An extremely halophilic proteobacterium combines a highly acidic proteome with a low cytoplasmic potassium content

Abstract

Halophilic archaea accumulate molar concentrations of KCl in their cytoplasm as an osmoprotectant and have evolved highly acidic proteomes that function only at high salinity. We examined osmoprotection in the photosynthetic Proteobacteria Halorhodospira halophila and Halorhodospira halochloris. Genome sequencing and isoelectric focusing gel electrophoresis showed that the proteome of H. halophila is acidic. In line with this finding, H. halophila accumulated molar concentrations of KCl when grown in high salt medium as detected by x-ray microanalysis and plasma emission spectrometry. This result extends the taxonomic range of organisms using KCl as a main osmoprotectant to the Proteobacteria. The closely related organism H. halochloris does not exhibit an acidic proteome, matching its inability to accumulate K(+). This observation indicates recent evolutionary changes in the osmoprotection strategy of these organisms. Upon growth of H. halophila in low salt medium, its cytoplasmic K(+) content matches that of Escherichia coli, revealing an acidic proteome that can function in the absence of high cytoplasmic salt concentrations. These findings necessitate a reassessment of two central aspects of theories for understanding extreme halophiles. First, we conclude that proteome acidity is not driven by stabilizing interactions between K(+) ions and acidic side chains but by the need for maintaining sufficient solvation and hydration of the protein surface at high salinity through strongly hydrated carboxylates. Second, we propose that obligate protein halophilicity is a non-adaptive property resulting from genetic drift in which constructive neutral evolution progressively incorporates weakly stabilizing K(+)-binding sites on an increasingly acidic protein surface.

FIGURE 1.

Effect of salt concentration on the growth of H. halophila (●) and H. halochloris (□). The dependence of the doubling time (a) and final absorbance (b) on the salinity of the growth medium is shown. c, typical growth curves of H. halophila at 5% (●), 15% (■), and 35% (▴) NaCl.

FIGURE 2.

H. halophila has an acidic proteome. a, proteomic distribution of calculated pI values for H. halophila (gray trace) and S. ruber (black trace) compared with three extremely halophilic halobacteria known to utilize K⁺ as an osmoprotectant (H. salinarum, H. marismortui, and N. pharaonis (middle three traces)) and three non-halophilic Proteobacteria (N. oceani, E. coli, and the purple phototroph R. sphaeroides (upper three traces), all three taxonomically fairly closely related to H. halophila). The pI distributions were normalized to the same total number of proteins. b, isoelectric focusing gel electrophoresis of total cell extracts from E. coli (lane 2), H. salinarum (lane 3), H. halophila (lane 4), and H. halochloris (lane 6) grown at 35% NaCl. Lane 1, 5, and 7 contain the indicated pI markers.

FIGURE 3.

H. halophila utilizes KCl as an osmoprotectant. a, x-ray microanalysis of the elemental composition of H. halophila (blue), H. halochloris (red), and H. salinarum (black) grown in the presence of 35% NaCl compared with that of E. coli (green). The cells were washed with isotonic ammonium sulfate solutions just prior to electron microscopy sample preparation. a.u., arbitrary units. b, the same set of samples as indicated in a were used to determine the cytoplasmic K⁺ concentrations (gray bars) by plasma emission spectrometry and of Cl⁻ concentrations (black bars) using a colorimetric assay.

FIGURE 4.

Effect of medium salinity on cytoplasmic KCl content and proteome acidity in Halorhodospira. a, dependence on medium salinity of cytoplasmic K⁺ (open symbols) and Cl⁻ (closed symbols) content of H. halophila (circles and solid lines), H. halochloris (squares and dashed lines), and H. salinarum (triangles and dotted lines) as determined by plasma emission spectrometry and calorimetrically, respectively. The typical K⁺ and Cl⁻ contents of E. coli near 200 mm are indicated by the arrow. b, elemental composition of H. halophila grown in media containing 5% (black), 15% (red), and 35% (blue) NaCl compared with that of E. coli (green). a.u., arbitrary units. c, effect of medium NaCl concentration (5, 15, and 35%) on proteome acidity of H. halophila as detected by isoelectric focusing gel electrophoresis.

FIGURE 5.

Effect of cell washing procedure on elemental analysis of H. halophila. The results of x-ray microanalysis of H. halophila cells obtained after washing the indicated cell samples with isotonic NaCl (a) and ammonium sulfate (b) solutions are depicted. a.u., arbitrary units.