Bacterial low temperature survival, ice nucleation proteins and ice-associating polymers
Ice Nucleation Protein , Antifreeze , Extracellular Polymer Substances , Low Temperature Survival
Microorganisms have developed ways to preserve cellular functions under low temperature conditions using a variety of biochemical adaptations including the modification of ice formation. In order to conduct a limited survey of microbial ice-associating strategies, a bacterial community associated with frost-exposed leaves was assessed by the construction of a 16S rDNA library, followed by the characterization of some isolates. Fifteen different species were identified based on their 16S rDNA. Among these, Pseudomonas syringae J6 had ice nucleation activity (INA), which promoted ice formation close to 0ºC, whereas Erwinia billingiae, Flavobacterium sp. and Sphingobacterium kitahiroshimense inhibited the recrystallization of small ice crystals at temperatures close to melting. The Erwinia billingiae isolate showed adhesive and swarming behaviour, which can be associated with biofilm formation. Visualization using negative staining, transmission electron microscopy and scanning electron microscopy confirmed the presence of flagella in addition to the presence of slimy biofilm architecture in these Erwina billingiae cultures. Subsequent purification of the extracellular polymeric substance followed by mass spectrometry allowed the identification of a putative outer membrane protein A, which may be involved in the protection of this bacterium to freeze-thaw cycles. To further explore bacterial ice nucleation activity, an ice nucleation protein was cloned from Pseudomonas borealis, a bacterium originating from tundra soil, using degenerative PCR and chromosome walking. The sequence of the putative ice nucleation protein gene (inaPb) was cloned and expressed in Escherichia coli, and its identification was confirmed in the recombinant cells. Although the INPPb was more divergent than other plant-related bacterial INPs, it retained the highly conserved, repetitive core region. The protein may fold so that it has two flat faces, one for protein-protein interactions and the other for ice binding. Expression of the INPPb coding region fused to jelly fish green fluorescent protein showed a temperature-dependent polarized distribution of the recombinant protein in E. coli. In summary, results from this thesis suggests that low temperature survival may be associated with a number of ice-associating adaptations including the presence of biofilm formation in Erwina billingiae amongst other bacteria, INA in P. borealis and INA-expressing recombinant E. coli.