Morphological Phenotypes, Cell Division, and Gene Expression of Escherichia coli under High Concentration of Sodium Sulfate

Abstract

Sodium and sulfate ions are among the suggested abundant ions on Europa, a moon of Jupiter. In order to investigate the potential habitability of Europa, we study the effects of sodium sulfate (Na₂SO₄) on a non-halophilic bacterium by subjecting Escherichia coli (E. coli) to a wide range of Na₂SO₄ concentrations (0-1.0 m). We discover that, as the concentration of sodium sulfate increases, the biomass doubling time increases and the cell growth is completely inhibited at 1.0 m Na₂SO₄. Furthermore, we find that E. coli exhibits three distinct morphological phenotypes-(i) shortened, (ii) normal, and (iii) elongated/filamented cells at 0.6 m and 0.8 m Na₂SO₄. We have examined the expression of different genes involved in sodium and sulfate transport (nhaA, nhaB, cysZ, sbp), osmotically driven transport of water (aqpZ), sulfate metabolism (cysN), fatty acid production (fabA), and a global transcriptional regulator (osmZ). Our results suggest that the expression of these genes is not affected significantly at high concentrations of sodium sulfate in the exponential growth phase. Using our experimental data and the existing data in the literature, we show that the osmotic pressure difference may play a major role in determining the growth inhibition of E. coli and B. subtilis at high concentrations of salt.

Keywords: cellular response of Escherichia coli under hyperosmolar stress; habitability of Europa; sodium sulfate.

Figure 1

Schematic of the cell membrane and membrane proteins such as ion channels, carrier proteins, and receptors. Cells interact with the external environment via membrane proteins.

Figure 2

Schematic of the experimental setup used to measure the optical density, OD${}_{488}$, of bacteria in real time.

Figure 3

(A) Linear-log plot of the growth curves of E. coli at different concentrations of Na₂SO₄. (B) Biomass doubling time, ${\tau }_d$, of bacteria as a function of salt concentration. The biomass doubling time, ${\tau }_d$, is calculated by fitting exponential curves through the data points (shown as solid lines in A) in the exponential growth phase. (C) Survival fraction of bacteria as a function of Na₂SO₄ concentration.

Figure 4

Representative images of cells at different concentrations (A) 0 m (control), (B) $0.2$ m, (C) $0.4$ m, (D) $0.6$ m, (E) $0.8$ m, and (F) $1.0$ m of Na₂SO₄.

Figure 5

Probability distribution function, $P(\ell )$, of cell length, ℓ, for Na₂SO₄ concentrations: (A) 0 m, (B) $0.2$ m, (C) $0.4$ m, (D) $0.6$ m, (E) 0.8 m, and (F) $1.0$ m. The cell length distribution exhibits large heterogeneities at high concentrations of sodium sulfate. For $1.0$ m Na₂SO₄, the bacterial cells do not exhibit any growth, and the cell length becomes smaller, presumably due to water efflux. Coefficient of variation (CV) of the probability distributions for all the salt concentrations are also shown.

Figure 6

(A) Average cell length, $〈\ell 〉$, as a function of sodium sulfate concentration. $〈\ell 〉$ decreases slightly at $0.2$ m and $0.4$ m, then it increases monotonically between $0.4$ m and $0.8$ m, and becomes smaller at $1.0$ m Na₂SO₄. (B) Variance of the cell length, ${\sigma }_{\ell }^2$, as a function of sodium sulfate concentration. The variance increases monotonically with Na₂SO₄ concentration up to $0.8$ m.

Figure 7

Time-lapse images of cells obtained at $0.8$ m Na₂SO₄ and subsequently grown on a thin LB-agar chamber without sodium sulfate.

Figure 8

Comparative expression of various genes involved in water transport (aqpZ), sodium transport (nhaA, nhaB), sulfate transport (cysZ, sbp), sulfate metabolism (cysN), a global transcriptional regulator (osmZ), and fatty acid production (fabA). We do not find significant changes in the expression of these genes ($〈\Delta \Delta C_t〉\le 0.5)$ at high salt concentration during the exponential growth phase. cysZ and nhaB exhibit slight upregulation within the error bar. However, this is not conclusive from our data due to large error bar with the $〈\Delta \Delta C_t〉\le 0.5$.

Figure 9

(A) Water activity, $a_W$, and (B) osmotic pressure, $\Pi$, as a function of concentration of aqueous solutions of sodium and magnesium sulfate at T = 37 ${}^{\circ }$C, computed using the Pitzer model.

Figure 10

Osmotic pressure, $\Pi$, as a function of concentration of aqueous solutions of various salts at T = 37 ${}^{\circ }$C, computed using the Pitzer model. We also show the osmotic pressures corresponding to the limiting salt concentrations of growth of E. coli and B. subtilis.