Putting life on ice: bacteria that bind to frozen water

Abstract

Ice-binding proteins (IBPs) are typically small, soluble proteins produced by cold-adapted organisms to help them avoid ice damage by either resisting or tolerating freezing. By contrast, the IBP of the Antarctic bacterium Marinomonas primoryensis is an extremely long, 1.5 MDa protein consisting of five different regions. The fourth region, a 34 kDa domain, is the only part that confers ice binding. Bioinformatic studies suggest that this IBP serves as an adhesin that attaches the bacteria to ice to keep it near the top of the water column, where oxygen and nutrients are available. Using temperature-controlled cells and a microfluidic apparatus, we show that M. primoryensis adheres to ice and is only released when melting occurs. Binding is dependent on the mobility of the bacterium and the functionality of the IBP domain. A polyclonal antibody raised against the IBP region blocks bacterial ice adhesion. This concept may be the basis for blocking biofilm formation in other bacteria, including pathogens. Currently, this IBP is the only known example of an adhesin that has evolved to bind ice.

Keywords: RTX adhesin; antifreeze proteins; biofilm; cold adaptation; ice-binding proteins; microfluidic cold finger.

Figure 1.

Schematic presentation of Marinomonas primoryensis bound to sea ice through MpIBP. Sea ice is represented by the hexagons. An enlargement of the binding location at the C-terminal end of MpIBP shows MpIBP_RIV (yellow), the ice-binding domain, flanked by RIII (green) and RV (purple). Only a few units of RII (out of approximately 120 repeats) are shown (orange). (Figure prepared using Inkscape (open source).)

Figure 2.

An illustration of the MCF. The microfluidic chip is placed on top of a cooling stage. A copper cold finger embedded in the middle of the channel functions as an independent cooling unit. An ice crystal is drawn as a circle in the channel below the tip of the cold finger. (Figure prepared using Google Sketchup.)

Figure 3.

Marinomonas primoryensis viewed by ultra-SEM. Scale bar, 1 µm.

Figure 4.

Accumulation of Marinomonas primoryensis on ice and blockage of this process by antibody to MpIBP_RIV. (a) Time-lapse images of ice grown in medium containing M. primoryensis in addition to rabbit pre-immune serum. (b) The same sequence of images as in (a) but with rabbit anti-MpIBP_RIV serum in place of the pre-immune serum. (c) Control series with Pseudomonas borealis in place of M. primoryensis. Bacteria in the crystal field in (c) are not attached to the ice (see the electronic supplementary material, movie S1). Scale bars, 10 µm.

Figure 5.

Accumulation of Marinomonas primoryensis on ice in microfluidics and the effects of anti-MpIBP_RIV and anti-MpIBP_RII. (a) Ice was held at a constant temperature slightly below the melting point in a solution containing fresh bacteria (OD ∼ 0.5) for 3 min before the image was taken. (b) Ice was held for 20 min at a constant temperature slightly below the melting point in a solution of bacteria treated overnight with anti-MpIBP_RIV serum (25% v/v). (c) Ice was held for 5 min at a constant temperature slightly below the melting point in a solution of bacteria treated overnight with anti-MpIBP_RII serum (25% v/v). The images in (i) and (ii) are focused on the top and bottom layers of the ice, respectively.

Figure 6.

Ice shaping and basal plane affinity by Marinomonas primoryensis. (a) Extensive accumulation of M. primoryensis on the ice surface changes crystal growth morphology from a flat disc to a more hexagonal shape. (b) A single ice crystal disc bound by M. primoryensis oriented with prism planes edge-on to the camera, showing bacterial accumulation mainly on the basal planes (indicated by red arrows). (c) Ice grown in an MCF chamber showing M. primoryensis preferentially accumulating on the basal plane (marked by the red arrow). Note: the ice contains more than one grain. The basal plane is identified by its faceting. The rough planes next to it are non-basal planes. Scale bars, 10 µm. (Online version in colour.)

Figure 7.

Marinomonas primoryensis are not incorporated into growing ice. Images (a–c) show advancing ice fronts on three adjacent ice grains growing in the MCF device at a rate of 0.5 µm s⁻¹. As the ice fronts advance (from top to bottom), more bacteria accumulate on their surfaces without being engulfed. Scale bars, 10 µm.

Figure 8.

(a) Congregation of Marinomonas primoryensis on ice. Ice grains in the MCF were grown rapidly in the presence of M. primoryensis. Many of the bacteria gathered in microcolonies at the interface between the ice and the microfluidic device (red arrows) or between grain boundaries. Some bacteria remained single on top of the ice (black arrows). (b) Magnification of the area in the top rectangle, showing two microcolonies. (c) Magnification of the area in the bottom rectangle, showing bacteria between adjacent ice grains. Scale bars, 10 µm.

Figure 9.

Analysis of Marinomonas primoryensis adhesion to ice over time and the effect of anti-MpIBP_RIV. The plot presents the distribution of bacteria between ice and medium over time. The ratios of ice-bound versus free bacteria were measured over time in the presence of anti-MpIBP_RIV serum (open circles and open triangles for solution and ice, respectively, dashed line) and pre-immune serum without anti-MpIBP_RIV (filled circles and filled triangles for solution and ice, respectively, solid line). At an OD_{600 nm} of 0.02, it takes 3 min for half the population to bind ice. Experiments with non-immunized serum or without serum showed indistinguishable results. The data are based on 20 independent experiments. Up to 6 min, n = 8–10; minute 7 and above, n = 3–4. Error bars represent standard error.

Figure 10.

Immunofluorescence of MpIBP. Immobilized Marinomonas primoryensis were immunolabelled with anti-MpIBP_RIV serum and visualized using fluorescently labelled goat anti-rabbit second antibody. (a) Anti-MpIBP_RIV serum. (b) Pre-immunized serum. Scale bar, 10 µm. Reaction with anti-MpIBP_RII anti-sera was previously demonstrated [19]. (Online version in colour.)