by Gemma Reguera
The Cold Side Of The Earth
In the midst of one of the worst Michigan winters on record, I felt inspired to learn more about how microbes cope with the cold (it is true: misery does indeed like company). So it happens that the polar vortex that has visited our northern states a few times this season has given us a glimpse at the subzero temperatures that prevail in the northern lands of our planet, sometimes all year round. The most northern areas are what we generally know as the Arctic, a treeless region that is either barren of vegetation or limited to tundra (shrubs, grass, lichens, etc.). As you continue going south, you enter the boreal region, characterized by its thick coniferous forests. The soils in these northern lands are called permafrost because they remain frozen for long periods of time. The almost permanent frozen state of these soils limits microbial activity and the turnover of organic matter. Not surprisingly, permafrost soils store approximately 25% of all of the planet’s soil organic matter.
For all their frigid circumstances, do not assume that permafrost microbes are in a perpetual state of hibernation. These frozen soils harbor a broad diversity of microbes. Furthermore, bulk measurements indicate that there is microbial metabolic activity despite the subzero temperature regimes. Just how much, we don’t know. Scientists fear that global warming will continue to thaw large areas of permafrost, promoting microbial activities and the turnover of the large quantities of organic matter trapped in the soils. This could lead to the release of substantial amounts of carbon into the atmosphere in the form of carbon dioxide and the greenhouse gas methane. However, with the limited information we have, it is difficult to predict how the permafrost microbial communities will respond to global warming.
The technical challenge: from ‘who they are’ to ‘what they do’
Microbial ecologists have developed a wide range of techniques to profile microbial communities and assess the abundance of specific phylogenetic groups so we can know ‘who is there’. However, measuring the metabolic activity of specific phylogenetic groups and their contribution to carbon cycling and biomineralization is more challenging. In a recent paper published in the ISME Journal, Tuorto et al. combined two powerful techniques to determine who is there and gain insights into what they are doing. They first used stable isotope probing (SIP) with the carbon isotope 13C to selectively label the DNA from metabolically active cells and distinguish it from the natural 12C-DNA of inactive, resident bacteria in the permafrost. To do so, they set up several permafrost soil microcosms (essentially small representatives of the larger habitat) and amended some with 13C-acetate before incubating them at temperatures ranging from 0 to -20°C. Metabolically active bacteria would metabolize the acetate to grow and support growth-related functions such as DNA replication. As a result, they would incorporate 13C isotope into the newly replicated DNA. By contrast, resident microbes running their metabolism at basal levels won’t be able to do this and will incorporate little or no 13C in their DNA. This approach allowed the team to selectively label the microbial DNA of the active (13C) and inactive (12C) permafrost bacteria in the same microcosm. As the two isotopes have different weights, they separated the two DNA fractions in a cesium chloride density gradient. Does this experiment ring the bell? A similar approach, but with N isotopes, was used in the Meselson-Stahl experiment to demonstrate the semiconservative nature of DNA replication!
This experiment relied on other clever technical details that I truly appreciated. As they did not know how much microbial activity they were going to detect in these inhospitable soils, they spiked the DNA extracted from the microcosms with archaeal DNA of Halobacterium salinarum labeled with 13C. This helped them locate the 13C-DNA band clearly in the gradient and facilitated its retrieval with a pipette. They also worried that some microbes could have problems metabolizing the 13C-acetate or incorporating the 13C isotope in their DNA and, thus, they would be underrepresented in the ‘active’ DNA pool. For this reason, they also extracted DNA from control microcosms to which they added 12C-acetate, which contains the natural carbon isotope. The challenges did not end here. The researchers used PCR to amplify the 16S rDNA sequences in the active and inactive DNA samples separated in the cesium chloride gradient. PCR can lead to the overestimation of the most abundant sequences and to the underestimation or no detection of the least represented target genes. Hence, they performed two consecutive rounds of PCR amplification and used aliquots collected at different cycles in the first PCR step as DNA templates for the second round. This allowed them to determine the lowest cycle number that yielded PCR products in both the heavy and light DNA bands, minimizing PCR bias towards the most abundant DNA sequences. After overcoming the myriad of technical challenges, they were ready to find out who was there and how active they were.
Some like it cold and others, really cold
After 6 months of incubation, the team confirmed that the 13C carbon had been incorporated into DNA extracted from the permafrost microcosms amended with 13C-acetate and separated it from the 12C (‘inactive’) DNA pool. After PCR-amplification of the 16S rDNA, they used a technique known as Terminal Restriction Fragment Length Polymorphism (TRFLP) to profile the microbial community and estimate the abundance of specific taxonomic groups. This technique relies on the fluorescent labeling of the PCR-amplified fragments at one end and their digestion with a restriction enzyme to generate small, end-labeled fragments. The size of the fragments is specific to a particular phylogenetic group (what they called operational taxonomic unit or OTU) and the fluorescence intensity gives an estimate of OTU abundance.
The first surprising finding was that a wide range of bacteria (152 OTUs in all) were able to metabolize acetate and replicate their DNA at subzero temperatures. Furthermore, there was no significant reduction in active OTUs with decreasing temperatures. Most (80%) of the OTUs were also identified in untreated permafrost samples, which were frozen at the beginning of the experiment and provided a measure of the microbial diversity in the original soils before acetate was metabolized. Hence, most of the original, resident bacteria were able to metabolize the acetate or grew using products derived from the metabolism of acetate by other bacteria. Even more interesting was the finding that half of the active OTUs had very specific temperature preferences, allowing the team to group these microbes according to the temperature range that supported their activity (see examples in Fig. 2). Group 1 (14% of the OTUs) was active at temperatures between 0 and -6°C, whereas group 2 (9% of OTUs) had an even narrower range between -6°C and -9°C. A third group of active microorganisms (13% OTUs) preferred colder temperatures from -9°C to -20°C and the last group, group 4 (15% OTUs), had a broad range spanning the experimental incubation temperatures (from 0 to -20°C). Thus, although the permafrost community was active across the temperature range, the microbes that drove those activities varied with the temperature.
Representative 16S rDNA sequences from each group were then cloned and sequenced to gain insights into the phylogenetic groups that are active in the permafrost at each temperature range. As shown in Fig. 3, the sequenced clones of active permafrost bacteria were associated with the same phyla (Acidobacteria, Actinobacteria, Chloroflexi, Gemmatimonadetes and Proteobacteria) and regardless of their preferred temperature. What changed was the type of microbe within the phylum. Unfortunately, all we know about these active microbes is that they are deeply branching 16S rDNA phylotypes distantly related to cultivated or uncultured bacteria from other cold environments. This is clearly the time in which microbiologists with ‘green’ thumbs should join the game and attempt to isolate these new microbes. After all, as my husband wisely reminds me often, ‘to know them, you have to grow them’. Interestingly, no clones were affiliated with the Firmicutes, although groups within this phylum had been detected in permafrost communities before. As this phylum includes spore-forming bacteria such as Bacillus and Clostridium, the authors speculate that they may persist in the permafrost in the dormant spore state. Alternatively, these microbes may not be able to metabolize acetate and their potential metabolic activity may have gone undetected.
Message from the cold
With a clever approach that combined classical and modern techniques, these researchers revealed an untapped diversity of metabolically active bacteria that was highly responsive to subzero temperature regimes. In general, most microbes were metabolically active within a narrow range of temperatures, suggesting that the microbial diversity in the permafrost fluctuates greatly with temperature shifts to maintain its productivity. These differential taxonomic patterns in response to temperature are easily interpreted in light of the competitive exclusion principle of ecological theory: if two microbes compete for the same resource under the same conditions (in this case, temperature), they cannot coexist. Hence, some microbes have become ‘specialists’, i.e., they have evolved a metabolism that maximizes resource utilization within a narrow range of temperatures, so as to outgrow competitors.
One resource that permafrost bacteria are likely to fight fiercely for is water. In frozen soils, water availability is restricted to a very thin, salty layer of liquid surrounding the soil particles. The concentration of solutes in this liquid layer is so high that psychrophilic microbes have evolved mechanisms for both thermo- and salt-adaptation. To access this water resource, psychrophilic microbes synthesize extracellular polymeric substances (EPS), which act as cryoprotectants and also facilitate attachment to the soil particles. In addition, EPS promotes water retention and nutrient absorption. Not surprisingly, studies show that decreases in temperature correlate well with increases in EPS synthesis and bacterial abundance in cold ecosystems. Changes in fatty acid composition have also been observed in these organisms, which likely allow them to modulate the properties of the lipid membrane to carry out membrane-associated functions despite the low water activity in their surroundings.
Of special significance is the finding that permafrost microbes are active across a wide range of subzero temperatures, suggesting that they play a major role in biogeochemical cycles in these cold ecosystems. Permafrost soils occupy approximately 20% of the Earth’s land surface. Hence, microbial activities in these soils are likely to impact our planet globally. It is difficult to assess how much these activities contribute to the release of greenhouse gases to the atmosphere because the need to use archaeal 13C-DNA to identify the 13C-DNA band in the cesium chloride gradient also precluded the team from investigating the contribution of the archaeal population (such as methanogens) to permafrost microbial activities and their responses to temperature shifts. Hence, future work needs to focus on these microbes as well.
Still, results from this study support the notion that the metabolic activity of permafrost communities is resilient to temperature variations. For this reason, it is reasonable to think that they could continue to be active, and perhaps be stimulated further, if global temperatures continue to rise and permafrost soils continue to melt. These conditions may also ‘wake up’ dormant microbes stimulating carbon turn over and gas emissions from these ecosystems in unpredictable ways. Despite these uncertainties, it is clear that permafrost microbial activities need to be accounted for in global climate models in order to improve their predictive value. But the real message of the story (at least the one I like the best) is that this is indeed a planet of microbes. Microbes are active everywhere we look, even in the coldest lands. Not sure I find comfort in it as I face the frigid Michigan temperatures, but as a microbiologist, I could not be happier.
Tuorto SJ, Darias P, McGuinness LR, Panikov N, Zhang T, Häggblom MM, Kerkhof LJ (2014). Bacterial genome replication at subzero temperatures in permafrost. The ISME journal, 8 (1), 139−149. PMID 23985750
Gemma is associate professor in the Department of Microbiology and Molecular Genetics, Michigan State University and an Associate Blogger at STC.