Conversations with Rick Cavicchioli: Antarctic clues to virus evolution

The image of the staid scientist holed up in the laboratory surrounded by similarly tempered collaborators is a quaint and ill-founded one. Not that they don’t knuckle down in the lab. They do. However, there’s a lot more old-fashioned, boots-on-the-ground, adventuring-to-distant-lands than you’d think. The same way there are photographers like James Nachtway and Don McCullin willing to go to great lengths for the perfect shot; dedicated scientists often put their safety on the line to study their subjects. Case in point, University of New South Wales Sydney’s Rick Cavicchioli.

During the course of his research on haloarchaea, Cavicchioli and his crew have ventured about as far from the workbench as possible. Antarctica. As a result of their tenacity, important and tantalizing insights into the evolution of viruses have emerged from extreme hypersaline conditions, promiscuous haloarchaea, and plasmids that take on membranes.

Professor Cavicchioli took the time to share his findings and images of his expeditions.

SCIENTIFIC INQUIRER: Let’s start with some background. What exactly are haloarchaea and where are they found?

Prof. Rick Cavicchioli

RICK CAVICCHIOLI: Haloarchaea are members of the Archaea, one of the three domains (major lineages) of life on Earth – the other two domains being Bacteria and Eucarya. Haloarchaea live in hypersaline environments where salt concentrations can be 5 molar – by comparison seawater is around 0.6 molar. Natural environments that haloarchaea are found in include the Dead Sea which borders Israel, the Great Salt Lake in Utah, and Lake Tyrrell in Australia. They can also be found on salted dried-fish and all around the world in salt crystallizer ponds in solar salterns that are used for collecting salt for human consumption – places like Alicante in Spain and Guerrero Negro in Mexico.

As a side point, note that despite a lot of looking, there are no known pathogens within the domain of life, Archaea – they are ‘friendly’ – this is probably a comforting thought when you consume salted fish!

The ability of haloarchaea to thrive in hypersaline environments is well illustrated by the red/pink/purple colouration that can be seen on the surface of some lakes, and particularly in solar salterns – the colour derives from the membrane pigments produced by the haloarchaea, and from Dunaliella (algae) that often grow as primary producers in these systems, using light energy to fix carbon dioxide for growth. Haloarchaea are photoheterotrophic microorganisms that grow by ‘remineralizing’ organic matter released from the phototrophic algae, gaining a certain amount of energy themselves by also using sunlight.

One of the remarkable features of haloarchaea is that they function by accumulating very high levels of intracellular salt – typically 3 molar chloride and 5 molar potassium. As such, all their cellular machinery has evolved to function, in fact require, high concentrations of salt. To put this in perspective – most cells would dehydrate and shrivel-up if placed in hypersaline lake water and their cellular components (e.g. enzymes) would not be active. By contrast, if haloarchaea were placed in fresh water, probably even marine water, they would swell-up and lyse.

So when studying environmental microorganisms like haloarchaea, it is very important to recognize that what is ‘normal’ and makes one type of organism ‘happy’, is often very different to what makes another one ‘happy’. When studying mechanisms of adaptation and ecology of the microorganisms that grow in the world’s diverse types of environments (hypersaline or otherwise), we must try to ‘look’ through the eyes of the resident microbes, and avoid our narrow anthropocentric perspective or the view learned from studies of model laboratory bacteria such as Escherichia coli.

SI: Can you describe the area of Antarctica where the water samples for the study were taken?

RC: Antarctica also contains hypersaline environments. In fact, the world’s saltiest water can be found in Don Juan Pond which is in the McMurdo Dry Valleys (about 100 km from McMurdo Station), with other salty water also present in Lake Vida, Lake Bonney and the outflow at Blood Falls. Across to the other side of Antarctica, 2,500 km away, is Davis Station which is located in the Vestfold Hills. This region, including the Rauer Islands (30 km from the Vestfold Hills), represents a coastal oasis containing hundreds of lake systems, most of which are marine-derived. Less than 10,000 years ago the area was marine, and as a result of isostatic rebound of the continent, the area rose slightly above sea level, leaving systems like Ace Lake, Organic Lake and Deep Lake cut-off from the ocean.

Deep Lake, which was isolated and became hypersaline about 3,500 year ago, has a number of claims to fame. It is the lowest accessible point on the Antarctic continent at about 50 m below sea level; measurement of water level in the lake commenced in 1976, with continual recording making it one of the longest non-weather records of the Antarctic environment; despite air temperatures dropping to -40°C and water to -20°C, the lake never freezes, making it the coldest aquatic environment on Earth that is known to support life.

SI: How was the research conducted in the field and then in the laboratory?

RC: Haloarchaea dominate the Deep Lake ecosystem. The first member of the Archaea isolated from a cold environment was Halorubrum lacusprofundi, from Deep Lake. The isolation of the species in the late 1980s was performed by Peter Franzmann and colleagues. Deep Lake is about 5 km from Davis Station, and laboratory facilities at the station enable research to be performed and samples cryogenically stored.

My group at UNSW Sydney first expeditioned to Antarctica in 2006. This followed years of laboratory work commencing in 1995, based on studies of Methanococcoides burtonii, a methanogen (also isolated by Peter Franzmann) from the anaerobic, hydrogen-sulfide rich, methane-saturated, bottom waters of Ace Lake. We had prodded and probed M. burtonii, examining how it had evolved an ability to function effectively in the cold. It proved an excellent model as various cousins of M. burtonii grow at temperatures of up to 122°C, so methanogens as a group are excellent for examining thermal adaptation.

The 2006/07 expedition, and subsequent expeditions to Davis in 2008/09 and an 18 month over-wintering expedition in 2013/15, enabled us to obtain samples primarily for metagenomic and metaproteomic studies. The ‘metaomic’ methods rely on the capturing of microbial biomass on filters, cryogenically preserving the samples in buffers that kill the cells, and performing DNA sequencing on the samples to determine gene sequences (metagenomics) and mass-spectrometry to determine protein sequences (metaproteomics). By capturing whole environmental samples, the analyses allow us to work out things like, which organisms are present, what functions they perform, and what microbial processes occur within the aquatic ecosystems.

SI: What did you discover?

RC: In 2013, we published a paper describing the presence of a low complexity community of haloarchaea in Deep Lake that are ‘promiscuous’ and undergo high levels of intergenera gene exchange. Haloarchaea in general appear to be fairly flexible with their DNA content, but the discovery we made set a new bench-mark for what haloarchaea were capable of transferring and inheriting in a natural environment.

In subsequent studies we kept an eye out for what mechanisms enable the gene exchange, and in 2015 we published a paper describing the interactions of the haloarchaea with viruses. By studying CRISPR spacers present in the haloarchaea and matching them to viruses we had identified, we established networks of host-virus interactions. CRISPRs are akin to primitive immune systems as they offer their host’s protection against invading forms of nucleic acids, such as viruses, and they preserve a memory of past invaders so that the cells can mount an effective response when the same type of invader, reinvades.

To gain a more detailed understanding of virus-host interactions, Susanne Erdmann joined my group in 2014 as an EMBO Fellow and set about isolating viruses in the laboratory that could infect the haloarchaea that we had isolated from Antarctica. Based on samples collected from the 2013-2015 expedition that included lakes from the Rauer Islands, Susanne identified many and varied ‘virus-like particles’ (VLPs). After sequencing the genomes of some of the purified VLPs she discovered one of the genomes was for a plasmid and not a virus. From her continued studies she determined that the plasmid-derived proteins went into the host membrane, formed specialized membrane vesicles that packaged the plasmid, and released plasmid-containing vesicles that were capable of infecting other cells. She recognized this as an important discovery because an ‘extracellular element’ (ECE) that is able to produce structures that encapsidate the ECE and is then capable of infecting other cells, is a defining property of a virus. As such, the discovery of a plasmid with these properties put to rest the last distinction between viruses and plasmids – in essence, blurring the boundaries for what in fact, if anything, distinguishes the two types of ECEs.

SI: Can you speculate as to why the haloarchaea plasmids employed protein envelopes?

RC: Our study found that the host organism encoded proteins that were similar to some of the plasmid proteins. Moreover, like the plasmid proteins, the host proteins were present in the vesicles. Susanne also discovered that the plasmid could integrate into the host’s genome, excise carrying long stretches of host DNA, package into vesicles and infect other cells. We therefore speculate that in the evolutionary past, an ECE picked up genes from its host as a means of making vesicles better tailored to its purpose of disseminating widely throughout the microbial population.

SI: How does your discovery bear similarities to the mechanisms by which modern viruses replicate? How might it shed light on how viruses evolved?

RC: Some of the proteins encoded by the plasmid and host appear to have structural similarity to proteins that form a cage-like structure around some types of mammalian transport vesicles. These vesicles transport ‘cargo’ within the golgi and to and from the endoplasmic reticulum, and some are known to be essential for the replication of viruses such as influenza and hepatitis C. So, we can see connections between systems present in Eucarya (mammals) and Archaea (haloarchaea), and we can see connections between plasmid-encoded genes and genes present in genome of the haloarchaeal host.

As members of all three cellular lineages of life (Archaea, Bacteria, Eucarya) make vesicles, it is not hard to conceive that vesicles could have, and still might serve similar purposes – i.e. as vehicles for infecting cells with plasmids. Moreover, compared to the morphology of membrane vesicles, many currently known viruses possess much more defined structures that encapsidate their nucleic acid (e.g. head-tailed viruses). It is therefore conceivable that ‘plasmid vesicles’ preceded structurally ‘sophisticated’ viruses, and may be the predecessor of some viruses such as those that use components of their hosts’ membranes (lipids) to form their viral particles (e.g. pleolipoviruses).

(Ed. Note: For more on Prof. Cavicchioli’s findings in relation to viral evolution read the article that accompanied his paper in Nature. [Read])

SI: Are there any similarities between haloarchaea plasmids and various bacterial plasmids?

RC: The haloarchaeal plasmids we describe, pR1SE and its derivatives, have typical plasmid replication and plasmid stability genes. Beyond the basic requirements for plasmid replication, pR1SE is special in encoding genes for plasmid vesicle function. I can see no reason why a bacterial plasmid couldn’t also harbour genes necessary for producing similar types of vesicles that allow packaging and dissemination of the plasmid.

It is often said, and it really is the case, that we have barely begun to learn about the diversity of the life on Earth, particularly the microbial world. What we have learned in the process is that evolution remains unperturbed by our propensity to pigeon-hole our few discoveries, and instead continues to offer up new aspects of life for us to interpret and fit into an amazing biological puzzle.

SI: On a more personal note, how did you come to a life in science? Did you always want to be a scientist?

RC: Way back I considered being an astronaut, pilot, physical education teacher, astronomer and archaeologist, but as High School came to a close I decided I wanted to study chemistry. I like to give some ‘credit’ to my High School teacher who ‘inspired’ me by warning that studying chemistry in school was one thing, but going onto university was another, and I should think very carefully before making such a decision – such was growing up in a provincial farming town.

Prior to this I spent my first 9 years of school in Australia’s outback, spending a great deal of my weekends out in the bush with my father looking for aboriginal artefacts and gazing up at the night stars pondering the what and whereof of life. I feel that the rich life experience my mother and father enabled me to experience, particularly through the outdoors, has provided me with life skills that are grounding and offer strong foundational support. To me, spontaneity is great, but clever decision-making leading to serendipitous eventuality is better. My experiences indicate that the best path to achievement is backing myself following contemplative reasoning, rather than impulsivity – at least most of the time!

After progressing between several Australian Universities for my undergraduate and postgraduate studies and postdoctoral research in the USA – during the process studying ethanol producing yeast, lignocellulose degrading rumen bacteria, and anaerobically respiring E. coli – I chose to study cold-adapted archaea using tools of the ‘genomics revolution’ (DNA sequencing). It has proven to be very rewarding and I rationalize that the work achieved by my group contributes to knowledge about an understudied lineage of life, the Archaea; environmental microorganisms – without them life on Earth would cease to exist; and life forms that dominate the planet – about 85% of life on Earth exists at or below 5°C, so our research is not only about Antarctica, but contributes to understanding the Earths cold biosphere.

SI: What is your least favorite part of being a scientist?

RC: Lack of resources and bureaucracies stymying our capacity to do work; dissemination of misinformation particularly to do with climate change; the world’s autocracies (government and commercial enterprises) attempting to quash scientific endeavour in order to feed their greedy self-interest; having a feeling of insufficient power to help achieve the changes we know science can deliver and realize the socioeconomic changes that must be made.

SI: What is the most important trait a researcher must possess?

RC: Single, most important trait – well ultimately, the ability to achieve strong scientific outcomes. As far as personal traits, I’d offer three: confidence, derived from having intellectual capacity matched with sufficient personal experience and wisdom to understand how to use it; passion, as the quintessential motivator; and flexibility, to recognize and do the most with available opportunities and find ways to create better opportunities.

For more information about Professor Cavicchioli’s research visit his group’s lab page.

The Nature Microbiology paper can be found here.

Here are more images of Prof. Cavicchioli and his group conducting research in Antarctica.

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