The genome of the square archaeon Haloquadratum walsbyi : life at the limits of water activity
© Bolhuis et al. 2006
Received: 17 March 2006
Accepted: 04 July 2006
Published: 04 July 2006
Skip to main content
© Bolhuis et al. 2006
Received: 17 March 2006
Accepted: 04 July 2006
Published: 04 July 2006
The square halophilic archaeon Haloquadratum walsbyi dominates NaCl-saturated and MgCl2 enriched aquatic ecosystems, which imposes a serious desiccation stress, caused by the extremely low water activity. The genome sequence was analyzed and physiological and physical experiments were carried out in order to reveal how H. walsbyi has specialized into its narrow and hostile ecological niche and found ways to cope with the desiccation stress.
A rich repertoire of proteins involved in phosphate metabolism, phototrophic growth and extracellular protective polymers, including the largest archaeal protein (9159 amino acids), a homolog to eukaryotic mucins, are amongst the most outstanding features. A relatively low GC content (47.9%), 15–20% less than in other halophilic archaea, and one of the lowest coding densities (76.5%) known for prokaryotes might be an indication for the specialization in its unique environment
Although no direct genetic indication was found that can explain how this peculiar organism retains its square shape, the genome revealed several unique adaptive traits that allow this organism to thrive in its specific and extreme niche.
Halophilic archaea (hereafter haloarchaea) predominate in NaCl-saturated aquatic ecosystems in which the salinity increases up to about ten-times the average seawater concentration. Further concentration of thalassic (seawater derived) hypersaline environments leads to the precipitation of magnesium salts thereby forming the absolute limit for life, since magnesium saturated waters (bitterns) are devoid of active life . And yet, up to this catastrophic event, haloarchaea are plentiful and reach population densities that rival the most productive natural aquatic environments known on Earth. Out of the more than 15 genera of haloarchaea, only one is responsible for the booming population explosion that follows the precipitation of NaCl. Square, non-motile, pigmented archaea dominate in most thalassic NaCl-saturated environments, reaching population densities of over 107 cells per ml. The two unique features of these cells are the wafer like rectangular shape and a cell thickness of not more than 0.1 μm. Already known since the early 1980s as Walsby's square bacterium , the organism resisted attempts to isolation for the next 25 years. However, in 2004, two strains of the square archaeon were independently isolated from a Spanish  and an Australian solar saltern . In their specific habitat these squares are challenged by the sub-lethal conditions of an extremely high MgCl2 concentration and high solar irradiance. The hygroscopic properties of the divalent Mg2+ ions dramatically decrease the water activity (Aw), a measure for the availability of free water molecules for biological processes [5, 6]. The Aw is 1.0 for pure water, 0.75 for a saturated NaCl solution and 0.3 for a saturated MgCl2 solution . The actual Aw of the MgCl2 enriched brines is unknown, but will decrease upon further concentration. Currently an Aw of 0.6 is recognized as the lower limit for life . This means that although the organism thrives in an aqueous environment it suffers severe desiccation stress. Special mechanisms are therefore required to maintain optimal water activity within the cell and at the cell surface. Concomitant with the extremely high salinity, the amount of dissolved oxygen decreases to near anoxia and some essential nutrients (e.g. phosphates) become unavailable due to complexation with Mg2+. Here, we present features from the genome of Haloquadratum walsbyi that might explain the worldwide success of this organism in saturated brines.
A consequence of the extremely high salinity is the decreased solubility of oxygen (about 20% of the amount of oxygen dissolved in freshwater). Low diffusion rates, relatively high temperatures, high oxygen consumption rates, and limited oxygenic photosynthesis leave the NaCl-saturated brines virtually anoxic. Moreover, complexation of essential nutrients with the excessive amounts of cations imposes an additional problem in acquiring sufficient sources of energy, nutrients and trace elements. Oligotrophic microorganisms are well adapted to nutrient limitation, e.g. by increasing the surface to volume ratio thereby optimizing the nutrient uptake capacity relative to cell volume. Most oligotrophes achieve a high surface to volume ratio (s/v) by reducing their cell diameter; H. walsbyi does so by extremely flattening itself . This strategy gives it what is probably the highest s/v ratio within the microbial world. Whereas spherical shaped microorganisms have to remain small in order to retain an optimal s/v ratio, the squares can become unlimitedly large since the s/v ratio solely depends on their thickness which in nature always appears to be very low (0.1 - 0.5 μm). In liquid cultures of H. walsbyi large cells of 40–40 μm and larger have been observed . In analogy to the oligotrophes, the high s/v ratio hints to a lifestyle in which membrane processes are of major importance.
Exceptional among archaea is the presence of a phosphoenolpyruvate (PEP) dependent phosphotransferase (PTS) system involved in the phosphorylation of dihydroxyacetone (DHA). PEP-PTS systems were so far only found in bacteria in which phosphorylation of substrates is coupled to their translocation over the membrane . In many bacteria DHA is phosphorylated by an ATP dependent dihydroxyacetone kinase (DhaK). However, some bacteria and H. walsbyi contain a unique cytosolic PEP-PTS dependent DhaK in which DHA is phosphorylated on the expense of PEP rather than ATP to give dihydroxyacetone-phosphate (DHAP) . DHA is translocated over the membrane via facilitated diffusion, a process that is driven by its concentration gradient. Maintenance of an inwardly-directed DHA gradient is achieved by phosphorylation of DHA by the PTS system in the cytosol rather than by a membrane associated PTS system. DHAP can be used as substrate for gluconeogenesis or glycolysis. In the glycolytic reaction DHAP is converted back to PEP resulting in the net generation of one molecule of pyruvate and one molecule of ATP for each molecule of DHA taken up (Fig. 5). Recent experimentation showed that H. walsbyi can grow on DHA as carbon and energy source (data not shown). Alternatively, DHAP is also an important intermediate in the formation of the stereoisomer sn-glycerol-1-phosphate which is part of the archaea-specific backbone of membrane lipids . Interestingly, dihydroxyacetone is a putative overflow product of glycerol metabolism in Salinibacter ruber, the dominant bacterium in crystallizer ponds . Metabolism of dihydroxyacetone by H. walsbyi might explain the observed synergistic effect on H. walsbyi colony formation when grown in association with S. ruber . In addition to DHA, H. walsbyi can grow on glycerol and pyruvate  but also on amino acids  for which all biosynthesis pathways are completely present. Glycerol and pyruvate are probably taken up by diffusion since specific uptake systems have not been identified. For the amino acids a large repertoire of amino acid uptake systems are present.
General features of the H. walsbyi genome
Replicon length (bp)
G+C content (%)
G+C of transposases (%)
Number of protein-coding genes
Average size of proteins
Percentage coding for proteins or RNAs
Average gene distance
Number of rRNA operons (16S, 23S, 5S)
Number of tRNAs
Number of other RNAs (7S, RNAseP)
Average pI of proteins
Similar to these microorganisms, H. walsbyi occupies a relatively stable but narrow ecological niche. However, nitrogen does not appear to be limiting in its natural habitat, and so we hypothesize that another factor, namely adaptation to the extremely high MgCl2 concentration, is responsible for the drift to an AT rich genome in H. walsbyi. Despite the presence of energy demanding cation efflux systems, the high external magnesium concentration will lead to an increase in the internal magnesium concentration that is higher than in other microorganisms. Magnesium ions are known to have a stabilizing effect on the DNA duplex, the secondary structure of RNA (Carter and Holbrook) and DNA-RNA heteroduplexes. In case of an already stable high-GC genome the additional stabilizing effect of magnesium might result in DNA rigidity that interferes with essential processes like DNA replication and transcription. We propose that the drift to an AT-rich genome might be induced as a long term evolutionary adaptation to this over-stabilization by magnesium and can be balanced by lowering the GC content of the genome.
A related peculiarity of the H. walsbyi genome is its remarkably low coding density (76%) as compared to other haloarchaea (86–91%) and prokaryotes in general . This is due to a very large average intergenic spacing of 289 bp mainly because of a high number of very long (> 1000 bp) intergenic regions. These long intergenic regions consist of non-coding DNA fragments, novel DNA repeat elements and pseudogenes, in most cases remnants of IS transposases. The low coding density, high number of pseudogenes and IS elements, and the drift towards a more AT rich genome may be signs that H. walsbyi is in a stage where it is undergoing genome shrinkage possibly due to its specialization into a very restrictive and specific environment with subsequent lack of growth competition from other species. Although saturated brines are present around the world and already exist since ancient geologic periods, competition with other microbes will be very relaxed in these physically limited environments, in a way similar to what happens with intracellular parasites or endosymbionts. The regular desiccation of these evaporative systems might act as evolutionary bottlenecks also favoring genome degradation .
The 47 kb plasmid PL47 has a homogeneous GC distribution, is similar in GC content to the chromosome (Table 1) and contains thirty-nine open reading frames. Most genes are hypothetical or conserved hypothetical. Of the identified genes, the majority encode proteins involved in plasmid maintenance, replication and restriction modification with the majority being of bacterial or viral (phage) descent rather than of archaeal descent. Probably these proteins are dedicated to the replication and maintenance of the plasmid itself. However, the plasmid replication protein RepH is not encoded on the plasmid but is located on the main chromosome. In addition, the plasmid does not contain a homolog of the CDC6 cell division control protein that is commonly found on the smaller replicons of other haloarchaea. The gene coverage (69%) of PL47 is even lower than that of the chromosome with an average gene distance of 371 bp.
In addition to its eye-catching shape, the square archaeon H. walsbyi is in many ways unique amongst haloarchaea. Its genome revealed a broad range of novel adaptive traits in both genome composition and protein sequences that may have contributed to this organism's domination in saturated brines. Further functional studies are required to test these assumptions. Finally, these findings provide clues about how life is possible in the 5 M MgCl2 containing Discovery basin in the Mediterranean deep sea that was recently shown to contain a unique microbial community  and possibly even in the proposed brines at the surface of Jupiter's moons Europa and Ganymede.
The Spanish isolate of the square halophilic archaeon Haloquadratum walsbyi strain HBSQ001 (DSM 16790) was grown to end exponential phase as described before . H. walsbyi was sequenced with 6.5-fold sequence coverage using a shotgun clone library (average insert size of 3 kb), and assembled with the PHRED-PHRAP-CONSED package . The sequence is of high quality (0.01 Errors/10 kb).
For gene prediction, REGANOR  from the annotation package GENDB  was used, which integrates results from CRITICA  and GLIMMER . The automatically predicted ORF set (3013 ORFs) was expert-curated resulting in a theoretical proteome of 2777 proteins. Curation involved sequence comparison to proteins from other halophiles (Halobacterium salinarum strain R1, ), Natronomonas pharaonis , Haloarcula marismortui  and public protein sequence databases. This permitted to identify additional small proteins and to improve the correctness of start codon assignments. tRNAs and other RNAs were predicted using tRNAscan  and BLAST  against other halophiles, respectively. Phylogenetic analysis of proteins was performed using the Microbial Genome Analysis System package MiGenAS [38, 39] and the MEGA3 phylogenetic tool software package [40, 41].
The genome can be accessed via HaloLex . General features and statistics on the genome of H. walsbyi are shown in Table S1. The main origin of replication is located in a highly conserved region and consists of a conserved stem-loop structure, and open reading frames encoding the conserved CDC6 cell division control protein, a signal sequence peptidase and DNA polymerase B . The sequence has been submitted to EMBL under the accession numbers [EMBL:AM180088, EMBL:AM180089] for the genome and plasmid PL47 respectively.
The RNA was extracted with peqGold RNAPure extraction solution (Peqlab Biotechnology) following the manufacturers instructions. After dissolving RNA in DEPC-H2O residual DNA was digested using the "DNA-free" kit (Ambion) following the manufacturers instructions. The quality of the RNA was checked using the 2100 Bioanalyzer (Agilent) and the RNA Nano LabChip (Agilent).
Total RNA was reverse transcribed into cDNA using SuperScript II (Invitrogen) following the manufacturer's instructions with 2 μg total RNA per reaction as template and the gene specific primers pcr4-rev and pcr7-rev, respectively.
The PCR reactions were performed using HotStarTaq (Qiagen) (50 μl per reaction) and 0.5 μl of the cDNA samples as template. The following temperature profile was applied on a Thermocycler T3 (Biometra): 95°C 15 min; 40× (95°C 30 sec, 60°C 30 sec, 72°C 50 sec (500 bp-PCRs) or 90 sec (1 kbp-PCRs). Subsequently the PCR reactions were analyzed by standard agarose gel electrophoresis.
pcr1-for: 5'-CAT TGG ATC GGT GTC TGC ACA GCA AC-3'
pcr1-rev: 5'-GCG CCG CTT GAA GGA GTT ATT TGC G-3'
pcr2-for: 5'-GAT CAC GCT CGA CGA CCT CG-3'
pcr2-rev: 5'-CGT TGA TGA CGC CAG CCT GC-3'
pcr3-for: 5'-CCA CTG GTC AGG TGA ATG CCT C-3'
pcr3-rev: 5'-CTT CCT GTC GCA TCC GAC TGG-3'
pcr4-for: 5'-GAC GCT ACT GCC ACC GGC GAT G-3'
pcr4-rev: 5'-GCA GAC CCG TGT TCG AAC CGT CC-3'
pcr5-for: 5'-GGA CTT GCT GGC ACG ATC GAC-3'
pcr5-rev: 5'-CTC CAG ATG TGC CAA CCT CGC-3'
pcr6-for: 5'-GCG GTT GAG TGG TAT CTT CAC C-3'
pcr6-rev: 5'-GCT ATC GGT GGC GGT GTC G-3'
pcr7-for: 5'-CTC CCC ATC CAG TAG TCG GTC ATT GG-3'
pcr7-rev: 5'-GAT TGT ATC CTC TCA AAT GCC CCG CTA AG-3'
We thank A. Mira and B. Poolman for critical reading the manuscript and helpful discussions, B-A Legault for support with phylogenetic analysis, H. Engelhardt for the electron tomographic image and R. Brouwer, T. Gillich, F. Schoetz, V. Hickmann for additional bioinformatics support and S. von Gronau for technical assistance. This work was supported by a grant to H.B. from the Netherlands Organization of Science NWO/ALW/NPP-851.20.023, and a grant from the GEMINI project QLK3-CT-2002-02056.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.