An Antarctic Extreme Halophile and Its Polyextremophilic Enzyme: Effects of Perchlorate Salts

Abstract

Effects of perchlorate salts prevalent on the surface of Mars are of significant interest to astrobiology from the perspective of potential life on the Red Planet. Halorubrum lacusprofundi, a cold-adapted halophilic Antarctic archaeon, was able to grow anaerobically on 0.04 M concentration of perchlorate. With increasing concentrations of perchlorate, growth was inhibited, with half-maximal growth rate in ca. 0.3 M NaClO₄ and 0.1 M Mg(ClO₄)₂ under aerobic conditions. Magnesium ions were also inhibitory for growth, but at considerably higher concentrations, with half-maximal growth rate above 1 M. For a purified halophilic β-galactosidase enzyme of H. lacusprofundi expressed in Halobacterium sp. NRC-1, 50% inhibition of catalytic activity was observed at 0.88 M NaClO₄ and 0.13 M Mg(ClO₄)₂. Magnesium ions were a more potent inhibitor of the enzyme than of cell growth. Steady-state kinetic analysis showed that Mg(ClO₄)₂ acts as a mixed inhibitor (K_I = 0.04 M), with magnesium alone being a competitive inhibitor (K_I = 0.3 M) and perchlorate alone acting as a very weak noncompetitive inhibitor (K_I = 2 M). Based on the estimated concentrations of perchlorate salts on the surface of Mars, our results show that neither sodium nor magnesium perchlorates would significantly inhibit growth and enzyme activity of halophiles. This is the first study of perchlorate effects on a purified enzyme. Key Words: Halophilic archaea-Perchlorate-Enzyme inhibition-Magnesium. Astrobiology 18, 412-418.

FIG. 1.

Anaerobic growth assay of Halorubrum lacusprofundi and Halobacterium sp. NRC-1. Graphs show the anaerobic growth of H. lacusprofundi (solid bars) and Halobacterium sp. NRC-1 (slashed bars) in the presence (gray) and absence (black) of 40 mM NaClO₄ 0 or 8 days after inoculation. Error bars indicate standard deviation (n = 3).

FIG. 2.

Growth rates of Halorubrum lacusprofundi and Halobacterium sp. NRC-1 in the presence of added MgCl₂, NaClO₄, or Mg(ClO₄)₂. Graphs show the percent growth rate for Halobacterium sp. NRC-1 (dashed) and H. lacusprofundi (solid) when various concentrations of MgCl₂ (black), NaClO₄ (dark gray), or Mg(ClO₄)₂ (light gray) were added to CM⁺ medium for Halobacterium sp. NRC-1 and ATCC 1682 for H. lacusprofundi.

FIG. 3.

Percent catalytic efficiency of β-galactosidase (pRK42) under the influence of different ions. Kinetic experiments were performed in various solutions using 10 μg/mL of enzyme per reaction at 50°C with ONPG concentrations of 1, 2.5, and 5 mM. Various molarities of NaCl were tested with no added magnesium or perchlorate (black). Ratios of sodium and magnesium chloride were tested (dotted gray). Ratios of sodium chloride and perchlorate were tested (dashed gray). Ratios of sodium chloride to magnesium perchlorate were tested (light gray). Concentrations higher than 0.18 M Mg(ClO₄)₂ showed no activity.

FIG. 4.

Dixon plot of MgCl₂ inhibition of Halorubrum lacusprofundi β-galactosidase. The inverse of V₀ for each ONPG concentration was plotted versus the concentrations of MgCl₂. Where the lines intersect is the –K_I. Kinetics were performed at 5 mM (dark gray), 2.5 mM (black), and 1 mM (light gray) ONPG in 0.0625–0.25 M MgCl₂ and 4.375–4 M NaCl at 50°C. Each reaction had a total volume of 500 μL with 10 μg/mL of enzyme. The value determined for K_I was 0.3 M.

FIG. 5.

Dixon plot of NaClO₄ inhibition of Halorubrum lacusprofundi β-galactosidase. The inverse of V₀ for each ONPG concentration was plotted versus the concentrations of NaClO₄. Where the lines intersect with each other and the x axis is the –K_I. Kinetics were performed at 5 mM (dark gray), 2.5 mM (black), and 1 mM (light gray) ONPG in 0.125–0.5 M NaClO₄ and 4.375–4 M NaCl at 50°C. Each reaction had a total volume of 500 μL with 10 μg/mL of enzyme. The value determined for K_I was 2 M.

FIG. 6.

Plot of Mg(ClO₄)₂ kinetics. The Lineweaver-Burk slope for each concentration of Mg(ClO₄)₂ was plotted against the concentration of the inhibitor using the alternative Dixon plot equation: . A linear regression was performed, and the x intercept represents the –K_I. Kinetics were performed at 5, 2.5, and 1 mM ONPG in 0.0625–0.225 M Mg(ClO₄)₂ and 4.375–4.05 M NaCl at 50°C. Each reaction had a total volume of 500 μL with 10 μg/mL of enzyme. The value determined for K_I was 0.04 M.