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

Some annelids produce sitosterol, a biomarker lipid more commonly found in plants. 

Abstract BackgroundStable 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. ResultsHere, 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. ConclusionsWe 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.