From satellites to peptides: Unraveling the microbial diversity of high-altitude Andean lakes by Tandem-Mass-Proteotyping

Kurzfassung

Introduction: Out of up to one trillion microbial species, only 104 have been cultured (1), indicating that the vast majority of microbial taxa remain undiscovered. In particular, the microbial diversity of extreme habitats is poorly investigated since they are usually hard to access. They represent surrogate habitats for extraterrestrial environments and are therefore very interesting for further research in regard to possible extraterrestrial life and the boundaries within which life can exist. High-altitude Andean lakes are an excellent example of extreme habitats representing extraterrestrial analogous habitats for lakes on early Mars (2). In comparison, they display multiple similarities such as elevated UV radiation, low and highly fluctuating air temperature, aridity, strong negative water balance, hydrothermal environment and more (2). Since microorganisms isolated from these habitats may be far related from already known species, techniques for taxonomical identification and accurate classification are necessary to explore Earth's biodiversity. Tandem mass spectrometry-based proteotyping was shown as a promising method for such purpose, representing a rapid, time and cost-effective option for the identification of environmental isolates (3). Methods: In this work, we cultivated, isolated and identified 66 different organisms from five different highaltitude Andean lakes. Environmental and chemical parameters comprising salt concentration, pH, temperature and UV-radiation were recorded. Cultivation was performed using common media for aquatic systems and after stress treatments to select extremophiles. Identification was done by 16S rRNA amplicon sequencing and LC-MS/MS proteotyping. It is the first study using tandem-massspectrometry as a tool for the identification of environmental extremophilic and especially halophilic isolates. Results: Sampled lakes showed diverse environmental characteristics including salt concentrations ranging from 0.2 to 27.9% salt and neutral to alkaline pH. According to the proteotyping approach, out of 66 isolates, 22 different families spanning 9 different classes were cultivated and identified. Most abundant classes were Gammaproteobacteria (30%), Actinomycetia (21%), Bacilli (19%), and Alphaproteobacteria (16%). Most of the identified species are known to be halotolerant/halophile, alkaliphile and/or adapted to cold temperatures. Proteotyping suggested the presence of 20 potentialnew species and 2 new genera, which were overlooked by the 16S rRNA amplicon sequencing approach only. Proteotyping and 16S rRNA amplicon sequencing identified 33 isolates as the same microbial species. Both methods yielded consistent genus identification for all isolates except for two isolates that potentially represented yet-unclassified organisms based on their peptidome. We highlighted that this approach is a suitable and benefitial option for the identification of microorganisms from extreme environments and preliminary functional characterization of poorly investigated, unsequenced microorganisms. Overall, we showed that proteotyping is beneficial for analyzing microbial communities of other extreme and extraterrestrial habitats.