Isolation of haloarchaea, growth patterns and Gram staining
We plated out each sample directly onto 30% SW agar plates containing 30% salts and 0.5% yeast extract, spread the soil particles evenly on the surface of the plate with a spatula, and incubated at 37°C. After 8 days, many pink colonies appeared around soil particles on plates of sample No.7 and No.12 (Fig. ) and a few colonies on plate of No.11 (Fig. ). Further incubation up to 30 days resulted in formation of pink to red colonies on plates of all soil samples, at least a few per plate, that were taken inside of the field. Sample No.12 gave extraordinarily high numbers of colonies and most colonies seem not associated with black soil particles visible with naked eyes. A few white to beige colonies appeared on sand sample No.5 taken at seashore. No colonies appeared from three seawater samples. After repeated transfers on fresh medium, 10 strains were isolated and subjected to the following characterizations.
Figure 3 Photographs of agar plates of samples No.12 (A) and No.11 (B). About 0.5 g of the sample was spread evenly on agar plate and incubated at 37°C for 30 days. Red colonies that appeared after 8 days incubation of plate No.11 (B) were marked with (more ...)
The 10 strains were cultivated in 25% SW liquid medium, and their growth patterns were observed at 37°C. Strains 10-1 and 14-1 grew very slowly, only after 2–3 days lag periods. On the other hand, strains 7-1, 7-2, 11-1, and 14-2 grew fast, with slight growth even after overnight culture. The final cell densities of the strain 7-1 and 7-2, however, were low, about one fifth of those of other 8 strains.
The 10 strains were pre-cultured in 25% SW liquid medium and streaked on agar plates of 25, 3, and 0.5% SW agar media, and incubated at 37°C for 10 days. No growth was observed on 3 and 0.5% SW agar plates.
Gram staining was done on stationary cells grown in 25% SW liquid medium. Strains 7-1, 7-2, 10-1, 10-2, and 11-2 stained negative, while strains 11-1, 13, 14-1, 14-2 were mixtures of cells that stained positive and negative. Most cells of the strain 12 stained gram positive.
The 16S rRNA gene sequences of strains 7-1, 7-2, and 12 showed highest similarities to that of Halogeometricum borinquense, 97.1, 97.0, and 94.6%, respectively. The sequences of strains 10-1, 10-2, 11-2, 13, and 14-1 were very close to that of Halococcus hamelinensis with 98.9–99.5% similarities. Strains 11-1 and 14-2 were most closely related to Haladaptatus paucihalophilus, with similarities of 97.5 and 97.6%, respectively. A phylogenetic tree reproduced by neighbor-joining method is shown in figure . We are in the process of fully characterizing these strains.
Figure 4 A neighbor-joining phylogenetic tree inferred from 16S rRNA gene sequences. Sequences of the 10 strains isolated in this study and those of the type strains of type species of all haloarchaeal genera validly published are comprised, as well as those of (more ...)
Survival rates in low salt solutions
In order to observe if the strains isolated from the salt field are tolerant to hypotonic solutions, three pellets of each strain cultivated in 25% SW liquid medium were suspended in sterile 30%, 3%, and 0.5% SW solutions, respectively, incubated at 37°C, and surviving cell numbers were measured every day. As shown in Fig. , at least 50% of the cells of all strains maintained viability in 30% SW solution for 9 days. Survival rates in 3% and 0.5% SW, however, varied considerably depending on the strains.
Figure 5 Survival rates of the 10 strains in SW solutions. The cells were suspended in the SW solutions containing 30% (●), 3% (○), and 0.5% (▲) salts, kept at 37°C, and viable cell numbers were counted every day. X-axis refers (more ...)
Two strains 7-1 and 7-2 died instantaneously upon suspension in 3% and 0.5% SW solutions, and the cells lysed leaving transparent solutions. Two reference strains, Halogeometricum borinquense JCM 10706T and Halococcus morrhuae JCM 8876T (= ATCC 17082T) also died instantly even in 3% SW solution. The cells of Hgm. borinquense lysed in 3 and 0.5% SW solutions, but the cell suspension of Hcc. morrhuae remained turbid (although red pigments had faded away), with even higher turbidity compared to the cell suspension in 30% SW. Microscopic observation of Hcc. morrhuae confirmed that the cells remained coccoid in 0.5% SW solution.
Viable cell numbers of strains 10-2 and 11-2 in 3% SW decreased to one tenth after 5 days, and cells died in 0.5% SW within a day. Strains 10-1, 12, 13, and 14-1 were slightly more resistant to 0.5% SW and maintained viabilities to some extents in 3% SW up to 9 days. The cell suspension of these strains in 3% and 0.5% SW remained turbid as in the case of Hcc. morrhuae, as was confirmed by microscopic observation.
Strains 11-1 and 14-2 possessed extraordinary strong tolerance to low salt conditions. About 20 and 34% of the cells, respectively, remained viable in 0.5% SW after 9 days incubation at 37°C. These cell suspensions again remained turbid, and no morphological change such as cell aggregation was observed.