Biofilm forming Haloarchaea from Deep Lake, Antarctica

Sabrina Fröls, Mike Dyall-Smith, Felicitas Pfeifer (2012) Biofilm formation by haloarchaea

Environmental Microbiology (doi:DOI: 10.1111/j.1462-2920.2012.02895.x)


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movie t-ADL on glass                 Fluorescent-stained t-ADL biofilm, showing 3D structure (AO stain, from S.Fröls).  Right: cells of the t-ADL isolate, bound firmly to a glass coverslip (phase contrast, MDS)

Here is a little background, from my perspective, on this wonderful study and its serendipitous story. We need to go back to 2007, when Dickson Oh was doing a B.Sc. honours year in my lab. He was isolating haloarchaea from various hypersaline lakes, including Deep Lake ( 68°33'34" S, 78°11'46" E), a very cold and hypersaline lake in the Vestfold Hills of Antarctica (see picture below). DL isolates and clonesDeep Lake is aptly named, as it is about 36 m deep, but is also unusual in several ways: it is hypersaline (saturated salt), has never been known to freeze, even at temperatures below -40°C, and the lake level is around 50 m below sea level. Despite the extreme conditions and the remote and barren location, there are microorganisms that live there. The green alga Dunaliella (Wright and Burton, 1981), and the haloarchaeon Hrr. lacusprofundi (Franzmann et al., 1988) were described in the 1980's. In 2000, 16S rRNA gene sequence data indicated that Deep Lake harboured a much greater diversity of microorganisms than the previous isolation studies had revealed, including haloarchaea that represented novel genera (Bowman et al., 2000). Given this intriguing history, I asked Rick Cavicchioli (Uni. NSW) for some water from this lake. He travelled there with a sampling team in late 2006 and sent us a sample. Dickson analysed this in 2007, and after a lot of work, both with 16S rRNA gene sequences, and cultivation, he and I managed to snap a picture of the microbial flora present at that time, and it was very strange. Most habitats show a diversity of microbial flora, but according to the sequence data we obtained there appeared to be only 3 dominant groups (genera), all haloarchaea, and in roughly equal proportions (see bar chart above). More remarkable was close similarity of the sequences belonging to each genus - with probably only one species per genus, and these species showing very limited diversity. Cultivation often gives a very biased view of microbial diversity, and this was true here also. Almost always it was the same type of organism that grew on plates, Halorubrum lacusprofundi - the organism that was isolated way back in 1988. However, Dickson was lucky to be in the right lab, because by using liquid media, he managed to recover an organism belonging to one of the other major genera, what we called then the t-ADL (for "true antarctic deep lake") group. We were unable to isolate any representative of ADL gp 1, the other dominant genus-level group.

 

One year later I was working at a Max-Planck Institute in Germany, and as I was looking down the microscope at cultures of the t-ADL strain, I noticed that the cells were sticking strongly to the plastic surface of cluster trays, forming a densely packed layer, a biofilm.

Deep LakeDEEP LAKE, 50m below sea level, and never freezes.
[photo: Damon Ward/Australian Antarctic Division]

This was unusual as haloarchaea are generally considered planktonic: they spend their time floating around in the water column. Many species are motile, have photosynthetic pigments, and contain gas vesicles for buoyancy, so why would they want to stick to surfaces. I threw a glass coverslip into a fresh t-ADL culture, and the cells stuck to that too!. Then I looked at some of the other Deep Lake isolates at hand. I had been isolating new strains of Hrr. lacusprofundi, and noticed that some of these formed good biofilms on plastic and glass surfaces, although their structures seemed to be different from those of t-ADL. Even on the surface of liquid media, Hrr. lacusprofundi formed films (picture below left).

 

traysI was fortunate to be working in Munich, because this is where the seredipity happened. I was invited to the annual archaeal conference in Frankfurt, where I heard about the work of Sabrina Fröls and Felicitas Pfeifer (TU Darmstadt). They were studying biofilm formation by the type strain of Halobacterium salinarum. This naturally led to an ongoing collaboration, where Sabrina examined the Deep Lake isolates, and could then compare the modes of biofilm formation by the different species. It is remarkable how many different haloarchaea can form biofilms. Previously only Haloferax volcanii was known to do this (Tripepi et al., 2010). Sabrina had developed a sensitive fluorescent assay for detecting and quantitating biofilms formed by extreme halophiles, and could then follow biofilm progression and 3D structure by confocal microscopy. She obtained many stunning images, such as the one at the top of this page, and discovered eDNA and glycoconjugates in the polymeric matrix between the cells. There is now no doubt that this mode of life is widespread among the haloarchaea, which raises many questions, such as the functional advantages of this lifestyle, the signals that control this phenomenon, and what proteins and surface structures mediate attachment? One tantalizing observation was that the t-ADL strain could readily lose its ability to attach to surfaces, presumably because there was a lack of selection for this phenotype in laboratory culture. A comparison the wild type and mutant genomes may very well pinpoint the genes involved. Sabrina and Felicitas are pictured below, while on the left is a mid-density biofilm of t-ADL, on the surface of a glass coverslip.

t-ADL dense
Sabrina Froels
Felicitas Pfeifer

Collecting water from Deep Lake is not a simple task. First you have to get there, with all your gear, and then the weather has to be good enough. Shown in the picture below are a sampling team in 2007, hurriedly working while the conditions are suitable (photo courtesy of Jeff Hoffman, J. Craig Venter Inst.). As you can see, the terrain around the lake is barren and rocky (the Vestfold Hills are generally snow and ice free), and the ocean is a couple of kilometers away. Personnel and equipment had to be lifted in and out by helicopter.

DL_samplingThe biofilm work has really only just begun with Hbt. salinarum and the Deep Lake isolates. As part of the continuing story, it is important to characterise and validly describe biofilm forming haloarchaea, such as the t-ADL isolate. The genome sequence has been completed at JGI (although I disagree with their contig assembly, but that is another story), and the taxonomic work has been done in collaboration with Heng-Lin Cui and colleagues (Jiangsu University, China). I have proposed to name this species "Halohasta litchfieldiae", in honour of Carol Litchfield, who spent a lifetime studying extremophiles in general, and halophiles in particular. In Carol's later years, she spent quite some time working with Bonnie Baxter on haloarchaea and haloviruses of the Great Salt Lake, Utah. What is her connection with Deep Lake? Well, nothing directly, but another species of "Halohasta" was recovered in China by Cui and colleagues, and in general, salt lakes around the world appear to be part of a 'global salt pond', as strains and viruses seem to move rapidly between them. In this way, Carol and Bonnie's work has added to the general knowledge of all salt lakes, including those in the Antarctic.

Mike D-S, October, 2012.

 

References

Bowman JP et al. (2000) The microbial composition of three limnologically disparate hypersaline Antarctic lakes. FEMS Microbiol Lett, 183:81-88.

Franzman PD et al. (1988) Halobacterium lacusprofundi sp. nov., a halophilic bacterium isolated from Deep Lake, Antarctica. System Appl Microbiol, 11:20-27.

Oh, D. (2007) Diversity of Haloarchaea across three Australian Solar Salterns and Deep Lake, Antarctica. B.Sc.(hons) thesis, Univ. Melb.

Tripepi M, Imam S, Pohlschroder M (2010) Haloferax volcanii flagella are required for motility but are not involved in PibD-dependent surface adhesion. J. Bact. 192:3093-3102.

Wright SW, Burton HR (1981) The biology of antarctic saline lakes. Hydrobiologia 82:319-338.