Publications

Taurine-respiring gut bacteria produce HS with ambivalent impact on host health. We report the isolation and ecophysiological characterization of a taurine-respiring mouse gut bacterium. Taurinivorans muris strain LT0009 represents a new widespread species that differs from the human gut sulfidogen Bilophila wadsworthia in its sulfur metabolism pathways and host distribution. T. muris specializes in taurine respiration in vivo, seemingly unaffected by mouse diet and genotype, but is dependent on other bacteria for release of taurine from bile acids. Colonization of T. muris in gnotobiotic mice increased deconjugation of taurine-conjugated bile acids and transcriptional activity of a sulfur metabolism gene-encoding prophage in other commensals, and slightly decreased the abundance of Salmonella enterica, which showed reduced expression of galactonate catabolism genes. Re-analysis of metagenome data from a previous study further suggested that T. muris can contribute to protection against pathogens by the commensal mouse gut microbiota. Together, we show the realized physiological niche of a key murine gut sulfidogen and its interactions with selected gut microbiota members.

Background

Jellyfish blooms represent a significant but largely overlooked source of labile organic matter (jelly-OM) in the ocean, characterized by a high protein content. Decaying jellyfish are important carriers for carbon export to the ocean’s interior. To accurately incorporate them into biogeochemical models, the interactions between microbes and jelly-OM have yet to be fully characterized. We conducted jelly-OM enrichment experiments in microcosms to simulate the scenario experienced by the coastal pelagic microbiome after the decay of a jellyfish bloom. We combined metagenomics, endo- and exo-metaproteomic approaches to obtain a mechanistic understanding on the metabolic network operated by the jelly-OM degrading bacterial consortium.

Results

Our analysis revealed that OM released during the decay of jellyfish blooms triggers a rapid shuffling of the taxonomic and functional profile of the pelagic bacterial community, resulting in a significant enrichment of protein/amino acid catabolism-related enzymes in the jelly-OM degrading community dominated by Pseudoalteromonadaceae, Alteromonadaceae and Vibrionaceae, compared to unamended control treatments. In accordance with the proteinaceous character of jelly-OM, Pseudoalteromonadaceae synthesized and excreted enzymes associated with proteolysis, while Alteromonadaceae contributed to extracellular hydrolysis of complex carbohydrates and organophosphorus compounds. In contrast, Vibrionaceae synthesized transporter proteins for peptides, amino acids and carbohydrates, exhibiting a cheater-type lifestyle, i.e. benefiting from public goods released by others. In the late stage of jelly-OM degradation, Rhodobacteraceae and Alteromonadaceae became dominant, growing on jelly-OM left-overs or bacterial debris, potentially contributing to the accumulation of dissolved organic nitrogen compounds and inorganic nutrients, following the decay of jellyfish blooms.

Conclusions

Our findings indicate that specific chemical and metabolic fingerprints associated with decaying jellyfish blooms are substantially different to those previously associated with decaying phytoplankton blooms, potentially altering the functioning and biogeochemistry of marine systems. We show that decaying jellyfish blooms are associated with the enrichment in extracellular collagenolytic bacterial proteases, which could act as virulence factors in human and marine organisms’ disease, with possible implications for marine ecosystem services. Our study also provides novel insights into niche partitioning and metabolic interactions among key jelly-OM degraders operating a complex metabolic network in a temporal cascade of biochemical reactions to degrade pulses of jellyfish-bloom-specific compounds in the water column.

Background

Heterotrophic microbes in the Southern Ocean are challenged by the double constraint of low concentrations of organic carbon (C) and iron (Fe). These essential elements are tightly coupled in cellular processes; however, the prokaryotic requirements of C and Fe under varying environmental settings remain poorly studied. Here, we used a combination of metatranscriptomics and metaproteomics to identify prokaryotic membrane transporters for organic substrates and Fe in naturally iron-fertilized and high-nutrient, low-chlorophyll waters of the Southern Ocean during spring and late summer.

Results

Pronounced differences in membrane transporter profiles between seasons were observed at both sites, both at the transcript and protein level. When specific compound classes were considered, the two approaches revealed different patterns. At the transcript level, seasonal patterns were only observed for subsets of genes belonging to each transporter category. At the protein level, membrane transporters of organic compounds were relatively more abundant in spring as compared to summer, while the opposite pattern was observed for Fe transporters. These observations suggest an enhanced requirement for organic C in early spring and for Fe in late summer. Mapping transcripts and proteins to 50 metagenomic-assembled genomes revealed distinct taxon-specific seasonal differences pointing to potentially opportunistic clades, such as Pseudomonadales and Nitrincolaceae, and groups with a more restricted repertoire of expressed transporters, such as Alphaproteobacteria and Flavobacteriaceae.

Conclusion

The combined investigations of C and Fe membrane transporters suggest seasonal changes in the microbial requirements of these elements under different productivity regimes. The taxon-specific acquisition strategies of different forms of C and Fe illustrate how diverse microbes could shape transcript and protein expression profiles at the community level at different seasons. Our results on the C- and Fe-related metabolic capabilities of microbial taxa provide new insights into their potential role in the cycling of C and Fe under varying nutrient regimes in the Southern Ocean.

The International Committee on Systematics of Prokaryotes (ICSP) discussed and rejected in 2020 a proposal to modify the International Code of Nomenclature of Prokaryotes to allow the use of gene sequences as type for naming prokaryotes. An alternative nomenclatural code, the (SeqCode), which considers genome sequences as type material for naming species, was published in 2022. Members of the ICSP subcommittee for the taxonomy of the phylum () consider that the use of gene sequences as type would benefit the taxonomy of microorganisms that are difficult to culture such as the chlamydiae and other strictly intracellular bacteria. We recommend the registration of new names of uncultured prokaryotes in the SeqCode registry.

Chlamydiae like Chlamydia trachomatis and Chlamydia psittaci are well-known human and animal pathogens. Yet, the chlamydiae are a much larger group of evolutionary ancient obligate intracellular bacteria that includes predominantly symbionts of protists and diverse animals. This makes them ideal model organisms to study evolutionary transitions from symbionts in microbial eukaryotes to pathogens of humans. To this end, comparative genome analysis has served as an important tool. Genome sequence data for many chlamydial lineages are, however, still lacking, hampering our understanding of their evolutionary history. Here, we determined the first high-quality draft genome sequence of the fish pathogen "Candidatus Clavichlamydia salmonicola", representing a separate genus within the human and animal pathogenic Chlamydiaceae. The "Ca. Clavichlamydia salmonicola" genome harbors genes that so far have been exclusively found in Chlamydia species suggesting that basic mechanisms important for the interaction with chordate hosts have evolved stepwise in the history of chlamydiae. Thus, the genome sequence of "Ca. Clavichlamydia salmonicola" allows to constrain candidate genes to further understand the evolution of chlamydial virulence mechanisms required to infect mammals.

Evolution experiments with free-living microbes have radically improved our understanding of genome evolution and how microorganisms adapt. Yet there is a paucity of such research focusing on strictly host-associated bacteria, even though they are widespread in nature. Here, we used the Acanthamoeba symbiont Protochlamydia amoebophila, a distant relative of the human pathogen Chlamydia trachomatis and representative of a large group of protist-associated environmental chlamydiae, as a model to study how obligate intracellular symbionts evolve and adapt to elevated temperature, a prerequisite for the pivotal evolutionary leap from protist to endothermic animal hosts. We established 12 replicate populations under two temperatures (20 °C, 30 °C) for 510 bacterial generations (38 months). We then used infectivity assays and pooled whole-genome resequencing to identify any evolved phenotypes and the molecular basis of adaptation in these bacteria. We observed an overall reduction in infectivity of the symbionts evolved at 30 °C, and we identified numerous nonsynonymous mutations and small indels in these symbiont populations, with several variants persisting throughout multiple time points and reaching high frequencies. This suggests that many mutations may have been beneficial and played an adaptive role. Mutated genes within the same temperature regime were more similar than those between temperature regimes. Our results provide insights into the molecular evolution of intracellular bacteria under the constraints of strict host dependance and highly structured populations and suggest that for chlamydial symbionts of protists, temperature adaptation was facilitated through attenuation of symbiont infectivity as a tradeoff to reduce host cell burden.

Previously we reported a microbiota-dependent caloric restriction (CR)-triggered increase in the levels of taurine and taurine-conjugated bile acids (BA) in the gut. Now, we show that restrictive diets, including intermittent fasting and fasting-mimicking diet, had a similar impact to CR. The type of cage bedding that CR mice were housed with affected the levels of BAs and taurine in the ileum. Removal of cage bedding neutralized CR phenotype in terms of taurine levels, BAs deconjugation, and fecal microbiota composition. Microbiota transplant from CR mice housed with bedding increased BAs deconjugation. Inhibition of bile salt hydrolase (BSH) prevented the increase in free taurine concentration while increasing taurine-conjugated BA levels. Ad libitum consumption of diets high in fiber increased the levels of taurine conjugates but did not elevate the levels of BAs. Dietary restriction is required to stimulate BAs secretion, while ingestion of fiber stimulates the capacity of microbiota to deconjugate BAs.

Introduction

Methods

Results

Conclusion

Aerobic nitrification is a key process in the global nitrogen cycle mediated by microorganisms. While nitrification has primarily been studied in near-neutral environments, this process occurs at a wide range of pH values, spanning ecosystems from acidic soils to soda lakes. Aerobic nitrification primarily occurs through the activities of ammonia-oxidising bacteria and archaea, nitrite-oxidising bacteria, and complete ammonia-oxidising (comammox) bacteria adapted to these environments. Here, we review the literature and identify knowledge gaps on the metabolic diversity, ecological distribution, and physiological adaptations of nitrifying microorganisms in acidic and alkaline environments. We emphasise that nitrifying microorganisms depend on a suite of physiological adaptations to maintain pH homeostasis, acquire energy and carbon sources, detoxify reactive nitrogen species, and generate a membrane potential at pH extremes. We also recognize the broader implications of their activities primarily in acidic environments, with a focus on agricultural productivity and nitrous oxide emissions, as well as promising applications in treating municipal wastewater.

Bacteria colonize plant roots and engage in reciprocal interactions with their hosts. However, the contribution of individual taxa or groups of bacteria to plant nutrition and fitness is not well characterized due to a lack of in situ evidence of bacterial activity. To address this knowledge gap, we developed an analytical approach that combines the identification and localization of individual bacteria on root surfaces via gold-based in situ hybridization with correlative NanoSIMS imaging of incorporated stable isotopes, indicative of metabolic activity. We incubated Kosakonia strain DS-1-associated, gnotobiotically grown rice plants with 15 N-N2 gas to detect in situ N2 fixation activity. Bacterial cells along the rhizoplane showed heterogeneous patterns of 15 N enrichment, ranging from the natural isotope abundance levels up to 12.07 at% 15 N (average and median of 3.36 and 2.85 at% 15 N, respectively, n = 697 cells). The presented correlative optical and chemical imaging analysis is applicable to a broad range of studies investigating plant-microbe interactions. For example, it enables verification of the in situ metabolic activity of host-associated commercialized strains or plant growth-promoting bacteria, thereby disentangling their role in plant nutrition. Such data facilitate the design of plant-microbe combinations for improvement of crop management.

Hot spring biofilms are stable, highly complex microbial structures. They form at dynamic redox and light gradients and are composed of microorganisms adapted to the extreme temperatures and fluctuating geochemical conditions of geothermal environments. In Croatia, a large number of poorly investigated geothermal springs host biofilm communities. Here, we investigated the microbial community composition of biofilms collected over several seasons at 12 geothermal springs and wells. We found biofilm microbial communities to be temporally stable and highly dominated by Cyanobacteria in all but one high-temperature sampling site (Bizovac well). Of the physiochemical parameters recorded, temperature had the strongest influence on biofilm microbial community composition. Besides Cyanobacteria, the biofilms were mainly inhabited by Chloroflexota, Gammaproteobacteria, and Bacteroidota. In a series of incubations with Cyanobacteria-dominated biofilms from Tuhelj spring and Chloroflexota- and Pseudomonadota-dominated biofilms from Bizovac well, we stimulated either chemoorganotrophic or chemolithotrophic community members, to determine the fraction of microorganisms dependent on organic carbon (in situ predominantly produced via photosynthesis) versus energy derived from geochemical redox gradients (here simulated by addition of thiosulfate). We found surprisingly similar levels of activity in response to all substrates in these two distinct biofilm communities, and observed microbial community composition and hot spring geochemistry to be poor predictors of microbial activity in the study systems.

Corals are associated with a variety of bacteria, which occur in the surface mucus layer, gastrovascular cavity, skeleton, and tissues. Some tissue-associated bacteria form clusters, termed cell-associated microbial aggregates (CAMAs), which are poorly studied. Here, we provide a comprehensive characterization of CAMAs in the coral . Combining imaging techniques, laser capture microdissection, and amplicon and metagenome sequencing, we show that (i) CAMAs are located in the tentacle tips and may be intracellular; (ii) CAMAs contain (Gammaproteobacteria) and (Chlamydiota) bacteria; (iii) may provide vitamins to its host and use secretion systems and/or pili for colonization and aggregation; (iv) and occur in distinct, but adjacent, CAMAs; and (v) may receive acetate and heme from neighboring . Our study provides detailed insight into coral endosymbionts, thereby improving our understanding of coral physiology and health and providing important knowledge for coral reef conservation in the climate change era.

Freshwater network ecosystems consist of interconnected lotic and lentic environments within the same catchment area. Using Plitvice Lakes as an example, we studied the changes in environmental conditions and microbial communities (bacteria and fungi) that occur with downstream flow. Water samples from tributaries, interlake streams, connections of the cascading lakes, and the Korana River, the main outflow of the system, were characterized using amplicon sequencing of bacterial 16S rRNA and fungal ITS2 genes. Our results show that different environmental conditions and bacterial and fungal communities prevail among the three stream types within the freshwater network ecosystem during multiple sampling seasons. Microbial community differences were also confirmed along the longitudinal gradient between the most distant sampling sites. The higher impact of “mass effect” was evident during spring and winter, while “species sorting” and “environmental selection” was more pronounced during summer. Prokaryotic community assembly was majorly influenced by deterministic processes, while fungal community assembly was highly dominated by stochastic processes, more precisely by the undominated fraction, which is not dominated by any process. Despite the differences between stream types, the microbial community of Plitvice Lakes is shown to be very stable by the core microbiome that makes up the majority of stream communities. Our results suggest microbial community succession along the river-lake continuum of microbial communities in small freshwater network ecosystems with developed tufa barriers.
IMPORTANCE Plitvice Lakes represent a rare freshwater ecosystem consisting of a complex network of lakes and waterfalls connecting them, as well as rivers and streams supplying water to the lake basin. The unique geomorphological, hydrological, biogeochemical, and biological phenomenon of Plitvice Lakes lies in the biodynamic process of forming tufa barriers. In addition to microbial communities, abiotic water factors also have a major influence on the formation of tufa. Therefore, it is important to understand how changes in environmental conditions and microbial community assembly affect the functioning of the ecosystem of a freshwater network with developed tufa barriers.

The recent discovery of Nitrospira species capable of complete ammonia oxidation (comammox) in non-marine natural and engineered ecosystems under mesothermal conditions has changed our understanding of microbial nitrification. However, little is known about the occurrence of comammox bacteria or their ability to survive in moderately thermal and/or hyperthermal habitats. Here, we report the wide distribution of comammox Nitrospira in five terrestrial hot springs at temperatures ranging from 36 to 80°C and provide metagenome-assembled genomes of 11 new comammox strains. Interestingly, the identification of dissimilatory nitrate reduction to ammonium (DNRA) in thermophilic comammox Nitrospira lineages suggests that they have versatile ecological functions as both sinks and sources of ammonia, in contrast to the described mesophilic comammox lineages, which lack the DNRA pathway. Furthermore, the in situ expression of key genes associated with nitrogen metabolism, thermal adaptation, and oxidative stress confirmed their ability to survive in the studied hot springs and their contribution to nitrification in these environments. Additionally, the smaller genome size and higher GC content, less polar and more charged amino acids in usage profiles, and the expression of a large number of heat shock proteins compared to mesophilic comammox strains presumably confer tolerance to thermal stress. These novel insights into the occurrence, metabolic activity, and adaptation of comammox Nitrospira in thermal habitats further expand our understanding of the global distribution of comammox Nitrospira and have significant implications for how these unique microorganisms have evolved thermal tolerance strategies.

Here, we present the complete genome sequence of Nitrospina watsonii 347, a nitrite-oxidizing bacterium isolated from the Black Sea at a depth of 100 m. The genome has a length of 3,011,914 bp with 2,895 predicted coding sequences. Its predicted metabolism is similar to that of Nitrospina gracilis with differences in defense against reactive oxygen species.

Marine sponges are critical components of marine benthic fauna assemblages, where their filter-feeding and reef-building capabilities provide bentho-pelagic coupling and crucial habitat. As potentially the oldest representation of a metazoan-microbe symbiosis, they also harbor dense, diverse, and species-specific communities of microbes, which are increasingly recognized for their contributions to dissolved organic matter (DOM) processing. Recent omics-based studies of marine sponge microbiomes have proposed numerous pathways of dissolved metabolite exchange between the host and symbionts within the context of the surrounding environment, but few studies have sought to experimentally interrogate these pathways. By using a combination of metaproteogenomics and laboratory incubations coupled with isotope-based functional assays, we showed that the dominant gammaproteobacterial symbiont, 'Candidatus Taurinisymbion ianthellae', residing in the marine sponge, Ianthella basta, expresses a pathway for the import and dissimilation of taurine, a ubiquitously occurring sulfonate metabolite in marine sponges. 'Candidatus Taurinisymbion ianthellae' incorporates taurine-derived carbon and nitrogen while, at the same time, oxidizing the dissimilated sulfite into sulfate for export. Furthermore, we found that taurine-derived ammonia is exported by the symbiont for immediate oxidation by the dominant ammonia-oxidizing thaumarchaeal symbiont, 'Candidatus Nitrosospongia ianthellae'. Metaproteogenomic analyses also suggest that 'Candidatus Taurinisymbion ianthellae' imports DMSP and possesses both pathways for DMSP demethylation and cleavage, enabling it to use this compound as a carbon and sulfur source for biomass, as well as for energy conservation. These results highlight the important role of biogenic sulfur compounds in the interplay between Ianthella basta and its microbial symbionts.

Chronic rhinosinusitis (CRS) is an inflammatory disease phenotypically classified by the absence (CRSsNP) or presence of nasal polyps (CRSwNP). The latter may also be associated with asthma and hypersensitivity towards non-steroidal anti-inflammatory drugs (NSAID) as a triad termed NSAID-exacerbated respiratory disease (N-ERD). The role of the microbiome in these disease entities with regard to the underlying inflammatory process and disease burden is yet not fully understood. To address this question, we measured clinical parameters and collected nasal samples (nasal mucosal fluids and microbiome swabs) of patients suffering from CRSsNP (n=20), CRSwNP (n=20) or N-ERD (n=20) as well as from patients without CRS (=disease controls, n=20). Importantly, all subjects refrained from taking local or systemic corticosteroids or immunosuppressants for at least two weeks prior to sampling. The nasal microbiome was analyzed using 16S rRNA gene amplicon sequencing, and levels of 33 inflammatory cytokines were determined in nasal mucosal fluids using the MSD platform.

Patients suffering from N-ERD and CRSwNP showed significantly worse smell perception and significantly higher levels of type 2 associated cytokines IL-5, IL-9, Eotaxin and CCL17. Across all 4 patient groups, Corynebacteria and Staphylococci showed the highest relative abundances. Although no significant difference in alpha and beta diversity was observed between the control and the CRS groups, pairwise testing revealed a higher relative abundance of Staphylococci in the middle meatus in N-ERD patients as compared to CRSwNP (p<0.001), CRSsNP (p<0.01) and disease controls (p<0.05) and of Lawsonella in patients suffering from CRSwNP in middle meatus and anterior naris in comparison to CRSsNP (p<0.0001 for both locations) and disease controls (p<0.01 and p<0.0001). Furthermore, we observed a positive correlation of Staphylococci with IL-5 (Pearson r=0.548) and a negative correlation for Corynebacteria and Eotaxin-3 (r=-0.540). Thus, in patients refraining from oral and nasal corticosteroid therapy for at least two weeks known to alter microbiome composition, we did not observe differences in microbiome alpha or beta diversity between various CRS entities and disease controls. However, our data suggest a close association between increased bacterial colonization with Staphylococci and decreased colonization by Corynebacteria as well as increased type 2 inflammation and disease burden.

Cable bacteria are centimeter-long filamentous bacteria that conduct electrons via internal wires, thus coupling sulfide oxidation in deeper, anoxic sediment with oxygen reduction in surface sediment. This activity induces geochemical changes in the sediment, and other bacterial groups appear to benefit from the electrical connection to oxygen. Here, we report that diverse bacteria swim in a tight flock around the anoxic part of oxygen-respiring cable bacteria and disperse immediately when the connection to oxygen is disrupted (by cutting the cable bacteria with a laser). Raman microscopy shows that flocking bacteria are more oxidized when closer to the cable bacteria, but physical contact seems to be rare and brief, which suggests potential transfer of electrons via unidentified soluble intermediates. Metagenomic analysis indicates that most of the flocking bacteria appear to be aerobes, including organotrophs, sulfide oxidizers, and possibly iron oxidizers, which might transfer electrons to cable bacteria for respiration. The association and close interaction with such diverse partners might explain how oxygen via cable bacteria can affect microbial communities and processes far into anoxic environments.

The oscillating redox conditions that characterize coastal sandy sediments foster microbial communities capable of respiring oxygen and nitrate simultaneously, thereby increasing the potential for organic matter remineralization, nitrogen (N)-loss and emissions of the greenhouse gas nitrous oxide. It is unknown to what extent these conditions also lead to overlaps between dissimilatory nitrate and sulfate respiration. Here, we show that sulfate and nitrate respiration co-occur in the surface sediments of an intertidal sand flat. Furthermore, we found strong correlations between dissimilatory nitrite reduction to ammonium (DNRA) and sulfate reduction rates. Until now, the nitrogen and sulfur cycles were assumed to be mainly linked in marine sediments by the activity of nitrate-reducing sulfide oxidisers. However, transcriptomic analyses revealed that the functional marker gene for DNRA (nrfA) was more associated with microorganisms known to reduce sulfate rather than oxidise sulfide. Our results suggest that when nitrate is supplied to the sediment community upon tidal inundation, part of the sulfate reducing community may switch respiratory strategy to DNRA. Therefore increases in sulfate reduction rate in-situ may result in enhanced DNRA and reduced denitrification rates. Intriguingly, the shift from denitrification to DNRA did not influence the amount of N2O produced by the denitrifying community. Our results imply that microorganisms classically considered as sulfate reducers control the potential for DNRA within coastal sediments when redox conditions oscillate and therefore retain ammonium that would otherwise be removed by denitrification, exacerbating eutrophication.

is a semi-wild cactus cultivated for its fruit. However, the cladodes are often discarded, wasting the potentially useful mucilage in them. The mucilage is composed primarily of heteropolysaccharides, characterized by their molar mass distribution, monosaccharide composition, structural features (by vibrational spectroscopy, FT IR, and atomic force microscopy, AFM), and fermentability by known saccharolytic commensal members of the gut microbiota. After fractionation with ion exchange chromatography, four polysaccharides were found: one neutral (composed mainly of galactose, arabinose, and xylose) and three acidic, with a galacturonic acid content from 10 to 35%. Their average molar masses ranged from 1.8 × 10 to 2.8 × 10 g·mol. Distinct structural features such as galactan, arabinan, xylan, and galacturonan motifs were present in the FT IR spectra. The intra- and intermolecular interactions of the polysaccharides, and their effect on the aggregation behavior, were shown by AFM. The composition and structural features of these polysaccharides were reflected in their prebiotic potential. and were not able to utilize them, whereas members of showed utilization capacity. The obtained data suggest a high economic potential for this species, with potential uses such as animal feed in arid areas, precise prebiotic, and symbiotic formulations, or as the carbon skeleton source in a green refinery. Our methodology can be used to evaluate the saccharides as the phenotype of interest, helping to guide the breeding strategy.

The role of the gut microbiome in developing Inflammatory Bowel Disease (IBD) in humans and dogs has received attention in recent years. Evidence suggests that IBD is associated with alterations in gut microbial composition, but further research is needed in veterinary medicine. The impact of IBD treatment on the gut microbiome needs to be better understood, especially in a breed-specific form of IBD in Yorkshire Terriers known as Yorkshire Terrier Enteropathy (YTE). This study aimed to investigate the difference in gut microbiome composition between YTE dogs during disease and remission and healthy Yorkshire Terriers. Our results showed a significant increase in specific taxa such as Clostridium sensu stricto 1, Escherichia-Shigella, and Streptococcus, and a decrease in Bacteroides, Prevotella, Alloprevotella, and Phascolarctobacterium in YTE dogs compared to healthy controls. No significant difference was found between the microbiome of dogs in remission and those with active disease, suggesting that the gut microbiome is affected beyond clinical recovery.

Electromicrobiology can be used to understand extracellular electron uptake in previously undescribed chemolithotrophs. Enrichment and characterization of the uncultivated electroautotroph "Candidatus Tenderia electrophaga" using electromicrobiology led to the designation of the order Tenderiales. Representative Tenderiales metagenome-assembled genomes (MAGs) have been identified in a number of environmental surveys, yet a comprehensive characterization of conserved genes for extracellular electron uptake has thus far not been conducted. Using comparative genomics, we identified conserved orthologous genes within the Tenderiales and nearest-neighbor orders important for extracellular electron uptake based on a previously proposed pathway from "Ca. Tenderia electrophaga." The Tenderiales contained a conserved cluster we designated uetABCDEFGHIJ, which encodes proteins containing features that would enable transport of extracellular electrons to cytoplasmic membrane-bound energy-transducing complexes such as two conserved cytochrome cbb3 oxidases. For example, UetJ is predicted to be an extracellular undecaheme c-type cytochrome that forms a heme wire. We also identified clusters of genes predicted to facilitate assembly and maturation of electron transport proteins, as well as cellular attachment to surfaces. Autotrophy among the Tenderiales is supported by the presence of carbon fixation and stress response pathways that could allow cellular growth by extracellular electron uptake. Key differences between the Tenderiales and other known neutrophilic iron oxidizers were revealed, including very few Cyc2 genes in the Tenderiales. Our results reveal a possible conserved pathway for extracellular electron uptake and suggest that the Tenderiales have an ecological role in coupling metal or mineral redox chemistry and the carbon cycle in marine and brackish sediments. IMPORTANCE Chemolithotrophic bacteria capable of extracellular electron uptake to drive energy metabolism and CO2 fixation are known as electroautotrophs. The recently described order Tenderiales contains the uncultivated electroautotroph "Ca. Tenderia electrophaga." The "Ca. Tenderia electrophaga" genome contains genes proposed to make up a previously undescribed extracellular electron uptake pathway. Here, we use comparative genomics to show that this pathway is well conserved among Tenderiales spp. recovered by metagenome-assembled genomes. This conservation extends to near neighbors of the Tenderiales but not to other well-studied chemolithotrophs, including iron and sulfur oxidizers, indicating that these genes may be useful markers of growth using insoluble extracellular electron donors. Our findings suggest that extracellular electron uptake and electroautotrophy may be pervasive among the Tenderiales, and the geographic locations from which metagenome-assembled genomes were recovered offer clues to their natural ecological niche.

Bacterial endosymbionts of the groups Wolbachia, Cardinium and Rickettsiaceae are well known for their diverse effects on their arthropod hosts, ranging from mutualistic relationships to reproductive phenotypes. Here, we analysed a unique system in which the dwarf spider Oedothorax gibbosus is co-infected with up to five different endosymbionts affiliated with Wolbachia, ‘Candidatus Tisiphia’ (formerly Torix group Rickettsia), Cardinium and Rhabdochlamydia. Using short-read genome sequencing data, we show that the endosymbionts are heterogeneously distributed among O. gibbosus populations and are frequently found co-infecting spider individuals. To study this intricate host–endosymbiont system on a genome-resolved level, we used long-read sequencing to reconstruct closed genomes of the Wolbachia, ‘Ca. Tisiphia’ and Cardinium endosymbionts. We provide insights into the ecology and evolution of the endosymbionts and shed light on the interactions with their spider host. We detected high quantities of transposable elements in all endosymbiont genomes and provide evidence that ancestors of the Cardinium, ‘Ca. Tisiphia’ and Wolbachia endosymbionts have co-infected the same hosts in the past. Our findings contribute to broadening our knowledge about endosymbionts infecting one of the largest animal phyla on Earth and show the usefulness of transposable elements as an evolutionary ‘contact-tracing’ tool.

Nitrite-oxidizing bacteria (NOB) catalyse the second nitrification step and are the main biological source of nitrate. The most diverse and widespread NOB genus is Nitrospira, which also contains complete ammonia oxidizers (comammox) that oxidize ammonia to nitrate. To date, little is known about the occurrence and biology of comammox and canonical nitrite oxidizing Nitrospira in extremely alkaline environments. Here, we studied the seasonal distribution and diversity, and the effect of short-term pH changes on comammox and canonical Nitrospira in sediments of two saline, highly alkaline lakes. We identified diverse canonical and comammox Nitrospira clade A-like phylotypes as the only detectable NOB during more than a year, suggesting their major importance for nitrification in these habitats. Gross nitrification rates measured in microcosm incubations were highest at pH 10 and considerably faster than reported for other natural, aquatic environments. Nitrification could be attributed to canonical and comammox Nitrospira and to Nitrososphaerales ammonia-oxidizing archaea. Furthermore, our data suggested that comammox Nitrospira contributed to ammonia oxidation at an extremely alkaline pH of 11. These results identify saline, highly alkaline lake sediments as environments of uniquely strong nitrification with novel comammox Nitrospira as key microbial players.

Marine and freshwater sponges harbor diverse communities of bacteria with vast potential to produce secondary metabolites that may play an important role in protecting the host from predators and infections. In this work, we initially used cultivation and metagenomics to investigate the microbial community of the freshwater sponge Spongilla lacustris collected in an Austrian lake. Representatives of 41 bacterial genera were isolated from the sponge sample and classified according to their 16S rRNA gene sequences. The genomes of 33 representative isolates and the 20 recovered metagenome-assembled genomes (MAGs) contained in total 306 secondary metabolite biosynthesis gene clusters (BGCs). Comparative 16S rRNA gene and genome analyses showed very little taxon overlap between the recovered isolates and the sponge community as revealed by cultivation-independent methods. Both culture-independent and -dependent analyses suggested high biosynthetic potential of the S. lacustris microbiome, which was confirmed experimentally even at the subspecies level for two isolates. To our knowledge, this is the most thorough description of the secondary metabolite production potential of a freshwater sponge microbiome to date. A large body of research is dedicated to marine sponges, filter-feeding animals harboring rich bacterial microbiomes believed to play an important role in protecting the host from predators and infections. Freshwater sponges have received so far much less attention with respect to their microbiomes, members of which may produce bioactive secondary metabolites with potential to be developed into drugs to treat a variety of diseases. In this work, we investigated the potential of bacteria associated with the freshwater sponge to biosynthesize diverse secondary metabolites. Using culture-dependent and -independent methods, we discovered over 300 biosynthetic gene clusters in sponge-associated bacteria and proved production of several compounds by selected isolates using genome mining. Our results illustrate the importance of a complex approach when dealing with microbiomes of multicellular organisms that may contain producers of medically important secondary metabolites.

The activities of different microbes in the cow rumen have been shown to modulate the host's ability to utilize plant biomass, while the host-rumen interface has received little attention. As datasets collected worldwide have pointed to Campylobacteraceae as particularly abundant members of the rumen epithelial microbiome, we targeted this group in a subset of seven cows with meta- and isolate genome analysis. We show that the dominant Campylobacteraceae lineage has recently speciated into two populations that were structured by genome-wide selective sweeps followed by population-specific gene import and recombination. These processes led to differences in gene expression and enzyme domain composition that correspond to the ability to utilize acetate, the main carbon source for the host, at the cost of inhibition by propionate. This trade-off in competitive ability further manifests itself in differential dynamics of the two populations in vivo. By exploring population-level adaptations that otherwise remain cryptic in culture-independent analyses, our results highlight how recent evolutionary dynamics can shape key functional roles in the rumen microbiome.

Simultaneous identification and metabolic analysis of microbes with single-cell resolution and high throughput are necessary to answer the question of "who eats what, when, and where" in complex microbial communities. Here, we present a mid-infrared photothermal-fluorescence in situ hybridization (MIP-FISH) platform that enables direct bridging of genotype and phenotype. Through multiple improvements of MIP imaging, the sensitive detection of isotopically labeled compounds incorporated into proteins of individual bacterial cells became possible, while simultaneous detection of FISH labeling with rRNA-targeted probes enabled the identification of the analyzed cells. In proof-of-concept experiments, we showed that the clear spectral red shift in the protein amide I region due to incorporation of C atoms originating from C-labeled glucose can be exploited by MIP-FISH to discriminate and identify C-labeled bacterial cells within a complex human gut microbiome sample. The presented methods open new opportunities for single-cell structure-function analyses for microbiology.

Infectious disease is widely considered to be a major driver of evolution. A preponderance of signatures of balancing selection at blood group-related genes is thought to be driven by inherent trade-offs in susceptibility to disease. B4galnt2 is subject to long-term balancing selection in house mice, where two divergent allele classes direct alternative tissue-specific expression of a glycosyltransferase in the intestine versus blood vessels. The blood vessel allele class leads to prolonged bleeding times similar to von Willebrand disease in humans, yet has been maintained for millions of years. Based on in vivo functional studies in inbred lab strains, it is hypothesized that the cost of prolonged bleeding times may be offset by an evolutionary trade-off involving susceptibility to a yet unknown pathogen(s). To identify candidate pathogens for which resistance could be mediated by B4galnt2 genotype, we here employed a novel "pathometagenomic" approach in a wild mouse population, which combines bacterial 16S rRNA gene-based community profiling with histopathology of gut tissue. Through subsequent isolation, genome sequencing and controlled experiments in lab mice, we show that the presence of the blood vessel allele is associated with resistance to a newly identified subspecies of Morganella morganii, a clinically important opportunistic pathogen. Given the increasing importance of zoonotic events, the approach outlined here may find useful application in the detection of emerging diseases in wild animal populations.

The incidence of nephrolithiasis is rising worldwide. Although it is a multifactorial disease, lifestyle plays a major role in its etiology. Another considerable factor could be an aberrant microbiome. In our observational single-center study, we aimed to investigate the composition of bacteria in kidney stones and urine focusing on patients with features of metabolic syndrome. Catheterized urine and kidney stones were collected prospectively from 100 consecutive patients undergoing endoscopic nephrolithotomy between 2020 and 2021 at our clinic. Microbiome composition was analyzed via 16S rRNA gene amplicon sequencing. Detection of bacteria was successful in 24% of the analyzed kidney stones. These patients had a prolonged length of stay compared to patients without verifiable bacteria in their stones (2.9 vs 1.5 days). Patients with features of metabolic syndrome were characterized by kidney stones colonized with classical gastrointestinal bacteria and displayed a significant enrichment of Enterococcaceae and Enterobacteriaceae. Stones of patients without features of metabolic syndrome characterized by Ureaplasma and Staphylococcaceae. Patients with bacteria in their kidney stones exhibit a longer length of stay, possibly due to more complex care. Patients presenting with features of metabolic syndrome displayed a distinct stone microbiome compared to metabolically fit patients. Understanding the role of bacteria in stone formation could enable targeted therapy, prevention of post-operative complications and new therapeutic strategies.

Chlamydiae is a bacterial phylum composed of obligate animal and protist endosymbionts. However, other members of the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum are primarily free living. How Chlamydiae transitioned to an endosymbiotic lifestyle is still largely unresolved. Here we reconstructed Planctomycetes-Verrucomicrobia-Chlamydiae species relationships and modelled superphylum genome evolution. Gene content reconstruction from 11,996 gene families suggests a motile and facultatively anaerobic last common Chlamydiae ancestor that had already gained characteristic endosymbiont genes. Counter to expectations for genome streamlining in strict endosymbionts, we detected substantial gene gain within Chlamydiae. We found that divergence in energy metabolism and aerobiosis observed in extant lineages emerged later during chlamydial evolution. In particular, metabolic and aerobic genes characteristic of the more metabolically versatile protist-infecting chlamydiae were gained, such as respiratory chain complexes. Our results show that metabolic complexity can increase during endosymbiont evolution, adding an additional perspective for understanding symbiont evolutionary trajectories across the tree of life.

Many marine sponges host highly diverse microbiomes that contribute to various aspects of host health. Although the putative function of individual groups of sponge symbionts has been increasingly described, the extreme diversity has generally precluded in-depth characterization of entire microbiomes, including identification of syntrophic partnerships. The Indo-Pacific sponge Ianthella basta is emerging as a model organism for symbiosis research, hosting only three dominant symbionts: a Thaumarchaeotum, a Gammaproteobacterium, and an Alphaproteobacterium and a range of other low abundance or transitory taxa. Here, we retrieved metagenome assembled genomes (MAGs) representing >90% of I. basta's microbial community, facilitating the metabolic reconstruction of the sponge's near complete microbiome. Through this analysis, we identified metabolic complementarity between microbes, including vitamin sharing, described the importance of low abundance symbionts, and characterized a novel microbe-host attachment mechanism in the Alphaproteobacterium. We further identified putative viral sequences, highlighting the role viruses can play in maintaining symbioses in I. basta through the horizontal transfer of eukaryotic-like proteins, and complemented this data with metaproteomics to identify active metabolic pathways in bacteria, archaea, and viruses. This data provide the framework to adopt I. basta as a model organism for studying host-microbe interactions and provide a basis for in-depth physiological experiments.