The genome of the square archaeon Haloquadratum walsbyi : life at the limits of water activity

Abstract

Background: 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.

Results: 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

Conclusion: 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.

Figure 1

H. walsbyi encodes halomucin, an extremely large protein homologous to mammalian mucins. GC content (C) and gene organization (B) of a 60.000 bp long region of the genome of H. walsbyi. This region contains the large, high GC gene encoding halomucin (hmu1) and the two capsular protein biosynthesis genes capC and capB. The domain organization of halomucin (A) reveals domains typically found in mammalian mucins. The letters D, S, V, G, and L indicate domains enriched for the corresponding amino acids. Halomucin expression is shown by detection of its mRNA via RT-PCR (D). cDNA was prepared by reverse transcription using primer sets numbered 1 to 7 the position of which correspond to the striped and numbered boxes in the hmu1 gene (B).

Figure 2

Phototrophic growth in H. walsbyi. (A) Protein phylogenetic tree showing the position of the "opsin"-homologs encoded by H. walsbyi, indicated as SquareBopI & II and SquareHop for the proton pumping bacteriorhodopsins and chloride pumping halorhodopsin respectively. Trees were constructed with the neighbor-joining method using 1000 bootstrap replicates. Bop, bacteriorhodopsins; Hop, halorhodopsins; SopI & II; sensory-rhodopsins. (B) Phototrophic growth of aerobic (▲) and anaerobic (■) cultures of H. walsbyi incubated in the light. (C) Photo-phosphorylation in H. walsbyi measured through light induced ATP generation (upper curve) and proton (H⁺) extrusion (lower curve). Phylogenetic analysis was performed using the Microbial Genome Analysis System package MiGenAS [38,39].

Figure 3

Electron Tomographic image of a single square cell of H, walsbyi. In agreement with previous observations, gas vesicles (GV) recognized by their spindle shape are found at the borders of the cell. The large number of circular electron-dense bodies are most likely poly-3-hydroxy-butyric acid (PHB) polymers consistent with Nile-Blue staining patterns [4]. The genes encoding PHB biosynthesis proteins have been identified (image by H. Engelhardt).

Figure 4

Phylogenetic tree showing the position of the three β-carotene 15,15' mono-oxygenase homologs HQ2020A, HQ2381A, HQ3007A of H. walsbyi. Trees were constructed with the neighbor-joining method using 1000 bootstrap replicates.

Figure 5

Phosphate metabolism in H. walsbyi. Scheme revealing the proteins involved in phosphate metabolism. The transporters PitA1-3, Phn2ECD, PstABC and phosphate storage enzyme polyphosphate kinase (Ppk) are common in several haloarchaea. Unique to H. walsbyi are the bacterial-type proteins involved in phosphonate metabolism, an ABC-type phosphonate uptake system plus phosphonate lyase, and the PTS dependent dihydroxyacetone kinase (DHAK). DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; Pi, phosphate ion; PEP, phospho enolpyruvate; PL, phospholipid.

Figure 6

Codon usage in halophilic archaea. Indicated is the percentage of G plus C or A plus T at the different codon positions showing the large difference between H. walsbyi and the other haloarchaea especially at the third codon position. Bars are H. walsbyi (blue), H. marismortui (red), N. pharaonis (yellow) and H. salinarum (purple).