Due to their fundamentally different biology, archaea are consistently overlooked in conventional microbiome surveys. Using amplicon sequencing, we evaluated methodological set-ups to detect archaea in samples from five different body sites: respiratory tract (nasal cavity), digestive tract (mouth, appendix, and stool) and skin. With optimized protocols, the detection of archaeal ribosomal sequence variants (RSVs) was increased from one (found in currently used, so-called “universal” approach) to 81 RSVs in a representative sample set. The results from this extensive primer-evaluation led to the identification of the primer pair combination 344f-1041R/519F-806R which performed superior for the analysis of the archaeome of gastrointestinal tract, oral cavity and skin. The proposed protocol might not only prove useful for analyzing the human archaeome in more detail but could also be used for other holobiont samples.

Forty years ago, archaea were described as a separate domain of life, distinct from bacteria and eukarya. Although it is known for quite a long time that methanogenic archaea are substantial components of the human gastrointestinal tract (GIT) and the oral cavity, the knowledge on the human archaeome is very limited. Various methodological problems contribute to the invisibility of the human archaeome, resulting in severe knowledge gaps and contradictory information. Similar to the bacteriome, the archaeal biogeography was found to be site-specific, forming (i) the thaumarchaeal skin landscape, (ii) the (methano)euryarchaeal GIT landscape, (iii) a mixed skin/GIT landscape in nose, and (iv) a woesearchaeal lung landscape, including numerous unknown archaeal clades. Compared with so-called universal microbiome approaches, archaea-specific protocols reveal a wide diversity and high quantity of archaeal signatures in various human tissues, with up to 1 : 1 ratios of bacteria and archaea in appendix and nose samples. The archaeome interacts closely with the bacteriome and the human body cells, whereas the roles of the human-associated archaea with respect to human health are only sparsely described. Methanogenic archaea and methane production were correlated with many health issues, including constipation, periodontitis and multiple sclerosis. However, one of the most burning questions — do archaeal pathogens exist? — still remains obscure to date.

15Due to their fundamentally different biology, archaea are consistently overlooked in conventional 16 microbiome surveys. Using amplicon sequencing, we evaluated methodological set-ups to detect 17 archaea in samples from five different body sites: respiratory tract (nose), digestive tract (mouth, 18 appendix, and stool) and skin. With the optimized protocols, the detection of archaeal ribosomal 19 sequence variants (RSVs) was increased from one (found in currently used, so-called "universal" 20 approach) to 81 RSVs in a representative sample set. In order to assess the archaeome diversity, a 21 specific archaea-targeting methodology is required, for which we propose a standard procedure. This 22 methodology might not only prove useful for analyzing the human archaeome in more detail but 23 could also be used for other holobionts' samples. 24 25 26 27

Background Human health is closely interconnected with its microbiome. Resilient microbiomes in, on, and around the human body will be key for safe and successful long-term space travel. However, longitudinal dynamics of microbiomes inside confined built environments are still poorly understood. Herein, we used the Hawaii Space Exploration Analog and Simulation IV (HI-SEAS IV) mission, a 1 year-long isolation study, to investigate microbial transfer between crew and habitat, in order to understand adverse developments which may occur in a future outpost on the Moon or Mars. Results Longitudinal 16S rRNA gene profiles, as well as quantitative observations, revealed significant differences in microbial diversity, abundance, and composition between samples of the built environment and its crew. The microbiome composition and diversity associated with abiotic surfaces was found to be rather stable, whereas the microbial skin profiles of individual crew members were highly dynamic, resulting in an increased microbiome diversity at the end of the isolation period. The skin microbiome dynamics were especially pronounced by a regular transfer of the indicator species Methanobrevibacter between crew members within the first 200 days. Quantitative information was used to track the propagation of antimicrobial resistance in the habitat. Together with functional and phenotypic predictions, quantitative and qualitative data supported the observation of a delayed longitudinal microbial homogenization between crew and habitat surfaces which was mainly caused by a malfunctioning sanitary facility. Conclusions This study highlights main routes of microbial transfer, interaction of the crew, and origins of microbial dynamics in an isolated environment. We identify key targets of microbial monitoring, and emphasize the need for defined baselines of microbiome diversity and abundance on surfaces and crew skin. Targeted manipulation to counteract adverse developments of the microbiome could be a highly important strategy to ensure safety during future space endeavors.

The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity.