Mr. Malcolm M. Zachariah Profile

Malcolm M. Zachariah

Doctoral Student

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Proceedings Article | 15 October 2012 Paper

Cryoprotection from lipoteichoic acid

Charles Rice, Amy Middaugh, Jason Wickham, Anthony Friedline, Kieth Thomas, Karen Johnson, Malcolm Zachariah, Ravindranth Garimella

Proceedings Volume 8521, 85210I (2012) https://doi.org/10.1117/12.970517

KEYWORDS: Bacteria, Liquids, Proteins, Crystals, Microorganisms, Veins, Luminescence, Biopolymers, Chemistry, Organisms

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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.

Proceedings Article | 23 September 2011 Paper

The nature of water within bacterial spores: protecting life in extreme environments

Charles Rice, Anthony Friedline, Karen Johnson, Malcolm Zachariah, Kieth Thomas

Proceedings Volume 8152, 81520V (2011) https://doi.org/10.1117/12.898766

KEYWORDS: Molecules, Proteins, Resistance, Spectroscopy, Thin film coatings, Ultraviolet radiation, Molecular spectroscopy, Liquids, Crystals, Chemistry

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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.

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