Expression and Partial Characterization of an Ice-Binding Protein from a Bacterium Isolated at a Depth of 3,519 m in the Vostok Ice Core, Antarctica

Abstract

Cryopreservation of microorganisms in ancient glacial ice is possible if lethal levels of macromolecular damage are not incurred and cellular integrity is not compromised via intracellular ice formation or recrystallization. Previously, a bacterium (isolate 3519-10) recovered from a depth of 3,519 m below the surface in the Vostok ice core was shown to secrete an ice-binding protein (IBP) that inhibits the recrystallization of ice. To explore the advantage that IBPs confer to ice-entrapped cells, experiments were designed to examine the expression of 3519-10's IBP gene and protein at different temperatures, assess the effect of the IBP on bacterial viability in ice, and determine how the IBP influences the physical structure of the ice. Total RNA isolated from cultures grown between 4 and 25°C and analyzed by reverse transcription-PCR indicated constitutive expression of the IBP gene. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 3519-10's extracellular proteins revealed a polypeptide of the predicted size of the 54-kDa IBP at all temperatures tested. In the presence of 100 μg mL(-1) of extracellular protein from 3519-10, the survival of Escherichia coli was increased by greater than 100-fold after 5 freeze-thaw cycles. Microscopic analysis of ice formed in the presence of the IBP indicated that per square millimeter field of view, there were ~5 times as many crystals as in ice formed in the presence of washed 3519-10 cells and non-IBP producing bacteria, and ~10 times as many crystals as in filtered deionized water. Presumably, the effect that the IBP has on bacterial viability and ice crystal structure is due to its activity as an inhibitor of ice recrystallization. A myriad of molecular adaptations are likely to play a role in bacterial persistence under frozen conditions, but the ability of 3519-10's IBP to control ice crystal structure, and thus the liquid vein network within the ice, may provide one explanation for its successful survival deep within the Antarctic ice sheet for thousands of years.

Keywords: freeze tolerance; ice-binding protein; polycrystalline ice; recrystallization inhibition.

Figure 1

Composite image of ice core section 3,519 viewed under cross-polarized illumination. The arrow is “up” and indicates the ice core orientation relative to the surface. The scale bar is 10 cm.

Figure 2

(A) Reverse transcription-PCR analysis of gene expression in 3519-10. The 876-bp bands in lanes 1, 4, 7, and 10 represents the presence of 16S rRNA; the 421 and 100 bp bands are fragments of RT-PCR IBP and cspA transcripts, respectively. The template for lanes 1–3 was RNA from a 4°C grown culture of 3519-10, 4–6 from 10°C, 7–9 from 15°C, and 10–12 from 25°C cultures. (B). Identical samples lacking a RT step. Lane assignments are the same as for (A). L is a low DNA mass ladder (Invitrogen).

Figure 3

(A) Image transect showing ice structure from the outside edge of the sample to center (left to right in image). The superimposed rectangle delineates a 1.5-mm by 10 mm area. (B) Ice structure formed from filtered deionized water. (C) Ice structure formed in the presence of extracellular proteins from 3519-10. Scale bars in (B) and (C) are 0.1 mm.

Figure 4

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracellular proteins from 3519-10. Lane 1 contains a 10- to 250-kDa protein ladder. Lanes 2–5 contain 10 μg total extracellular protein harvested from cultures grown at 4, 10, 15, and 25°C respectively. Lane 6 contains 10 μg of BSA (66 kDa). The arrow indicates the expected migration of the IBP based on the predicted size of 54 kDa.

Figure 5

Fraction of E. coli surviving four freeze-thaw cycles in the presence of 100 μg mL⁻¹ of extracellular proteins harvested from a culture of 3519-10 grown at 4°C (⋄) and 25°C (□), as well as 100 μg mL⁻¹ BSA(Δ). The bars represent the SE of triplicate samples; the frozen control (◦) is the average of six replicates from two experiments.

Figure 6

Number of crystals per square millimeter for each ice sample (error bars are 1 σ). From left to right: ices made with filtered deionized water, washed 3519-10 cells, washed Paenisporosarcina isolate B5 cells, and 3519-10’s extracellular proteins.