185 Biodata of Henk Bolhuis, Author of “Walsby's Square Archaeon; It's

Biodata of Henk Bolhuis, author of “Walsby’s Square Archaeon; It’s Hip to be Square, But Even More Hip to be Culturable” Dr. Henk Bolhuis is currently a post doctoral researcher at the Department of Microbial Ecology of the University of Groningen, The Netherlands. He obtained his Ph.D. from the University of Groningen in 1996. After a post doc position at the Max Planck institute for Biochemistry in Martinsried, Germany, he returned to the University of Groningen. Dr. Bolhuis scientific interests are in the areas of: microbial evolution with the emphasis on halophilic Archaea, microbial diversity in extreme environments and marine ecosystems and microbial genomics. E-mail: [email protected] 185 N. Gunde-Cimerman et al. (eds.), Adaptation to Life at High Salt Concentrations in Archaea, Bacteria, and Eukarya, 185-199. © 2005 Springer. Printed in the Netherlands. WALSBY’S SQUARE ARCHAEON It’s Hip To Be Square, But Even More Hip To Be Culturable HENK BOLHUIS Lab for Microbial Ecology, Centre of Ecological and Evolutionary Studies, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands “The mask that you wore - My fingers would explore The costume of control - Excitement soon unfolds” Jim Morrison, Easy Ride 1. Historical Background Twenty-five years ago Anthony Walsby and his wife Fausta paid a short visit to Eilat in Israel, principally to look at the Solar Lake on the Sinai Peninsula. Wolfgang Krumbein, who at that moment was working in Eilat, invited them for a two-day trip to Ophira and the Sabkha Gavish in the south of Sinai. Walsby collected samples from salty puddles that were used by Bedouin to collect solar salt. Upon return to the Marine Science Department in Menai Bridge, North Wales, he analysed the samples by light microscope in search for gas-vacuolate cyanobacteria. It was then that he saw the square 'bacteria' for the first time (Walsby, 1980). The original report of this discovery, which he published in Nature, was received with some scepticism since at that time cells with that unique shape were unknown. It is now well established that these square structures are living micro-organisms although they are still the only known examples with such a perfectly square morphology. Walsby told me that in his manuscript he originally proposed to name the organism "Quadra fausta", for his wife, but with the pretence that "fausta" (Latin "fortunate") reflected the serendipity of the discovery. However, one of the referees suggested not to name it until it was in axenic culture and so this name was left out of the final 1980 publication. Walsby said he spent several months trying to isolate the squares, but without success. Thereafter, many others tried to isolate these intriguing squares for further characterisation. Until now nobody succeeded, despite the fact that these square cells were quite large, easily recognised by microscopy, and highly abundant in crystalliser ponds, which were easily accessible. This enigmatic organism stubbornly remained in the long list of unculturables. 2. The Unculturables Hampered by the fact that many micro-organisms are not easily cultivated, and due to the introduction of novel molecular biological tools, microbial ecological research has 188 HENK BOLHUIS shifted from the more traditional cultivation based analysis of microbial communities to a state of the art molecular discipline. The molecular approach has focused largely on the conserved 16S rRNA gene encoding the prokaryotic ribosomal small subunit RNA molecule, as a molecular identifier for micro-organisms (Woese and Fox, 1977). One of the major conclusions to be drawn from the culture independent analysis of various ecosystems is that the extant microbial diversity is far greater than previously thought. Analysis based on 16S rRNA gene sequence comparisons led to the discovery of many novel microbial taxa and species. Nevertheless, these studies also have important limitations. Often they do not get beyond assigning putative relationships with other organisms and speculations on their eco-physiological role are based on similarities with related cultivated species, which is very unreliable since microbes can be very diverse in their physiology and genetic makeup even within a narrow range of variation of the 16S rRNA gene sequence (Rodríguez-Valera, 2002). Therefore cultivation is still the method of choice to fully understand the physiology and complex ecological interactions in which micro-organisms engage. However a common problem in microbiology is the fact that a large number of micro-organisms that are present in an ecosystem resist cultivation with the conventional tools. Many of these “unculturables” are well known to microbiologist either by molecular detection, visual observation or by their biochemical signatures of reactions they catalyse. Unfortunately, the species that seem less important for the ecosystem functioning are relatively easily cultivated, whereas the ecologically relevant and dominant organisms often resist cultivation. Nevertheless, improvement of cultivation techniques and insight in their specific requirements from physiological studies led to a number of recent successes in which micro-organisms previously thought to be unculturable were isolated. Well known examples are species of “Pelagibacter“ belonging to the abundant marine SAR11 cluster of bacteria (Morris et al., 2002; Rappé et al., 2002) and the anaerobic ammonium oxidising planktomycetes (anammox) (Strous et al., 1999). In this overview I will summarise our current knowledge about one of the most intriguing micro-organism that for long could not be cultivated, the enigmatic halophilic square archaeon, better known as “Walsby’s Square Bacterium”. The square archaeon has recently been isolated in both my lab in Groningen, The Netherlands (Bolhuis et al., 2004) and in the lab of Mike Dyall-Smith, Melbourne, Australia (Burns et al., 2004b). Although the subject of this review has become widely know as Walsby’s Square Bacterium, I will refer hereafter only to Walsby’s Square Archaeon since phylogenetically this organism belongs to the domain of Archaea and further confusion about its phylogenetic position should be prevented. 3. Culture Independent Characterisation of Walsby’s Square Archaeon As described above, the square Archaea are found in hypersaline ecosystems. These include naturally formed or man-made ecosystems in which seawater evaporates, leading to a concentration and sequential precipitation of calcium carbonate and calcium sulphate, and leaving a hypersaline sodium chloride rich brine. Further evaporation and concentration of the brine result in the precipitation of sodium chloride (halite), where WALSBY’S SQUARE ARCHAEON 189 after a dense magnesium chloride brine develops (bittern) that eventually becomes hostile to life (Oren, 1994). It is especially at the last stage of halite formation and before the brines get sterile, where the squares dominate (Benlloch et al., 1995). The original finding place of Walsby’s Square Archaeon in the Sinai Peninsula was visited by Walther Stoeckenius in August 1980 who described it as a pool of which most of the 500 m diameter depression was evaporated. At that time of the year, it solely consisted of a 10 to 15 meter wide and 150 m long pool that was not more than 20-50 cm deep (Stoeckenius, 1981). However as Walsby remarked he suspected that Stoeckenius would not have had to travel this far and that similar organisms are likely to be found in other hypersaline ecosystems as well (A. Walsby, personal communication). This was indeed true since Stoeckenius later found square Archaea close to his own lab in the San Francisco Bay salterns (Stoeckenius, 1981). It is currently known that these organisms are widely distributed and can be found in a variety of hypersaline environments that are close or above sodium chloride saturation such as the well studied crystalliser ponds in Spain (Benlloch et al., 1995, 1996, 2001, 2002; Bolhuis et al., 2004), near San Francisco Bay and in Baja California (Stoeckenius, 1981), Australia (Burns et al., 2004a, 2004b), Israel (Oren, 1993) and in Lake Saxkoye, Russia (Oren, 1994). In the absence of molecular tools and isolation procedures, the square archaeon has initially mainly been studied by (electron-) microscopy or on basis of physiological tests on samples that were naturally enriched in the abundantly present square Archaea. However, since none of these samples were pure, caution had to be made in drawing conclusions from the experiments. Below I will summarise a number of the initial observations and physiological test. 3.1. STRUCTURAL FEATURES 3.1.1. The Cell Envelope In order to provide evidence to criticasters that the square cells were indeed living micro-organisms rather than some crystalline artefacts, Walsby conducted an electron microscopic analysis of the cells in collaboration with Ken Parkes. They showed that the surface of the squares was covered with ~20 nm particles that were arranged in a regular hexagonal lattice later to be identified as a surface layer (S-layer) and a characteristic feature for haloarchaea (Parkes and Walsby, 1981). The presence of this S-layer in the square cells led Walsby to his, as was shown later correct, assumption that the squares were related to haloarchaea (Walsby, 1980). In general, S-layers are formed from identical protein subunits arranged into a monolayer of simple and repetitive patterns (Eichler, 2003). Archaeal S-layers are dominated by glycosylated proteins that, like other haloarchaeal

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