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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 using¹⁸O heavy water labeling in a 63-species community derived from human fecal samples grown on media simulating two different diets. Activity could be quantified on average for 27 species per sample, with 9 species showing significantly higher activity on a high protein diet, as compared to a high fiber diet. Surprisingly, among the species with increased activity on high protein were severalBacteroidesspecies known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics.

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.

 

Microbially induced carbonate precipitation (MICP) is a bio-mediated ground improvement technique that can increase soil stiffness and produce cohesion within granular material. Most experimental investigations on MICP-treated soils are performed on idealized granular materials. Evaluating a narrow range of particle sizes dismisses the potential influence of soil fabric on MICP treatment efficiency. Therefore, little is known regarding the influence of soil fabric on the level of improvement achievable post-MICP treatment. We investigate the influence of the coefficient of uniformity (Cu) on the level of improvement that can be obtained from MICP treatment. This study couples unconfined compression testing with microscale observations obtained from x-ray computed tomography (CT) of two sand mixtures with different Cu values. A soil column and CT specimen of each sand mixture were prepared and received the same number of MICP- injections. The shear wave velocity (Vs) of the soil columns was monitored to evaluate the increase in soil stiffness over time. After MICP treatment, the bio-cemented columns were subjected to unconfined compressive strength testing. Results indicate that for a similar mass of carbonate, the soil with a larger Cu experienced a greater increase in Vs but a lower maximum unconfined compressive strength. Through CT imaging, the soil with a smaller Cu was observed to have a more uniform distribution of carbonate within the sand matrix whereas the soil with a larger Cu has more sporadic MICP trends. This study elucidates the influence of soil fabric on the level of improvement that can be achieved through MICP treatment and assesses the reliability of x-ray CT scanning of MICP-treated sands with moderate carbonate content. 

Feathers are arguably the most complex integumentary structures in the entire animal kingdom. The evolutionary origins of feathers are still debated, but growing evidence from both molecular studies in extinct theropods [1–8] and living birds (e.g., [9–18]), as well as numerous fossil discoveries of structures morphologically consistent with feathers (e.g., [4,19–25]) indicate that feathers arose from filamentous structures first identifed in some theropod dinosaurs and birds more than 160 million years ago (e.g., [2,26,27]). However, some data suggest that integumentary structures similar to those from which feathers derived may have been present at the base of Dinosauria [28,29] or perhaps, the base of Archosauria ([30,31] and references therein). Because modern feathers are not biomineralized in life (contra [32,33]) their persistence in the fossil record is counterintuitive, but critical. The impressions of feathers in sediments surrounding skeletal elements led to the identification of Archaeopteryx as the first bird [34,35], but there was no organic trace with this specimen to suggest that any original material remained. However, the first specimen attributed to Archaeopteryx was a single, isolated feather [36]. This specimen presented differently from feather impressions surrounding the skeletal remains, instead visualized as a carbonized trace clearly distinct from the embedding sediments, suggesting that taphonomic processes resulting in preservation differed between the isolated feather and the skeletal specimen. The environmental factors resulting in these different modes of preservation remain relatively unexplored. 

Abstract 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. 

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