Extremely halophilic bacteria in crystallizer ponds from solar salterns

Abstract

It is generally assumed that hypersaline environments with sodium chloride concentrations close to saturation are dominated by halophilic members of the domain Archaea, while Bacteria are not considered to be relevant in this kind of environment. Here, we report the high abundance and growth of a new group of hitherto-uncultured Bacteria in crystallizer ponds (salinity, from 30 to 37%) from multipond solar salterns. In the present study, these Bacteria constituted from 5 to 25% of the total prokaryotic community and were affiliated with the Cytophaga-Flavobacterium-Bacteroides phylum. Growth was demonstrated in saturated NaCl. A provisional classification of this new bacterial group as "Candidatus Salinibacter gen. nov." is proposed. The perception that Archaea are the only ecologically relevant prokaryotes in hypersaline aquatic environments should be revised.

FIG. 1

Phylogenetic tree based on 16S rDNA sequences from the complete EHB sequences and all almost-complete sequences of the CFB phylum available (over 370) and representatives of the domain Bacteria. The multifurcation indicates a topology that could not be unambiguously resolved. The phylogenetic position of EHB sequences did not differ in any of the treeing approaches. The bar indicates 10% of estimated sequence divergence. Sequence accession numbers: Chlorobium limicola, Y10640; Halomonas aquamarina, M93352; Haloanaerobacter lacunaris, X89075; Haloanaerobium lacusroseus, L39787; E. halophila, M26630; R. marinus, X77140; R. obamensis, X95071; and Spirulina subsalsa, AB003166.

FIG. 2

Identification by FISH of Bacteria in samples from crystallizer ponds. Identical microscopic fields were visualized with an epifluorescence microscope using filter sets specific for DAPI (a and c) and the fluorochromes used for probe labeling (b and d). (a and b) Cells from the crystallizer in Majorca hybridized with Cy3-labeled probe EHB412. (c and d) Cells from CR-30 (Alicante) hybridized with fluorescein-labeled probe EHB586 and Cy3-labeled probe EHB1451. Bar, 5 μm.

FIG. 3

Growth curve at 37°C of the halophilic bacteria detected with probe EHB412. Liquid media containing 0.1% (wt/vol) yeast extract and a salt mixture (“sw” in reference 27) at concentrations (vol/vol) of 20% (circles), 25% (inverted triangles), 30% (squares), and 30% plus NaCl up to saturation (triangles) were inoculated with crystallizer water (1% [vol/vol]) and incubated at 37°C. EHB were detected by hybridization with probe EHB412 (filled symbols). Empty symbols, total DAPI counts.