Diversity of Haloquadratum and other haloarchaea in three, geographically distant, Australian saltern crystallizer ponds.

Dickson Oh, Kate Porter, Brendan Russ, David Burns and Mike Dyall-Smith. Extremophiles (published online 20 Dec, 2009)

 This study was initiated in 2007 to try to understand more about the diversity, or rather the lack of diversity, of square haloarchaea, ie. Haloquadratum species. In 2004 we were able to grow this fascinating organism, which has cells that are exquisitely thin, geometrically regular squares. It was formally described in 2007. We have since recovered several other isolates from different salt lakes but the 16S rRNA data indicated that the genus contains only one species, and that all were extremely closely related. For example, the 16S rRNA gene sequence of the Australian type strain (C23), when compared to the corresponding sequence of a Spanish isolate, showed only 2 base differences, yet they were isolated over 16000 km apart, by different laboratories, using different methods. lab photo early 2009

Dickson Oh came from Singapore to Melbourne in 2007 to do his B.Sc. honours year with me. Aided by David Burns (who isolated the C23 type strain), Kate Porter and Brendan Russ, Dickson was able to recover 16S rRNA gene sequences and isolates from water samples taken from saltern crystallizer ponds in Queensland, South Australia and Victoria. The picture at left shows (L-R) me, Dickson, Kate, Dave and Brendan, at the beginning of 2007. The results showed that Haloquadratum sequences were all very close, much more so for other genera of haloarchaea found in the very same lakes/crystallizer ponds. A comparison with published sequence data from Spain, Turkey, Tunisia, Israel, and Peru confirmed that these organisms are extremely closely related to each other, forming one species with a restricted diversity. The puzzle that is now presented is how to explain such uniformity across such huge distances. One possibility is a global dispersal mechanism that spreads the most successful strain to suitable sites all over the world. This is believed to occur for many Bacteria, particularly those that live in soil or in the oceans. However, Haloquadratum walsbyi cells are very fragile, and require very high salt concentrations. They are usually found in isolated ponds, and even if close to the sea, they cannot tolerate the 3.5% salt concentration of seawater. They do not form resistant spores. There are still two possibilities that could be tested: (1) they could become entrapped in minute salt crystals that are then carried long distances by air currents, and/or (2) they could be carried on the exterior surfaces of migratory birds.

The actual study included cultivation independent and cultivation dependent aspects. For the former work, cellular DNA was extracted and rRNA genes PCR amplified, cloned and fully sequenced. For the latter work, Dickson prepared enormous numbers of plates and liquid extinction (dilution) cluster trays that needed to be incubated for > 12 weeks. The pictures below show some examples of these on his bench.

lab pic early 2009
Plates ready for inoculation

Dilution trays after lengthy incubation. Pink wells have haloarchaea

Colonies appearing on DBCM2 plates. Some show distinct cloudiness around them (why?)

To analyse the sequence data accurately, and to obtain community statistics that could be compared to other studies, I used a recently available suite of tools that are freely available from Pat Schloss, and called MOTHUR. With this I could output diversity indices, the 'heat map' diagram below, and many other useful values. Together with the phylogenetic tree reconstructions generated in the ARB package, I was able to summarise the important features of the sequence data and output them in a readily interpretable form. I can recommend both packages (and they work on Macs!).

Apart from the primary interest in the diversity of Haloquadratum, there were a number of other important results from this study. One was the recovery of two new isolates of Hqr. walsbyi, including one with distinctively large ges vesicles (Bajool9, currently under study). A second was the isolation of novel haloarchaea belonging to a new genus, and the third was the detection of sequences that represent many new taxa of haloarchaea, including one that is above the genus level! The latter I've termed the MSP8 clade, after a couple of sequences recovered in 1998 by Grant et al. in an alkaline soda lake in eastern Africa. These aspects are currently being followed up.heat map

The 'heat map' shown nearby depicts the 'species-level' taxa that are shared or not-shared between the 3 Australian sample sites (Bajool in Qld., Dry Creek in SA, and Lara in Vic.). The two top lines are Halorubrum and Haloquadratum species, respectively (shared by all 3 sites). The red colour indicates dominance, and it is no suprise that Haloquadratum is dominant at all sites. But apart from these two genera there are many other taxa that are unique to a single site (the lines do not extend horizontally to an adjacent site), and many of these are novel, and remain to be isolated in a laboratory and their properties determined. The take home message is that there is still much to learn about the microbial communities of salt lakes.

In addition to this published study of Haloquadratum diversity, Dickson also studied the microbial diversity of Deep Lake, Antarctica. This is a hypersaline lake that has an average annual temperature of -20C! A water sample was provided by Rick Cavicchioli of the UNSW, and gave us a fascinating view of the types of microorganisms that have to deal with a combination of saturating salt, below zero C temperatures, and oligotrophic conditions. Much more is to come from this initial work, including a novel haloarchaeal genus, and the the genomic sequencing of the major prokaryotic groups (recently funded by JGI). This was a remarkably successful honours year, and even though the lab was closed in 2008, I was able to continue working with the data and strains in Germany, resulting in the 2010 publication and submission of the tADL isolate to JGI for sequencing.

Mike Dyall-Smith, Dez 2009

Update: by 2012, the Oh et al. (2010) paper had been cited 12 times, and the genome sequence of the antarctic haloarchaeon tADL, a dominant member of the Deep Lake microbial community, had been completed by JGI. It was also included in a study of biofilm formation, as it attaches strongly to glass and plastic (Fröls et al., 2012). A novel strain of Hqr. walsbyi (Bajool 9) had been deposited in culture collections and sent to other laboratories. Not bad for an honours year.