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.
Origin of life theories have produced numerous schools of thought, two of which are termed RNA World
and Snowball Earth. Complex organic molecules can be produced by RNA catalysis. This chemistry would be
hindered by ice formation that would sequester moecules in the frozen water matrix. We present experimental data
showing that RNA retains antifreeze properties that would enable the exchange of reactants/products of the catalytic
reactions.
Recent studies from our lab demonstrated that teichoic acid is surrounded by liquid water at -40 °C. The size
and shape of the liquid water pockets has been visualized with fluorescence microscopy images of aqueous Rhodamine-
B solutions. The long, thin channels surround ice crystals with a size of 5-20 microns. Subsequent studies show that B.
subtilis Gram-positive bacteria are sequestered into large pockets without added teichoic acid. Here, the ice crystals are
orders of manitude larger. When bacteria are mixed with teichoic acid solutions, the distribution of bacteria changes
dramatically. The smaller ice crystals allow the bacteria to align in the thin channels of liquid water seen with teichoic
acid only. The role of teichoic acid in the freeze tolerance was examined with live/dead fluorescence assays of bacteria
mixed with teichoic acid. These quantitative assays were used to determine if teichoic acid acts in a synergetic fashion
to enhance the survivability of E. coli, a gram-negative species which lacks teichoic acid. Additionally, we have
obtained B. subtilis mutants lacking wall-associated teichoic acids to evaluate cryoprotection compared to the wild-type
strain.
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,
although, with a few exceptions, antifreeze proteins are missing. Survival has been attributed to external factors,
such as high salt concentration (brine veins) and adhesion to particulates or ice crystal defects. Teichoic acid is a
phosphodiester polymer ubiquitous in Gram positive bacteria, composing 50% of the mass of the bacterial cell wall
and excreted into the extracellular space of biofilm communities. We have found that when bound to the
peptidoglycan cell wall (wall teichoic acid) or as a free molecule (lipoteichoic acid), teichoic acid is surrounded by
liquid water at temperatures significantly below freezing. Using solid-state NMR, we are unable to collect 31P
CPMAS spectra for frozen solutions of lipoteichoic acid at temperatures above -60 °C. For wall teichoic acid in
D2O, signals are not seen above -30 °C. These results can be explained by the presence of liquid water, which
permits rapid molecular motion to remove 1H/31P dipolar coupling. 2H quadrupole echo NMR spectroscopy reveals
that both liquid and solid water are present. We 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.
Conference Committee Involvement (4)
Instruments, Methods, and Missions for Astrobiology XVI
27 August 2013 | San Diego, California, United States
Instruments, Methods, and Missions for Astrobiology XV
14 August 2012 | San Diego, California, United States
Instruments, Methods, and Missions for Astrobiology XIV
23 August 2011 | San Diego, California, United States
Instruments, Methods, and Missions for Astrobiology XIII
3 August 2010 | San Diego, California, United States
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