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The phylum “Candidatus Omnitrophica” (candidate division OP3) is ubiquitous in anaerobic habitats but is currently characterized only by draft genomes from metagenomes and single cells. We had visualized cells of the phylotype OP3 LiM in methanogenic cultures on limonene as small epibiotic cells. In this study, we enriched OP3 cells by double density gradient centrifugation and obtained the first closed genome of an apparently clonal OP3 cell population by applying metagenomics and PCR for gap closure. Filaments of acetoclastic Methanosaeta, the largest morphotype in the culture community, contained empty cells, cells devoid of rRNA or of both rRNA and DNA, and dead cells according to transmission electron microscopy (TEM), thin-section TEM, scanning electron microscopy (SEM), catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), and LIVE/DEAD imaging. OP3 LiM cells were ultramicrobacteria (200 to 300 nm in diameter) and showed two physiological stages in CARD-FISH fluorescence signals: strong signals of OP3 LiM cells attached to Bacteria and to Archaea indicated many rRNA molecules and an active metabolism, whereas free-living OP3 cells had weak signals. Metaproteomics revealed that OP3 LiM lives with highly expressed secreted proteins involved in depolymerization and uptake of macromolecules and an active glycolysis and energy conservation by the utilization of pyruvate via a pyruvate:ferredoxin oxidoreductase and an Rnf complex (ferredoxin:NAD oxidoreductase). Besides sugar fermentation, a nucleotidyl transferase may contribute to energy conservation by phosphorolysis, the phosphate-dependent depolymerization of nucleic acids. Thin-section TEM showed distinctive structures of predation. Our study demonstrated a predatory metabolism for OP3 LiM cells, and therefore, we propose the name “Candidatus Velamenicoccus archaeovorus” gen. nov., sp. nov., for OP3 LiM. IMPORTANCE Epibiotic bacteria are known to live on and off bacterial cells. Here, we describe the ultramicrobacterial anaerobic epibiont OP3 LiM living on Archaea and Bacteria. We detected sick and dead cells of the filamentous archaeon Methanosaeta in slowly growing methanogenic cultures. OP3 LiM lives as a sugar fermenter, likely on polysaccharides from outer membranes, and has the genomic potential to live as a syntroph. The predatory lifestyle of OP3 LiM was supported by its genome, the first closed genome for the phylum “Candidatus Omnitrophica,” and by images of cell-to-cell contact with prey cells. We propose naming OP3 LiM “Candidatus Velamenicoccus archaeovorus.” Its metabolic versatility explains the ubiquitous presence of “Candidatus Omnitrophica” 3 in anoxic habitats and gives ultramicrobacterial epibionts an important role in the recycling and remineralization of microbial biomass. The removal of polysaccharides from outer membranes by ultramicrobacteria may also influence biological interactions between pro- and eukaryotes.

Chemosynthetic symbioses occur worldwide in marine habitats, but comprehensive physiological studies of chemoautotrophic bacteria thriving on animals are scarce. Stilbonematinae are coated by thiotrophic Gammaproteobacteria. As these nematodes migrate through the redox zone, their ectosymbionts experience varying oxygen concentrations. However, nothing is known about how these variations affect their physiology. Here, by applying omics, Raman microspectroscopy, and stable isotope labeling, we investigated the effect of oxygen on “Candidatus Thiosymbion oneisti.” Unexpectedly, sulfur oxidation genes were upregulated in anoxic relative to oxic conditions, but carbon fixation genes and incorporation of 13C-labeled bicarbonate were not. Instead, several genes involved in carbon fixation were upregulated under oxic conditions, together with genes involved in organic carbon assimilation, polyhydroxyalkanoate (PHA) biosynthesis, nitrogen fixation, and urea utilization. Furthermore, in the presence of oxygen, stress-related genes were upregulated together with vitamin biosynthesis genes likely necessary to withstand oxidative stress, and the symbiont appeared to proliferate less. Based on its physiological response to oxygen, we propose that “Ca. T. oneisti” may exploit anaerobic sulfur oxidation coupled to denitrification to proliferate in anoxic sand. However, the ectosymbiont would still profit from the oxygen available in superficial sand, as the energy-efficient aerobic respiration would facilitate carbon and nitrogen assimilation. IMPORTANCE Chemoautotrophic endosymbionts are famous for exploiting sulfur oxidization to feed marine organisms with fixed carbon. However, the physiology of thiotrophic bacteria thriving on the surface of animals (ectosymbionts) is less understood. One longstanding hypothesis posits that attachment to animals that migrate between reduced and oxic environments would boost sulfur oxidation, as the ectosymbionts would alternatively access sulfide and oxygen, the most favorable electron acceptor. Here, we investigated the effect of oxygen on the physiology of “Candidatus Thiosymbion oneisti,” a gammaproteobacterium which lives attached to marine nematodes inhabiting shallow-water sand. Surprisingly, sulfur oxidation genes were upregulated under anoxic relative to oxic conditions. Furthermore, under anoxia, the ectosymbiont appeared to be less stressed and to proliferate more. We propose that animal-mediated access to oxygen, rather than enhancing sulfur oxidation, would facilitate assimilation of carbon and nitrogen by the ectosymbiont.

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., 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.

Large surface-to-volume ratios provide optimal nutrient uptake conditions for small microorganisms in oligotrophic habitats. The surface area can be increased with appendages. Here, we describe chains of interconnecting vesicles protruding from cells of strain Hel3_A1_48, affiliating with Formosa spp. within the Flavobacteriia and originating from coastal free-living bacterioplankton. The chains were up to 10 μm long and had vesicles emanating from the outer membrane with a single membrane and a size of 80 to 100 nm by 50 to 80 nm. Cells extruded membrane tubes in the exponential phase, whereas vesicle chains dominated on cells in the stationary growth phase. This formation is known as pearling, a physical morphogenic process in which membrane tubes protrude from liposomes and transform into chains of interconnected vesicles. Proteomes of whole-cell membranes and of detached vesicles were dominated by outer membrane proteins, including the type IX secretion system and surface-attached peptidases, glycoside hydrolases, and endonucleases. Fluorescein-labeled laminarin stained the cells and the vesicle chains. Thus, the appendages provide binding domains and degradative enzymes on their surfaces and probably storage volume in the vesicle lumen. Both may contribute to the high abundance of these Formosa-affiliated bacteria during laminarin utilization shortly after spring algal blooms. IMPORTANCE Microorganisms produce membrane vesicles. One synthesis pathway seems to be pearling that describes the physical formation of vesicle chains from phospholipid vesicles via extended tubes. Bacteria with vesicle chains had been observed as well as bacteria with tubes, but pearling was so far not observed. Here, we report the observation of, initially, tubes and then vesicle chains during the growth of a flavobacterium, suggesting biopearling of vesicle chains. The flavobacterium is abundant during spring bacterioplankton blooms developing after algal blooms and has a special set of enzymes for laminarin, the major storage polysaccharide of microalgae. We demonstrated with fluorescently labeled laminarin that the vesicle chains bind laminarin or contain laminarin-derived compounds. Proteomic analyses revealed surface-attached degradative enzymes on the outer membrane vesicles. We conclude that the large surface area and the lumen of vesicle chains may contribute to the ecological success of this marine bacterium.

Enclosure experiments are frequently used to investigate the impact of changing environmental conditions on microbial assemblages. Yet, how the incuba- tion itself challenges complex bacterial communities is thus far unknown. In this study, metaproteomic profiling, 16S rRNA gene analyses, and cell counts were com- bined to evaluate bacterial communities derived from marine, mesohaline, and oli- gohaline conditions after long-term batch incubations. Early in the experiment, the three bacterial communities were highly diverse and differed significantly in their compositions. Manipulation of the enclosures with terrigenous dissolved organic car- bon resulted in notable differences compared to the control enclosures at this early phase of the experiment. However, after 55 days, bacterial communities in the ma- nipulated and the control enclosures under marine and mesohaline conditions were all dominated by gammaproteobacterium Spongiibacter. In the oligohaline enclo- sures, actinobacterial cluster I of the hgc group (hgc-I) remained abundant in the late phase of the incubation. Metaproteome analyses suggested that the ability to use outer membrane-based internal energy stores, in addition to the previously de- scribed grazing resistance, may enable the gammaproteobacterium Spongiibacter to prevail in long-time incubations. Under oligohaline conditions, the utilization of ex- ternal recalcitrant carbon appeared to be more important (hgc-I). Enclosure experi- ments with complex natural microbial communities are important tools to investi- gate the effects of manipulations. However, species-specific properties, such as individual carbon storage strategies, can cause manipulation-independent effects and need to be considered when interpreting results from enclosures. This study was financially supported by the SAW-funded ATKiM project, which provided funds to D. P. R. Herlemann, C. Meeske, K. Jürgens, S. Markert, and T. Schweder. D. P. R. Herlemann was also supported by the European Regional Develop- ment Fund/Estonian Research Council funded Mobilitas Plus Top Researcher grant MOBTT24. We thank the crew and captain of the RV Meteor (M86, M87) for support during the research cruise. The computations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) at the PDC Centre for High Performance Computing (PDC-HPC) and Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX). We thank Jana Matulla for excellent technical assis- tance and Stephan Fuchs for his help and advice in MS database construction. We also thank Stefan E. Heiden for valuable help with the CDD BLAST analyses. This study was financially supported by the SAW-funded ATKiM project, which provided funds to D. P. R. Herlemann, C. Meeske, K. Jürgens, S. Markert, and T. Schweder. D. P. R. Herlemann was also supported by the European Regional Develop- ment Fund/Estonian Research Council funded Mobilitas Plus Top Researcher grant MOBTT24. We thank the crew and captain of the RV Meteor (M86, M87) for support during the research cruise. The computations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) at the PDC Centre for High Performance Computing (PDC-HPC) and Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX). We thank Jana Matulla for excellent technical assis- tance and Stephan Fuchs for his help and advice in MS database construction. We also thank Stefan E. Heiden for valuable help with the CDD BLAST analyses.