Bacterial adaptation to the cold: In situ activities of extracellular enzymes in the North Water Polynya and characterization of a cold-active aminopeptidase from Colwellia psychrerythraea strain 34H

The objective of this dissertation was to advance a fundamental understanding of the strategies marine bacteria employ to survive at low temperatures, specifically through the use of cold-active extracellular enzymes (EE). I used a combination of field- and laboratory-based research approaches to investigate factors that may influence the activity of bacterial EE in stably-cold marine environments. Chapters two and three describe the results of experiments performed while participating in the international North Water Polynya Study. The results of this field campaign significantly increased the number of psychrophilic EE activities (thermal optima ≤15°C) documented in natural habitats (12 of 42 cases, with optima to unprecedented lows of 10°C), established an important role for EE in influencing the content of organic matter despite the near-0°C conditions, and suggested that sinking aggregates preferentially select for psychrophilic bacteria and cold-active EE relative to surrounding seawater. These field studies also underscored the need to establish a model EE-producing psychrophilic bacterium to better understand the factors affecting bacterial expression, activity, and hydrolytic lifetime of EE.; The material in chapter four describes the characterization of marine psychrophile Colwellia psychrerythraea strain 34H, isolated from the near-freezing sediments of the Northeast Water Polynya, Greenland. Strain 34H was chosen as the model organism for my studies because it consistently yielded an extract of cell-free aminopeptidase with activity optimized at 20°C, the lowest optimum yet reported for cell-free EEA from a pure culture. The purification, characterization and structural studies of the cold-active M1-family aminopeptidase (MAP) expressed by strain 34H are reported in chapter five. In agreement with previous studies of cold-active enzymes, MAP displayed high activity at low temperatures (∼0°C), low thermal optima for activity (19°C), low stability against denaturing agents, and amino acid changes which hypothetically lead to increased structural flexibility when compared to mesophilic homologs. Additional research indicated that extrinsic stabilizing factors, such as exopolymeric substances, significantly affected the activity and lifetime of MAP. Overall, this dissertation enhanced knowledge of activity characteristics and structure-function relationships of cold-active enzymes in general, while indicating the importance of extrinsic stabilizing factors for maintaining cold-active EEA in the environment.