In a concept study the ability to induce heterogeneous ice formation by Bacterial Ghosts (BGs) from Escherichia coli carrying ice nucleation protein InaZ from Pseudomonas syringae in their outer membrane was investigated by a droplet-freezing assay of ultra-pure water. As determined by the median freezing temperature and cumulative ice nucleation spectra it could be demonstrated that both the living recombinant E. coli and their corresponding BGs functionally display InaZ on their surface. Under the production conditions chosen both samples belong to type II ice-nucleation particles inducing ice formation at a temperature range of between ¡5.6 C and ¡6.7 C, respectively. One advantage for the application of such BGs over their living recombinant mother bacteria is that they are non-living native cell envelopes retaining the biophysical properties of ice nucleation and do no longer represent genetically modified organisms (GMOs).

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.

<p><strong>Abstract.</strong> An N-terminal truncated form of the ice nucleation protein (INP) of <i>Pseudomonas syringae</i> lacking the transport sequence for the localization of InaZ in the outer membrane was fused to N- and C- terminal inner membrane (IM) anchors and expressed in <i>Escherichia coli</i> C41. The ice nucleation (IN) activity of the corresponding living recombinant <i>E. coli</i> catalyzing heterogeneous ice formation of super-cooled water at high subzero temperatures was tested by droplet freezing assay. Median freezing temperature (T₅₀) of the parental living <i>E. coli</i> C41 cells without INP was detected at &amp;minus;20.1&amp;thinsp;&amp;deg;C and with inner membrane anchored INPs at T₅₀ value between &amp;minus;7&amp;thinsp;&amp;deg;C and &amp;minus;9&amp;thinsp;&amp;deg;C demonstrating that IM anchored INPs facing the luminal IM site are able to induce IN from the inside of the bacterium almost similar to bacterial INPs located at the outer membrane. Bacterial Ghosts (BGs) derived from the different constructs showed first droplet freezing values between &amp;minus;6&amp;thinsp;&amp;deg;C and &amp;minus;8&amp;thinsp;&amp;deg;C whereas C41 BGs alone without carrying IM anchored INPs exhibit a T₅₀ of &amp;minus;18.9&amp;thinsp;&amp;deg;C. The more efficient IN of INP-BGs compared to their living parental strains can be explained by the free access of IM anchored INP constructs to ultrapure water filling the inner space of the BGs. The cell killing rate of -NINP carrying <i>E. coli</i> at subzero temperatures is higher when compared to survival rates of the parental C41 strain.</p>