1. Introduction
The geophysical transformation accompanied by climatic changes during the Earth’s evolution for the last ~4 billion years resulted in the establishment of a wide range of ecosystems on this planet, and various lines of evidence suggest that microbial life has existed and evolved during the last ~3.5 billion years of Earth’s history [
1,
2]. These ecosystems, diverse in their physicochemical properties and changing over time, have required that life develop novel metabolic strategies for the exploitation of the numerous niches available across the planet. The functioning of microorganisms at the cellular and community levels is constrained by a set of physicochemical parameters within the ecosystems they inhabit [
3]. Some of these ecosystems are categorized as “extreme habitats” where the inhabiting organisms have adapted to environmental parameters often considered inimical to the maintenance of life-functions for others [
4]. Given that over 70% of Earth maintains near- or below-freezing temperatures, the cold ecospheres constitute the largest “physical extremes” for microbial communities to inhabit, manifest adaptive attributes, and drive key biological and geochemical processes [
5,
6].
Within the cryosphere, the Antarctic continent offers perennially cold, subzero temperatures as well as other challenges, such as oligotrophy, intense winds, aridity, and high solar UV radiation (during the austral summer months). Thus, all organisms, including microorganisms, living on this icy continent must possess various adaptive traits to sustain life [
7]. Early studies of soil microbiology in the McMurdo Dry Valleys relied upon culture-dependent methods [
8], however the diversity and distribution of microorganisms in various Antarctic ecosystems were not more fully explored until the advent of DNA-based culture-independent methods [
5,
6,
7,
9,
10,
11,
12,
13,
14,
15]. Recently, the applications of shotgun sequencing of the metagenome have enabled the elucidation of both taxonomic identities as well as crucial adaptive genetic traits necessary for microorganisms to cope with the environmentally imposed physical and nutritional extremes on this icy continent [
16,
17,
18,
19,
20,
21].
Although about 98% of the Antarctic landmass is covered by an ice sheet with a mean thickness of 2.16 km (maximum thickness up to ~4.78 km), a number of ice-free “oases” exist where various open-water lakes, perennially-ice covered lakes, and ponds largely support life [
22,
23]. Among these lakes, the perennially ice-covered lakes are particularly interesting to explore microbial communities and their adaptive strategies due to their unique physical, chemical, and limnological features [
24]. The permanent ice cover (~3–5 m) of these lakes restricts wind-driven mixing of the water column, exchange of atmospheric gases, deposition of sediments, and light penetration [
25,
26]. In addition, most of these lakes manifest a stable water column with strong chemical stratification and minimal vertical mixing [
27,
28]. Lake Untersee is one of the largest (11.4 km
2) and deepest (>160 m) perennially ice-covered ultraoligotrophic freshwater lakes in Antarctica. This lake is located in the Grüber Mountains of Central Queen Maud Land in East Antarctica and is partly dammed by the Anuchin Glacier [
29,
30,
31,
32]. The water column in Lake Untersee is well-mixed due to the temperature gradient (~0 °C on the top and 4 °C at the bottom) [
29], contains a high concentration (150%) of dissolved oxygen, and harbors benthic photosynthetic microbial mats [
30]. In addition, the high pH gradient, ranging between 9.8 and 12.1, unusual dynamics of temperature and water circulation, high methane content in some locations, and ultraoligotrophic conditions offer unique challenges to the microbial communities in this lake [
29,
30,
33].
In this study, we have used the shotgun metagenomics approach along with bioinformatics tools to explore the microbial community compositions and genetic signatures for cold-responsive genes that code for cold-induced proteins (CIPs) and cold-associated general stress-responsive proteins (CASPs) in microbial communities of Lake Untersee water and sediment samples. In addition, we have used seven publicly available shotgun metagenome datasets of various other Antarctic lake and soil samples to compare and contrast the metagenomic profile of microbial communities and cold-responsive stress genes in Lake Untersee water and sediment samples.
4. Discussion
The rapid advancements of culture-independent NGS have revolutionized our understanding of the microbial communities and their functional genes in a wide range of ecosystems, including the polar environments [
58,
59,
60]. By using this approach, the bacterial metabolic genes for adaptation to cold temperature environments have been studied in cyanobacterial mats in Arctic and Antarctic ice shelves [
21], microbial mats from Antarctic Lake Joyce [
18], and permafrost samples from Alaska [
61]. In order to obtain collective insights into the microbial distributions and abundances of genes associated with CIPs and CASPs, we have analyzed metagenomes from Lake Untersee water and sediment samples for comparison with selected publicly available soils and water metagenomes from diverse ecosystems in the Antarctic continent.
In general, the microbiota of all metagenomes used in this study showed mostly comparable taxonomic compositions. For example, Proteobacteria and Bacteroidetes were the abundant phyla, whereas phylum Actinobacteria, although varied in their abundances, were found in all metagenomes. The microbial taxa and cold-adaptive traits found in our samples have also been reported previously in diverse Antarctic soil, sediment, and aquatic ecosystems [
21,
62,
63,
64,
65]. Despite the similarities in microbial composition across all metagenomes, noticeable differences at the genus level were found when compared between the water and the combined soil and lake sediment metagenomes. The water metagenomes had relatively higher abundances of
Prochlorococcus and
Thiomicrospira than the soil and lake sediment metagenomes, which was also reported in several sub-zero Antarctic lakes [
15].
Prochlorococcus and
Thiomicrospira are known to be one of the key contributors to Antarctic aquatic ecosystems as they are characterized as photosynthetic organisms and autotrophic sulfur-oxidizing gammaproteobacterium, respectively [
15]. In contrast, the soil and lake sediment metagenomes showed relatively higher abundances of
Myxococcus,
Anaeromyxobacter, and
Haliangium than the water metagenomes.
Myxococcus,
Anaeromyxobacter, and
Haliangium have been previously found in other Antarctic soil samples [
66]; and
Gloeobacter has been reported in Antarctic sediment samples [
67]. Interestingly, myxobacteria (such as
Myxococcus) have been generally considered to be mesophilic soil microbes [
68]; however, the first psychrophilic myxobacteria were identified in soil samples in Antarctic McMurdo Dry Valleys and South Victoria Land [
68]. A few other studies have also reported
Anaeromyxobacter, Haliangium, and
Gloeobacter in Antarctic soil and sediment ecosystems.
The cold-adaptive traits in water metagenomes revealed a generally similar distribution of CIPs, including a high number of genes associated with DNA replication (GyrA, RecA, and DnaA), protein folding (chaperone proteins DnaJ and DnaK), protein biosynthesis (translation initiation factor), and the transcription termination protein NusA. Like the water metagenomes, the soil and lake sediment metagenomes also showed similar CIP distributions in all samples. Within the cold stress proteins (cold shock family of proteins), CspA was found to be highly abundant in each water and combined soil and lake sediment metagenome. At low temperatures, cold stress proteins are expressed quickly and remain active to stabilize the mRNA, thus helping proper protein folding and allowing bacteria to adapt their physiology to the cold temperature environments [
17,
18,
21,
69,
70]. Particularly, CspA is known to function as an RNA chaperone, destabilizing the secondary structures of mRNA necessary for the expression of the cold-inducible proteins and enhancing the expression of GyrA [
18,
71,
72]. Moreover, CspA, CspC, and CspE act as transcriptional anti-terminators, allowing alternative mechanisms for the regulation of other CIPs, such as NusA, IF2, RbfA, and PNPase [
73]. Although each water and combined soil and lake sediment metagenome showed a lower abundance of cold-responsive stress proteins as compared to DnaA, DnaK, DnaJ, DNA topoisomerases, and recombination factors, these proteins have been observed to be highly abundant, particularly in Antarctic and Arctic ecosystems. This is due to their role in helping bacteria maintain steady-state cellular metabolism, growth, and division in order to cope with the consistent cold environments [
18,
21].
In the presence of cold stress, bacterial cell membranes undergo decreased membrane fluidity but an increase in permeability. It has been reported that FAD offsets membrane stiffness by modifying the existing fatty acid chain structures of the cell membrane [
74,
75,
76,
77]. Moreover, TS has been characterized to be involved in numerous stress-related processes and predicted to function in the restriction of oxidative damage, cryopreservation, and cell membrane protection [
78,
79,
80,
81]. The combined soil and lake sediment metagenomes showed more genes related to FAD and TS than the water metagenomes. Especially, FAD and TS were more abundant in the MS_soil and the MDV_soil metagenomes, implying that protective responses are needed for bacteria surviving in the open soil ecosystems due to the fluctuations in temperatures, desiccation, and poor nutrient availability. H-NS is known as a nucleoid-associated DNA binding protein [
82] and a regulator of the expression of various cold shock genes [
83,
84,
85,
86]. In our study, H-NS was only found in the LU_water and the LU_sediment metagenomes. This may support the presence of almost the entire cold-shock family of proteins (CspA, CspB, CspC, CspD, CspE, CspG, and antifreeze proteins) in the metagenomes of Lake Untersee.
The distribution of CASPs in each water and combined soil and lake sediment metagenome showed genes associated with the regulation of GyrB (DNA gyrase) and glutamate biosynthesis. DNA gyrase is known to play an important role in regulating DNA topology during transcription and manifests higher activity at cold temperatures [
73,
77,
87]. Glutamate, glycine, choline, and betaine are known cryo- and osmoprotectants [
21,
88], thus supporting our results of heightened glutamate synthesis genes observed in all metagenomes. Furthermore, choline and betaine biosynthesis allow bacteria to increase osmolality, thus helping to protect against cold-related damage to the cell structure and function [
89]. A high representation of glutamate biosynthesis in all metagenomes used in this study might reflect the high osmotic stress present across the Antarctic continent. Additionally, the high number of genes associated with choline and betaine biosynthesis found in the AL_water and NB_water metagenomes as opposed to the LU_water metagenome may be due to the relatively higher salt concentrations in the Ace and Newcomb Bay lakes as compared to the freshwater of Lake Untersee. EPS also plays an important role in cryoprotection against ice crystal damage and high salinity [
19,
21]. A relatively higher abundance of genes associated with EPS biosynthesis was found in Lake Untersee and the combined soil and lake sediment metagenomes than in Ace Lake and Newcomb Bay Lake metagenomes. This may be due to a relatively higher abundance of Cyanobacteria, which are known to produce a copious amount of EPS [
21]. All water and combined soil and lake sediment metagenomes had a noteworthy distribution of tRNA dihydrouridine synthase, which is known to help maintain conformational flexibility and dynamic motion of tRNA at cold temperatures [
90]. Thus, an abundance of sequences for tRNA dihydrouridine synthase in our metagenomes indicates an adaptive advantage in microorganisms inhabiting the Antarctic environment.
In a previous study, the shotgun metagenomics approach was applied to the microbial communities of a laboratory culture of
Euplotes focardii, a psychrophilic marine ciliate collected from sediments in Terra Nova Bay, Antarctica [
91]. These microbial communities were considered to be representative of the Antarctic sample upon collection, and, similar to this study, showed a heightened distribution of the phyla Proteobacteria followed by Bacteroidetes. Functional analysis demonstrated ice binding and antifreeze proteins and proteins involved in the oxidative stress response, which supported the postulated underlying genetic capacity for adaptation to their consistently cold and oxygen-rich environment. Interestingly, antibiotic treatment of the ciliate cultures showed a reduction in the proliferation of
E. focardii, which was attributed to the loss of key biogeochemical (carbon and nitrogen) and nutrient cycling performed by the associated microbiota. As such, the various cold-responsive stress genes (CIPs and CASPs) observed in the extreme Antarctic ecosystems of this study demonstrate crucial microbial adaptations to cold stress, allowing for both their persistence and possible sustenance of other inhabiting organisms that are metabolically restricted by the cold stress.
The mechanisms of bacterial genetic adaptation in low- and subzero-temperature environments have been well-reported [
63,
69]. Our analyses included the updated list of CIPs and CASPs found in microbial metagenomes. These proteins have been filtered from the metagenomics datasets by using bioinformatics tools to achieve a comprehensive outlook of microbial community composition and mechanisms to cope with cold and other stresses present in Antarctica. Overall, noticeable differences were found in the microbial taxa distribution and various cold- and stress-related functions among all Antarctic metagenomes. However, the key genes necessary for adaptation in the continuous low- and subzero-temperature environment were well-represented across all Antarctic metagenomes used in this study. Moreover, the permanently ice-covered Lake Untersee metagenomes had high abundances of sequences for cold-responsive stress proteins and H-NS, indicating that this lake environment poses comparatively greater survival challenges to the inhabiting microbial communities.