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  1. Abstract

    A limited number of bacteria are able to colonize the nuclei of eukaryotes. ‘CandidatusEndonucleobacter’ infects the nuclei of deep-sea mussels, where it replicates to ≥80,000 bacteria per nucleus and causes nuclei to swell to 50 times their original size. How these parasites are able to replicate and avoid apoptosis is not known. Dual RNA-sequencing transcriptomes of infected nuclei isolated using laser-capture microdissection revealed that ‘CandidatusEndonucleobacter’ does not obtain most of its nutrition from nuclear DNA or RNA. Instead, ‘CandidatusEndonucleobacter’ upregulates genes for importing and digesting sugars, lipids, amino acids and possibly mucin from its host. It likely prevents apoptosis of host cells by upregulating 7–13 inhibitors of apoptosis, proteins not previously seen in bacteria. Comparative phylogenetic analyses revealed that ‘Ca. Endonucleobacter’ acquired inhibitors of apoptosis through horizontal gene transfer from their hosts. Horizontal gene transfer from eukaryotes to bacteria is assumed to be rare, but may be more common than currently recognized.

     
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    Free, publicly-accessible full text available November 1, 2025
  2. Hockett, Kevin Loren (Ed.)
    ABSTRACT

    Synthetic microbial communities (SynComs) are a valuable tool to study community assembly patterns, host–microbe interactions, and microbe–microbe interactions in a fully controllable setting. Constructing the SynCom inocula for plant–microbe experiments can be time-consuming and difficult because a large number of isolates with different medium requirements and growth rates are grown in parallel and mixed to appropriate titers. A potential workaround to assembling fresh SynCom inocula for every experiment could be to prepare and freeze SynComs on a large scale, creating ready-to-use inocula. The objective of this study was to compare the reproducibility, stability, and colonization ability of freshly prepared versus frozen SynCom inocula. We used a community of seven species known to colonize maize roots. The results from inoculation with the frozen SynCom were as consistent as those of standardizedde novoconstruction of fresh SynCom. Our results indicate that creating frozen SynCom inocula for repeated use in experiments not only saves time but could also improve cross-experiment reproducibility. Although this approach was only validated with one SynCom, it demonstrates a principle that can be tested for improving approaches in constructing other SynComs.

    IMPORTANCE

    Synthetic communities (SynComs) are an invaluable tool to characterize and model plant–microbe interactions. Multimember SynComs approximate intricate real-world interactions between plants and their microbiome, but the complexity and time required for their construction increase enormously for each additional member added to the SynCom. Therefore, researchers who study a diversity of microbiomes using SynComs are looking for ways to simplify the use of SynComs. In this manuscript, we evaluate the feasibility of creating ready-to-use freezer stocks of a well-studied seven-member SynCom for maize roots. The frozen ready-to-use SynCom stocks work according to the principle of “just add buffer and apply to sterilized seeds or seedlings” and thus can save time applied in multiple days of laborious growing and combining of multiple microorganisms. We show that ready-to-use SynCom stocks provide comparable results to those of freshly constructed SynComs and thus allow for significant time savings when working with SynComs.

     
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    Free, publicly-accessible full text available December 12, 2024
  3. Abstract

    Arbuscular mycorrhizal symbiosis (AM) is a beneficial trait originating with the first land plants, which has subsequently been lost by species scattered throughout the radiation of plant diversity to the present day, including the modelArabidopsis thaliana. To explore if elements of this apparently beneficial trait are still present and could be reactivated we generatedArabidopsisplants expressing a constitutively active form ofInteracting Protein of DMI3, a key transcription factor that enables AM within the Common Symbiosis Pathway, which was lost fromArabidopsisalong with the AM host trait. We characterize the transcriptomic effect of expressingIPD3inArabidopsiswith and without exposure to the AM fungus (AMF)Rhizophagus irregularis, and compare these results to the AM modelLotus japonicusand itsipd3knockout mutantcyclops-4. Despite its long history as a non-AM species, restoringIPD3in the form of its constitutively active DNA-binding domain toArabidopsisaltered expression of specific gene networks. Surprisingly, the effect of expressingIPD3inArabidopsisand knocking it out inLotuswas strongest in plants not exposed to AMF, which is revealed to be due to changes inIPD3genotype causing a transcriptional state, which partially mimics AMF exposure in non-inoculated plants. Our results indicate that molecular connections to symbiosis machinery remain in place in this nonAM species, with implications for both basic science and the prospect of engineering this trait for agriculture.

     
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  4. Abstract

    Bathymodioline mussels rely on thiotrophic and/or methanotrophic chemosynthetic symbionts for nutrition, yet, secondary heterotrophic symbionts are often present and play an unknown role in the fitness of the organism. The bathymodioline Idas mussels that thrive in gas seeps and on sunken wood in the Mediterranean Sea and the Atlantic Ocean, host at least six symbiont lineages that often co-occur. These lineages include the primary symbionts chemosynthetic methane- and sulfur-oxidizing gammaproteobacteria, and the secondary symbionts, Methylophagaceae, Nitrincolaceae and Flavobacteriaceae, whose physiology and metabolism are obscure. Little is known about if and how these symbionts interact or exchange metabolites. Here we curated metagenome-assembled genomes of Idas modiolaeformis symbionts and used genome-centered metatranscriptomics and metaproteomics to assess key symbiont functions. The Methylophagaceae symbiont is a methylotrophic autotroph, as it encoded and expressed the ribulose monophosphate and Calvin-Benson-Bassham cycle enzymes, particularly RuBisCO. The Nitrincolaceae ASP10-02a symbiont likely fuels its metabolism with nitrogen-rich macromolecules and may provide the holobiont with vitamin B12. The Urechidicola (Flavobacteriaceae) symbionts likely degrade glycans and may remove NO. Our findings indicate that these flexible associations allow for expanding the range of substrates and environmental niches, via new metabolic functions and handoffs.

     
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  5. Some annelids produce sitosterol, a biomarker lipid more commonly found in plants. 
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  6. Metaproteomics is a powerful tool for the characterization of metabolism, physiology, and functional interactions in microbial communities, including plant-associated microbiota. However, the metaproteomic methods that have been used to study plant-associated microbiota are very laborious and require large amounts of plant tissue, hindering wider application of these methods. We optimized and evaluated different protein extraction methods for metaproteomics of plant-associated microbiota in two different plant species ( Arabidopsis and maize). Our main goal was to identify a method that would work with low amounts of input material (40 to 70 mg) and that would maximize the number of identified microbial proteins. We tested eight protocols, each comprising a different combination of physical lysis method, extraction buffer, and cell-enrichment method on roots from plants grown with synthetic microbial communities. We assessed the performance of the extraction protocols by liquid chromatography-tandem mass spectrometry–based metaproteomics and found that the optimal extraction method differed between the two species. For Arabidopsis roots, protein extraction by beating whole roots with small beads provided the greatest number of identified microbial proteins and improved the identification of proteins from gram-positive bacteria. For maize, vortexing root pieces in the presence of large glass beads yielded the greatest number of microbial proteins identified. Based on these data, we recommend the use of these two methods for metaproteomics with Arabidopsis and maize. Furthermore, detailed descriptions of the eight tested protocols will enable future optimization of protein extraction for metaproteomics in other dicot and monocot plants. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license . 
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  7. Abstract Background

    Stable isotope probing (SIP) approaches are a critical tool in microbiome research to determine associations between species and substrates, as well as the activity of species. The application of these approaches ranges from studying microbial communities important for global biogeochemical cycling to host-microbiota interactions in the intestinal tract. Current SIP approaches, such as DNA-SIP or nanoSIMS allow to analyze incorporation of stable isotopes with high coverage of taxa in a community and at the single cell level, respectively, however they are limited in terms of sensitivity, resolution or throughput.

    Results

    Here, we present an ultra-sensitive, high-throughput protein-based stable isotope probing approach (Protein-SIP), which cuts cost for labeled substrates by 50–99% as compared to other SIP and Protein-SIP approaches and thus enables isotope labeling experiments on much larger scales and with higher replication. The approach allows for the determination of isotope incorporation into microbiome members with species level resolution using standard metaproteomics liquid chromatography-tandem mass spectrometry (LC–MS/MS) measurements. At the core of the approach are new algorithms to analyze the data, which have been implemented in an open-source software (https://sourceforge.net/projects/calis-p/). We demonstrate sensitivity, precision and accuracy using bacterial cultures and mock communities with different labeling schemes. Furthermore, we benchmark our approach against two existing Protein-SIP approaches and show that in the low labeling range used our approach is the most sensitive and accurate. Finally, we measure translational activity using18O heavy water labeling in a 63-species community derived from human fecal samples grown on media simulating two different diets. Activity could be quantified on average for 27 species per sample, with 9 species showing significantly higher activity on a high protein diet, as compared to a high fiber diet. Surprisingly, among the species with increased activity on high protein were severalBacteroidesspecies known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics.

    Conclusions

    We demonstrate that our Protein-SIP approach allows for the ultra-sensitive (0.01 to 10% label) detection of stable isotopes of elements found in proteins, using standard metaproteomics data.

     
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  8. Abstract Background

    Many animals live in intimate associations with a species-rich microbiome. A key factor in maintaining these beneficial associations is fidelity, defined as the stability of associations between hosts and their microbiota over multiple host generations. Fidelity has been well studied in terrestrial hosts, particularly insects, over longer macroevolutionary time. In contrast, little is known about fidelity in marine animals with species-rich microbiomes at short microevolutionary time scales, that is at the level of a single host population. Given that natural selection acts most directly on local populations, studies of microevolutionary partner fidelity are important for revealing the ecological and evolutionary processes that drive intimate beneficial associations within animal species.

    Results

    In this study on the obligate symbiosis between the gutless marine annelidOlavius algarvensisand its consortium of seven co-occurring bacterial symbionts, we show that partner fidelity varies across symbiont species from strict to absent over short microevolutionary time. Using a low-coverage sequencing approach that has not yet been applied to microbial community analyses, we analysed the metagenomes of 80O. algarvensisindividuals from the Mediterranean and compared host mitochondrial and symbiont phylogenies based on single-nucleotide polymorphisms across genomes. Fidelity was highest for the two chemoautotrophic, sulphur-oxidizing symbionts that dominated the microbial consortium of allO. algarvensisindividuals. In contrast, fidelity was only intermediate to absent in the sulphate-reducing and spirochaetal symbionts with lower abundance. These differences in fidelity are likely driven by both selective and stochastic forces acting on the consistency with which symbionts are vertically transmitted.

    Conclusions

    We hypothesize that variable degrees of fidelity are advantageous forO. algarvensisby allowing the faithful transmission of their nutritionally most important symbionts and flexibility in the acquisition of other symbionts that promote ecological plasticity in the acquisition of environmental resources.

     
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  9. Gralnick, Jeffrey A. (Ed.)
    ABSTRACT Field studies are central to environmental microbiology and microbial ecology, because they enable studies of natural microbial communities. Metaproteomics, the study of protein abundances in microbial communities, allows investigators to study these communities “ in situ ,” which requires protein preservation directly in the field because protein abundance patterns can change rapidly after sampling. Ideally, a protein preservative for field deployment works rapidly and preserves the whole proteome, is stable in long-term storage, is nonhazardous and easy to transport, and is available at low cost. Although these requirements might be met by several protein preservatives, an assessment of their suitability under field conditions when targeted for metaproteomic analyses is currently lacking. Here, we compared the protein preservation performance of flash freezing and the preservation solution RNA later using the marine gutless oligochaete Olavius algarvensis and its symbiotic microbes as a test case. In addition, we evaluated long-term RNA later storage after 1 day, 1 week, and 4 weeks at room temperature (22°C to 23°C). We evaluated protein preservation using one-dimensional liquid chromatography-tandem mass spectrometry. We found that RNA later and flash freezing preserved proteins equally well in terms of total numbers of identified proteins and relative abundances of individual proteins, and none of the test time points was altered, compared to time zero. Moreover, we did not find biases against specific taxonomic groups or proteins with particular biochemical properties. Based on our metaproteomic data and the logistical requirements for field deployment, we recommend RNA later for protein preservation of field-collected samples targeted for metaproteomic analyses. IMPORTANCE Metaproteomics, the large-scale identification and quantification of proteins from microbial communities, provide direct insights into the phenotypes of microorganisms on the molecular level. To ensure the integrity of the metaproteomic data, samples need to be preserved immediately after sampling to avoid changes in protein abundance patterns. In laboratory setups, samples for proteomic analyses are most commonly preserved by flash freezing; however, liquid nitrogen or dry ice is often unavailable at remote field locations, due to their hazardous nature and transport restrictions. Our study shows that RNA later can serve as a low-hazard, easy-to-transport alternative to flash freezing for field preservation of samples for metaproteomic analyses. We show that RNA later preserves the metaproteome equally well, compared to flash freezing, and protein abundance patterns remain stable during long-term storage for at least 4 weeks at room temperature. 
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