

Comparative proteomics of related symbiotic mussel species reveals high variability of host-symbiont interactions.

Deep-sea Bathymodiolus mussels and their chemoautotrophic symbionts are well-studied representatives of mutualistic host-microbe associations. However, how host-symbiont interactions vary on the molecular level between related host and symbiont species remains unclear. Therefore, we compared the host and symbiont metaproteomes of Pacific B. thermophilus, hosting a thiotrophic symbiont, and Atlantic B. azoricus, containing two symbionts, a thiotroph and a methanotroph. We identified common strategies of metabolic support between hosts and symbionts, such as the oxidation of sulfide by the host, which provides a thiosulfate reservoir for the thiotrophic symbionts, and a cycling mechanism that could supply the host with symbiont-derived amino acids. However, expression levels of these processes differed substantially between both symbioses. Backed up by genomic comparisons, our results furthermore revealed an exceptionally large repertoire of attachment-related proteins in the B. thermophilus symbiont. These findings imply that host-microbe interactions can be quite variable, even between closely related systems.

Horizontal acquisition of a patchwork Calvin cycle by symbiotic and free-living Campylobacterota (formerly Epsilonproteobacteria).

Most autotrophs use the Calvin-Benson-Bassham (CBB) cycle for carbon fixation. In contrast, all currently described autotrophs from the Campylobacterota (previously Epsilonproteobacteria) use the reductive tricarboxylic acid cycle (rTCA) instead. We discovered campylobacterotal epibionts ("Candidatus Thiobarba") of deep-sea mussels that have acquired a complete CBB cycle and may have lost most key genes of the rTCA cycle. Intriguingly, the phylogenies of campylobacterotal CBB cycle genes suggest they were acquired in multiple transfers from Gammaproteobacteria closely related to sulfur-oxidizing endosymbionts associated with the mussels, as well as from Betaproteobacteria. We hypothesize that "Ca. Thiobarba" switched from the rTCA cycle to a fully functional CBB cycle during its evolution, by acquiring genes from multiple sources, including co-occurring symbionts. We also found key CBB cycle genes in free-living Campylobacterota, suggesting that the CBB cycle may be more widespread in this phylum than previously known. Metatranscriptomics and metaproteomics confirmed high expression of CBB cycle genes in mussel-associated "Ca. Thiobarba". Direct stable isotope fingerprinting showed that "Ca. Thiobarba" has typical CBB signatures, suggesting that it uses this cycle for carbon fixation. Our discovery calls into question current assumptions about the distribution of carbon fixation pathways in microbial lineages, and the interpretation of stable isotope measurements in the environment.

Host-Microbe Interactions in the Chemosynthetic Riftia pachyptila Symbiosis.

The deep-sea tubeworm Riftia pachyptila lacks a digestive system but completely relies on bacterial endosymbionts for nutrition. Although the symbiont has been studied in detail on the molecular level, such analyses were unavailable for the animal host, because sequence information was lacking. To identify host-symbiont interaction mechanisms, we therefore sequenced the Riftia transcriptome, which served as a basis for comparative metaproteomic analyses of symbiont-containing versus symbiont-free tissues, both under energy-rich and energy-limited conditions. Our results suggest that metabolic interactions include nutrient allocation from symbiont to host by symbiont digestion and substrate transfer to the symbiont by abundant host proteins. We furthermore propose that Riftia maintains its symbiont by protecting the bacteria from oxidative damage while also exerting symbiont population control. Eukaryote-like symbiont proteins might facilitate intracellular symbiont persistence. Energy limitation apparently leads to reduced symbiont biomass and increased symbiont digestion. Our study provides unprecedented insights into host-microbe interactions that shape this highly efficient symbiosis.IMPORTANCE All animals are associated with microorganisms; hence, host-microbe interactions are of fundamental importance for life on earth. However, we know little about the molecular basis of these interactions. Therefore, we studied the deep-sea Riftia pachyptila symbiosis, a model association in which the tubeworm host is associated with only one phylotype of endosymbiotic bacteria and completely depends on this sulfur-oxidizing symbiont for nutrition. Using a metaproteomics approach, we identified both metabolic interaction processes, such as substrate transfer between the two partners, and interactions that serve to maintain the symbiotic balance, e.g., host efforts to control the symbiont population or symbiont strategies to modulate these host efforts. We suggest that these interactions are essential principles of mutualistic animal-microbe associations.

Two intracellular and cell type-specific bacterial symbionts in the placozoan Trichoplax H2.

Placozoa is an enigmatic phylum of simple, microscopic, marine metazoans1,2. Although intracellular bacteria have been found in all members of this phylum, almost nothing is known about their identity, location and interactions with their host3-6. We used metagenomic and metatranscriptomic sequencing of single host individuals, plus metaproteomic and imaging analyses, to show that the placozoan Trichoplax sp. H2 lives in symbiosis with two intracellular bacteria. One symbiont forms an undescribed genus in the Midichloriaceae (Rickettsiales)7,8 and has a genomic repertoire similar to that of rickettsial parasites9,10, but does not seem to express key genes for energy parasitism. Correlative image analyses and three-dimensional electron tomography revealed that this symbiont resides in the rough endoplasmic reticulum of its host's internal fibre cells. The second symbiont belongs to the Margulisbacteria, a phylum without cultured representatives and not known to form intracellular associations11-13. This symbiont lives in the ventral epithelial cells of Trichoplax, probably metabolizes algal lipids digested by its host and has the capacity to supplement the placozoan's nutrition. Our study shows that one of the simplest animals has evolved highly specific and intimate associations with symbiotic, intracellular bacteria and highlights that symbioses can provide access to otherwise elusive microbial dark matter.

More Is Not Always Better: Evaluation of 1D and 2D-LC-MS/MS Methods for Metaproteomics.

Metaproteomics, the study of protein expression in microbial communities, is a versatile tool for environmental microbiology. Achieving sufficiently high metaproteome coverage to obtain a comprehensive picture of the activities and interactions in microbial communities is one of the current challenges in metaproteomics. An essential step to maximize the number of identified proteins is peptide separation via liquid chromatography (LC) prior to mass spectrometry (MS). Thorough optimization and comparison of LC methods for metaproteomics are, however, currently lacking. Here, we present an extensive development and test of different 1D and 2D-LC approaches for metaproteomic peptide separations. We used fully characterized mock community samples to evaluate metaproteomic approaches with very long analytical columns (50 and 75 cm) and long gradients (up to 12 h). We assessed a total of over 20 different 1D and 2D-LC approaches in terms of number of protein groups and unique peptides identified, peptide spectrum matches (PSMs) generated, the ability to detect proteins of low-abundance species, the effect of technical replicate runs on protein identifications and method reproducibility. We show here that, while 1D-LC approaches are faster and easier to set up and lead to more identifications per minute of runtime, 2D-LC approaches allow for a higher overall number of identifications with up to >10,000 protein groups identified. We also compared the 1D and 2D-LC approaches to a standard GeLC workflow, in which proteins are pre-fractionated via gel electrophoresis. This method yielded results comparable to the 2D-LC approaches, however with the drawback of a much increased sample preparation time. Based on our results, we provide recommendations on how to choose the best LC approach for metaproteomics experiments, depending on the study aims.

Effects of hypoxia-reoxygenation stress on mitochondrial proteome and bioenergetics of the hypoxia-tolerant marine bivalve Crassostrea gigas.

Mitochondria are key intracellular targets of hypoxia-reoxygenation (H/R) stress due to their central role in generation of ATP and reactive oxygen species (ROS). Intertidal oysters Crassostrea gigas are adapted to frequent H/R cycles and maintain aerobic function despite frequent oxygen fluctuations. To gain insight into the molecular mechanisms of H/R tolerance, we assessed changes in mitochondrial respiration and (phospho)proteome of C. gigas during hypoxia and recovery. Oyster mitochondria maintained OXPHOS capacity despite a decline in cytochrome c oxidase activity during H/R stress. Rearrangements of the mitochondrial proteome during H/R stress involved upregulation of mitochondrial electron transport system and iron-binding proteins, and suppression of the pathways that channel electrons to ubiquinone, possibly as a mechanism to limit ROS production. H/R stress led to upregulation of a mitophagic activator PGAM5 and dephosphorylation of metalloendopeptidase OMA1, indicating stimulation of mitochondrial quality control mechanisms. Changes in abundance and phosphorylation levels of key proteins involved in mitochondrial protein homeostasis indicate suppression of protein synthesis during hypoxia, likely as an energy-saving mechanism, and its subsequent reactivation during reoxygenation. Thus, shifts in the mitochondrial (phospho-)proteome might play an important role in H/R stress resistance of oysters ensuring mitochondrial integrity and function during oxygen fluctuations. SIGNIFICANCE: Hypoxia-reoxygenation (H/R) stress elicits shifts in proteome and phosphoproteome of mitochondria in a hypoxia-tolerant model bivalve, oyster Crassostrea gigas, upregulating electron transport system, limiting electron flow to ubiquinone and activating mitochondrial quality control and protein homeostasis mechanisms. These findings provide insights into the potential role of proteomic shifts in adaptive response to H/R stress and serve as an important benchmark to understand the mechanisms of mitochondrial sensitivity to hypoxia and reoxygenation.

Centrifugation-Based Enrichment of Bacterial Cell Populations for Metaproteomic Studies on Bacteria-Invertebrate Symbioses.

Owing to high sample complexity, metaproteomic investigations on bacteria-animal symbioses with two or more uncultured partners can be challenging. A selective isolation or enrichment of distinct (sub-)populations within those consortia can solve this problem. Subsequent discrete proteomic analyses benefit from increased sample purity and higher proteome coverage for each of the individual organisms. Here, we describe centrifugation-based methods that allow for a separation of the host and its bacterial symbiont population(s), or even for an enrichment of distinct symbiotic cell cycle stages in the deep-sea mussels Bathymodiolus azoricus and B. thermophilus, the gutless oligochaete Olavius algarvensis and the deep-sea tube worm Riftia pachyptila, respectively.

Adaptive mechanisms that provide competitive advantages to marine bacteroidetes during microalgal blooms.

Polysaccharide degradation by heterotrophic microbes is a key process within Earth's carbon cycle. Here, we use environmental proteomics and metagenomics in combination with cultivation experiments and biochemical characterizations to investigate the molecular details of in situ polysaccharide degradation mechanisms during microalgal blooms. For this, we use laminarin as a model polysaccharide. Laminarin is a ubiquitous marine storage polymer of marine microalgae and is particularly abundant during phytoplankton blooms. In this study, we show that highly specialized bacterial strains of the Bacteroidetes phylum repeatedly reached high abundances during North Sea algal blooms and dominated laminarin turnover. These genomically streamlined bacteria of the genus Formosa have an expanded set of laminarin hydrolases and transporters that belonged to the most abundant proteins in the environmental samples. In vitro experiments with cultured isolates allowed us to determine the functions of in situ expressed key enzymes and to confirm their role in laminarin utilization. It is shown that laminarin consumption of Formosa spp. is paralleled by enhanced uptake of diatom-derived peptides. This study reveals that genome reduction, enzyme fusions, transporters, and enzyme expansion as well as a tight coupling of carbon and nitrogen metabolism provide the tools, which make Formosa spp. so competitive during microalgal blooms.

Metaproteomics method to determine carbon sources and assimilation pathways of species in microbial communities.

Measurements of stable carbon isotope ratios (δ13C) are widely used in biology to address questions regarding food sources and metabolic pathways used by organisms. The analysis of these so-called stable isotope fingerprints (SIFs) for microbes involved in biogeochemical cycling and microbiota of plants and animals has led to major discoveries in environmental microbiology. Currently, obtaining SIFs for microbial communities is challenging as the available methods either only provide low taxonomic resolution, such as the use of lipid biomarkers, or are limited in throughput, such as nanoscale secondary ion MS imaging of single cells. Here we present "direct protein-SIF" and the Calis-p software package (https://sourceforge.net/projects/calis-p/), which enable high-throughput measurements of accurate δ13C values for individual species within a microbial community. We benchmark the method using 20 pure culture microorganisms and show that the method reproducibly provides SIF values consistent with gold-standard bulk measurements performed with an isotope ratio mass spectrometer. Using mock community samples, we demonstrate that SIF values can also be obtained for individual species within a microbial community. Finally, a case study of an obligate bacteria-animal symbiosis shows that direct protein-SIF confirms previous physiological hypotheses and can provide unexpected insights into the symbionts' metabolism. This confirms the usefulness of this approach to accurately determine δ13C values for different species in microbial community samples.

Nitrogen fixation in a chemoautotrophic lucinid symbiosis.

The shallow water bivalve Codakia orbicularis lives in symbiotic association with a sulfur-oxidizing bacterium in its gills. The endosymbiont fixes CO2 and thus generates organic carbon compounds, which support the host's growth. To investigate the uncultured symbiont's metabolism and symbiont-host interactions in detail we conducted a proteogenomic analysis of purified bacteria. Unexpectedly, our results reveal a hitherto completely unrecognized feature of the C. orbicularis symbiont's physiology: the symbiont's genome encodes all proteins necessary for biological nitrogen fixation (diazotrophy). Expression of the respective genes under standard ambient conditions was confirmed by proteomics. Nitrogenase activity in the symbiont was also verified by enzyme activity assays. Phylogenetic analysis of the bacterial nitrogenase reductase NifH revealed the symbiont's close relationship to free-living nitrogen-fixing Proteobacteria from the seagrass sediment. The C. orbicularis symbiont, here tentatively named 'Candidatus Thiodiazotropha endolucinida', may thus not only sustain the bivalve's carbon demands. C. orbicularis may also benefit from a steady supply of fixed nitrogen from its symbiont-a scenario that is unprecedented in comparable chemoautotrophic symbioses.