Date of Award

8-2014

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Gary S. Sayler

Committee Members

Elizabeth M. Fozo, Alice C. Layton

Abstract

Bacterial response to cold shock and cold adaptation is not fully understood. While several cold inducible genes have been identified in mesophilic orangisms, the roles they play in cold acclimation remain unclear. Few studies emphasize cold acclimation of psychrotrophic or psychrophilic bacteria. Available cold acclimation studies look at a limited number of genes involved in cold stress, and fewer studies compare genes involved in cold shock and cold adaptation. As “omics” technologies become more readily available, looking at whole cell response to cold stress is more achievable. This study uses genomic and transcriptomic approaches to advance the understanding of cold shock and cold adaptation in Pseudomonas fluorescens HK44. Genomic mining was performed using the RAST online database to identify genes that may be used during cold shock and cold adaptation. After defining temperature growth ranges for P. fluorescens HK44, RNA was harvested from cells grown at 4˚C, 25˚C and cells shifted from 25˚C to 4˚C for 30 minutes during exponential growth to generate transcriptomic libraries. The Illumina High Seq was used to generate raw sequencing reads, before analysis was carried out in CLC Bio. Growth studies indicate that P. fluorescens HK44 is a psychrotroph with a growth range between 4˚C and 32˚C. Genomic mining of the HK44 genome identified multiple copies of the cspA gene, where phylogenetic analysis suggests the number of cspA gene copies present in a genome is indicative of the temperature classification of Pseudomonas species. Genes surrounding cold shock protein genes were investigated for their potential role in cold acclimation and indicate that cryoprotectants and/or membrane modifications may occur in response to cold stress in HK44. Transcriptomic data indicated that two cspA genes were differentially expressed; cspA 1 during the cold adapted vs cold shock experiment, and cspA 2 during the cold shock 1 experiment. Expression of the genes surrounding cold shock protein genes indicate that cells respond to low temperature by regulating genes that may allow for a) the accumulation of metabolites that can be transformed into cryoprotectants and b) the production of alternative phospholipids to be incorporated into the membrane in order to increase fluidity.

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