Haloquadratum walsbyi

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A Microbial Biorealm page on the genus Haloquadratum walsbyi


Higher order taxa

cellular organisms; Archaea; Euryarchaeota; Halobacteria; Halobacteriales; Halobacteriaceae; Haloquadratum; Haloquadratum walsbyi

(from NCBI



Description and significance

The square halophilic archaeon Haloquadratum walsbyi was first discovered by A.E. Walsby in 1980. First found in water from a salt pool near the Red Sea, Haloquadratum walsbyi was soon found in many salt lakes around the world, making it an archaeon of specific interest because of this abundance in hypersaline ecosystems, which suggests that it plays an important ecological role. A.E. Walsby detected Haloquadratum walsbyi by "conventional microscopy in brine samples collected from a salt crust forming the surface of a hypersaline pool on the Sinai Peninsula" (1).

The cells of "Walsby's square archaeon" possess a unique square-like shape, unlike the spherical or cylindrical shape of many organisms. Easily recognizable for its perfect square morphology, Haloquadratum walsbyi has sharp edges and acute straight corners. (3)

Despite their abundance in salt lakes around the world, cultivation of Haloquadratum walsbyi has been very difficult, and it has been marked an unculturable organism. In "Isolation and cultivation of Walsby's square archaon," the first cultivation of the organism is discussed. (1)

It is important for this organism to be studied because of its halophilicity which suggests that Haloquadratum walsbyi plays an important ecological role in high-salt content habitats. The genome sequence gives insight in the molecular, ecological & physiological properties of the organism, including its square shape and its ability to survive in the hypersaline environments it is found in and the high UV radiation it is exposed to. Additionally, studying the genome sequence might enable us to understand the metabolic properties which allow Haloquadratum walsbyi to live in such an environment and which initially made the organism difficult to cultivate. (2)

Genome structure

Haloquadratum walsbyi was found to be a member of a novel genus within the family of Halobateriaceae, based on its 16S RNA sequence. The 3.1 Mb large genome has been sequenced and is discussed in "The genome of the square archaeon Haloquadratum walsbyi: life at the limits of water activity" by Bolhuis et al (2).

The genome of the Haloquadratum walsbyi strain studied by Bolhuis is comprised of a 3,132,494 bp chromosome (which has 2738 open reading frames) and a 46,867 bp plasmid (which has 39 open reading frames). The genome has a well conserved haloarchael region, which contains the origin of replication and the cell division control protein CDC6_1. (2)

Its genome has a GC content of 47.9%, which is remarkably low compared to the generally high GC content of other haloarchaea (usually 60-70%). This GC content is constant throughout the genome. It is thought that the lowered GC content is compensated by the large number of photolyases which help it cope with the high levels of UV irradiation which it encounters in the shallow coastal lagoons it inhabits. Although its GC content differs from most other haloarchaea, most of the typical haloarchael proteins that Haloquadratum walsbyi encodes were found to be highly conserved at the level of the amino acid sequence. (2)

Notable about H.walsbyi's genome include the following: its low coding density at 75% (compared to the 86-91% of other haloarchaea), its expression of a water enriched capsule, the fact that it encodes two bacteriorhodopsin proteins, as well as its utilization of a dihydroxyacetone via phophoenolpyruvate dependent phosphotransferase system. (2)

The 47 kb plasmid of H.walsbyi, which similarly has a constant GC distribution, contains many genes--many of which are “hypothetical or conserved hypothetical” (2). The majority of the identified genes encode proteins that are involved in the maintenance, replication, and restriction modification of the plasmid. Interestingly, most of these are found to be of bacterial or viral (phage) descent instead of archaeal descent. (2)

Cell structure and metabolism

The very thin and flat Haloquadratum walsbyi cells measure between 1 to 5 micrometers in thickness and have a unique square-like shape. They form sheets of 2D arrays of 10 or more cells. Its surface area is very large compared to its volume due to this structure, which is thought to be helpful in nutrient exchange with the environment (4). Accordingly to Walsby (10), “its shape is probably determined by the pattern in which the cell envelope particles assemble.”

The cells are very fragile and when in sheets, contacts between cells are easily broken. The gas vesicles within Haloquadratum walsbyi are easily collapsed even by gentle pressure. Its cells stained Gram-negative and were found to grow optimally in media with 18% salts around neutral pH (6.5-7.0). Its optimum growth temperature was found to be 45 degrees Celsius and its minimum growth temperature at 25-30 degrees Celsius. (4)

Although it was found to be non-motile, another feature of H.walsbyi (which might contribute to its ability to control its location) is the abundance of intracellular refractile bodies, which were identified as gas vesicles. These gas vesicles were also found to be easily collapsed by gentle pressure. The production of these gas vesicles are thought to provide H.walsbyi with control over its location in its habitat, as suggested by Legault et al. (7). PHA storage granules have also been found in H.walsbyi. (4)

Haloquadratum walsbyi are aerobic heterotrophs, which only use oxygen as a final electron acceptor (4) and can neither use nitrate nor DMSO as alternative acceptors nor grow anaerobically using L-arginine. It was found that H.walsbyi grows best on pyruvate as its sole carbon source (4). H.walsbyi did not produce acid from carbohydrate utilization and was found to not produce beta-galactosidase (4).


Haloquadratum walsbyi was first collected by Anthony Walsby from hypersaline waters in the south of Sinai, but they are found in hypersaline waters all over the world. Ecosystems (both natural and man-made) in which sea water evaporates cause the concentration and precipitation of the compounds calcium carbonate and calcium sulphate. This leaves behind a hypersaline brine that is rich in sodium chloride. These crystalliser ponds further evaporate and concentrate the brines, causing the precipitation of halite (sodium chloride). Dense magnesium chloride brine then develops, but at the last stage of the formation of halite, Haloquadratum walsbyi is seen dominating the ponds. This occurs before the magnesium chloride brines become sterile. At this point, Haloquadratum walsbyi constitutes up to 80% of the microbial biomass.

The fact that Haloquadratum walsbyi is found in such environments (and thrives here) and cannot be cultivated unless in medium that contains a high molarity of MgCl and NaCl indicates its status as one of the most halophilic organisms.

Pathology and Antibiotic Resistance

Nothing was found about how this organism causes disease, although it was found that H.walsbyi have sensitivity and resistance to multiple antibiotics. It is sensitive to the following antibiotics: “anisomycin, choloramphenicol, erythromycin, novobiocin, rifampicin, simvastatin, and tetracycline” (4). It is resistant to the following antibiotics: ampicillin, bacitracin, cycloheximide, kanamycin, mycostatin, neomycin, and streptomycin” (4).

Application to Biotechnology

It is not known if this organism produces any useful compounds or enzymes. However, in Legault’s article “Environmental genomics of Haloquadratum walsbyi in a saltern crystallizer indicates a large pool of accessory genes in an otherwise incoherent species” (7), an accessory gene pool was found that was studied for functional analysis. In the accessory sequence pool, the core functional categories of transcription, translation, and energy metabolism were found to be underrepresented, while peripheral functional categories (e.g. signal transduction, gene regulation) were found to be overrepresented. Additionally, IS-encoded transposases and cell-envelope components were identified in larger amounts than usually found. (7)

Current Research

The findings about its accessory gene pool, particularly the high level of IS elements and phage-related genes, perhaps point to the bacterial adaptation of H.walsbyi, as well as its pathogenecity and its involvement in horizontal gene transfer. The transcriptional regulators and signal transduction genes could possibly produce diversification that allows the organism to respond to environmental changes, helping H.walsbyi optimize the exploitation of resources in various conditions. (7)

Information about the genome has pointed to clues about how life is possible in the hypersaline environments H.walsbyi is found in and possibly even point to the possibility of brines that are proposed to live in the “surface of Jupiter’s moons Europa and Ganymede.” (2)

The phylogenomic analysis of the proteins produced by H.walsbyi along with those of Archaea are being studied as well as their role in the use of the process of methanogenesis by scientists in Canada (5).

The high tolerance of ultraviolent light in haloarchaea is also being studied at the genomic level by scientists in China (11).


1. Bolhuis, H., Poele E.M., and Rodríguez-Valera, F. (2004) "Isolation and cultivation of Walsby's square archaeon." Environ. Microbiol. 6, 1287-1291

2. Bolhuis, H., Palm, P., Wende, A., Falb, M., Rampp, M., Rodriguez-Valera, F., Pfeiffer, F., and Oesterhelt, D. "The genome of the square archaeon Haloquadratum walsbyi : life at the limits of water activity." BMC Genomics (2006) 7:169.

3. Burns DG, Camakaris HM, Janssen PH, Dyall-Smith ML. "Cultivation of Walsby's square haloarchaeon." FEMS Microbiol Lett. 2004 Sep 15;238(2):469-73.

4. Burns DG, Janssen PH, Itoh T, Kamekura M, Li Z, Jensen G, Rodriguez-Valera F, Bolhuis H, Dyall-Smith ML. "Haloquadratum walsbyi gen. nov., sp. nov., the square haloarchaeon of Walsby, isolated from saltern crystallizers in Australia and Spain." Int J Syst Evol Microbiol. 2007 Feb;57(Pt 2):387-92.

5. Gao B, Gupta RS. "Phylogenomic analysis of proteins that are distinctive of Archaea and its main subgroups and the origin of methanogenesis." BMC Genomics. 2007 Mar 29;8:86.

6. Kessel M, Cohen Y. "Ultrastructure of square bacteria from a brine pool in Southern Sinai." J Bacteriol. 1982 May;150(2):851-60.

7. Legault BA, Lopez-Lopez A, Alba-Casado JC, Doolittle WF, Bolhuis H, Rodriguez-Valera F, Papke RT. Environmental genomics of "Haloquadratum walsbyi" in a saltern crystallizer indicates a large pool of accessory genes in an otherwise coherent species. BMC Genomics. 2006 Jul 4;7:171

8. Pasić L, Bartual SG, Ulrih NP, Grabnar M, Velikonja BH. "Diversity of halophilic archaea in the crystallizers of an Adriatic solar saltern." FEMS Microbiol Ecol. 2005 Nov 1;54(3):491-8. Epub 2005 Jul 25.

9. Stoeckenius W. "Walsby's square bacterium: fine structure of an orthogonal procaryote." J Bacteriol. 1981 Oct;148(1):352-60.

10. Walsby A. E. "A square bacterium" Nature 283, 69-71. 03 January 1980. doi:10.1038/283069a0

11. Zhou P, Wen J, Oren A, Chen M, Wu M. "Genomic survey of sequence features for ultraviolet tolerance in haloarchaea (family Halobacteriaceae)." Genomics. 2007 May 9

Edited by student of Rachel Larsen and Kit Pogliano

Edited KMG