Survival secrets of Deep Lake

Deep Lake, in Antarctica's Vestfold Hills, harbours a unique flora of salt-loving Archaea that survive at temperatures way below freezing. Professor Rick Cavicchioli's team at UNSW has been plumbing the lake's mysteries.

In the depths of an east Antarctic winter, Deep Lake, in the Vestfold Hills, remains permanently free of ice, even when its surface water temperature drops to minus 20° Celsius.

With a salt concentration of 270 gL^-1, it is the saltiest of more than 300 lakes and ponds in the Vestfold Hills, which were originally below sea level before isostatic rebound of the continental crust caused the sea to retreat around 3000 to 5000 years ago.

Hypersaline and intensely cold, Deep Lake is surely one of the most hostile aquatic environments on the planet. But in its isolated waters, a simple yet remarkable microbial community, dominated by cold-adapted haloarchaea, has evolved.

Molecular biologist Professor Rick Cavicchioli and his team at the University of NSW have been plumbing the lake’s mysteries and Cavicchioli says the lake, which is up to 36 metres deep, is “archaea top to bottom”.

The lake’s archaea are not merely salt-tolerant; Cavicchioli says they require high salt concentrations to survive.

Other microbes, including bacteria and single-celled algae, such as the halophyte Dunaliella, are mere bit players.

At July’s annual conference of the Australian Society of Microbiology in Melbourne, Cavicchioli will describe what he and his Australian and US colleagues have found in the Deep Lake community.

A dominant heterotroph

Four types of haloarchaea, representing four different genera, make up 72% of Deep Lake’s microbial community.

A metagenomic analysis showed that the four genera share about 85% nucleotide identity within their 16S ribosomal RNA genes and average about 73% nucleotide identity across their genomes.

Cavicchioli’s team isolated the lake’s biota by taking water samples obtained at depths of 5, 12, 24 and 36 metres and passing them through progressively finer pore size filters of 20-3.0, 3.0-0.8, 0.8-0.1 micrometres. Remarkably, there was almost no change in species composition through the full depth of the water column or across the different filter sizes.

Two undescribed strains of rchaea, tADL and DL31, accounted for 44% and 18% of the lake’s community, respectively. Halorubrum lacusprofundi represents about 10% of the lake’s community and strain DL1 comprises 0.3%.

The bimodal lifestyle of the tADL strain probably explains its dominance in the Deep Lake community. It has a flagellum, and intracellular gas vehicles that provide buoyancy, allowing it to plumb the full depth and breath of the frigid water column.

In the austral summer, it ascends towards the surface to benefit from the warmer temperatures and therefore grow faster and produce more progeny, as well as use the spoils from the growth of the phototrophic Dunaliella. Because Dunaliella accumulate glycerol as an osmoprotectant during summer and the long, dark Antarctic winter, Cavicchioli says tADL’s heterotrophic lifestyle will be facilitated by the provision of glycerol as a carbon and energy source from dead and lysed Dunaliella cells.

An agent of change

Using 454 amplicon sequencing of the 16S rRNA gene, Cavicchioli’s colleagues at the US Department of Energy’s Joint Genome Institute in Walnut Creek, California, have genotyped the four archaea and shown they represent distinct genera.

Nevertheless, a comparative genomic analysis has revealed a surprising degree of shared genetic identity.

In a simple, closed system, where one might expect to find relative genetic stasis, tADL works as an agent for evolutionary change. tADL exchanges DNA with other haloarchaea in the lake. And as the most abundant member of the community, it plays a key role in the distribution of DNA.

Cavicchioli says tADL shares genes from a specific region of its single replicon genome with relatively small, dynamic regions of the genomes of the other haloarchaea, all of which have multiple replicons.

By localising the ‘promiscuous’ regions of the genome to distinct locations, each species retains its own genomic signature. These genomic signatures are important because they provide the basis for conferring specialisation, enabling each type to occupy an environmental niche.

“If you compare Deep Lake with a hypersaline lake in outback Australia, or some other temperate or tropical location, the microbial biota would be very different, and probably more diverse,” Cavicchioli said.

Living with high salt

The microbial community present in hypersaline systems throughout the world reflects the nature of local nutrient inputs and the type of salts present. Cavicchioli says neighbouring lakes that are not as salty in the Vestfold Hills also tend to have more diverse communities than Deep Lake, including a more diverse consortium of bacteria.

“The relative lack of diversity in Deep Lake tells us this environment was selected for particular types of organisms that can cope with the extreme conditions present in the lake, and do very well - they out-compete everything else,” he said.

“We believe that, in Deep Lake, the archaea that now live there have specific requirements for the very high levels of salt, and they came to dominate very soon after the lake became isolated.

“As for niche partitioning, if you have organisms exchanging DNA when rates of cell division are very low, because of the extreme cold, it doesn’t take long for a high degree of homogenisation to develop. But because individual species are competing for different types of nutrients, ecotypes emerge that don’t directly compete with each other, which leads to niche partitioning, despite their broad genetic similarities.

“The interesting thing about haloarchaea is that they all accumulate high levels of intracellular salt, whereas most bacteria exclude salt, between 3- and 5-molar potassium and chloride,” Cavicchioli said.

“You need pretty special proteins, which have a high level of negative charge, to neutralise the salt and maintain function at that sort of salt concentration.”

DNA exchange

Cavicchioli says while the lake waters are dark for much of the year, due to the low angle of the sun, they are actually very clear, allowing visible light to penetrate the full depth of the water column. Calculations of UV levels at different depths have shown that shorter-wavelength UV light is more strongly attenuated by organic material with increasing depth.

“The very high level of UV during summer is likely to impact on the haloarchaea in the upper waters, and we expect they have evolved mechanisms for coping with this extreme level of radiation,” Cavicchioli said.

Cavicchioli says while the mechanisms of genetic exchange between the different haloarchaea are unknown, it is clear from the genomic analyses that the exchanges involve very large nucleotide chunks that undergo recombination after transfer - one possibility is that viruses specialised to infect haloarchaea could be acting as vectors, another is that they transfer DNA via mechanisms involving cell-cell contact.

The exchange of DNA is likely to be mediated by transposons, with selection acting to see that DNA integrated in particular regions of the recipients’ genomes.

Potential treatment for membrane biofouling

Cavicchioli says the ability of Deep Lake’s haloarchaea to function in conditions of extreme cold and salinity raises the possibility that some of their enzymes could find low-temperature industrial applications.

“We’ve considered the possibility of using Antarctic haloarchaeal enzymes for facilitating water recycling processes by employing them to treat biofouling that occurs on membrane filters,” he said.

“Most of the work in this field is driven by engineers, not biologists - if the flux rate goes down, engineers typically tackle the problem by scaling up the surface area of the filters and using bleach and sodium hydroxide to try and clean them, which are pretty nasty chemicals and often not very effective.

“With salt-stable, cold-active enzymes from haloarchaea that are inherently biodegradable, you would still get a high reaction rate in normal cold water, so there would be savings with regards to heating costs, and importantly, environmental benefits through less consumption of fossil fuels for energy generation and less generation of waste.”

Professor Cavicchioli heads a world-leading laboratory in research into the molecular basis of cold adaptation in archaea at UNSW’s School of Biotechnology and Biomolecular sciences in Sydney. He has made major contributions to the field of archaeal cold adaptation through studies of archaeal proteins, intracellular solutes, tRNA, lipids, gene regulation, transcriptomics, comparative genomics and proteomics, and to the broader fields of extremophiles, archaeal biology and cold adapted proteins. His team's focus on cold and extreme adaptation underpins a biotechnology program aimed at developing enzymes with enhanced performances, with applications to a broad range of industries.