Ice-nucleating bacteria control the order and dynamics of interfacial water

Abstract

Ice-nucleating organisms play important roles in the environment. With their ability to induce ice formation at temperatures just below the ice melting point, bacteria such as Pseudomonas syringae attack plants through frost damage using specialized ice-nucleating proteins. Besides the impact on agriculture and microbial ecology, airborne P. syringae can affect atmospheric glaciation processes, with consequences for cloud evolution, precipitation, and climate. Biogenic ice nucleation is also relevant for artificial snow production and for biomimetic materials for controlled interfacial freezing. We use interface-specific sum frequency generation (SFG) spectroscopy to show that hydrogen bonding at the water-bacteria contact imposes structural ordering on the adjacent water network. Experimental SFG data and molecular dynamics simulations demonstrate that ice-active sites within P. syringae feature unique hydrophilic-hydrophobic patterns to enhance ice nucleation. The freezing transition is further facilitated by the highly effective removal of latent heat from the nucleation site, as apparent from time-resolved SFG spectroscopy.

Keywords: Ice nucleation; Ice protein; Pseudomonas Syringae; sum frequency generation.

Fig. 1. SFG spectra of P. syringae.

(A to E) SFG spectra of P. syringae bacteria lysate, which contain ice-active inaZ proteins (A) and control substances (B to E) in contact with water at different temperatures. INPs increase the water signal with decreasing temperatures, whereas control substances leave the water signal unchanged. a.u., arbitrary units.

Fig. 2. Analysis of temperature-dependent SFG spectra for P. syringae at a water surface.

(A) Spectra for RT and 5°C along with two fitting components related to more weakly and strongly hydrogen-bonded water within the broad water spectrum. CH and nonresonant fitting components are not included here. (B) Plots of the amplitudes obtained from fits to the SFG spectra from RT to 5°C. The mode related to more strongly hydrogen-bonded water increases strongly, whereas the weakly hydrogen-bonded water mode remains almost unchanged.

Fig. 3. MD simulation of the inaZ ice-active site.

(A) The top view illustrates the ladder-type regions of groups of amino acids on the IN dimer. The side view shows an MD snapshot of the water structure at the IN site. Together, side chains and clathrate water form a template for ice nucleation. Threonine, purple; serine, yellow; alanine, blue; tyrosine, green; glutamic acid, orange; glycine, white. (B) Calculated SFG spectra for regions with different amino acids present. Thr- and Ser-rich areas leave the water signal intensity unchanged, whereas there is a clear trend toward a stronger water signal near glutamic acid– and serine-rich regions. (C) Integrated SFG intensity for the IN site regions at 270 and 300 K. The increased intensity at regions 1 and 6 indicates more ordered water near the perimeter of the IN site.

Fig. 4. Energy transfer processes at the water-INP interface.

(A) Time-resolved difference sum frequency spectrum for the water–P. syringae interface after excitation with a 2470-cm⁻¹ pump pulse near the weakly H-bonded water resonances (dashed line). The signal bleach is very intense in the low-frequency water peak related to strongly H-bonded water. This shows that energy transfer is very rapid and efficient. For clarity, any spectral changes due to thermal effects have been removed. (B) Time-dependent bleach integrated over two spectral regions, 2330 to 2430 cm⁻¹ (strongly H-bonded) and 2480 to 2580 cm⁻¹ (weakly H-bonded). Fits of the data using a coupled differential equation model reveal extremely efficient (80 ± 50 fs) energy transfer between more weakly and more strongly H-bonded water molecules. (C) Time-resolved populations of the more weakly and more strongly H-bonded water molecules extracted from the coupled differential equations. The states become populated from the excitation pulse, energy transfer, and decay to the ground states (not plotted). For the water–P. syringae interface, the more strongly H-bonded state’s population is higher than that of the initially excited peak, which proves extremely efficient energy transfer.