Thesis

Role of a small heat-shock protein in the development of Nostoc punctiforme

Nostoc punctiforme is a filamentous cyanobacterium with the ability to differentiate into three different cell types: nitrogen fixing heterocysts, motile hormogonia and spoke-like akinetes. Akinetes are able to survive a variety of harsh conditions such as lack of nutrients, cold, heat, desiccation, etc. Previous microarray data identified NpF0784 encoding a small heat-shock protein (sHSP) as a potential gene involved in akinetes induction in N. punctiforme. This sHSP protein is highly conserved within the Nostocales and shows decreased homology with other members within the phylum Cyanobacteria. GFP transcriptional reporter strains confirmed the array results showing increased expression from NpF0784 in akinetes and also following exposure to environmental stresses such as high salt, high osmotic pressure, and in response to pH extremes of 6 and 11. A second DNA microarray experiment using the zwf model system of akinetes was conducted to see if there was differential gene expression due to the deletion of this sHSP. Only 8 photosynthetic genes were up-regulated >2-fold, whereas 44 genes were down-regulated in the sHSP mutant. The genes with lower expression in the mutant strain included dnaK and groL, indicating this sHSP could play a role in the functioning of these chaperones. Other down-regulated genes included a PfpI-family intracellular protease, as well as two encoding putative transcriptional regulators. To discern between direct and indirect action by this sHSP, a bacterial two-hybrid system was employed to find proteins directly interacting with NpF0784. Screening of specific chaperones hypothesized to interact with this sHSP led to the identification of two interacting proteins; a protein similar to a ClpB-like ATP-dependent chaperone and itself. Survival studies comparing wile-type and NpF0784 deletion strains following exposure to high salt or osmotic stress showed reduced survival for the mutant strain. Together these data support the hypothesis that NpF0784 encodes a homo-mulimeric sHSP that is important for normal gene regulation during cell development as well as helping cells cope with certain stresses via interaction with a ClpB-like chaperone.

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