Exploring Andean High-Altitude Lake Extremophiles through Advanced Proteotyping

Abstract

Quickly identifying and characterizing isolates from extreme environments is currently challenging while very important to explore the Earth's biodiversity. As these isolates may, in principle, be distantly related to known species, techniques are needed to reliably identify the branch of life to which they belong. Proteotyping these environmental isolates by tandem mass spectrometry offers a rapid and cost-effective option for their identification using their peptide profiles. In this study, we document the first high-throughput proteotyping approach for environmental extremophilic and halophilic isolates. Microorganisms were isolated from samples originating from high-altitude Andean lakes (3700-4300 m a.s.l.) in the Chilean Altiplano, which represent environments on Earth that resemble conditions on other planets. A total of 66 microorganisms were cultivated and identified by proteotyping and 16S rRNA gene amplicon sequencing. Both the approaches revealed the same genus identification for all isolates except for three isolates possibly representing not yet taxonomically characterized organisms based on their peptidomes. Proteotyping was able to indicate the presence of two potentially new genera from the families of Paracoccaceae and Chromatiaceae/Alteromonadaceae, which have been overlooked by 16S rRNA amplicon sequencing approach only. The paper highlights that proteotyping has the potential to discover undescribed microorganisms from extreme environments.

Keywords: Altiplano; Atacama Desert; extremophiles; halophiles; high-altitude Andean lakes; tandem mass spectrometry proteotyping.

Figure 1

Sample locations in the Atacama in Chile. Remodified image from Boy et al., with the inclusion of sampling locations. Image was created with QGIS. Rain distribution is indicated by color grading and corresponds to 0 (brown) to 155 mm p.a. (green).

Figure 2

Overview of the experimental workflow. Samples were taken from five different HAAL, and microorganisms were cultivated with diverse techniques. Pure isolates were obtained and further analyzed according to their 16S rRNA sequence and peptide profile. For 16S rRNA sequencing, DNA was extracted, amplified, purified, and further sequenced and blasted against a nucleotide database provided by NCBI. For MS/MS proteotyping, isolates were cultivated in liquid matter, and proteins were digested with the SP3 microplate procedure. Peptides were analyzed with MS/MS and proteotyped according to an in-house pipeline. Figure was created with Biorender.com.

Figure 3

16S rRNA-based tree of SM33 and related sequences within the NCBI database. Numbers given at the branches indicate the amount of substitution per nucleotide position. Nucleotide tree is based on 1325 positions.

Figure 4

16S rRNA-based phylogenetic tree of SS13 and the closest sequences within the NCBI database. The numbers on the branches represent the substitutions per nucleotide position. In total, 1341 basepairs were compared.

Figure 5

Phylogenetic reconstruction based on MLSA of specific conserved proteins: rpoA, rpoB’, rpoB, secY, and chaperonin GroEL. The final data set consisted of a total of 3,768 positions. Aquisalimonas asiatica DSM 18102^T was used as the rooted outgroup. To classify SS13, three different species from three different genera were included. The scale bar represents 0.050 substitutions per position.