Freezing from the inside: Ice nucleation in Escherichia coli and Escherichia coli ghosts by inner membrane bound ice nucleation protein InaZ

Abstract

Ice nucleation (IN) active bacteria such as Pseudomonas syringae promote the growth of ice crystals more effectively than any material known. Using the specialized ice nucleation protein (INP) InaZ, P. syringae-the well studied epiphytic plant pathogen-attacks plants by frost damage and, likewise fascinating, drives ice nucleation within clouds when airborne in the atmosphere by linkage to the Earth's water cycle. While ice nucleation proteins play a tremendous role for life on the planet, the molecular details of their activity on the bacterial membrane surface are largely unknown. Bacterial ghosts (BGs) derived from Escherichia coli can be used as simplified model systems to study the mode of action of InaZ. In this work, the authors used BGs to study the role of InaZ localization on the luminal side of the bacterial inner membrane. Naturally, P. syringae INPs are displayed on the surface of the outer membrane; so in contrast, the authors engineered an N-terminal truncated form of inaZ lacking the transport sequence for anchoring of InaZ on the outer membrane. This construct was fused to N-and C-terminal inner membrane anchors and expressed in Escherichia coli C41. The IN activity of the corresponding living recombinant E. coli catalyzing interfacial ice formation of supercooled water at high subzero temperatures was tested by a droplet-freezing assay and surface spectroscopy. The median freezing temperature (T 50 ) of the parental living E. coli C41 cells without INP was detected at −20.1 °C and with inner membrane anchored INPs at a T 50 value between −7 and −9 °C, demonstrating that the induction of IN from the inside of the bacterium by inner membrane anchored INPs facing the luminal inner membrane side is very similar to IN induced by bacterial INPs located at the outer membrane. Bacterial ghosts derived from these different constructs showed first droplet freezing values between −6 and −8 °C, whereas E. coli C41 BGs alone without carrying inner membrane anchored INPs exhibit a T 50 of −18.9 °C. Sum frequency generation spectroscopy showed structural ordered water at the BG/water interface, which increased close to the water melting point. Together, this indicates that the more efficient IN of INP-BGs compared to their living parental strains can be explained by the free access of inner membrane anchored INP constructs to ultrapure water filling the inner space of the BGs.

Ice nucleation proteins self-assemble into large fibres to trigger freezing at near 0 °C

“…The activity of the filtered supernatant was only a few degrees lower than that of whole bacteria (T 50 = –9.6 °C) ( Fig. 5A ), which agrees with the results of Kassmannhuber et al (28). This indicates that large INP structures are present within the bacterial cytoplasm.…”

“…Nor is there any difference in activity if the N-terminal INP domain and linker region are present or not (Fig. 7) (8,28).…”

“…Remarkably, INPs with N-terminal truncations are only slightly less active, suggesting that assembly on the cell surface is not mandatory, and it can occur in the cytoplasm whether anchored to the inner surface of the plasma membrane or free in solution (27,28).…”

“…When expressed recombinantly in E. coli , INPs have full activity suggesting that multimers are still able to form and that they are the product of self-assembly. Remarkably, INPs with N-terminal truncations are only slightly less active, suggesting that assembly on the cell surface is not mandatory, and it can occur in the cytoplasm whether anchored to the inner surface of the plasma membrane or free in solution (27, 28).…”

Water-organizing motif continuity is critical for potent ice nucleation protein activity

“…It should be noted that the AlphaFold model predicts less than 3 degrees of rotation between adjacent coils; this relatively small amount of twist may be eliminated by the zippering together of neighboring solenoids to form a flat surface without unduly straining the structure. Recently, an InaZ deletion construct without its N-terminal domain was expressed in E. coli , leading to cytoplasmic INP retention, yet the ice nucleation temperature was only 2–3 °C lower than that of full-length InaZ 47 . This suggests that large INP assemblies were able to form in the cytoplasm and efficiently nucleate ice without a membrane for support.…”