@article{hornstein_charles_franklin_edwards_vintila_kleiner_sederoff_2024, title={<i>IPD3</i>, a master regulator of arbuscular mycorrhizal symbiosis, affects genes for immunity and metabolism of non-host <i>Arabidopsis</i> when restored long after its evolutionary loss}, volume={114}, ISSN={["1573-5028"]}, url={https://doi.org/10.1007/s11103-024-01422-3}, DOI={10.1007/s11103-024-01422-3}, abstractNote={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 model Arabidopsis thaliana . To explore if elements of this apparently beneficial trait are still present and could be reactivated we generated Arabidopsis plants expressing a constitutively active form of Interacting Protein of DMI3 , a key transcription factor that enables AM within the Common Symbiosis Pathway, which was lost from Arabidopsis along with the AM host trait. We characterize the transcriptomic effect of expressing IPD3 in Arabidopsis with and without exposure to the AM fungus (AMF) Rhizophagus irregularis , and compare these results to the AM model Lotus japonicus and its ipd3 knockout mutant cyclops-4 . Despite its long history as a non-AM species, restoring IPD3 in the form of its constitutively active DNA-binding domain to Arabidopsis altered expression of specific gene networks. Surprisingly, the effect of expressing IPD3 in Arabidopsis and knocking it out in Lotus was strongest in plants not exposed to AMF, which is revealed to be due to changes in IPD3 genotype 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.}, number={2}, journal={PLANT MOLECULAR BIOLOGY}, author={Hornstein, Eli D. and Charles, Melodi and Franklin, Megan and Edwards, Brianne and Vintila, Simina and Kleiner, Manuel and Sederoff, Heike}, year={2024}, month={Apr} }
 @article{ratinskaia_malavin_zvi-kedem_vintila_kleiner_rubin-blum_2024, title={Metabolically-versatileCa.Thiodiazotropha symbionts of the deep-sea lucinid clamLucinoma kazanihave the genetic potential to fix nitrogen}, url={https://doi.org/10.1101/2024.04.05.588213}, DOI={10.1101/2024.04.05.588213}, abstractNote={Lucinid clams are one of the most diverse and widespread symbiont-bearing animal groups in both shallow and deep-sea chemosynthetic habitats. Lucicnids harbor Ca . Thiodiazotropha symbionts that can oxidize inorganic and organic substrates such as hydrogen sulfide and formate to gain energy. The interplay between these key metabolic functions, nutrient uptake and biotic interactions in Ca. Thiodiazotropha is not fully understood. We collected Lucinoma kazani individuals from next to a deep-sea brine pool in the eastern Mediterranean Sea, at a depth of 1150 m and used Oxford Nanopore and Illumina sequencing to obtain high-quality genomes of their Ca. Thiodiazotropha gloverae symbiont. The genomes served as the basis for transcriptomic and proteomic analyses to characterize the in situ gene expression, metabolism and physiology of the symbionts. We found genes needed for N2 fixation in the deep-sea symbiont genome, which, to date, were only found in shallow-water Ca. Thiodiazotropha. However, we did not detect the expression of these genes and thus the potential role of nitrogen fixation in this symbiosis remains to be determined. We also found the high expression of carbon fixation and sulfur oxidation genes, which indicates chemolithoautotrophy as the key physiology of Ca . Thiodiazotropha. However, we also detected the expression of pathways for using methanol and formate as energy sources. Our findings highlight the key traits these microbes maintain to support the nutrition of their hosts and interact with them.}, author={Ratinskaia, Lina and Malavin, Stas and Zvi-Kedem, Tal and Vintila, Simina and Kleiner, Manuel and Rubin-Blum, Maxim}, year={2024}, month={Apr} }
 @article{petrone_bartlett_jiang_korenek_vintila_tenekjian_yancy_david_kleiner_2024, title={Metaproteomics and DNA metabarcoding as tools to assess dietary intake in humans}, url={https://doi.org/10.1101/2024.04.09.588275}, DOI={10.1101/2024.04.09.588275}, abstractNote={Objective biomarkers of food intake are a sought-after goal in nutrition research. The majority of biomarker development to date has focused on metabolites detected in blood, urine, skin or hair, but detection of consumed foods in stool has also been shown to be possible via DNA sequencing. An additional food macromolecule in stool that harbors sequence information is protein. However, the use of protein as an intake biomarker has only been explored to a very limited extent. Here, we evaluate and compare measurement of residual food-derived DNA and protein in stool as potential biomarkers of intake. We performed a pilot study of DNA sequencing-based metabarcoding (FoodSeq) and mass spectrometry-based metaproteomics in five individuals' stool sampled in short, longitudinal bursts accompanied by detailed diet records (n=27 total samples). Dietary data provided by stool DNA, stool protein, and written diet record independently identified a strong within-person dietary signature, identified similar food taxa, and had significantly similar global structure in two of the three pairwise comparisons between measurement techniques (DNA-to-protein and DNA-to-diet record). Metaproteomics identified proteins including myosin, ovalbumin, and beta-lactoglobulin that differentiated food tissue types like beef from dairy and chicken from egg, distinctions that were not possible by DNA alone. Overall, our results lay the groundwork for development of targeted metaproteomic assays for dietary assessment and demonstrate that diverse molecular components of food can be leveraged to study food intake using stool samples.}, author={Petrone, Brianna L. and Bartlett, Alexandria and Jiang, Sharon and Korenek, Abigail and Vintila, Simina and Tenekjian, Christine and Yancy, William S., Jr. and David, Lawrence A. and Kleiner, Manuel}, year={2024}, month={Apr} }
 @article{michellod_bien_birgel_violette_kleiner_fearn_zeidler_gruber-vodicka_dubilier_liebeke_2023, title={De novo phytosterol synthesis in animals}, volume={380}, ISSN={["1095-9203"]}, url={https://doi.org/10.1126/science.add7830}, DOI={10.1126/science.add7830}, abstractNote={Sterols are lipids that regulate multiple processes in eukaryotic cells, and are essential components of cellular membranes. Sterols are currently assumed to be kingdom specific, with phytosterol synthesis restricted to plants while animals are only able to synthesize cholesterol. Here, we challenge this assumption by demonstrating that the marine annelids Olavius and Inanidrilus synthesize the phytosterol sitosterol de novo. Using multi-omics, high-resolution metabolite imaging, heterologous gene expression and enzyme assays, we characterized the biosynthetic pathway of sitosterol and showed that it is the most abundant sterol in these worms (60%). We show that phytosterol synthesis partially overlaps with cholesterol synthesis and involves a non-canonical sterol methyltransferase (SMT), C24-SMT, an essential enzyme for sitosterol synthesis in plants, but not known from bilaterians. Our comparative phylogenetic analyses of C24-SMT homologs revealed that these are widely distributed across annelids and other animal phyla, including sponges and rotifers. Our findings show that phytosterol synthesis and use is not restricted to the plant kingdom, and indicate that the evolution of sterols in animals is more complex than previously assumed.}, number={6644}, journal={SCIENCE}, author={Michellod, Dolma and Bien, Tanja and Birgel, Daniel and Violette, Marlene and Kleiner, Manuel and Fearn, Sarah and Zeidler, Caroline and Gruber-Vodicka, Harald R. and Dubilier, Nicole and Liebeke, Manuel}, year={2023}, month={May}, pages={520–526} }
 @article{parnell_pal_awan_vintila_houdinet_hawkes_balint-kurti_wagner_kleiner_2023, title={Effective seed sterilization methods require optimization across maize genotypes}, url={https://doi.org/10.1101/2023.12.14.571779}, DOI={10.1101/2023.12.14.571779}, abstractNote={Studies of plant-microbe interactions using synthetic microbial communities (SynComs) often require the removal of seed-associated microbes by seed sterilization prior to inoculation to provide gnotobiotic growth conditions. A diversity of seed sterilization protocols have been developed in the past and have been used on different plant species with various amounts of validation. From these studies it has become clear that each plant species requires its own optimized sterilization protocol. It has, however, so far not been tested if the same protocol works equally well for different varieties and seed sources of one plant species. We evaluated six seed sterilization protocols on two different varieties (Sugar Bun & B73) of maize. All unsterilized maize seeds showed fungal growth upon germination on filter paper, highlighting the need for a sterilization protocol. A short sterilization protocol with hypochlorite and ethanol was sufficient to prevent fungal growth on Sugar Bun germinants, however a longer protocol with heat treatment and germination in fungicide was needed to obtain clean B73 germinants. This difference may have arisen from the effect of either genotype or seed source. We then tested the protocol that performed best for B73 on three additional maize genotypes from four sources. Seed germination rates and fungal contamination levels varied widely by genotype and geographic source of seeds. Our study shows that consideration of both variety and seed source is important when optimizing sterilization protocols and highlights the importance of including seed source information in plant-microbe interaction studies that use sterilized seeds.}, author={Parnell, J. Jacob and Pal, Gaurav and Awan, Ayesha and Vintila, Simina and Houdinet, Gabriella and Hawkes, Christine V. and Balint-Kurti, Peter J. and Wagner, Maggie R. and Kleiner, Manuel}, year={2023}, month={Dec} }
 @article{swift_kolp_carmichael_ford_hansen_sikes_kleiner_wagner_2023, title={Environmental legacy effects impact maize growth and microbiome assembly under drought stress}, url={https://doi.org/10.1101/2023.04.11.536405}, DOI={10.1101/2023.04.11.536405}, abstractNote={Background and Aims As the climate changes, plants and their associated microbiomes face greater water limitation and increased frequency of drought. Historical environmental patterns can leave a legacy effect on soil and root-associated microbiomes, but the impact of this conditioning on future drought performance is poorly understood. Precipitation gradients provide a means to assess these legacy effects. Methods We collected soil microbiomes from four native prairies across a steep precipitation gradient in Kansas, USA. Seedlings of two Zea mays genotypes were inoculated with each soil microbiome in a factorial drought experiment. We investigated plant phenotypic and root microbiome responses to drought and modeled relationships between plant growth metrics and climatic conditions from the soil microbiome origin sites. Results Drought caused plants to accumulate shoot mass more slowly and achieve greater root/shoot mass ratios. Drought restructured the bacterial root-associated microbiome via depletion of Pseudomonadota and enrichment of Actinomycetota, whereas the fungal microbiome was largely unaffected. An environmental legacy effect on prairie soil microbiomes influenced plants’ drought responses: counterintuitively, prairie soil inocula from historically wetter locations increased shoot biomass under drought more than inocula from historically drier prairie soils. Conclusion We demonstrated links between soil microbiome legacy effects and plant performance under drought, suggesting that future drying climates may condition soils to negatively impact plant performance.}, author={Swift, Joel F. and Kolp, Matthew R. and Carmichael, Amanda and Ford, Natalie E. and Hansen, Paige M. and Sikes, Benjamin A. and Kleiner, Manuel and Wagner, Maggie R.}, year={2023}, month={Apr} }
 @article{parnell_vintila_tang_wagner_kleiner_2023, title={Evaluation of ready-to-use freezer stocks of a synthetic microbial community for maize root colonization}, volume={12}, ISSN={["2165-0497"]}, url={https://doi.org/10.1128/spectrum.02401-23}, DOI={10.1128/spectrum.02401-23}, abstractNote={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 standardized de novo construction 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. 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.}, journal={MICROBIOLOGY SPECTRUM}, author={Parnell, J. Jacob and Vintila, Simina and Tang, Clara and Wagner, Maggie R. and Kleiner, Manuel}, editor={Hockett, Kevin LorenEditor}, year={2023}, month={Dec} }
 @article{parnell_vintila_tang_wagner_kleiner_2023, title={Evaluation of ready-to-use freezer stocks of a synthetic microbial community for maize root colonization}, url={https://doi.org/10.1101/2023.05.10.540175}, DOI={10.1101/2023.05.10.540175}, abstractNote={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 media 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 pre-make and freeze SynComs on a large scale, creating ready-to-use stock 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 standardized de novo construction 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 increases 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 multiple days of laborious growing and combining of multiple microorganisms. We show that ready-to-use SynCom stocks provide comparable results to freshly constructued SynComs and thus allow for large time savings when working with SynComs.}, author={Parnell, J. Jacob and Vintila, Simina and Tang, Clara and Wagner, Maggie R. and Kleiner, Manuel}, year={2023}, month={May} }
 @article{bartlett_blakeley-ruiz_richie_theriot_kleiner_2023, title={Large Quantities of Bacterial DNA and Protein in Common Dietary Protein Source Used in Microbiome Studies}, url={https://doi.org/10.1101/2023.12.07.570621}, DOI={10.1101/2023.12.07.570621}, abstractNote={Diet has been shown to greatly impact the intestinal microbiota. To understand the role of individual dietary components, defined diets with purified components are frequently used in diet-microbiota studies. Many of the frequently used defined diets use purified casein as the protein source. Previous work indicated that this casein contains microbial DNA potentially impacting results of microbiome studies. Other diet-based microbially derived molecules that may impact microbiome measurements, such as proteins detected by metaproteomics, have not been determined for casein. Additionally, other protein sources used in microbiome studies have not been characterized for their microbial content. We used metagenomics and metaproteomics to identify and quantify microbial DNA and protein in a casein-based defined diet to better understand potential impacts on metagenomic and metaproteomic microbiome studies. We further tested six additional defined diets with purified protein sources with an integrated metagenomic-metaproteomic approach and show that contaminating microbial protein is unique to casein within the tested set as microbial protein was not identified in diets with other protein sources. We also illustrate the contribution of diet-derived microbial protein in diet-microbiota studies by metaproteomic analysis of stool samples from germ-free mice (GF) and mice with a conventional microbiota (CV) following consumption of diets with casein and non-casein protein. This study highlights a potentially confounding factor in diet-microbiota studies that must be considered through evaluation of the diet itself within a given study. Importance Many diets used in diet-microbiota studies use casein as the source of dietary protein. We found large quantities of microbial DNA and protein in casein-based diets. This microbial DNA and protein are resilient to digestion as it is present in fecal samples of mice consuming casein-based diets. This contribution of diet-derived microbial DNA and protein to microbiota measurements may influence results and conclusions and must therefore be considered in diet-microbiota studies. We tested additional dietary protein sources and did not detect microbial DNA or protein. Our findings highlight the necessity of evaluating diet samples in diet-microbiota studies to ensure that potential microbial content of the diet can be accounted for in microbiome measurements.}, author={Bartlett, Alexandria and Blakeley-Ruiz, J. Alfredo and Richie, Tanner and Theriot, Casey M. and Kleiner, Manuel}, year={2023}, month={Dec} }
 @article{kleiner_polerecky_lott_bergin_häusler_liebeke_wentrup_musat_kuypers_dubilier_2023, title={Mechanism of high energy efficiency of carbon fixation by sulfur-oxidizing symbionts revealed by single-cell analyses and metabolic modeling}, url={https://doi.org/10.1101/2023.11.25.568684}, DOI={10.1101/2023.11.25.568684}, abstractNote={In chemosynthetic symbioses between marine invertebrates and autotrophic sulfur-oxidizing bacteria the symbionts feed their host by producing organic compounds from CO2 using reduced sulfur compounds as an energy source. One such symbiosis, the gutless marine worm Olavius algarvensis harbors at least five bacterial symbionts of which four have the genetic potential for an autotrophic metabolism. In this study we combined single-cell analyses of CO2 fixation, CO2 release and bulk uptake, with measurements of O2 respiration, sulfur content, and polyhydroxyalkanoate content, as well as mathematical modelling to investigate how energy derived from sulfur oxidation drives carbon fluxes within the symbiosis and between the holobiont and its habitat. We found that under aerobic conditions without external energy sources only the primary symbiont, Ca. Thiosymbion algarvensis, fixed carbon. This symbiont relied on internal sulfur storage for energy production. Our model showed that the apparent efficiency of carbon fixation driven by sulfur oxidation in the symbiosis was higher than thermodynamically feasible if only stored sulfur was considered as source of energy and reducing equivalents. The model and additional calculations showed that reducing equivalents must be derived from a different source than energy. We identified the large amounts of polyhdroxyalkanoate stored by the symbiont as the likely source of reducing equivalents for carbon fixation in the symbiont which boosts the yield of sulfur-driven carbon fixation. The model also showed that heterotrophic carbon fixation by host tissue is not negligible and has to be considered when assessing transfer of carbon from the symbionts to the host.}, author={Kleiner, M. and Polerecky, L. and Lott, C. and Bergin, C. and Häusler, S. and Liebeke, M. and Wentrup, C. and Musat, N. and Kuypers, M. M. M. and Dubilier, N.}, year={2023}, month={Nov} }
 @article{zvi-kedem_vintila_kleiner_tchernov_rubin-blum_2023, title={Metabolic handoffs between multiple symbionts may benefit the deep-sea bathymodioline mussels}, url={https://doi.org/10.1101/2023.02.09.527947}, DOI={10.1101/2023.02.09.527947}, abstractNote={Bathymodioline mussels rely on thiotrophic and 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, including the primary, chemosynthetic methane- and sulfur-oxidizing gammaproteobacteria, and the secondary Methylophagaceae, Nitrincolaceae and Flavobacteraceae symbionts, whose physiology and metabolism are obscure. Little is known about whether and how these symbionts interact or exchange metabolites. Here we curated metagenome-assembled genomes of Idas modiolaeformis symbionts and used genomecentered 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 Flavobacteriaceae Urechidicola 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.}, author={Zvi-Kedem, Tal and Vintila, Simina and Kleiner, Manuel and Tchernov, Dan and Rubin-Blum, Maxim}, year={2023}, month={Feb} }
 @article{zvi-kedem_vintila_kleiner_tchernov_rubin-blum_2023, title={Metabolic handoffs between multiple symbionts may benefit the deep-sea bathymodioline mussels}, volume={3}, ISSN={["2730-6151"]}, DOI={10.1038/s43705-023-00254-4}, abstractNote={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.}, number={1}, journal={ISME COMMUNICATIONS}, author={Zvi-Kedem, Tal and Vintila, Simina and Kleiner, Manuel and Tchernov, Dan and Rubin-Blum, Maxim}, year={2023}, month={May} }
 @article{hornstein_charles_franklin_edwards_vintila_kleiner_sederoff_2023, title={Re-engineering a lost trait:IPD3, a master regulator of arbuscular mycorrhizal symbiosis, affects genes for immunity and metabolism of non-host Arabidopsis when restored long after its evolutionary loss}, url={https://doi.org/10.1101/2023.03.06.531368}, DOI={10.1101/2023.03.06.531368}, abstractNote={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 model Arabidopsis thaliana. To explore why an apparently beneficial trait would be repeatedly lost, we generated Arabidopsis plants expressing a constitutively active form of Interacting Protein of DMI3, a key transcription factor that enables AM within the Common Symbiosis Pathway, which was lost from Arabidopsis along with the AM host trait. We characterize the transcriptomic effect of expressing IPD3 in Arabidopsis with and without exposure to the AM fungus (AMF) Rhizophagus irregularis, and compare these results to the AM model Lotus japonicus and its ipd3 knockout mutant cyclops-4. Despite its long history as a non-AM species, restoring IPD3 in the form of its constitutively active DNA-binding domain to Arabidopsis altered expression of specific gene networks. Surprisingly, the effect of expressing IPD3 in Arabidopsis and knocking it out in Lotus was strongest in plants not exposed to AMF, which is revealed to be due to changes in IPD3 genotype causing a transcriptional state which partially mimics AMF exposure in non-inoculated plants. Our results indicate that despite the long interval since loss of AM and IPD3 in Arabidopsis, 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.}, author={Hornstein, Eli D. and Charles, Melodi and Franklin, Megan and Edwards, Brianne and Vintila, Simina and Kleiner, Manuel and Sederoff, Heike}, year={2023}, month={Mar} }
 @article{kleiner_kouris_violette_d'angelo_liu_korenek_tolic_sachsenberg_mccalder_lipton_et al._2023, title={Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes}, volume={11}, ISSN={["2049-2618"]}, DOI={10.1186/s40168-022-01454-1}, abstractNote={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.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 using 18O 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 several Bacteroides species known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics.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.}, number={1}, journal={MICROBIOME}, author={Kleiner, Manuel and Kouris, Angela and Violette, Marlene and D'Angelo, Grace and Liu, Yihua and Korenek, Abigail and Tolic, Nikola and Sachsenberg, Timo and McCalder, Janine and Lipton, Mary S. S. and et al.}, year={2023}, month={Feb} }
 @article{blakeley-ruiz_kleiner_2022, title={Considerations for constructing a protein sequence database for metaproteomics}, volume={20}, ISSN={["2001-0370"]}, url={http://dx.doi.org/10.1016/j.csbj.2022.01.018}, DOI={10.1016/j.csbj.2022.01.018}, abstractNote={Mass spectrometry-based metaproteomics has emerged as a prominent technique for interrogating the functions of specific organisms in microbial communities, in addition to total community function. Identifying proteins by mass spectrometry requires matching mass spectra of fragmented peptide ions to a database of protein sequences corresponding to the proteins in the sample. This sequence database determines which protein sequences can be identified from the measurement, and as such the taxonomic and functional information that can be inferred from a metaproteomics measurement. Thus, the construction of the protein sequence database directly impacts the outcome of any metaproteomics study. Several factors, such as source of sequence information and database curation, need to be considered during database construction to maximize accurate protein identifications traceable to the species of origin. In this review, we provide an overview of existing strategies for database construction and the relevant studies that have sought to test and validate these strategies. Based on this review of the literature and our experience we provide a decision tree and best practices for choosing and implementing database construction strategies.}, journal={COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL}, publisher={Elsevier BV}, author={Blakeley-Ruiz, J. Alfredo and Kleiner, Manuel}, year={2022}, pages={937–952} }
 @article{michellod_bien_birgel_jensen_kleiner_fearn_zeidler_gruber-vodicka_dubilier_liebeke_2022, title={De novo phytosterol synthesis in animals}, volume={4}, url={https://doi.org/10.1101/2022.04.22.489198}, DOI={10.1101/2022.04.22.489198}, abstractNote={Abstract Sterols are lipids that regulate multiple processes in eukaryotic cells, and are essential components of cellular membranes. Sterols are currently assumed to be kingdom specific, with phytosterol synthesis restricted to plants while animals are only able to synthesize cholesterol. Here, we challenge this assumption by demonstrating that the marine annelids Olavius and Inanidrilus synthesize the phytosterol sitosterol de novo . Using multi-omics, high-resolution metabolite imaging, heterologous gene expression and enzyme assays, we show that sitosterol is the most abundant (60%) sterol in these animals and characterize its biosynthetic pathway. We show that phytosterol synthesis partially overlaps with cholesterol synthesis and involves a non-canonical C-24 sterol methyltransferase (C 24 -SMT). C 24 -SMT is an essential enzyme for sitosterol synthesis in plants, but not known from animals with bilateral symmetry (bilaterians). Our comparative phylogenetic analyses of C 24 -SMT homologs revealed that these are widely distributed across annelids and other animal phyla, including sponges and rotifers. Our findings show that phytosterol synthesis and use is not restricted to the plant kingdom, and indicate that the evolution of sterols in animals is more complex than previously assumed.}, publisher={Cold Spring Harbor Laboratory}, author={Michellod, Dolma and Bien, Tanja and Birgel, Daniel and Jensen, Marlene and Kleiner, Manuel and Fearn, Sarah and Zeidler, Caroline and Gruber-Vodicka, Harald R and Dubilier, Nicole and Liebeke, Manuel}, year={2022}, month={Apr} }
 @misc{bartlett_kleiner_2022, title={Dietary protein and the intestinal microbiota: An understudied relationship}, volume={25}, ISSN={["2589-0042"]}, DOI={10.1016/j.isci.2022.105313}, abstractNote={Diet has a profound impact on the microbial community in the gastrointestinal tract, the intestinal microbiota, to the benefit or detriment of human health. To understand the influence of diet on the intestinal microbiota, research has focused on individual macronutrients. Some macronutrients (e.g. fiber) have been studied in great detail and have been found to strongly influence the intestinal microbiota. The relationship between dietary protein, a vital macronutrient, and the intestinal microbiota has gone largely unexplored. Emerging evidence suggests that dietary protein strongly impacts intestinal microbiota composition and function and that protein-microbiota interactions can have critical impacts on host health. In this review, we focus on recent studies investigating the impact of dietary protein quantity and source on the intestinal microbiota and resulting host health consequences. We highlight major open questions critical to understanding health outcomes mediated by interactions between dietary protein and the microbiota.}, number={11}, journal={ISCIENCE}, author={Bartlett, Alexandria and Kleiner, Manuel}, year={2022}, month={Nov} }
 @article{beck_kleiner_garrell_2022, title={Elucidating Plant-Microbe-Environment Interactions Through Omics-Enabled Metabolic Modelling Using Synthetic Communities}, volume={13}, ISSN={["1664-462X"]}, DOI={10.3389/fpls.2022.910377}, abstractNote={With a growing world population and increasing frequency of climate disturbance events, we are in dire need of methods to improve plant productivity, resilience, and resistance to both abiotic and biotic stressors, both for agriculture and conservation efforts. Microorganisms play an essential role in supporting plant growth, environmental response, and susceptibility to disease. However, understanding the specific mechanisms by which microbes interact with each other and with plants to influence plant phenotypes is a major challenge due to the complexity of natural communities, simultaneous competition and cooperation effects, signalling interactions, and environmental impacts. Synthetic communities are a major asset in reducing the complexity of these systems by simplifying to dominant components and isolating specific variables for controlled experiments, yet there still remains a large gap in our understanding of plant microbiome interactions. This perspectives article presents a brief review discussing ways in which metabolic modelling can be used in combination with synthetic communities to continue progress toward understanding the complexity of plant-microbe-environment interactions. We highlight the utility of metabolic models as applied to a community setting, identify different applications for both flux balance and elementary flux mode simulation approaches, emphasize the importance of ecological theory in guiding data interpretation, and provide ideas for how the integration of metabolic modelling techniques with big data may bridge the gap between simplified synthetic communities and the complexity of natural plant-microbe systems.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Beck, Ashley E. and Kleiner, Manuel and Garrell, Anna-Katharina}, year={2022}, month={Jun} }
 @article{salvato_vintila_finkel_dangl_kleiner_2022, title={Evaluation of Protein Extraction Methods for Metaproteomic Analyses of Root-Associated Microbes}, volume={35}, ISSN={["1943-7706"]}, url={https://doi.org/10.1094/MPMI-05-22-0116-TA}, DOI={10.1094/MPMI-05-22-0116-TA}, abstractNote={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 (40-70 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 LC-MS/MS-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 highest 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 highest 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.}, number={11}, journal={MOLECULAR PLANT-MICROBE INTERACTIONS}, author={Salvato, Fernanda and Vintila, Simina and Finkel, Omri M. and Dangl, Jeffery L. and Kleiner, Manuel}, year={2022}, month={Nov}, pages={977–988} }
 @article{sato_wippler_wentrup_ansorge_sadowski_gruber-vodicka_dubilier_kleiner_2022, title={Fidelity varies in the symbiosis between a gutless marine worm and its microbial consortium}, volume={10}, ISSN={["2049-2618"]}, DOI={10.1186/s40168-022-01372-2}, abstractNote={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.In this study on the obligate symbiosis between the gutless marine annelid Olavius algarvensis and 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 80 O. algarvensis individuals 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 all O. algarvensis individuals. 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.We hypothesize that variable degrees of fidelity are advantageous for O. algarvensis by 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. Video Abstract.}, number={1}, journal={MICROBIOME}, author={Sato, Yui and Wippler, Juliane and Wentrup, Cecilia and Ansorge, Rebecca and Sadowski, Miriam and Gruber-Vodicka, Harald and Dubilier, Nicole and Kleiner, Manuel}, year={2022}, month={Oct} }
 @article{smith_salvato_garikipati_kleiner_septer_2021, title={Activation of the Type VI Secretion System in the Squid Symbiont Vibrio fischeri Requires the Transcriptional Regulator TasR and the Structural Proteins TssM and TssA}, volume={203}, ISSN={["1098-5530"]}, DOI={10.1128/JB.00399-21}, abstractNote={Interbacterial weapons like the T6SS are often located on mobile genetic elements, and their expression is highly regulated. We found that two conserved structural proteins are required for T6SS expression in Vibrio fischeri. ABSTRACT Bacteria have evolved diverse strategies to compete for a niche, including the type VI secretion system (T6SS), a contact-dependent killing mechanism. T6SSs are common in bacterial pathogens, commensals, and beneficial symbionts, where they affect the diversity and spatial structure of host-associated microbial communities. Although T6SS gene clusters are often located on genomic islands (GIs), which may be transferred as a unit, the regulatory strategies that promote gene expression once the T6SS genes are transferred into a new cell are not known. We used the squid symbiont Vibrio fischeri to identify essential regulatory factors that control expression of a strain-specific T6SS encoded on a GI. We found that a transcriptional reporter for this T6SS is active only in strains that contain the T6SS-encoding GI, suggesting the GI encodes at least one essential regulator. A transposon screen identified seven mutants that could not activate the reporter. These mutations mapped exclusively to three genes on the T6SS-containing GI that encode two essential structural proteins (a TssA-like protein and TssM) and a transcriptional regulator (TasR). Using T6SS reporters, reverse transcription-PCR (RT-PCR), competition assays, and differential proteomics, we found that all three genes are required for expression of many T6SS components, except for the TssA-like protein and TssM, which are constitutively expressed. Based on these findings, we propose a model whereby T6SS expression requires conserved structural proteins, in addition to the essential regulator TasR, and this ability to self-regulate may be a strategy to activate T6SS expression upon transfer of T6SS-encoding elements into a new bacterial host. IMPORTANCE Interbacterial weapons like the T6SS are often located on mobile genetic elements, and their expression is highly regulated. We found that two conserved structural proteins are required for T6SS expression in Vibrio fischeri. These structural proteins also contain predicted GTPase and GTP binding domains, suggesting their role in promoting T6SS expression may involve sensing the energetic state of the cell. Such a mechanism would provide a direct link between T6SS activation and cellular energy levels, providing a “checkpoint” to ensure the cell has sufficient energy to build such a costly weapon. Because these regulatory factors are encoded within the T6SS gene cluster, they are predicted to move with the genetic element to activate T6SS expression in a new host cell.}, number={21}, journal={JOURNAL OF BACTERIOLOGY}, author={Smith, Stephanie and Salvato, Fernanda and Garikipati, Aditi and Kleiner, Manuel and Septer, Alecia N.}, year={2021}, month={Nov} }
 @article{hinzke_kleiner_meister_schlueter_hentschker_pane-farre_hildebrandt_felbeck_sievert_bonn_et al._2021, title={Bacterial symbiont subpopulations have different roles in a deep-sea symbiosis}, volume={10}, ISSN={["2050-084X"]}, url={https://europepmc.org/articles/PMC7787665}, DOI={10.7554/elife.58371}, abstractNote={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.}, journal={ELIFE}, publisher={eLife Sciences Publications, Ltd}, author={Hinzke, Tjorven and Kleiner, Manuel and Meister, Mareike and Schlueter, Rabea and Hentschker, Christian and Pane-Farre, Jan and Hildebrandt, Petra and Felbeck, Horst and Sievert, Stefan M. and Bonn, Florian and et al.}, year={2021}, month={Jan} }
 @article{bossche_kunath_schallert_schaepe_abraham_armengaud_arntzen_bassignani_benndorf_fuchs_et al._2021, title={Critical Assessment of MetaProteome Investigation (CAMPI): a multi-laboratory comparison of established workflows}, volume={12}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-021-27542-8}, abstractNote={Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.}, number={1}, journal={NATURE COMMUNICATIONS}, author={Bossche, Tim and Kunath, Benoit J. and Schallert, Kay and Schaepe, Stephanie S. and Abraham, Paul E. and Armengaud, Jean and Arntzen, Magnus O. and Bassignani, Ariane and Benndorf, Dirk and Fuchs, Stephan and et al.}, year={2021}, month={Dec} }
 @article{jensen_wippler_kleiner_2021, title={Evaluation of RNAlater as a Field-Compatible Preservation Method for Metaproteomic Analyses of Bacterium-Animal Symbioses}, volume={9}, ISSN={["2165-0497"]}, url={https://doi.org/10.1128/Spectrum.01429-21}, DOI={10.1128/Spectrum.01429-21}, abstractNote={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. 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 RNAlater using the marine gutless oligochaete Olavius algarvensis and its symbiotic microbes as a test case. In addition, we evaluated long-term RNAlater 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 RNAlater 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 RNAlater 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 RNAlater can serve as a low-hazard, easy-to-transport alternative to flash freezing for field preservation of samples for metaproteomic analyses. We show that RNAlater 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.}, number={2}, journal={MICROBIOLOGY SPECTRUM}, publisher={American Society for Microbiology}, author={Jensen, Marlene and Wippler, Juliane and Kleiner, Manuel}, editor={Gralnick, Jeffrey A.Editor}, year={2021}, month={Oct} }
 @article{jensen_wippler_kleiner_2021, title={Evaluation of RNAlater™ as a field-compatible preservation method for metaproteomic analyses of bacteria-animal symbioses}, volume={6}, url={https://doi.org/10.1101/2021.06.16.448770}, DOI={10.1101/2021.06.16.448770}, abstractNote={Field studies are central to environmental microbiology and microbial ecology as they enable studies of natural microbial communities. Metaproteomics, the study of protein abundances in microbial communities, allows to study these communities ‘in situ’ which requires protein preservation directly in the field as 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 non-hazardous 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 in field conditions when targeted for metaproteomics is currently lacking. Here, we compared the protein preservation performance of flash freezing and the preservation solution RNAlater™ using the marine gutless oligochaete Olavius algarvensis and its symbiotic microbes as a test case. In addition, we evaluated long-term RNAlater™ storage after 1 day, 1 week and 4 weeks at room temperature (22-23 °C). We evaluated protein preservation using one dimensional liquid chromatography tandem mass spectrometry (1D-LC-MS/MS). We found that RNAlater™ and flash freezing preserved proteins equally well in terms of total number of identified proteins or relative abundances of individual proteins and none of the test time points were altered compared to t0. Moreover, we did not find biases against specific taxonomic groups or proteins with particular biochemical properties. Based on our metaproteomics data and the logistical requirements for field deployment we recommend RNAlater™ for protein preservation of field-collected samples when targeted for metaproteomcis. Importance Metaproteomics, the large-scale identification and quantification of proteins from microbial communities, provides 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 set-ups 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 its hazardous nature and transport restrictions. Our study shows that RNAlater™ can serve as a low hazard, easy to transport alternative to flash freezing for field preservation of samples for metaproteomics. We show that RNAlater™ preserves the metaproteome equally well as compared to flash freezing and protein abundance patterns remain stable during long-term storage for at least 4 weeks at room temperature.}, publisher={Cold Spring Harbor Laboratory}, author={Jensen, Marlene and Wippler, Juliane and Kleiner, Manuel}, year={2021}, month={Jun} }
 @article{mordant_kleiner_2021, title={Evaluation of Sample Preservation and Storage Methods for Metaproteomics Analysis of Intestinal Microbiomes}, volume={9}, ISSN={["2165-0497"]}, url={https://doi.org/10.1128/Spectrum.01877-21}, DOI={10.1128/Spectrum.01877-21}, abstractNote={ABSTRACT A critical step in studies of the intestinal microbiome using meta-omics approaches is the preservation of samples before analysis. Preservation is essential for approaches that measure gene expression, such as metaproteomics, which is used to identify and quantify proteins in microbiomes. Intestinal microbiome samples are typically stored by flash-freezing and storage at −80°C, but some experimental setups do not allow for immediate freezing of samples. In this study, we evaluated methods to preserve fecal microbiome samples for metaproteomics analyses when flash-freezing is not possible. We collected fecal samples from C57BL/6 mice and stored them for 1 and 4 weeks using the following methods: flash-freezing in liquid nitrogen, immersion in RNAlater, immersion in 95% ethanol, immersion in a RNAlater-like buffer, and combinations of these methods. After storage, we extracted protein and prepared peptides for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis to identify and quantify peptides and proteins. All samples produced highly similar metaproteomes, except for ethanol-preserved samples that were distinct from all other samples in terms of protein identifications and protein abundance profiles. Flash-freezing and RNAlater (or RNAlater-like treatments) produced metaproteomes that differed only slightly, with less than 0.7% of identified proteins differing in abundance. In contrast, ethanol preservation resulted in an average of 9.5% of the identified proteins differing in abundance between ethanol and the other treatments. Our results suggest that preservation at room temperature in RNAlater or an RNAlater-like solution performs as well as freezing for the preservation of intestinal microbiome samples before metaproteomics analyses. IMPORTANCE Metaproteomics is a powerful tool to study the intestinal microbiome. By identifying and quantifying a large number of microbial, dietary, and host proteins in microbiome samples, metaproteomics provides direct evidence of the activities and functions of microbial community members. A critical step for metaproteomics workflows is preserving samples before analysis because protein profiles are susceptible to fast changes in response to changes in environmental conditions (air exposure, temperature changes, etc.). This study evaluated the effects of different preservation treatments on the metaproteomes of intestinal microbiome samples. In contrast to prior work on preservation of fecal samples for metaproteomics analyses, we ensured that all steps of sample preservation were identical so that all differences could be attributed to the preservation method.}, number={3}, journal={MICROBIOLOGY SPECTRUM}, publisher={American Society for Microbiology}, author={Mordant, Angie and Kleiner, Manuel}, editor={Khursigara, Cezar M.Editor}, year={2021}, month={Dec} }
 @article{sato_wippler_wentrup_ansorge_sadowski_gruber-vodicka_dubilier_kleiner_2021, title={Fidelity varies in the symbiosis between a gutless marine worm and its microbial consortium}, volume={1}, url={https://doi.org/10.1101/2021.01.30.428904}, DOI={10.1101/2021.01.30.428904}, abstractNote={Abstract In obligate symbioses, partner fidelity plays a central role in maintaining the association over evolutionary time. Fidelity has been well studied in hosts with only a few symbionts, but little is known about how fidelity is maintained in obligate associations with multiple co-occurring symbionts. Here, we show that partner fidelity varies from strict to absent in a gutless marine annelid and its consortium of co-occurring symbionts that provide it with nutrition. We sequenced the metagenomes of 80 Olavius algarvensis individuals from the Mediterranean, and compared host mitochondrial and symbiont phylogenies based on single nucleotide polymorphisms across genomes, using a low-coverage sequencing approach that has not yet been applied to microbial community analyses. Fidelity was strongest for the two chemoautotrophic, sulphur-oxidizing symbionts that dominated the microbial consortium in all host individuals. In contrast, fidelity was only intermediate to absent in the sulphate-reducing and spirochaetal symbionts, which occurred in lower abundance and were not always present in all host individuals. We propose that variable degrees of fidelity are advantageous for these hosts by 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.}, publisher={Cold Spring Harbor Laboratory}, author={Sato, Yui and Wippler, Juliane and Wentrup, Cecilia and Ansorge, Rebecca and Sadowski, Miriam and Gruber-Vodicka, Harald and Dubilier, Nicole and Kleiner, Manuel}, year={2021}, month={Jan} }
 @misc{salvato_hettich_kleiner_2021, title={Five key aspects of metaproteomics as a tool to understand functional interactions in host-associated microbiomes}, volume={17}, ISSN={["1553-7374"]}, url={https://doi.org/10.1371/journal.ppat.1009245}, DOI={10.1371/journal.ppat.1009245}, abstractNote={Host-associated microbial communities (microbiomes) play critical roles in human, animal, and plant health and development. However, interactions between the host, members of the microbiome, and invading pathogens are in most cases still poorly understood. Such interactions are multidimensional [1] and can alter the taxonomic composition and/or the functional metabolic activities of the microbiome in response to disease or treatment conditions. For example, after 2 days of antibiotic treatment, the mouse gut microbiome is altered and more susceptible to invasion by the pathogen Clostridioides difficile [2]. Studies of these multidimensional interactions have been fueled by the ability to use high-throughput sequencing of phylogenetic marker genes to profile microbial community composition and shotgun metagenomics to profile functional potential [3]. However, many protein-coding genes predicted from metagenomes are not necessarily expressed under a given condition, and thus, it is difficult to assess the activities and functional interactions in microbial communities based on DNA sequencing data alone [4]. The physiological and pathological processes expressed in these communities under specific conditions are better reflected by the abundances of transcripts or proteins [5,6]. In this Pearl, we provide a brief introduction to metaproteomics, which is a tool for the large-scale analysis of proteins in microbiomes that allows researchers to address a diversity of questions related to functions and interactions in microbiomes [7]. The term “metaproteomics” was first used in 2004 for “the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time” [8], and since then, a large array of metaproteomics approaches have been developed [7]. Our objective in this Pearl is to highlight what we feel are 5 essential elements to be considered for a metaproteomics research campaign and to introduce nonexpert readers to the topic without going into too much technical detail.}, number={2}, journal={PLOS PATHOGENS}, publisher={Public Library of Science (PLoS)}, author={Salvato, Fernanda and Hettich, Robert L. and Kleiner, Manuel}, editor={Hogan, Deborah A.Editor}, year={2021}, month={Feb} }
 @article{mankowski_kleiner_erséus_leisch_sato_volland_hüttel_wentrup_woyke_wippler_et al._2021, title={Highly variable fidelity drives symbiont community composition in an obligate symbiosis}, volume={4}, url={https://doi.org/10.1101/2021.04.28.441735}, DOI={10.1101/2021.04.28.441735}, abstractNote={Many animals are obligately associated with microbial symbionts that provide essential services such as nutrition or protection against predators. It is assumed that in such obligate associations fidelity between the host and its symbionts must be high to ensure the evolutionary success of the symbiosis. We show here that this is not the case in marine oligochaete worms, despite the fact that they are so dependent on their bacterial symbionts for their nutrition and waste recycling that they have lost their digestive and excretory systems. Our metagenomic analyses of 64 gutless oligochaete species from around the world revealed highly variable levels of fidelity not only across symbiont lineages, but also within symbiont clades. We hypothesize that in gutless oligochaetes, selection within host species for locally adapted and temporally stable symbiont communities leads to varying levels of symbiont fidelity and shuffles the composition of symbiont assemblages across geographic and evolutionary scales.}, publisher={Cold Spring Harbor Laboratory}, author={Mankowski, Anna and Kleiner, Manuel and Erséus, Christer and Leisch, Nikolaus and Sato, Yui and Volland, Jean-Marie and Hüttel, Bruno and Wentrup, Cecilia and Woyke, Tanja and Wippler, Juliane and et al.}, year={2021}, month={Apr} }
 @article{maggie sogin_kleiner_borowski_gruber-vodicka_dubilier_2021, title={Life in the Dark: Phylogenetic and Physiological Diversity of Chemosynthetic Symbioses}, volume={75}, ISSN={["1545-3251"]}, DOI={10.1146/annurev-micro-051021-123130}, abstractNote={Possibly the last discovery of a previously unknown major ecosystem on Earth was made just over half a century ago, when researchers found teaming communities of animals flourishing two and a half kilometers below the ocean surface at hydrothermal vents. We now know that these highly productive ecosystems are based on nutritional symbioses between chemosynthetic bacteria and eukaryotes and that these chemosymbioses are ubiquitous in both deep-sea and shallow-water environments. The symbionts are primary producers that gain energy from the oxidation of reduced compounds, such as sulfide and methane, to fix carbon dioxide or methane into biomass to feed their hosts. This review outlines how the symbiotic partners have adapted to living together. We first focus on the phylogenetic and metabolic diversity of these symbioses and then highlight selected research directions that could advance our understanding of the processes that shaped the evolutionary and ecological success of these associations. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.}, journal={ANNUAL REVIEW OF MICROBIOLOGY, VOL 75, 2021}, author={Maggie Sogin, E. and Kleiner, Manuel and Borowski, Christian and Gruber-Vodicka, Harald R. and Dubilier, Nicole}, year={2021}, pages={695–718} }
 @article{wagner_tang_salvato_clouse_bartlett_vintila_phillips_sermons_hoffmann_balint-kurti_et al._2021, title={Microbe-dependent heterosis in maize}, volume={118}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.2021965118}, DOI={10.1073/pnas.2021965118}, abstractNote={Hybrids account for nearly all commercially planted varieties of maize and many other crop plants because crosses between inbred lines of these species produce first-generation [F1] offspring that greatly outperform their parents. The mechanisms underlying this phenomenon, called heterosis or hybrid vigor, are not well understood despite over a century of intensive research. The leading hypotheses-which focus on quantitative genetic mechanisms (dominance, overdominance, and epistasis) and molecular mechanisms (gene dosage and transcriptional regulation)-have been able to explain some but not all of the observed patterns of heterosis. Abiotic stressors are known to impact the expression of heterosis; however, the potential role of microbes in heterosis has largely been ignored. Here, we show that heterosis of root biomass and other traits in maize is strongly dependent on the belowground microbial environment. We found that, in some cases, inbred lines perform as well by these criteria as their F1 offspring under sterile conditions but that heterosis can be restored by inoculation with a simple community of seven bacterial strains. We observed the same pattern for seedlings inoculated with autoclaved versus live soil slurries in a growth chamber and for plants grown in steamed or fumigated versus untreated soil in the field. In a different field site, however, soil steaming increased rather than decreased heterosis, indicating that the direction of the effect depends on community composition, environment, or both. Together, our results demonstrate an ecological phenomenon whereby soil microbes differentially impact the early growth of inbred and hybrid maize.}, number={30}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Wagner, Maggie R. and Tang, Clara and Salvato, Fernanda and Clouse, Kayla M. and Bartlett, Alexandria and Vintila, Simina and Phillips, Laura and Sermons, Shannon and Hoffmann, Mark and Balint-Kurti, Peter J. and et al.}, year={2021}, month={Jul} }
 @article{ataeian_vadlamani_haines_mosier_dong_kleiner_strous_hawley_2021, title={Proteome and strain analysis of cyanobacterium Candidatus "Phormidium alkaliphilum" reveals traits for success in biotechnology}, volume={24}, ISSN={["2589-0042"]}, url={https://doi.org/10.1016/j.isci.2021.103405}, DOI={10.1016/j.isci.2021.103405}, abstractNote={Cyanobacteria encompass a diverse group of photoautotrophic bacteria with important roles in nature and biotechnology. Here we characterized Candidatus "Phormidium alkaliphilum," an abundant member in alkaline soda lake microbial communities globally. The complete, circular whole-genome sequence of Ca. "P. alkaliphilum" was obtained using combined Nanopore and Illumina sequencing of a Ca. "P. alkaliphilum" consortium. Strain-level diversity of Ca. "P. alkaliphilum" was shown to contribute to photobioreactor robustness under different operational conditions. Comparative genomics of closely related species showed that adaptation to high pH was not attributed to specific genes. Proteomics at high and low pH showed only minimal changes in gene expression, but higher productivity in high pH. Diverse photosystem antennae proteins, and high-affinity terminal oxidase, compared with other soda lake cyanobacteria, appear to contribute to the success of Ca. "P. alkaliphilum" in photobioreactors and biotechnology applications.}, number={12}, journal={ISCIENCE}, publisher={Elsevier BV}, author={Ataeian, Maryam and Vadlamani, Agasteswar and Haines, Marianne and Mosier, Damon and Dong, Xiaoli and Kleiner, Manuel and Strous, Marc and Hawley, Alyse K.}, year={2021}, month={Dec} }
 @article{kleiner_kouris_jensen_grace_liu_korenek_tolić_sachsenberg_mccalder_lipton_et al._2021, title={Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes}, volume={3}, url={https://doi.org/10.1101/2021.03.29.437612}, DOI={10.1101/2021.03.29.437612}, abstractNote={Stable isotope probing (SIP) approaches are a critical tool in microbiome research to determine associations between species and substrates. 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, are limited in terms of sensitivity, resolution or throughput. Here we present an ultra-sensitive, high-throughput protein-based stable isotope probing approach (Protein-SIP), which cuts cost for labeled substrates by ∼90% as compared to other SIP and Protein-SIP approaches and thus enables isotope labeling experiments on much larger scales and with higher replication. It allows for the determination of isotope incorporation into microbiome members with species level resolution using standard metaproteomics LC-MS/MS measurements. The analysis has been implemented as an open-source application (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 using 18O 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 several Bacteroides species known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber based prebiotics. In summary, 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.}, publisher={Cold Spring Harbor Laboratory}, author={Kleiner, Manuel and Kouris, Angela and Jensen, Marlene and Grace, D’Angelo and Liu, Yihua and Korenek, Abigail and Tolić, Nikola and Sachsenberg, Timo and McCalder, Janine and Lipton, Mary S. and et al.}, year={2021}, month={Mar} }
 @article{ponnudurai_heiden_sayavedra_hinzke_kleiner_hentschker_felbeck_sievert_schlüter_becher_et al._2020, title={Comparative proteomics of related symbiotic mussel species reveals high variability of host–symbiont interactions}, url={https://doi.org/10.1038/s41396-019-0517-6}, DOI={10.1038/s41396-019-0517-6}, abstractNote={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.}, journal={The ISME Journal}, author={Ponnudurai, Ruby and Heiden, Stefan E and Sayavedra, Lizbeth and Hinzke, Tjorven and Kleiner, Manuel and Hentschker, Christian and Felbeck, Horst and Sievert, Stefan M and Schlüter, Rabea and Becher, Dörte and et al.}, year={2020}, month={Feb} }
 @article{speare_smith_salvato_kleiner_septer_2020, title={Environmental Viscosity Modulates Interbacterial Killing during Habitat Transition}, volume={11}, ISSN={["2150-7511"]}, DOI={10.1128/mBio.03060-19}, abstractNote={Bacteria often engage in interference competition to gain access to an ecological niche, such as a host. However, little is known about how the physical environment experienced by free-living or host-associated bacteria influences such competition. We used the bioluminescent squid symbiont Vibrio fischeri to study how environmental viscosity impacts bacterial competition. Our results suggest that upon transition from a planktonic environment to a host-like environment, V. fischeri cells activate their type VI secretion system, a contact-dependent interbacterial nanoweapon, to eliminate natural competitors. This work shows that competitor cells form aggregates under host-like conditions, thereby facilitating the contact required for killing, and reveals how V. fischeri regulates a key competitive mechanism in response to the physical environment. ABSTRACT Symbiotic bacteria use diverse strategies to compete for host colonization sites. However, little is known about the environmental cues that modulate interbacterial competition as they transition between free-living and host-associated lifestyles. We used the mutualistic relationship between Eupyrmna scolopes squid and Vibrio fischeri bacteria to investigate how intraspecific competition is regulated as symbionts move from the seawater to a host-like environment. We recently reported that V. fischeri uses a type VI secretion system (T6SS) for intraspecific competition during host colonization. Here, we investigated how environmental viscosity impacts T6SS-mediated competition by using a liquid hydrogel medium that mimics the viscous host environment. Our data demonstrate that although the T6SS is functionally inactive when cells are grown under low-viscosity liquid conditions similar to those found in seawater, exposure to a host-like high-viscosity hydrogel enhances T6SS expression and sheath formation, activates T6SS-mediated killing in as little as 30 min, and promotes the coaggregation of competing genotypes. Finally, the use of mass spectrometry-based proteomics revealed insights into how cells may prepare for T6SS competition during this habitat transition. These findings, which establish the use of a new hydrogel culture condition for studying T6SS interactions, indicate that V. fischeri rapidly responds to the physical environment to activate the competitive mechanisms used during host colonization. IMPORTANCE Bacteria often engage in interference competition to gain access to an ecological niche, such as a host. However, little is known about how the physical environment experienced by free-living or host-associated bacteria influences such competition. We used the bioluminescent squid symbiont Vibrio fischeri to study how environmental viscosity impacts bacterial competition. Our results suggest that upon transition from a planktonic environment to a host-like environment, V. fischeri cells activate their type VI secretion system, a contact-dependent interbacterial nanoweapon, to eliminate natural competitors. This work shows that competitor cells form aggregates under host-like conditions, thereby facilitating the contact required for killing, and reveals how V. fischeri regulates a key competitive mechanism in response to the physical environment.}, number={1}, journal={MBIO}, author={Speare, Lauren and Smith, Stephanie and Salvato, Fernanda and Kleiner, Manuel and Septer, Alecia N.}, year={2020} }
 @article{sato_wippler_wentrup_dubilier_kleiner_2020, title={High-Quality Draft Genome Sequences of Two Deltaproteobacterial Endosymbionts, Delta1a and Delta1b, from the Uncultured Sva0081 Clade, Assembled from Metagenomes of the Gutless Marine Worm Olavius algarvensis}, volume={9}, ISSN={["2576-098X"]}, DOI={10.1128/MRA.00276-20}, abstractNote={Here, we present high-quality metagenome-assembled genome sequences of two closely related deltaproteobacterial endosymbionts from the gutless marine worm Olavius algarvensis (Annelida). The first is an improved draft genome sequence of the previously described sulfate-reducing symbiont Delta1. The second is from a closely related, recently discovered symbiont of O. algarvensis. ABSTRACT Here, we present high-quality metagenome-assembled genome sequences of two closely related deltaproteobacterial endosymbionts from the gutless marine worm Olavius algarvensis (Annelida). The first is an improved draft genome sequence of the previously described sulfate-reducing symbiont Delta1. The second is from a closely related, recently discovered symbiont of O. algarvensis.}, number={16}, journal={MICROBIOLOGY RESOURCE ANNOUNCEMENTS}, author={Sato, Yui and Wippler, Juliane and Wentrup, Cecilia and Dubilier, Nicole and Kleiner, Manuel}, year={2020}, month={Apr} }
 @article{sato_wippler_wentrup_woyke_dubilier_kleiner_2020, title={High-Quality Draft Genome Sequences of the Uncultured Delta3 Endosymbiont (Deltaproteobacteria) Assembled from Metagenomes of the Gutless Marine Worm Olavius algarvensis}, volume={9}, ISBN={2576-098X}, DOI={10.1128/MRA.00704-20.}, abstractNote={["Here, we present two high-quality, draft metagenome-assembled genomes of deltaproteobacterial OalgDelta3 endosymbionts from the gutless marine worm ", {:i=>"Olavius algarvensis"}, " Their 16S rRNA gene sequences share 98% identity with Delta3 endosymbionts of related host species ", {:i=>"Olavius ilvae"}, " (GenBank accession no. AJ620501) and ", {:i=>"Inanidrilus exumae"}, " (GenBank accession no. FM202060), for which no symbiont genomes are available."]}, number={31}, journal={MICROBIOLOGY RESOURCE ANNOUNCEMENTS}, author={Sato, Yui and Wippler, Juliane and Wentrup, Cecilia and Woyke, Tanja and Dubilier, Nicole and Kleiner, Manuel}, year={2020}, month={Jul} }
 @article{sato_wippler_wentrup_woyke_dubilier_kleiner_2020, title={High-Quality Draft Genome Sequences of the Uncultured Delta3 Endosymbiont (Deltaproteobacteria) Assembled from Metagenomes of the Gutless Marine Worm Olavius algarvensis}, volume={9}, ISSN={["2576-098X"]}, url={https://doi.org/10.1128/MRA.00704-20}, DOI={10.1128/MRA.00704-20}, abstractNote={Here, we present two high-quality, draft metagenome-assembled genomes of deltaproteobacterial OalgDelta3 endosymbionts from the gutless marine worm Olavius algarvensis. Their 16S rRNA gene sequences share 98% identity with Delta3 endosymbionts of related host species Olavius ilvae (GenBank accession no. AJ620501) and Inanidrilus exumae (GenBank accession no. FM202060), for which no symbiont genomes are available. ABSTRACT Here, we present two high-quality, draft metagenome-assembled genomes of deltaproteobacterial OalgDelta3 endosymbionts from the gutless marine worm Olavius algarvensis. Their 16S rRNA gene sequences share 98% identity with Delta3 endosymbionts of related host species Olavius ilvae (GenBank accession no. AJ620501) and Inanidrilus exumae (GenBank accession no. FM202060), for which no symbiont genomes are available.}, number={31}, journal={MICROBIOLOGY RESOURCE ANNOUNCEMENTS}, publisher={American Society for Microbiology}, author={Sato, Yui and Wippler, Juliane and Wentrup, Cecilia and Woyke, Tanja and Dubilier, Nicole and Kleiner, Manuel}, editor={Stewart, Frank J.Editor}, year={2020}, month={Jul} }
 @article{assie_leisch_meier_gruber-vodicka_tegetmeyer_meyerdierks_kleiner_hinzke_joye_saxton_et al._2020, title={Horizontal acquisition of a patchwork Calvin cycle by symbiotic and free-living Campylobacterota (formerly Epsilonproteobacteria)}, volume={14}, ISSN={["1751-7370"]}, url={https://doi.org/10.1038/s41396-019-0508-7}, DOI={10.1038/s41396-019-0508-7}, abstractNote={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.}, number={1}, journal={ISME JOURNAL}, author={Assie, Adrien and Leisch, Nikolaus and Meier, Dimitri V and Gruber-Vodicka, Harald and Tegetmeyer, Halina E. and Meyerdierks, Anke and Kleiner, Manuel and Hinzke, Tjorven and Joye, Samantha and Saxton, Matthew and et al.}, year={2020}, month={Jan}, pages={104–122} }
 @article{hinzke_kleiner_meister_schlüter_hentschker_pané-farré_hildebrandt_felbeck_sievert_bonn_et al._2020, title={Metabolic differences between symbiont subpopulations in the deep-sea tubeworm Riftia pachyptila}, volume={4}, url={https://doi.org/10.1101/2020.04.08.032177}, DOI={10.1101/2020.04.08.032177}, abstractNote={The hydrothermal vent tube worm Riftia pachyptila lives in intimate symbiosis with intracellular sulfur-oxidizing gammaproteobacteria. Although the symbiont population consists of a single 16S rRNA phylotype, bacteria in the same host animal exhibit a remarkable degree of metabolic diversity: They simultaneously utilize two carbon fixation pathways and various energy sources and electron acceptors. Whether these multiple metabolic routes are employed in the same symbiont cells, or rather in distinct symbiont subpopulations, was unclear. As Riftia symbionts vary considerably in cell size and shape, we enriched individual symbiont cell sizes by density gradient centrifugation in order to test whether symbiont cells of different sizes show different metabolic profiles. Metaproteomic analysis and statistical evaluation using clustering and random forests, supported by microscopy and flow cytometry, strongly suggest that Riftia symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: Small symbionts actively divide and may establish cellular symbiont-host interaction, as indicated by highest abundance of the cell division key protein FtsZ and highly abundant chaperones and porins in this initial phase. Large symbionts, on the other hand, apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Highest abundance of enzymes for CO2 fixation, carbon storage and biosynthesis in large symbionts indicates that in this late differentiation stage the symbiont’s metabolism is efficiently geared towards the production of organic material. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.}, publisher={Cold Spring Harbor Laboratory}, author={Hinzke, Tjorven and Kleiner, Manuel and Meister, Mareike and Schlüter, Rabea and Hentschker, Christian and Pané-Farré, Jan and Hildebrandt, Petra and Felbeck, Horst and Sievert, Stefan M. and Bonn, Florian and et al.}, year={2020}, month={Apr} }
 @article{wagner_tang_salvato_clouse_bartlett_sermons_hoffmann_balint-kurti_kleiner_2020, title={Microbe-dependent heterosis in maize}, volume={5}, url={https://doi.org/10.1101/2020.05.05.078766}, DOI={10.1101/2020.05.05.078766}, abstractNote={Significance Almost all grain crops grown on commercial farms are hybrid cultivars because these hybrid plants are reliably healthier, larger, and more productive than their inbred parent lines. The widespread and valuable phenomenon of hybrid superiority is called heterosis. Despite over a century of intensive research into heterosis, it is unclear how or why hybrid genomes give rise to superior phenotypes. Most hypotheses and research thus far have focused on genetic and physiological mechanisms of heterosis. In contrast, this article presents evidence for a microbe-driven mechanism of heterosis, whereby the activity of live soil microbes affects the expression of heterosis. This finding will open lines of research that could advance our understanding of heterosis. Hybrids account for nearly all commercially planted varieties of maize and many other crop plants because crosses between inbred lines of these species produce first-generation [F1] offspring that greatly outperform their parents. The mechanisms underlying this phenomenon, called heterosis or hybrid vigor, are not well understood despite over a century of intensive research. The leading hypotheses—which focus on quantitative genetic mechanisms (dominance, overdominance, and epistasis) and molecular mechanisms (gene dosage and transcriptional regulation)—have been able to explain some but not all of the observed patterns of heterosis. Abiotic stressors are known to impact the expression of heterosis; however, the potential role of microbes in heterosis has largely been ignored. Here, we show that heterosis of root biomass and other traits in maize is strongly dependent on the belowground microbial environment. We found that, in some cases, inbred lines perform as well by these criteria as their F1 offspring under sterile conditions but that heterosis can be restored by inoculation with a simple community of seven bacterial strains. We observed the same pattern for seedlings inoculated with autoclaved versus live soil slurries in a growth chamber and for plants grown in steamed or fumigated versus untreated soil in the field. In a different field site, however, soil steaming increased rather than decreased heterosis, indicating that the direction of the effect depends on community composition, environment, or both. Together, our results demonstrate an ecological phenomenon whereby soil microbes differentially impact the early growth of inbred and hybrid maize.}, publisher={Cold Spring Harbor Laboratory}, author={Wagner, Maggie R. and Tang, Clara and Salvato, Fernanda and Clouse, Kayla M. and Bartlett, Alexandria and Sermons, Shannon and Hoffmann, Mark and Balint-Kurti, Peter J. and Kleiner, Manuel}, year={2020}, month={May} }
 @article{kleiner_bushnell_sanderson_hooper_duerkop_2020, title={Microbial DNA on the move: sequencing based detection and analysis of transduced DNA in pure cultures and microbial communities}, volume={1}, url={https://doi.org/10.1101/2020.01.15.908442}, DOI={10.1101/2020.01.15.908442}, abstractNote={Horizontal gene transfer (HGT) plays a central role in microbial evolution. Our understanding of the mechanisms, frequency and taxonomic range of HGT in polymicrobial environments is limited, as we currently rely on historical HGT events inferred from genome sequencing and studies involving cultured microorganisms. We lack approaches to observe ongoing HGT in microbial communities. To address this knowledge gap, we developed a DNA sequencing based “transductomics” approach that detects and characterizes microbial DNA transferred via transduction. We validated our approach using model systems representing a range of transduction modes and show that we can detect numerous classes of transducing DNA. Additionally, we show that we can use this methodology to obtain insights into DNA transduction among all major taxonomic groups of the intestinal microbiome. This work extends the genomic toolkit for the broader study of mobile DNA within microbial communities and could be used to understand how phenotypes spread within microbiomes. Significance Statement Microbes can rapidly evolve new capabilities by acquiring genes from other organisms through a process called horizontal gene transfer (HGT). HGT occurs via different routes, one of which is by the transfer of DNA carried by microbe infecting viruses (phages) or virus-like agents. This process is called transduction and has primarily been studied in the lab using pure cultures or indirectly in environmental communities by analyzing signatures in microbial genomes revealing past transduction events. The transductomics approach that we present here, allows for the detection and characterization of genes that are potentially transferred between microbes in complex microbial communities at the time of measurement and thus provides insights into real-time ongoing horizontal gene transfer.}, publisher={Cold Spring Harbor Laboratory}, author={Kleiner, Manuel and Bushnell, Brian and Sanderson, Kenneth E. and Hooper, Lora V. and Duerkop, Breck A.}, year={2020}, month={Jan} }
 @article{kleiner_bushnell_sanderson_hooper_duerkop_2020, title={Transductomics: sequencing-based detection and analysis of transduced DNA in pure cultures and microbial communities}, volume={8}, ISSN={["2049-2618"]}, url={https://europepmc.org/articles/PMC7667829}, DOI={10.1186/s40168-020-00935-5}, abstractNote={Abstract Background Horizontal gene transfer (HGT) plays a central role in microbial evolution. Our understanding of the mechanisms, frequency, and taxonomic range of HGT in polymicrobial environments is limited, as we currently rely on historical HGT events inferred from genome sequencing and studies involving cultured microorganisms. We lack approaches to observe ongoing HGT in microbial communities. Results To address this knowledge gap, we developed a DNA sequencing-based “transductomics” approach that detects and characterizes microbial DNA transferred via transduction. We validated our approach using model systems representing a range of transduction modes and show that we can detect numerous classes of transducing DNA. Additionally, we show that we can use this methodology to obtain insights into DNA transduction among all major taxonomic groups of the intestinal microbiome. Conclusions The transductomics approach that we present here allows for the detection and characterization of genes that are potentially transferred between microbes in complex microbial communities at the time of measurement and thus provides insights into real-time ongoing horizontal gene transfer. This work extends the genomic toolkit for the broader study of mobile DNA within microbial communities and could be used to understand how phenotypes spread within microbiomes.}, number={1}, journal={MICROBIOME}, publisher={Springer Science and Business Media LLC}, author={Kleiner, Manuel and Bushnell, Brian and Sanderson, Kenneth E. and Hooper, Lora V. and Duerkop, Breck A.}, year={2020}, month={Nov} }
 @article{zorz_sharp_kleiner_gordon_pon_dong_strous_2019, title={A shared core microbiome in soda lakes separated by large distances}, volume={10}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-019-12195-5}, abstractNote={In alkaline soda lakes, concentrated dissolved carbonates establish productive phototrophic microbial mats. Here we show how microbial phototrophs and autotrophs contribute to this exceptional productivity. Amplicon and shotgun DNA sequencing data of microbial mats from four Canadian soda lakes indicate the presence of > 2,000 species of Bacteria and Eukaryotes. We recover metagenome-assembled-genomes for a core microbiome of < 100 abundant bacteria, present in all four lakes. Most of these are related to microbes previously detected in sediments of Asian alkaline lakes, showing that common selection principles drive community assembly from a globally distributed reservoir of alkaliphile biodiversity. Detection of > 7,000 proteins show how phototrophic populations allocate resources to specific processes and occupy complementary niches. Carbon fixation proceeds by the Calvin-Benson-Bassham cycle, in Cyanobacteria, Gammaproteobacteria, and, surprisingly, Gemmatimonadetes. Our study provides insight into soda lake ecology, as well as a template to guide efforts to engineer biotechnology for carbon dioxide conversion.}, journal={NATURE COMMUNICATIONS}, author={Zorz, Jackie K. and Sharp, Christine and Kleiner, Manuel and Gordon, Paul M. K. and Pon, Richard T. and Dong, Xiaoli and Strous, Marc}, year={2019}, month={Sep} }
 @article{jaeckle_seah_tietjen_leisch_liebeke_kleiner_berg_gruber-vodicka_2019, title={Chemosynthetic symbiont with a drastically reduced genome serves as primary energy storage in the marine flatworm Paracatenula}, volume={116}, ISSN={["1091-6490"]}, url={https://doi.org/10.1073/pnas.1818995116}, DOI={10.1073/pnas.1818995116}, abstractNote={Significance Animals typically store their primary energy reserves in specialized cells. Here, we show that in the small marine flatworm Paracatenula, this function is performed by its bacterial chemosynthetic symbiont. The intracellular symbiont occupies half of the biomass in the symbiosis and has a highly reduced genome but efficiently stocks up and maintains carbon and energy, particularly sugars. The host rarely digests the symbiont cells to access these stocks. Instead, the symbionts appear to provide the bulk nutrition by secreting outer-membrane vesicles. This is in contrast to all other described chemosynthetic symbioses, where the hosts continuously digest full cells of a small and ideally growing symbiont population that cannot provide a long-term buffering capacity during nutrient limitation. Hosts of chemoautotrophic bacteria typically have much higher biomass than their symbionts and consume symbiont cells for nutrition. In contrast to this, chemoautotrophic Candidatus Riegeria symbionts in mouthless Paracatenula flatworms comprise up to half of the biomass of the consortium. Each species of Paracatenula harbors a specific Ca. Riegeria, and the endosymbionts have been vertically transmitted for at least 500 million years. Such prolonged strict vertical transmission leads to streamlining of symbiont genomes, and the retained physiological capacities reveal the functions the symbionts provide to their hosts. Here, we studied a species of Paracatenula from Sant’Andrea, Elba, Italy, using genomics, gene expression, imaging analyses, as well as targeted and untargeted MS. We show that its symbiont, Ca. R. santandreae has a drastically smaller genome (1.34 Mb) than the symbiont´s free-living relatives (4.29–4.97 Mb) but retains a versatile and energy-efficient metabolism. It encodes and expresses a complete intermediary carbon metabolism and enhanced carbon fixation through anaplerosis and accumulates massive intracellular inclusions such as sulfur, polyhydroxyalkanoates, and carbohydrates. Compared with symbiotic and free-living chemoautotrophs, Ca. R. santandreae’s versatility in energy storage is unparalleled in chemoautotrophs with such compact genomes. Transmission EM as well as host and symbiont expression data suggest that Ca. R. santandreae largely provisions its host via outer-membrane vesicle secretion. With its high share of biomass in the symbiosis and large standing stocks of carbon and energy reserves, it has a unique role for bacterial symbionts—serving as the primary energy storage for its animal host.}, number={17}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Jaeckle, Oliver and Seah, Brandon K. B. and Tietjen, Malin and Leisch, Nikolaus and Liebeke, Manuel and Kleiner, Manuel and Berg, Jasmine S. and Gruber-Vodicka, Harald R.}, year={2019}, month={Apr}, pages={8505–8514} }
 @article{rubin-blum_dubilier_kleiner_2019, title={Genetic evidence for two carbon fixation pathways (the Calvin-Benson-Bassham Cycle and the Reverse Tricarboxylic Acid Cycle) in symbiotic and free-living bacteria}, volume={4}, ISSN={["2379-5042"]}, url={https://doi.org/10.1128/mSphere.00394-18}, DOI={10.1128/msphere.00394-18}, abstractNote={Primary production on Earth is dependent on autotrophic carbon fixation, which leads to the incorporation of carbon dioxide into biomass. Multiple metabolic pathways have been described for autotrophic carbon fixation, but most autotrophic organisms were assumed to have the genes for only one of these pathways. Our finding of a cultivable bacterium with two carbon fixation pathways in its genome, the rTCA and the CBB cycle, opens the possibility to study the potential benefits of having these two pathways and the interplay between them. Additionally, this will allow the investigation of the unusual and potentially very efficient mechanism of electron flow that could drive the rTCA cycle in these autotrophs. Such studies will deepen our understanding of carbon fixation pathways and could provide new avenues for optimizing carbon fixation in biotechnological applications. ABSTRACT Very few bacteria are able to fix carbon via both the reverse tricarboxylic acid (rTCA) and the Calvin-Benson-Bassham (CBB) cycles, such as symbiotic, sulfur-oxidizing bacteria that are the sole carbon source for the marine tubeworm Riftia pachyptila, the fastest-growing invertebrate. To date, the coexistence of these two carbon fixation pathways had not been found in a cultured bacterium and could thus not be studied in detail. Moreover, it was not clear if these two pathways were encoded in the same symbiont individual, or if two symbiont populations, each with one of the pathways, coexisted within tubeworms. With comparative genomics, we show that Thioflavicoccus mobilis, a cultured, free-living gammaproteobacterial sulfur oxidizer, possesses the genes for both carbon fixation pathways. Here, we also show that both the CBB and rTCA pathways are likely encoded in the genome of the sulfur-oxidizing symbiont of the tubeworm Escarpia laminata from deep-sea asphalt volcanoes in the Gulf of Mexico. Finally, we provide genomic and transcriptomic data suggesting a potential electron flow toward the rTCA cycle carboxylase 2-oxoglutarate:ferredoxin oxidoreductase, via a rare variant of NADH dehydrogenase/heterodisulfide reductase in the E. laminata symbiont. This electron-bifurcating complex, together with NAD(P)+ transhydrogenase and Na+ translocating Rnf membrane complexes, may improve the efficiency of the rTCA cycle in both the symbiotic and the free-living sulfur oxidizer. IMPORTANCE Primary production on Earth is dependent on autotrophic carbon fixation, which leads to the incorporation of carbon dioxide into biomass. Multiple metabolic pathways have been described for autotrophic carbon fixation, but most autotrophic organisms were assumed to have the genes for only one of these pathways. Our finding of a cultivable bacterium with two carbon fixation pathways in its genome, the rTCA and the CBB cycle, opens the possibility to study the potential benefits of having these two pathways and the interplay between them. Additionally, this will allow the investigation of the unusual and potentially very efficient mechanism of electron flow that could drive the rTCA cycle in these autotrophs. Such studies will deepen our understanding of carbon fixation pathways and could provide new avenues for optimizing carbon fixation in biotechnological applications.}, number={1}, journal={mSphere}, publisher={American Society for Microbiology}, author={Rubin-Blum, M. and Dubilier, N. and Kleiner, M.}, editor={Hallam, Steven J.Editor}, year={2019}, pages={e00394–18} }
 @article{hinzke_kleiner_breusing_felbeck_häsler_sievert_schlüter_rosenstiel_reusch_schweder_et al._2019, title={Host-Microbe Interactions in the Chemosynthetic Riftia pachyptila Symbiosis.}, volume={10}, url={https://europepmc.org/articles/PMC6918071}, DOI={10.1128/mBio.02243-19}, abstractNote={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. ABSTRACT 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.}, number={6}, journal={mBio}, publisher={American Society for Microbiology}, author={Hinzke, Tjorven and Kleiner, Manuel and Breusing, Corinna and Felbeck, Horst and Häsler, Robert and Sievert, Stefan M. and Schlüter, Rabea and Rosenstiel, Philip and Reusch, Thorsten B. H. and Schweder, Thomas and et al.}, editor={Distel, Daniel and Ruby, Edward G.Editors}, year={2019}, month={Dec} }
 @article{hinzke_kleiner_breusing_felbeck_häsler_sievert_schlüter_rosenstiel_reusch_schweder_et al._2019, title={Host-microbe interactions in the chemosyntheticRiftia pachyptilasymbiosis}, volume={5}, url={https://doi.org/10.1101/651323}, DOI={10.1101/651323}, abstractNote={Abstract 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 enabled comparative metaproteomic analyses of symbiont-containing versus symbiont-free tissues, both under energy-rich and energy-limited conditions. We demonstrate that metabolic interactions include nutrient allocation from symbiont to host by symbiont digestion, and substrate transfer to the symbiont by abundant host proteins. Our analysis further suggests 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.}, publisher={Cold Spring Harbor Laboratory}, author={Hinzke, Tjorven and Kleiner, Manuel and Breusing, Corinna and Felbeck, Horst and Häsler, Robert and Sievert, Stefan M. and Schlüter, Rabea and Rosenstiel, Philip and Reusch, Thorsten B. H. and Schweder, Thomas and et al.}, year={2019}, month={May} }
 @article{kleiner_2019, title={Metaproteomics: Much More than Measuring Gene Expression in Microbial Communities}, volume={4}, ISSN={["2379-5077"]}, url={https://doi.org/10.1128/mSystems.00115-19}, DOI={10.1128/mSystems.00115-19}, abstractNote={Metaproteomics is the large-scale identification and quantification of proteins from microbial communities and thus provides direct insight into the phenotypes of microorganisms on the molecular level. Initially, metaproteomics was mainly used to assess the “expressed” metabolism and physiology of microbial community members. ABSTRACT Metaproteomics is the large-scale identification and quantification of proteins from microbial communities and thus provides direct insight into the phenotypes of microorganisms on the molecular level. Initially, metaproteomics was mainly used to assess the “expressed” metabolism and physiology of microbial community members. However, recently developed metaproteomic tools allow quantification of per-species biomass to determine community structure, in situ carbon sources of community members, and the uptake of labeled substrates by community members. In this perspective, I provide a brief overview of the questions that we can currently address, as well as new metaproteomics-based approaches that we and others are developing to address even more questions in the study of microbial communities and plant and animal microbiota. I also highlight some areas and technologies where I anticipate developments and potentially major breakthroughs in the next 5 years and beyond.}, number={3}, journal={MSYSTEMS}, publisher={American Society for Microbiology}, author={Kleiner, Manuel}, year={2019} }
 @article{hinzke_kouris_hughes_strous_kleiner_2019, title={More Is Not Always Better: Evaluation of 1D and 2D-LC-MS/MS Methods for Metaproteomics}, volume={10}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2019.00238}, abstractNote={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.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Hinzke, Tjorven and Kouris, Angela and Hughes, Rebecca-Ayme and Strous, Marc and Kleiner, Manuel}, year={2019}, month={Feb} }
 @article{seah_antony_huettel_zarzycki_borzyskowski_erb_kouris_kleiner_liebeke_dubilier_et al._2019, title={Sulfur-Oxidizing Symbionts without Canonical Genes for Autotrophic CO2 Fixation}, volume={10}, ISSN={["2150-7511"]}, url={https://doi.org/10.1128/mBio.01112-19}, DOI={10.1128/mBio.01112-19}, abstractNote={Many animals and protists depend on symbiotic sulfur-oxidizing bacteria as their main food source. These bacteria use energy from oxidizing inorganic sulfur compounds to make biomass autotrophically from CO2, serving as primary producers for their hosts. Here we describe a clade of nonautotrophic sulfur-oxidizing symbionts, “Candidatus Kentron,” associated with marine ciliates. They lack genes for known autotrophic pathways and have a carbon stable isotope fingerprint heavier than other symbionts from similar habitats. Instead, they have the potential to oxidize sulfur to fuel the uptake of organic compounds for heterotrophic growth, a metabolic mode called chemolithoheterotrophy that is not found in other symbioses. Although several symbionts have heterotrophic features to supplement primary production, in Kentron they appear to supplant it entirely. ABSTRACT Since the discovery of symbioses between sulfur-oxidizing (thiotrophic) bacteria and invertebrates at hydrothermal vents over 40 years ago, it has been assumed that autotrophic fixation of CO2 by the symbionts drives these nutritional associations. In this study, we investigated “Candidatus Kentron,” the clade of symbionts hosted by Kentrophoros, a diverse genus of ciliates which are found in marine coastal sediments around the world. Despite being the main food source for their hosts, Kentron bacteria lack the key canonical genes for any of the known pathways for autotrophic carbon fixation and have a carbon stable isotope fingerprint that is unlike other thiotrophic symbionts from similar habitats. Our genomic and transcriptomic analyses instead found metabolic features consistent with growth on organic carbon, especially organic and amino acids, for which they have abundant uptake transporters. All known thiotrophic symbionts have converged on using reduced sulfur to gain energy lithotrophically, but they are diverse in their carbon sources. Some clades are obligate autotrophs, while many are mixotrophs that can supplement autotrophic carbon fixation with heterotrophic capabilities similar to those in Kentron. Here we show that Kentron bacteria are the only thiotrophic symbionts that appear to be entirely heterotrophic, unlike all other thiotrophic symbionts studied to date, which possess either the Calvin-Benson-Bassham or the reverse tricarboxylic acid cycle for autotrophy. IMPORTANCE Many animals and protists depend on symbiotic sulfur-oxidizing bacteria as their main food source. These bacteria use energy from oxidizing inorganic sulfur compounds to make biomass autotrophically from CO2, serving as primary producers for their hosts. Here we describe a clade of nonautotrophic sulfur-oxidizing symbionts, “Candidatus Kentron,” associated with marine ciliates. They lack genes for known autotrophic pathways and have a carbon stable isotope fingerprint heavier than other symbionts from similar habitats. Instead, they have the potential to oxidize sulfur to fuel the uptake of organic compounds for heterotrophic growth, a metabolic mode called chemolithoheterotrophy that is not found in other symbioses. Although several symbionts have heterotrophic features to supplement primary production, in Kentron they appear to supplant it entirely.}, number={3}, journal={MBIO}, publisher={American Society for Microbiology}, author={Seah, Brandon K. B. and Antony, Chakkiath Paul and Huettel, Bruno and Zarzycki, Jan and Borzyskowski, Lennart Schada and Erb, Tobias J. and Kouris, Angela and Kleiner, Manuel and Liebeke, Manuel and Dubilier, Nicole and et al.}, editor={Giovannoni, Stephen J.Editor}, year={2019} }
 @article{seah_antony_huettel_zarzycki_borzyskowski_erb_kouris_kleiner_liebeke_dubilier_et al._2019, title={Sulfur-oxidizing symbionts without canonical genes for autotrophic CO2fixation}, volume={2}, url={https://doi.org/10.1101/540435}, DOI={10.1101/540435}, abstractNote={Abstract Since the discovery of symbioses between sulfur-oxidizing (thiotrophic) bacteria and invertebrates at hydrothermal vents over 40 years ago, it has been assumed that autotrophic fixation of CO 2 by the symbionts drives these nutritional associations. In this study, we investigated Candidatus Kentron, the clade of symbionts hosted by Kentrophoros , a diverse genus of ciliates which are found in marine coastal sediments around the world. Despite being the main food source for their hosts, Kentron lack the key canonical genes for any of the known pathways for autotrophic fixation, and have a carbon stable isotope fingerprint unlike other thiotrophic symbionts from similar habitats. Our genomic and transcriptomic analyses instead found metabolic features consistent with growth on organic carbon, especially organic and amino acids, for which they have abundant uptake transporters. All known thiotrophic symbionts have converged on using reduced sulfur to generate energy lithotrophically, but they are diverse in their carbon sources. Some clades are obligate autotrophs, while many are mixotrophs that can supplement autotrophic carbon fixation with heterotrophic capabilities similar to those in Kentron. We have shown that Kentron are the only thiotrophic symbionts that appear to be entirely heterotrophic, unlike all other thiotrophic symbionts studied to date, which possess either the Calvin-Benson-Bassham or reverse tricarboxylic acid cycles for autotrophy. Significance Statement Many animals and protists depend on symbiotic sulfur-oxidizing bacteria as their main food source. These bacteria use energy from oxidizing inorganic sulfur compounds to make biomass autotrophically from CO 2 , serving as primary producers for their hosts. Here we describe apparently non-autotrophic sulfur symbionts called Kentron, associated with marine ciliates. They lack genes for known autotrophic pathways, and have a carbon stable isotope fingerprint heavier than other symbionts from similar habitats. Instead they have the potential to oxidize sulfur to fuel the uptake of organic compounds for heterotrophic growth, a metabolic mode called chemolithoheterotrophy that is not found in other symbioses. Although several symbionts have heterotrophic features to supplement primary production, in Kentron they appear to supplant it entirely.}, publisher={Cold Spring Harbor Laboratory}, author={Seah, Brandon K. B. and Antony, Chakkiath Paul and Huettel, Bruno and Zarzycki, Jan and Borzyskowski, Lennart Schada and Erb, Tobias J. and Kouris, Angela and Kleiner, Manuel and Liebeke, Manuel and Dubilier, Nicole and et al.}, year={2019}, month={Feb} }
 @article{gruber-vodicka_leisch_kleiner_hinzke_liebeke_mcfall-ngai_hadfield_dubilier_2019, title={TheTrichoplaxmicrobiome: the simplest animal lives in an intimate symbiosis with two intracellular bacteria}, volume={3}, url={https://doi.org/10.1101/568287}, DOI={10.1101/568287}, abstractNote={Placozoa is an enigmatic phylum of simple, microscopic, marine metazoans. Although intracellular bacteria have been found in all members of this phylum, almost nothing is known about their identity, location and interactions with their host. We used metagenomic and metatranscriptomic sequencing of single host individuals, plus metaproteomic and imaging analyses, to show that the placozoan Trichoplax H2 lives in symbiosis with two intracellular bacteria. One symbiont forms a new genus in the Midichloriaceae (Rickettsiales) and has a genomic repertoire similar to that of rickettsial parasites, but does not appear to express key genes for energy parasitism. Correlative microscopy and 3-D electron tomography revealed that this symbiont resides in an unusual location, the rough endoplasmic reticulum of its host’s internal fiber cells. The second symbiont belongs to the Margulisbacteria, a phylum without cultured representatives and not known to form intracellular associations. This symbiont lives in the ventral epithelial cells of Trichoplax, likely metabolizes algal lipids digested by its host, and has the capacity to supplement the placozoan’s nutrition. Our study shows that even the simplest animals known have evolved highly specific and intimate associations with symbiotic, intracellular bacteria, and highlights that symbioses with microorganisms are a basal trait of animal life.}, publisher={Cold Spring Harbor Laboratory}, author={Gruber-Vodicka, Harald R. and Leisch, Nikolaus and Kleiner, Manuel and Hinzke, Tjorven and Liebeke, Manuel and McFall-Ngai, Margaret and Hadfield, Michael G. and Dubilier, Nicole}, year={2019}, month={Mar} }
 @article{gruber-vodicka_leisch_kleiner_hinzke_liebeke_mcfall-ngai_hadfield_dubilier_2019, title={Two intracellular and cell type-specific bacterial symbionts in the placozoan Trichoplax H2}, volume={4}, ISSN={["2058-5276"]}, DOI={10.1038/s41564-019-0475-9}, abstractNote={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.}, number={9}, journal={NATURE MICROBIOLOGY}, author={Gruber-Vodicka, Harald R. and Leisch, Nikolaus and Kleiner, Manuel and Hinzke, Tjorven and Liebeke, Manuel and McFall-Ngai, Margaret and Hadfield, Michael G. and Dubilier, Nicole}, year={2019}, month={Sep}, pages={1465–1474} }
 @article{kleiner_dong_hinzke_wippler_thorson_mayer_strous_2018, title={A metaproteomics method to determine carbon sources and assimilation pathways of species in microbial communities}, volume={1}, url={https://doi.org/10.1101/245290}, DOI={10.1101/245290}, abstractNote={Measurements of the carbon stable isotope ratio (δ13C) are widely used in biology to address major questions regarding food sources and metabolic pathways used by organisms. Measurement of these so called stable carbon isotope fingerprints (SIFs) for microbes involved in biogeochemical cycling and microbiota of plants and animals have led to major discoveries in environmental microbiology. Currently, obtaining SIFs for microbial communities is challenging as the available methods either only provide limited taxonomic resolution, such as with the use of lipid biomarkers, or are limited in throughput, such as NanoSIMS 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 show that SIF values can also be obtained for individual species within a microbial community. Finally, a case study of an obligate bacteria-animal symbiosis showed that direct Protein-SIF confirms previous physiological hypotheses and can provide unexpected new insights into the symbionts’ metabolism. This confirms the usefulness of this new approach to accurately determine δ13C values for different species in microbial community samples. Significance To understand the roles that microorganisms play in diverse environments such as the open ocean and the human intestinal tract, we need an understanding of their metabolism and physiology. A variety of methods such as metagenomics and metaproteomics exist to assess the metabolism of environmental microorganisms based on gene content and gene expression. These methods often only provide indirect evidence for which substrates are used by a microorganism in a community. The direct Protein-SIF method that we developed allows linking microbial species in communities to the environmental carbon sources they consume by determining their stable carbon isotope signature. Direct Protein-SIF also allows assessing which carbon fixation pathway is used by autotrophic microorganisms that directly assimilate CO2.}, publisher={Cold Spring Harbor Laboratory}, author={Kleiner, Manuel and Dong, Xiaoli and Hinzke, Tjorven and Wippler, Juliane and Thorson, Erin and Mayer, Bernhard and Strous, Marc}, year={2018}, month={Jan} }
 @article{petersen_kemper_gruber-vodicka_cardini_van der geest_kleiner_bulgheresi_mußmann_herbold_seah_et al._2018, title={Author Correction: Chemosynthetic symbionts of marine invertebrate animals are capable of nitrogen fixation}, volume={3}, ISSN={2058-5276}, url={http://dx.doi.org/10.1038/S41564-018-0196-5}, DOI={10.1038/S41564-018-0196-5}, abstractNote={In this Article, the completeness and number of contigs for draft genomes from two individuals of Laxus oneistus are incorrect in the main text, although the correct information is included in Table 1. The original and corrected versions of the relevant sentence are shown in the correction notice.}, number={8}, journal={Nature Microbiology}, publisher={Springer Science and Business Media LLC}, author={Petersen, Jillian M. and Kemper, Anna and Gruber-Vodicka, Harald and Cardini, Ulisse and van der Geest, Matthijs and Kleiner, Manuel and Bulgheresi, Silvia and Mußmann, Marc and Herbold, Craig and Seah, Brandon K. B. and et al.}, year={2018}, month={Jun}, pages={961–961} }
 @article{hinzke_kleiner_markert_2018, series={Methods in Molecular Biology}, title={Centrifugation-based enrichment of bacterial cell populations for metaproteomic studies on bacteria-invertebrate symbioses}, volume={1841}, DOI={10.1007/978-1-4939-8695-8_22}, abstractNote={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.}, journal={Springer}, author={Hinzke, T. and Kleiner, M. and Markert, S.}, editor={Becher, D.Editor}, year={2018}, pages={319–334}, collection={Methods in Molecular Biology} }
 @article{zorz_kozlowski_stein_strous_kleiner_2018, title={Comparative Proteomics of Three Species of Ammonia-Oxidizing Bacteria.}, url={http://europepmc.org/abstract/med/29867847}, DOI={10.3389/fmicb.2018.00938}, abstractNote={Ammonia-oxidizing bacteria (AOB) are important members of terrestrial, marine, and industrial microbial communities and play a fundamental role in the Nitrogen cycle within these systems. They are responsible for the first step of nitrification, ammonia oxidation to nitrite. Although AOB are widespread and essential to environmental and industrial systems, where they regularly experience fluctuations in ammonia availability, no comparative studies of the physiological response of diverse AOB species at the protein level exist. In the present study, we used 1D-LC-MS/MS proteomics to compare the metabolism and physiology of three species of ammonia AOB, Nitrosomonas europaea, Nitrosospira multiformis, and Nitrosomonas ureae, under ammonia replete and ammonia starved conditions. Additionally, we compared the expression of orthologous genes to determine the major differences in the proteome composition of the three species. We found that approximately one-third of the predicted proteome was expressed in each species and that proteins for the key metabolic processes, ammonia oxidation and carbon fixation, were among the most abundant. The red copper protein, nitrosocyanin was highly abundant in all three species hinting toward its possible role as a central metabolic enzyme in AOB. The proteomic data also allowed us to identify pyrophosphate-dependent 6-phosphofructokinase as the potential enzyme replacing the Calvin-Benson-Bassham cycle enzyme Fructose-1,6-bisphosphatase missing in N. multiformis and N. ureae. Additionally, between species, there were statistically significant differences in the expression of many abundant proteins, including those related to nitrogen metabolism (nitrite reductase), motility (flagellin), cell growth and division (FtsH), and stress response (rubrerythrin). The three species did not exhibit a starvation response at the proteome level after 24 h of ammonia starvation, however, the levels of the RuBisCO enzyme were consistently reduced after the starvation period, suggesting a decrease in capacity for biomass accumulation. This study presents the first published proteomes of N. ureae and N. multiformis, and the first comparative proteomics study of ammonia-oxidizing bacteria, which gives new insights into consistent metabolic features and differences between members of this environmentally and industrially important group.}, journal={Frontiers in microbiology}, author={Zorz, JK and Kozlowski, JA and Stein, LY and Strous, M and Kleiner, M}, year={2018} }
 @article{assié_leisch_meier_gruber-vodicka_tegetmeyer_meyerdirks_kleiner_hinzke_joye_saxton_et al._2018, title={Horizontal acquisition of a patchwork Calvin cycle by symbiotic and free-living Campylobacterota (formerly Epsilonproteobacteria)}, volume={10}, url={https://doi.org/10.1101/437616}, DOI={10.1101/437616}, abstractNote={Abstract Although the majority of known autotrophs use the Calvin-Benson-Bassham (CBB) cycle for carbon fixation, 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 lost key genes of the rTCA cycle. Intriguingly, the phylogenies of campylobacterotal CBB 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 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, additional evidence that it uses this cycle for carbon fixation. Our discovery calls into question current assumptions about the distribution of carbon fixation pathways across the tree of life, and the interpretation of stable isotope measurements in the environment.}, publisher={Cold Spring Harbor Laboratory}, author={Assié, Adrien and Leisch, Nikolaus and Meier, Dimitri V. and Gruber-Vodicka, Harald and Tegetmeyer, Halina E. and Meyerdirks, Anke and Kleiner, Manuel and Hinzke, Tjorven and Joye, Samantha and Saxton, Matthew and et al.}, year={2018}, month={Oct} }
 @article{kleiner_dong_hinzke_wippler_thorson_mayer_strous_2018, title={Metaproteomics method to determine carbon sources and assimilation pathways of species in microbial communities}, volume={115}, ISSN={["0027-8424"]}, url={http://europepmc.org/abstract/med/29844191}, DOI={10.1073/pnas.1722325115}, abstractNote={Significance To understand the roles that microorganisms play in diverse environments such as the open ocean or the human intestinal tract, we need an understanding of their metabolism and physiology. A variety of methods such as metagenomics and metaproteomics exist to assess the metabolism of environmental microorganisms based on gene content and gene expression. These methods often only provide indirect evidence for which substrates are used by a microorganism in a community. The direct protein stable isotope fingerprint (SIF) method that we developed allows linking microbial species in communities to the environmental carbon sources they consume by determining their stable carbon isotope signature. Direct protein-SIF also allows assessing which carbon fixation pathway is used by autotrophic microorganisms that directly assimilate CO2. 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.}, number={24}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Kleiner, Manuel and Dong, Xiaoli and Hinzke, Tjorven and Wippler, Juliane and Thorson, Erin and Mayer, Bernhard and Strous, Marc}, year={2018}, month={Jun}, pages={E5576–E5584} }
 @article{duerkop_kleiner_paez-espino_zhu_bushnell_hassell_winter_kyrpides_hooper_2018, title={Murine colitis reveals a disease-associated bacteriophage community}, volume={3}, ISSN={["2058-5276"]}, url={https://doi.org/10.1038/s41564-018-0210-y}, DOI={10.1038/s41564-018-0210-y}, abstractNote={The dysregulation of intestinal microbial communities is associated with inflammatory bowel diseases (IBD). Studies aimed at understanding the contribution of the microbiota to inflammatory diseases have primarily focused on bacteria, yet the intestine harbours a viral component dominated by prokaryotic viruses known as bacteriophages (phages). Phage numbers are elevated at the intestinal mucosal surface and phages increase in abundance during IBD, suggesting that phages play an unidentified role in IBD. We used a sequence-independent approach for the selection of viral contigs and then applied quantitative metagenomics to study intestinal phages in a mouse model of colitis. We discovered that during colitis the intestinal phage population is altered and transitions from an ordered state to a stochastic dysbiosis. We identified phages specific to pathobiotic hosts associated with intestinal disease, whose abundances are altered during colitis. Additionally, phage populations in healthy and diseased mice overlapped with phages from healthy humans and humans with IBD. Our findings indicate that intestinal phage communities are altered during inflammatory disease, establishing a platform for investigating phage involvement in IBD.}, number={9}, journal={NATURE MICROBIOLOGY}, author={Duerkop, Breck A. and Kleiner, Manuel and Paez-Espino, David and Zhu, Wenhan and Bushnell, Brian and Hassell, Brian and Winter, Sebastian E. and Kyrpides, Nikos C. and Hooper, Lora V}, year={2018}, month={Sep}, pages={1023–1031} }
 @article{fida_voordouw_ataeian_kleiner_okpala_mand_voordouw_2018, title={Synergy of Sodium Nitroprusside and Nitrate in Inhibiting the Activity of Sulfate Reducing Bacteria in Oil-Containing Bioreactors}, volume={9}, ISSN={["1664-302X"]}, url={http://europepmc.org/abstract/med/29867883}, DOI={10.3389/fmicb.2018.00981}, abstractNote={Sodium nitroprusside (SNP) disrupts microbial biofilms through the release of nitric oxide (NO). The actions of SNP on bacteria have been mostly limited to the genera Pseudomonas, Clostridium, and Bacillus. There are no reports of its biocidal action on sulfate-reducing bacteria (SRB), which couple the reduction of sulfate to sulfide with the oxidation of organic electron donors. Here, we report the inhibition and kill of SRB by low SNP concentrations [0.05 mM (15 ppm)] depending on biomass concentration. Chemical reaction of SNP with sulfide did not compromise its efficacy. SNP was more effective than five biocides commonly used to control SRB. Souring, the SRB activity in oil reservoirs, is often controlled by injection of nitrate. Control of SRB-mediated souring in oil-containing bioreactors was inhibited by 4 mM (340 ppm) of sodium nitrate, but required only 0.05 mM (15 ppm) of SNP. Interestingly, nitrate and SNP were found to be highly synergistic with 0.003 mM (1 ppm) of SNP and 1 mM (85 ppm) of sodium nitrate being sufficient in inhibiting souring. Hence, using SNP as an additive may greatly increase the efficacy of nitrate injection in oil reservoirs.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Fida, Tekle T. and Voordouw, Johanna and Ataeian, Maryam and Kleiner, Manuel and Okpala, Gloria and Mand, Jaspreet and Voordouw, Gerrit}, year={2018}, month={May} }
 @article{kleiner_thorson_sharp_dong_liu_li_strous_2017, title={Assessing species biomass contributions in microbial communities via metaproteomics}, volume={8}, ISSN={["2041-1723"]}, url={https://www.nature.com/articles/s41467-017-01544-x}, DOI={10.1038/s41467-017-01544-x}, abstractNote={Microbial community structure can be analyzed by quantifying cell numbers or by quantifying biomass for individual populations. Methods for quantifying cell numbers are already available (e.g., fluorescence in situ hybridization, 16-S rRNA gene amplicon sequencing), yet high-throughput methods for assessing community structure in terms of biomass are lacking. Here we present metaproteomics-based methods for assessing microbial community structure using protein abundance as a measure for biomass contributions of individual populations. We optimize the accuracy and sensitivity of the method using artificially assembled microbial communities and show that it is less prone to some of the biases found in sequencing-based methods. We apply the method to communities from two different environments, microbial mats from two alkaline soda lakes, and saliva from multiple individuals. We show that assessment of species biomass contributions adds an important dimension to the analysis of microbial community structure.}, journal={NATURE COMMUNICATIONS}, publisher={Cold Spring Harbor Laboratory}, author={Kleiner, Manuel and Thorson, Erin and Sharp, Christine E. and Dong, Xiaoli and Liu, Dan and Li, Carmen and Strous, Marc}, year={2017}, month={Nov} }
 @article{kleiner_thorson_sharp_dong_liu_li_strous_2017, title={Assessing species biomass contributions in microbial communities via metaproteomics}, volume={4}, url={https://doi.org/10.1101/130575}, DOI={10.1101/130575}, abstractNote={Abstract Assessment of microbial community composition is the cornerstone of microbial ecology. Microbial community composition can be analyzed by quantifying cell numbers or by quantifying biomass for individual populations. However, as cell volumes can differ by orders of magnitude, these two approaches yield vastly different results. Methods for quantifying cell numbers are already available (e.g. fluorescence in situ hybridization, 16S rRNA gene amplicon sequencing), yet methods for assessing community composition in terms of biomass are lacking. We developed metaproteomics based methods for assessing microbial community composition using protein abundance as a measure for biomass contributions of individual populations. We optimized the accuracy and sensitivity of the method using artificially assembled microbial communities and found that it is less prone to some of the biases found in sequencing-based methods. We applied the method using communities from two different environments, microbial mats from two alkaline soda lakes and saliva from multiple individuals.}, publisher={Cold Spring Harbor Laboratory}, author={Kleiner, Manuel and Thorson, Erin and Sharp, Christine E. and Dong, Xiaoli and Liu, Dan and Li, Carmen and Strous, Marc}, year={2017}, month={Apr} }
 @article{dong_kleiner_sharp_thorson_li_liu_strous_2017, title={Fast and Simple Analysis of MiSeq Amplicon Sequencing Data with MetaAmp}, volume={8}, DOI={10.3389/fmicb.2017.01461}, abstractNote={Microbial community profiling by barcoded 16S rRNA gene amplicon sequencing currently has many applications in microbial ecology. The low costs of the parallel sequencing of multiplexed samples, combined with the relative ease of data processing and interpretation (compared to shotgun metagenomes) have made this an entry-level approach. Here we present the MetaAmp pipeline for processing of SSU rRNA gene and other non-coding or protein-coding amplicon sequencing data by investigators that are inexperienced with bioinformatics procedures. It accepts single-end or paired-end sequences in fasta or fastq format from various sequencing platforms. It includes read quality control, and merging of forward and reverse reads of paired-end reads. It makes use of UPARSE, Mothur, and the SILVA database for clustering, removal of chimeric reads, taxonomic classification and generation of diversity metrics. The pipeline has been validated with a mock community of known composition. MetaAmp provides a convenient web interface as well as command line interface. It is freely available at: http://ebg.ucalgary.ca/metaamp. Since its launch two years ago, MetaAmp has been used >2,800 times, by many users worldwide.}, journal={Frontiers in Microbiology}, publisher={Frontiers Media SA}, author={Dong, Xiaoli and Kleiner, Manuel and Sharp, Christine E. and Thorson, Erin and Li, Carmen and Liu, Dan and Strous, Marc}, year={2017}, month={Aug} }
 @article{dong_kleiner_sharp_thorson_li_liu_strous_2017, title={Fast and simple analysis of MiSeq amplicon sequencing data with MetaAmp}, volume={4}, DOI={10.1101/131631}, abstractNote={Abstract Microbial community profiling by barcoded 16S rRNA gene amplicon sequencing currently has many applications in microbial ecology. The low costs of the parallel sequencing of multiplexed samples, combined with the relative ease of data processing and interpretation (compared to shotgun metagenomes) have made this an entry-level approach. Here we present the MetaAmp pipeline for processing of SSU rRNA gene and other non-coding or protein-coding amplicon sequencing data by investigators that are inexperienced with bioinformatics procedures. It accepts single-end or paired-end sequences in fasta or fastq format from various sequencing platforms. It includes read quality control, and merging of forward and reverse reads of paired-end reads. It makes use of UPARSE, Mothur, and the SILVA database for clustering, removal of chimeric reads, taxonomic classification and generation of diversity metrics. The pipeline has been validated with a mock community of known composition. MetaAmp provides a convenient web interface as well as command line interface. It is freely available at: http://ebg.ucalgary.ca/metaamp . Since its launch two years ago, MetaAmp has been used >2,800 times, by many users worldwide.}, publisher={Cold Spring Harbor Laboratory}, author={Dong, Xiaoli and Kleiner, Manuel and Sharp, Christine E. and Thorson, Erin and Li, Carmen and Liu, Dan and Strous, Marc}, year={2017}, month={Apr} }
 @article{genome sequence of the sulfur-oxidizing bathymodiolus thermophilus gill endosymbiont._2017, url={http://europepmc.org/abstract/med/28878861}, DOI={10.1186/s40793-017-0266-y}, abstractNote={Bathymodiolus thermophilus, a mytilid mussel inhabiting the deep-sea hydrothermal vents of the East Pacific Rise, lives in symbiosis with chemosynthetic Gammaproteobacteria within its gills. The intracellular symbiont population synthesizes nutrients for the bivalve host using the reduced sulfur compounds emanating from the vents as energy source. As the symbiont is uncultured, comprehensive and detailed insights into its metabolism and its interactions with the host can only be obtained from culture-independent approaches such as genomics and proteomics. In this study, we report the first draft genome sequence of the sulfur-oxidizing symbiont of B. thermophilus, here tentatively named Candidatus Thioglobus thermophilus. The draft genome (3.1 Mb) harbors 3045 protein-coding genes. It revealed pathways for the use of sulfide and thiosulfate as energy sources and encodes the Calvin-Benson-Bassham cycle for CO2 fixation. Enzymes required for the synthesis of the tricarboxylic acid cycle intermediates oxaloacetate and succinate were absent, suggesting that these intermediates may be substituted by metabolites from external sources. We also detected a repertoire of genes associated with cell surface adhesion, bacteriotoxicity and phage immunity, which may perform symbiosis-specific roles in the B. thermophilus symbiosis.}, journal={Standards in Genomic Sciences}, year={2017} }
 @article{ponnudurai_kleiner_sayavedra_petersen_moche_otto_becher_takeuchi_satoh_dubilier_et al._2017, title={Metabolic and physiological interdependencies in the Bathymodiolus azoricus symbiosis}, volume={11}, url={https://doi.org/10.1038/ismej.2016.124}, DOI={10.1038/ismej.2016.124}, abstractNote={The hydrothermal vent mussel Bathymodiolus azoricus lives in an intimate symbiosis with two types of chemosynthetic Gammaproteobacteria in its gills: a sulfur oxidizer and a methane oxidizer. Despite numerous investigations over the last decades, the degree of interdependence between the three symbiotic partners, their individual metabolic contributions, as well as the mechanism of carbon transfer from the symbionts to the host are poorly understood. We used a combination of proteomics and genomics to investigate the physiology and metabolism of the individual symbiotic partners. Our study revealed that key metabolic functions are most likely accomplished jointly by B. azoricus and its symbionts: (1) CO2 is pre-concentrated by the host for carbon fixation by the sulfur-oxidizing symbiont, and (2) the host replenishes essential biosynthetic TCA cycle intermediates for the sulfur-oxidizing symbiont. In return (3), the sulfur oxidizer may compensate for the host's putative deficiency in amino acid and cofactor biosynthesis. We also identified numerous 'symbiosis-specific' host proteins by comparing symbiont-containing and symbiont-free host tissues and symbiont fractions. These proteins included a large complement of host digestive enzymes in the gill that are likely involved in symbiont digestion and carbon transfer from the symbionts to the host.}, number={2}, journal={The ISME Journal}, publisher={Springer Science and Business Media LLC}, author={Ponnudurai, Ruby and Kleiner, Manuel and Sayavedra, Lizbeth and Petersen, Jillian M and Moche, Martin and Otto, Andreas and Becher, Dörte and Takeuchi, Takeshi and Satoh, Noriyuki and Dubilier, Nicole and et al.}, year={2017}, month={Feb}, pages={463–477} }
 @misc{metaproteomics of phototrophic biomats from two soda lakes in the canadian rocky mountains_2017, url={http://www.ebi.ac.uk/pride/archive/projects/PXD006343}, year={2017} }
 @article{kleiner_2017, title={Normalization of metatranscriptomic and metaproteomic data for differential gene expression analyses: The importance of accounting for organism abundance}, volume={5}, url={https://doi.org/10.7287/peerj.preprints.2846v1}, DOI={10.7287/peerj.preprints.2846v1}, abstractNote={Metatranscriptomics and metaproteomics make it possible to measure gene expression in microbial communities. So far these approaches were mostly used to get a general overview of the dominant metabolism and physiologies of community members. Recently, environmental microbiologists have started using metatranscriptomics and metaproteomics to look at gene expression differences between different environments or conditions. This has been mostly done by using makeshift adaptations of pure culture focused differential transcriptomics and proteomics approaches. However, since meta-omics data has many more variables attached to it as compared to pure culture derived data, such makeshift adaptations are problematic at best. One particular challenge is posed by the data normalization strategies used to account for technical and biological variables in meta-omic data. Here I discuss the most common normalization strategy for transcriptomic and proteomic data and why it is not valid by itself for meta-omic data. I provide logical proof that variation in species abundances between samples is an additional variable that must be accounted for during normalization of meta-omic data. Finally, I show how the existing normalization methods for transcriptomic and proteomic data can be augmented to be applicable to meta-omic data.}, journal={PeerJ Preprints}, publisher={PeerJ}, author={Kleiner, M.}, year={2017}, pages={e2846v1} }
 @article{kleiner_2017, title={Normalization of metatranscriptomic and metaproteomic data for differential gene expression analyses: The importance of accounting for organism abundance}, volume={3}, url={https://doi.org/10.7287/peerj.preprints.2846}, DOI={10.7287/peerj.preprints.2846}, abstractNote={Metatranscriptomics and metaproteomics make it possible to measure gene expression in microbial communities. So far these approaches were mostly used to get a general overview of the dominant metabolism and physiologies of community members. Recently, environmental microbiologists have started using metatranscriptomics and metaproteomics to look at gene expression differences between different environments or conditions. This has been mostly done by using makeshift adaptations of pure culture focused differential transcriptomics and proteomics approaches. However, since meta-omics data has many more variables attached to it as compared to pure culture derived data, such makeshift adaptations are problematic at best. One particular challenge is posed by the data normalization strategies used to account for technical and biological variables in meta-omic data. Here I discuss the most common normalization strategy for transcriptomic and proteomic data and why it is not valid by itself for meta-omic data. I provide logical proof that variation in species abundances between samples is an additional variable that must be accounted for during normalization of meta-omic data. Finally, I show how the existing normalization methods for transcriptomic and proteomic data can be augmented to be applicable to meta-omic data.}, publisher={PeerJ}, author={Kleiner, Manuel}, year={2017}, month={Mar} }
 @misc{quantification of mock microbial communities with metagenomes, 16s rrna gene amplicons and metaproteomics_2017, url={http://www.ebi.ac.uk/pride/archive/projects/PXD006118}, year={2017} }
 @misc{re-analysis of an ultra-deep and quantitative saliva proteome_2017, url={http://www.ebi.ac.uk/pride/archive/projects/PXD006366}, year={2017} }
 @article{rubin-blum_antony_borowski_sayavedra_pape_sahling_bohrmann_kleiner_redmond_valentine_et al._2017, title={Short-chain alkanes fuel mussels and sponge Cycloclasticus symbionts from deep-sea gas and oil seeps}, volume={2}, url={https://doi.org/10.1038/nmicrobiol.2017.93}, DOI={10.1038/nmicrobiol.2017.93}, abstractNote={Cycloclasticus bacteria are ubiquitous in oil-rich regions of the ocean and are known for their ability to degrade polycyclic aromatic hydrocarbons (PAHs). In this study, we describe Cycloclasticus that have established a symbiosis with Bathymodiolus heckerae mussels and poecilosclerid sponges from asphalt-rich, deep-sea oil seeps at Campeche Knolls in the southern Gulf of Mexico. Genomic and transcriptomic analyses revealed that, in contrast to all previously known Cycloclasticus, the symbiotic Cycloclasticus appears to lack the genes needed for PAH degradation. Instead, these symbionts use propane and other short-chain alkanes such as ethane and butane as carbon and energy sources, thus expanding the limited range of substrates known to power chemosynthetic symbioses. Analyses of short-chain alkanes in the environment of the Campeche Knolls symbioses revealed that these are present at high concentrations (in the μM to mM range). Comparative genomic analyses revealed high similarities between the genes used by the symbiotic Cycloclasticus to degrade short-chain alkanes and those of free-living Cycloclasticus that bloomed during the Deepwater Horizon oil spill. Our results indicate that the metabolic versatility of bacteria within the Cycloclasticus clade is higher than previously assumed, and highlight the expanded role of these keystone species in the degradation of marine hydrocarbons. Cycloclasticus bacterial symbionts of mussels and sponges that live in deep-sea gas and oil seeps are capable of using short-chain alkanes as their primary energy source, providing further insight into chemosynthetic symbioses.}, journal={Nature Microbiology}, publisher={Springer Nature}, author={Rubin-Blum, Maxim and Antony, Chakkiath Paul and Borowski, Christian and Sayavedra, Lizbeth and Pape, Thomas and Sahling, Heiko and Bohrmann, Gerhard and Kleiner, Manuel and Redmond, Molly C. and Valentine, David L. and et al.}, year={2017}, pages={17093} }
 @misc{bathymodiolus azoricus host-symbiont proteomics_2016, url={http://www.ebi.ac.uk/pride/archive/projects/PXD004061}, year={2016} }
 @article{petersen_kemper_gruber-vodicka_cardini_geest_kleiner_bulgheresi_mußmann_herbold_seah_et al._2016, title={Chemosynthetic sulphur-oxidizing symbionts of marine invertebrate animals are capable of nitrogen fixation}, volume={2}, url={https://doi.org/10.1038/nmicrobiol.2016.195}, DOI={10.1038/nmicrobiol.2016.195}, abstractNote={Abstract Chemosynthetic symbioses are partnerships between invertebrate animals and chemosynthetic bacteria. The latter are the primary producers, providing most of the organic carbon needed for the animal host's nutrition. We sequenced genomes of the chemosynthetic symbionts from the lucinid bivalve Loripes lucinalis and the stilbonematid nematode Laxus oneistus . The symbionts of both host species encoded nitrogen fixation genes. This is remarkable as no marine chemosynthetic symbiont was previously known to be capable of nitrogen fixation. We detected nitrogenase expression by the symbionts of lucinid clams at the transcriptomic and proteomic level. Mean stable nitrogen isotope values of Loripes lucinalis were within the range expected for fixed atmospheric nitrogen, further suggesting active nitrogen fixation by the symbionts. The ability to fix nitrogen may be widespread among chemosynthetic symbioses in oligotrophic habitats, where nitrogen availability often limits primary productivity.}, journal={Nature Microbiology}, publisher={Springer Nature}, author={Petersen, Jillian M. and Kemper, Anna and Gruber-Vodicka, Harald and Cardini, Ulisse and Geest, Matthijs and Kleiner, Manuel and Bulgheresi, Silvia and Mußmann, Marc and Herbold, Craig and Seah, Brandon K.B. and et al.}, year={2016}, pages={16195} }
 @article{zimmermann_wentrup_sadowski_blazejak_gruber-vodicka_kleiner_ott_cronholm_de_erséus_et al._2016, title={Closely coupled evolutionary history of ecto- and endosymbionts from two distantly-related animal phyla}, volume={25}, url={http://europepmc.org/abstract/med/26826340}, DOI={10.1111/mec.13554}, abstractNote={The level of integration between associated partners can range from ectosymbioses to extracellular and intracellular endosymbioses, and this range has been assumed to reflect a continuum from less intimate to evolutionarily highly stable associations. In this study, we examined the specificity and evolutionary history of marine symbioses in a group of closely related sulphur‐oxidizing bacteria, called Candidatus Thiosymbion, that have established ecto‐ and endosymbioses with two distantly related animal phyla, Nematoda and Annelida. Intriguingly, in the ectosymbiotic associations of stilbonematine nematodes, we observed a high degree of congruence between symbiont and host phylogenies, based on their ribosomal RNA (rRNA) genes. In contrast, for the endosymbioses of gutless phallodriline annelids (oligochaetes), we found only a weak congruence between symbiont and host phylogenies, based on analyses of symbiont 16S rRNA genes and six host genetic markers. The much higher degree of congruence between nematodes and their ectosymbionts compared to those of annelids and their endosymbionts was confirmed by cophylogenetic analyses. These revealed 15 significant codivergence events between stilbonematine nematodes and their ectosymbionts, but only one event between gutless phallodrilines and their endosymbionts. Phylogenetic analyses of 16S rRNA gene sequences from 50 Cand. Thiosymbion species revealed seven well‐supported clades that contained both stilbonematine ectosymbionts and phallodriline endosymbionts. This closely coupled evolutionary history of marine ecto‐ and endosymbionts suggests that switches between symbiotic lifestyles and between the two host phyla occurred multiple times during the evolution of the Cand. Thiosymbion clade, and highlights the remarkable flexibility of these symbiotic bacteria.}, number={13}, journal={Molecular Ecology}, author={Zimmermann, J and Wentrup, C and Sadowski, M and Blazejak, A and Gruber-Vodicka, H and Kleiner, M and Ott, J and Cronholm, B and De, Wit P and Erséus, C and et al.}, year={2016}, pages={3203–3223} }
 @misc{differential proteomics of lenisia limosa (breviatea) and its epibiotic arcobacter symbiont_2016, url={http://www.ebi.ac.uk/pride/archive/projects/PXD003275}, year={2016} }
 @article{hamann_gruber-vodicka_kleiner_tegetmeyer_riedel_littmann_chen_milucka_viehweger_becker_et al._2016, title={Environmental Breviatea harbor mutualistic Arcobacter epibionts}, volume={534}, url={http://europepmc.org/abstract/med/27279223}, DOI={10.1038/nature18297}, abstractNote={The cultivation of Lenisia limosa, a newly discovered breviate protist, symbiotically colonized by relatives of the animal-associated bacterium Arcobacter. This study describes a previously unknown type of mutualistic symbiosis between a newly discovered breviate protist (Lenisia limosa) and the animal-associated bacterium Arcobacter spp. Marc Strous and colleagues show that the symbiosis is driven by the transfer of hydrogen and is mutualistic, providing benefits to both partners. The symbiont induces expression of a novel NAD(P)H-accepting hydrogenase, offering a fitness gain to the breviate as the symbiont acts as a hydrogen scavenger. In turn, the breviate induces expression of proteins in the symbiont that seem to be related to classical bacterial virulence factors, acting in this instance to promote the mutualistic relationship. Breviatea form a lineage of free living, unicellular protists, distantly related to animals and fungi1,2. This lineage emerged almost one billion years ago, when the oceanic oxygen content was low, and extant Breviatea have evolved or retained an anaerobic lifestyle3,4. Here we report the cultivation of Lenisia limosa, gen. et sp. nov., a newly discovered breviate colonized by relatives of animal-associated Arcobacter. Physiological experiments show that the association of L. limosa with Arcobacter is driven by the transfer of hydrogen and is mutualistic, providing benefits to both partners. With whole-genome sequencing and differential proteomics, we show that an experimentally observed fitness gain of L. limosa could be explained by the activity of a so far unknown type of NAD(P)H-accepting hydrogenase, which is expressed in the presence, but not in the absence, of Arcobacter. Differential proteomics further reveal that the presence of Lenisia stimulates expression of known ‘virulence’ factors by Arcobacter. These proteins typically enable colonization of animal cells during infection5, but may in the present case act for mutual benefit. Finally, re-investigation of two currently available transcriptomic data sets of other Breviatea4 reveals the presence and activity of related hydrogen-consuming Arcobacter, indicating that mutualistic interaction between these two groups of microbes might be pervasive. Our results support the notion that molecular mechanisms involved in virulence can also support mutualism6, as shown here for Arcobacter and Breviatea.}, number={7606}, journal={Nature}, author={Hamann, E. and Gruber-Vodicka, H. and Kleiner, M. and Tegetmeyer, H.E. and Riedel, D. and Littmann, S. and Chen, J. and Milucka, J. and Viehweger, B. and Becker, K.W. and et al.}, year={2016}, pages={254–258} }
 @article{schimak_kleiner_wetzel_liebeke_dubilier_fuchs_2016, title={MiL-FISH: Multi-labelled oligonucleotides for fluorescence in situ hybridisation improve visualization of bacterial cells}, volume={82}, url={http://europepmc.org/abstract/med/26475101}, DOI={10.1128/AEM.02776-15}, abstractNote={ABSTRACT Fluorescence in situ hybridization (FISH) has become a vital tool for environmental and medical microbiology and is commonly used for the identification, localization, and isolation of defined microbial taxa. However, fluorescence signal strength is often a limiting factor for targeting all members in a microbial community. Here, we present the application of a multilabeled FISH approach (MiL-FISH) that (i) enables the simultaneous targeting of up to seven microbial groups using combinatorial labeling of a single oligonucleotide probe, (ii) is applicable for the isolation of unfixed environmental microorganisms via fluorescence-activated cell sorting (FACS), and (iii) improves signal and imaging quality of tissue sections in acrylic resin for precise localization of individual microbial cells. We show the ability of MiL-FISH to distinguish between seven microbial groups using a mock community of marine organisms and its applicability for the localization of bacteria associated with animal tissue and their isolation from host tissues using FACS. To further increase the number of potential target organisms, a streamlined combinatorial labeling and spectral imaging-FISH (CLASI-FISH) concept with MiL-FISH probes is presented here. Through the combination of increased probe signal, the possibility of targeting hard-to-detect taxa and isolating these from an environmental sample, the identification and precise localization of microbiota in host tissues, and the simultaneous multilabeling of up to seven microbial groups, we show here that MiL-FISH is a multifaceted alternative to standard monolabeled FISH that can be used for a wide range of biological and medical applications.}, number={1}, journal={Applied and Environmental Microbiology}, author={Schimak, M.P. and Kleiner, M. and Wetzel, S. and Liebeke, M. and Dubilier, N. and Fuchs, B.}, year={2016}, pages={62–70} }
 @misc{proteomics of candidatus thiodiazotropha endoloripes, the chemosynthetic symbiont of the lucinid clam loripes lucinalis from the bay of fetovaia_2016, url={http://www.ebi.ac.uk/pride/archive/projects/PXD004536}, year={2016} }
 @misc{proteomics of cycloclasticus endosymbiont of deep-sea mussel bathymodiolus heckerae from campeche knolls, southern gulf of mexico_2016, url={http://www.ebi.ac.uk/pride/archive/projects/PXD005351}, year={2016} }
 @article{yu_kleiner_velicer_2016, title={Spontaneous Reversions of an Evolutionary Trait Loss Reveal Regulators of a Small RNA That Controls Multicellular Development in Myxobacteria}, volume={198}, DOI={10.1128/jb.00389-16}, abstractNote={ABSTRACT Lost traits can reevolve, but the probability of trait reversion depends partly on a trait's genetic complexity. Myxobacterial fruiting body development is a complex trait controlled by the small RNA (sRNA) Pxr, which blocks development under conditions of nutrient abundance. In developmentally proficient strains of Myxococcus xanthus, starvation relaxes the inhibition by Pxr, thereby allowing development to proceed. In contrast, the lab-evolved strain OC does not develop because it fails to relay an early starvation signal that alleviates inhibition by Pxr. A descendant of OC, strain PX, previously reevolved developmental proficiency via a mutation in pxr that inactivates its function. A single-colony screen was used to test whether reversion of OC to developmental proficiency occurs only by mutation of pxr or might also occur through alternative regulatory loci. Five spontaneous mutants of OC that exhibited restored development were isolated, and all five showed defects in Pxr synthesis, structure, or processing, including one that incurred an eight-nucleotide deletion in pxr. Two mutations occurred in the σ54 response regulator (RR) gene MXAN_1078 (named pxrR here), immediately upstream of pxr. PxrR was found to positively regulate pxr transcription, presumably via the σ54 promoter of pxr. Two other mutations were identified in a histidine kinase (HK) gene (MXAN_1077; named pxrK here) immediately upstream of pxrR. Evolutionarily, the rate of trait restoration documented in this study suggests that reversion of social defects in natural microbial populations may be common. Molecularly, these results suggest a mechanism by which the regulatory functions of an HK-RR two-component signaling system and an sRNA are integrated to control initiation of myxobacterial development. IMPORTANCE Many myxobacteria initiate a process of multicellular fruiting body development upon starvation, but key features of the regulatory network controlling the transition from growth to development remain obscure. Previous work with Myxococcus xanthus identified the first small RNA (sRNA) regulator (Pxr) known to serve as a gatekeeper in this life history transition, as it blocks development when nutrients are abundant. In the present study, a screen for spontaneous mutants of M. xanthus was developed that revealed a two-component system operon (encoding a histidine kinase and a σ54 response regulator) associated with the production and processing of Pxr sRNA. This discovery broadens our knowledge of early developmental gene regulation and also represents an evolutionary integration of two-component signaling and sRNA gene regulation to control a bacterial social trait.}, number={23}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Yu, Yuen-Tsu N. and Kleiner, Manuel and Velicer, Gregory J.}, editor={Mullineaux, C. W.Editor}, year={2016}, month={Sep}, pages={3142–3151} }
 @article{wippler_kleiner_lott_gruhl_abraham_giannone_young_hettich_dubilier_2016, title={Transcriptomic and proteomic insights into innate immunity and adaptations to a symbiotic lifestyle in the gutless marine worm Olavius algarvensis}, volume={17}, DOI={10.1186/s12864-016-3293-y}, abstractNote={The gutless marine worm Olavius algarvensis has a completely reduced digestive and excretory system, and lives in an obligate nutritional symbiosis with bacterial symbionts. While considerable knowledge has been gained of the symbionts, the host has remained largely unstudied. Here, we generated transcriptomes and proteomes of O. algarvensis to better understand how this annelid worm gains nutrition from its symbionts, how it adapted physiologically to a symbiotic lifestyle, and how its innate immune system recognizes and responds to its symbiotic microbiota.Key adaptations to the symbiosis include (i) the expression of gut-specific digestive enzymes despite the absence of a gut, most likely for the digestion of symbionts in the host's epidermal cells; (ii) a modified hemoglobin that may bind hydrogen sulfide produced by two of the worm's symbionts; and (iii) the expression of a very abundant protein for oxygen storage, hemerythrin, that could provide oxygen to the symbionts and the host under anoxic conditions. Additionally, we identified a large repertoire of proteins involved in interactions between the worm's innate immune system and its symbiotic microbiota, such as peptidoglycan recognition proteins, lectins, fibrinogen-related proteins, Toll and scavenger receptors, and antimicrobial proteins.We show how this worm, over the course of evolutionary time, has modified widely-used proteins and changed their expression patterns in adaptation to its symbiotic lifestyle and describe expressed components of the innate immune system in a marine oligochaete. Our results provide further support for the recent realization that animals have evolved within the context of their associations with microbes and that their adaptive responses to symbiotic microbiota have led to biological innovations.}, number={1}, journal={BMC Genomics}, publisher={Springer Nature}, author={Wippler, Juliane and Kleiner, Manuel and Lott, Christian and Gruhl, Alexander and Abraham, Paul E. and Giannone, Richard J. and Young, Jacque C. and Hettich, Robert L. and Dubilier, Nicole}, year={2016}, month={Nov} }
 @misc{transcriptomic and proteomic insights into innate immunity and adaptations to a symbiotic lifestyle in the gutless marine worm olavius algarvensis_2016, url={https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=77bbe652de0b4439b9a9e89016f99c93}, year={2016} }
 @article{sayavedra_kleiner_ponnudurai_wetzel_pelletier_barbe_satoh_shoguchi_fink_breusing_et al._2015, title={Abundant toxin-related genes in the genomes of beneficial symbionts from deep-sea hydrothermal vent mussels}, volume={4}, url={http://europepmc.org/abstract/med/26371554}, DOI={10.7554/eLife.07966}, abstractNote={Bathymodiolus mussels live in symbiosis with intracellular sulfur-oxidizing (SOX) bacteria that provide them with nutrition. We sequenced the SOX symbiont genomes from two Bathymodiolus species. Comparison of these symbiont genomes with those of their closest relatives revealed that the symbionts have undergone genome rearrangements, and up to 35% of their genes may have been acquired by horizontal gene transfer. Many of the genes specific to the symbionts were homologs of virulence genes. We discovered an abundant and diverse array of genes similar to insecticidal toxins of nematode and aphid symbionts, and toxins of pathogens such as Yersinia and Vibrio. Transcriptomics and proteomics revealed that the SOX symbionts express the toxin-related genes (TRGs) in their hosts. We hypothesize that the symbionts use these TRGs in beneficial interactions with their host, including protection against parasites. This would explain why a mutualistic symbiont would contain such a remarkable ‘arsenal’ of TRGs. DOI: http://dx.doi.org/10.7554/eLife.07966.001}, journal={eLife}, author={Sayavedra, L. and Kleiner, M. and Ponnudurai, R. and Wetzel, S. and Pelletier, E. and Barbe, V. and Satoh, N. and Shoguchi, E. and Fink, D. and Breusing, C. and et al.}, year={2015}, pages={e07966} }
 @article{kleiner_hooper_duerkop_2015, title={Evaluation of methods to purify virus-like particles for metagenomic sequencing of intestinal viromes}, volume={16}, url={http://europepmc.org/abstract/med/25608871}, DOI={10.1186/s12864-014-1207-4}, abstractNote={Viruses are a significant component of the intestinal microbiota in mammals. In recent years, advances in sequencing technologies and data analysis techniques have enabled detailed metagenomic studies investigating intestinal viromes (collections of bacteriophage and eukaryotic viral nucleic acids) and their potential contributions to the ecology of the microbiota. An important component of virome studies is the isolation and purification of virus-like particles (VLPs) from intestinal contents or feces. Several methods have been applied to isolate VLPs from intestinal samples, yet to our knowledge, the efficiency and reproducibility between methods have not been explored. A rigorous evaluation of methods for VLP purification is critical as many studies begin to move from descriptive analyses of virus diversity to studies striving to quantitatively compare viral abundances across many samples. Therefore, reproducible VLP purification methods which allow for high sample throughput are needed. Here we compared and evaluated four methods for VLP purification using artificial intestinal microbiota samples of known bacterial and viral composition.We compared the following four methods of VLP purification from fecal samples: (i) filtration + DNase, (ii) dithiothreitol treatment + filtration + DNase, (iii) filtration + DNase + PEG precipitation and (iv) filtration + DNase + CsCl density gradient centrifugation. Three of the four tested methods worked well for VLP purification. We observed several differences between methods related to the removal efficiency of bacterial and host DNAs and biases against specific phages. In particular the CsCl density gradient centrifugation method, which is frequently used for VLP purification, was most efficient in removing host derived DNA, but also showed strong discrimination against specific phages and showed a lower reproducibility of quantitative results.Based on our data we recommend the use of methods (i) or (ii) for large scale studies when quantitative comparison of viral abundances across samples is required. The CsCl density gradient centrifugation method, while being excellently suited to achieve highly purified samples, in our opinion, should be used with caution when performing quantitative studies.}, number={7}, journal={BMC Genomics}, author={Kleiner, M. and Hooper, L.V. and Duerkop, B.A.}, year={2015}, pages={7} }
 @article{kleiner_wentrup_holler_lavik_harder_lott_littmann_kuypers_dubilier_2015, title={Use of carbon monoxide and hydrogen by a bacteria-animal symbiosis from seagrass sediments}, volume={17}, url={http://europepmc.org/abstract/med/26013766}, DOI={10.1111/1462-2920.12912}, abstractNote={Summary The gutless marine worm O lavius algarvensis lives in symbiosis with chemosynthetic bacteria that provide nutrition by fixing carbon dioxide (CO 2) into biomass using reduced sulfur compounds as energy sources. A recent metaproteomic analysis of the O . algarvensis symbiosis indicated that carbon monoxide (CO) and hydrogen (H 2) might also be used as energy sources. We provide direct evidence that the O . algarvensis symbiosis consumes CO and H 2. Single cell imaging using nanoscale secondary ion mass spectrometry revealed that one of the symbionts, the γ3‐symbiont, uses the energy from CO oxidation to fix CO 2. Pore water analysis revealed considerable in‐situ concentrations of CO and H 2 in the O . algarvensis environment, Mediterranean seagrass sediments. Pore water H 2 concentrations (89–2147 nM) were up to two orders of magnitude higher than in seawater, and up to 36‐fold higher than previously known from shallow‐water marine sediments. Pore water CO concentrations (17–51 nM) were twice as high as in the overlying seawater (no literature data from other shallow‐water sediments are available for comparison). Ex‐situ incubation experiments showed that dead seagrass rhizomes produced large amounts of CO. CO production from decaying plant material could thus be a significant energy source for microbial primary production in seagrass sediments.}, number={12}, journal={Environmental Microbiology}, author={Kleiner, M. and Wentrup, C. and Holler, T. and Lavik, G. and Harder, J. and Lott, C. and Littmann, S. and Kuypers, M.M.M. and Dubilier, N.}, year={2015}, pages={5023–5035} }
 @article{winkel_pjevac_kleiner_littmann_meyerdierks_amann_mußmann_2014, title={Identification and activity of acetate-assimilating bacteria in diffuse fluids venting from deep-sea hydrothermal systems}, volume={90}, url={http://europepmc.org/abstract/med/25244359}, DOI={10.1111/1574-6941.12429}, abstractNote={Diffuse hydrothermal fluids often contain organic compounds such as hydrocarbons, lipids, and organic acids. Microorganisms consuming these compounds at hydrothermal sites are so far only known from cultivation-dependent studies. To identify potential heterotrophs without prior cultivation, we combined microbial community analysis with short-term incubations using (13)C-labeled acetate at two distinct hydrothermal systems. We followed cell growth and assimilation of (13)C into single cells by nanoSIMS combined with fluorescence in situ hybridization (FISH). In 55 °C-fluids from the Menez Gwen hydrothermal system/Mid-Atlantic Ridge, a novel epsilonproteobacterial group accounted for nearly all assimilation of acetate, representing the first aerobic acetate-consuming member of the Nautiliales. In contrast, Gammaproteobacteria dominated the (13) C-acetate assimilation in incubations of 37 °C-fluids from the back-arc hydrothermal system in the Manus Basin/Papua New Guinea. Here, 16S rRNA gene sequences were mostly related to mesophilic Marinobacter, reflecting the high content of seawater in these fluids. The rapid growth of microorganisms upon acetate addition suggests that acetate consumers in diffuse fluids are copiotrophic opportunists, which quickly exploit their energy sources, whenever available under the spatially and temporally highly fluctuating conditions. Our data provide first insights into the heterotrophic microbial community, catalyzing an under-investigated part of microbial carbon cycling at hydrothermal vents.}, number={3}, journal={FEMS Microbiology Ecology}, author={Winkel, M. and Pjevac, P. and Kleiner, M. and Littmann, S. and Meyerdierks, A. and Amann, R. and Mußmann, M.}, year={2014}, pages={731–746} }
 @article{kleiner_young_shah_verberkmoes_dubilier_2013, title={Metaproteomics Reveals Abundant Transposase Expression in Mutualistic Endosymbionts}, volume={4}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000321187400018&KeyUID=WOS:000321187400018}, DOI={10.1128/mBio.00223-13}, abstractNote={ABSTRACT Transposases, enzymes that catalyze the movement of mobile genetic elements, are the most abundant genes in nature. While many bacteria encode an abundance of transposases in their genomes, the current paradigm is that the expression of transposase genes is tightly regulated and generally low due to its severe mutagenic effects. In the current study, we detected the highest number of transposase proteins ever reported in bacteria, in symbionts of the gutless marine worm Olavius algarvensis with metaproteomics. At least 26 different transposases from 12 different families were detected, and genomic and proteomic analyses suggest that many of these are active. This high expression of transposases indicates that the mechanisms for their tight regulation have been disabled or no longer exist. IMPORTANCE The expansion of transposable elements (TE) within the genomes of host-restricted symbionts and pathogens plays an important role in their emergence and evolution and might be a key mechanism for adaptation to the host environment. However, little is known so far about the underlying causes and evolutionary mechanisms of this TE expansion. The current model of genome evolution in host-restricted bacteria explains TE expansion within the confines of the paradigm that transposase expression is always low. However, recent work failed to verify this model. Based on our data, we hypothesize that increased transposase expression, which has not previously been described, may play a role in TE expansion, and could be one explanation for the sometimes very rapid emergence and evolution of new obligate symbionts and pathogens from facultative ones. The expansion of transposable elements (TE) within the genomes of host-restricted symbionts and pathogens plays an important role in their emergence and evolution and might be a key mechanism for adaptation to the host environment. However, little is known so far about the underlying causes and evolutionary mechanisms of this TE expansion. The current model of genome evolution in host-restricted bacteria explains TE expansion within the confines of the paradigm that transposase expression is always low. However, recent work failed to verify this model. Based on our data, we hypothesize that increased transposase expression, which has not previously been described, may play a role in TE expansion, and could be one explanation for the sometimes very rapid emergence and evolution of new obligate symbionts and pathogens from facultative ones.}, number={3}, journal={Mbio}, author={Kleiner, Manuel and Young, Jacque C. and Shah, Manesh and VerBerkmoes, Nathan C. and Dubilier, Nicole}, year={2013} }
 @article{kleiner_petersen_dubilier_2012, title={Convergent and divergent evolution of metabolism in sulfur-oxidizing symbionts and the role of horizontal gene transfer}, volume={15}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000311181000012&KeyUID=WOS:000311181000012}, DOI={10.1016/j.mlb.2012.09.003}, number={5}, journal={Current Opinion in Microbiology}, author={Kleiner, Manuel and Petersen, Jillian M. and Dubilier, Nicole}, year={2012}, pages={621–631} }
 @article{kleiner_petersen_dubilier_2012, title={Convergent and divergent evolution of metabolism in sulfur-oxidizing symbionts and the role of horizontal gene transfer}, volume={15}, ISSN={1369-5274}, url={http://dx.doi.org/10.1016/j.mib.2012.09.003}, DOI={10.1016/j.mib.2012.09.003}, abstractNote={Symbioses between marine invertebrates and autotrophic sulfur-oxidizing bacteria have evolved from multiple lineages within the Gammaproteobacteria in a striking example of convergent evolution. These GammaSOX symbionts all perform the same basic function: they provide their hosts with nutrition through the fixation of CO2 into biomass using reduced sulfur compounds as an energy source. However, our review of recent –omics based studies and genome mining for this study revealed that the GammaSOX symbionts diverge in many other metabolic capabilities and functions, and we show how these divergences could reflect adaptations to different hosts and habitat conditions. Our phylogenetic analyses of key metabolic genes in GammaSOX symbionts revealed that these differed markedly from 16S rRNA phylogenies. We hypothesize that horizontal gene transfer (HGT) would explain many of these incongruencies, and conclude that HGT may have played a significant role in shaping the metabolic evolution of GammaSOX symbionts.}, number={5}, journal={Current Opinion in Microbiology}, publisher={Elsevier BV}, author={Kleiner, Manuel and Petersen, Jillian M and Dubilier, Nicole}, year={2012}, month={Oct}, pages={621–631} }
 @article{kleiner_wentrup_lott_teeling_wetzel_young_chang_shah_verberkmoes_zarzycki_et al._2012, title={Metaproteomics of a gutless marine worm and its symbiotic microbial community reveal unusual pathways for carbon and energy use}, volume={109}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000304090600007&KeyUID=WOS:000304090600007}, DOI={10.1073/pnas.1121198109}, abstractNote={Low nutrient and energy availability has led to the evolution of numerous strategies for overcoming these limitations, of which symbiotic associations represent a key mechanism. Particularly striking are the associations between chemosynthetic bacteria and marine animals that thrive in nutrient-poor environments such as the deep sea because the symbionts allow their hosts to grow on inorganic energy and carbon sources such as sulfide and CO2. Remarkably little is known about the physiological strategies that enable chemosynthetic symbioses to colonize oligotrophic environments. In this study, we used metaproteomics and metabolomics to investigate the intricate network of metabolic interactions in the chemosynthetic association between Olavius algarvensis, a gutless marine worm, and its bacterial symbionts. We propose previously undescribed pathways for coping with energy and nutrient limitation, some of which may be widespread in both free-living and symbiotic bacteria. These pathways include (i) a pathway for symbiont assimilation of the host waste products acetate, propionate, succinate and malate; (ii) the potential use of carbon monoxide as an energy source, a substrate previously not known to play a role in marine invertebrate symbioses; (iii) the potential use of hydrogen as an energy source; (iv) the strong expression of high-affinity uptake transporters; and (v) as yet undescribed energy-efficient steps in CO2 fixation and sulfate reduction. The high expression of proteins involved in pathways for energy and carbon uptake and conservation in the O. algarvensis symbiosis indicates that the oligotrophic nature of its environment exerted a strong selective pressure in shaping these associations.}, number={19}, journal={Proceedings of the National Academy of Sciences of the United States of America}, author={Kleiner, Manuel and Wentrup, Cecilia and Lott, Christian and Teeling, Hanno and Wetzel, Silke and Young, Jacque and Chang, Yun-Juan and Shah, Manesh and VerBerkmoes, Nathan C. and Zarzycki, Jan and et al.}, year={2012}, pages={E1173–E1182} }
 @article{markert_gardebrecht_felbeck_sievert_klose_becher_albrecht_thurmer_daniel_kleiner_et al._2011, title={Status quo in physiological proteomics of the uncultured Riftia pachyptila endosymbiont}, volume={11}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000293269100015&KeyUID=WOS:000293269100015}, DOI={10.1002/pmic.201100059}, abstractNote={Riftia pachyptila, the giant deep‐sea tube worm, inhabits hydrothermal vents in the Eastern Pacific ocean. The worms are nourished by a dense population of chemoautotrophic bacterial endosymbionts. Using the energy derived from sulfide oxidation, the symbionts fix CO2 and produce organic carbon, which provides the nutrition of the host. Although the endosymbionts have never been cultured, cultivation‐independent techniques based on density gradient centrifugation and the sequencing of their (meta‐) genome enabled a detailed physiological examination on the proteomic level. In this study, the Riftia symbionts' soluble proteome map was extended to a total of 493 identified proteins, which allowed for an explicit description of vital metabolic processes such as the energy‐generating sulfide oxidation pathway or the Calvin cycle, which seems to involve a reversible pyrophosphate‐dependent phosphofructokinase. Furthermore, the proteomic view supports the hypothesis that the symbiont uses nitrate as an alternative electron acceptor. Finally, the membrane‐associated proteome of the Riftia symbiont was selectively enriched and analyzed. As a result, 275 additional proteins were identified, most of which have putative functions in electron transfer, transport processes, secretion, signal transduction and other cell surface‐related functions. Integrating this information into complex pathway models a comprehensive survey of the symbiotic physiology was established.}, number={15}, journal={Proteomics}, author={Markert, S. and Gardebrecht, A. and Felbeck, H. and Sievert, S. M. and Klose, J. and Becher, D. and Albrecht, D. and Thurmer, A. and Daniel, R. and Kleiner, M. and et al.}, year={2011}, pages={3106–3117} }
 @article{kleiner_woyke_ruehland_dubilier_2011, title={The Olavius algarvensis Metagenome Revisited: Lessons Learned from the Analysis of the Low-Diversity Microbial Consortium of a Gutless Marine Worm}, DOI={10.1002/9781118010549.ch32}, abstractNote={This chapter contains sections titled: Introduction Method Discussion: Challenges of Analyzing Co-Occurring Symbionts in Very Small Hosts Major Findings of the Study in Brief The Metagenome Revisited Conclusions Internet Resources for the O. Algarvensis Metagenome References}, journal={Handbook of Molecular Microbial Ecology II}, publisher={Wiley-Blackwell}, author={Kleiner, Manuel and Woyke, Tanja and Ruehland, Caroline and Dubilier, Nicole}, year={2011}, month={Nov}, pages={319–333} }