Antifreeze proteins in fish, plants, and insects provide protection to a few degrees below freezing. Microbes have been found to survive at even lower temperatures, and with a few exceptions, antifreeze proteins are missing. We show that lipoteichoic acid (LTA), a biopolymer in the cell wall of Gram-positive bacteria, can be added to B. subtilis cultures and increase freeze tolerance. At 1 % w/v, LTA enables a 50 % survival rate, similar to the results obtained with 1 % w/v glycerol as measured with the resazurin cell viability assay. In the absence of added LTA or glycerol, a very small number of B. subtilis cells survive freezing. This suggests that an innate freeze tolerance mechanism exists. While cryoprotection can be provided by extracellular polymeric substances, our data demonstrate a role for LTA in cryoprotection. Currently, the exact mode of action for LTA cryoprotection is unknown. With a molecular weight of 3–5 kDa, it is unlikely to enter the cell cytoplasm. However, low temperature microscopy data show small ice crystals aligned along channels of liquid water. Our observations suggest that teichoic acids could protect liquid water within biofilms and planktonic bacteria, augmenting the role of brine while also raising the possibility for survival without brine present.

Dormant bacterial spores are able to survive long periods of time without nutrients, withstand harsh environmental conditions, and germinate into metabolically active bacteria when conditions are favorable. Numerous factors influence this hardiness, including the spore structure and the presence of compounds to protect DNA from damage. It is known that the water content of the spore core plays a role in resistance to degradation, but the exact state of water inside the core is a subject of discussion. Two main theories present themselves: either the water in the spore core is mostly immobile and the core and its components are in a glassy state, or the core is a gel with mobile water around components which themselves have limited mobility. Using deuterium solid-state NMR experiments, we examine the nature of the water in the spore core. Our data show the presence of unbound water, bound water, and deuterated biomolecules that also contain labile deuterons. Deuterium-hydrogen exchange experiments show that most of these deuterons are inaccessible by external water. We believe that these unreachable deuterons are in a chemical bonding state that prevents exchange. Variable-temperature NMR results suggest that the spore core is more rigid than would be expected for a gel-like state. However, our rigid core interpretation may only apply to dried spores whereas a gel core may exist in aqueous suspension. Nonetheless, the gel core, if present, is inaccessible to external water.

Numerous chemical additives lower the freezing point of water, but life at sub-zero temperatures is sustained by a limited number of biological cryoprotectants. Antifreeze proteins in fish, plants, and insects provide protection to a few degrees below freezing. Microbes have been found to survive at even lower temperatures, and with a few exceptions, antifreeze proteins are missing. Survival has been attributed to external factors, such as the high salt concentration of brine veins and adhesion to particulates or ice crystal defects. We have discovered an endogenous cryoprotectant in the cell wall of bacteria, lipoteichoic acid biopolymers. Adding 1% LTA to bacteria cultures immediately prior to freezing provides 50% survival rate, similar to the results obtained with 1% glycerol. In the absence of an additive, bacterial survival is negligible as measured with the resazurin cell viability assay. The mode of action for LTA cryoprotection is unknown. With a molecular weight of 3-5 kDa, it is unlikely to enter the cell cytoplasm. Our observations suggest that teichoic acids could provide a shell of liquid water around biofilms and planktonic bacteria, removing the need for brine veins to prevent bacterial freezing

The bacterial spore is a formidable container of life, protecting the vital contents from chemical attack, antimicrobial agents, heat damage, UV light degradation, and water dehydration. The exact role of the spore components remains in dispute. Nevertheless, water molecules are important in each of these processes. The physical state of water within the bacterial spore has been investigated since the early 1930's. The water is found two states, free or bound, in two different areas, core and non-core. It is established that free water is accessible to diffuse and exchange with deuterated water and that the diffusible water can access all areas of the spore. The presence of bound water has come under recent scrutiny and has been suggested the water within the core is mobile, rather than bound, based on the analysis of deuterium relaxation rates. Using an alternate method, deuterium quadrupole-echo spectroscopy, we are able to distinguish between mobile and immobile water molecules. In the absence of rapid motion, the deuterium spectrum of D2O is dominated by a broad line, whose line shape is used as a characteristic descriptor of molecular motion. The deuterium spectrum of bacterial spores reveals three distinct features: the broad peak of immobilized water, a narrow line of water in rapid motion, and a signal of intermediate width. This third signal is assigned this peak from partially deuterated proteins with the spore in which N-H groups have undergone exchange with water deuterons to form N-D species. As a result of these observations, the nature of water within the spore requires additional explanation to understand how the spore and its water preserve life.