Thesis

Identification of sigma / anti-sigma factor regulatory pairs in the cyanobacterium Nostoc punctiforme

Nostoc punctiforme is a filamentous cyanobacterium that has an immense potential to be used as a platform to produce biofuels and other high-value compounds. Their ability to synthesize lipid droplets and other valuable metabolites while eliminating the requirement for land also considerably increases their commercial prospective. N. punctiforme grows using plant-like oxygenic photosynthesis and can also differentiate into spore-like akinetes, nitrogen-fixing heterocysts, or motile hormogonia in response to stress. This unique ability to adapt to environmental stresses by differentiation also makes them a valuable model for studying cellular differentiation. A subset of the genes involved in stress responses are hypothesized to be regulated by the twelve alternative sigma factor subunits of RNA polymerase, which were previously identified in the genome (along with the housekeeping sigma, SigA). The sigma factors are known to be post translationally regulated by the anti-sigma factors, that are hypothesized to sequester the sigma factors until stress induces their release. However, unlike the sigma factors, the anti-sigma factors are well conserved at the primary sequence level. Thus, not much is known about anti-sigma factors in general or about the specific sigma/anti-sigma interactions in N. punctiforme. Seven putative anti-sigma factors were identified in N. punctiforme by sequence homology. We screened for the interaction of all thirteen sigma factors with these putative anti-sigma factors using a bacterial 2-hybrid assay (BacTH). Each of the anti-sigma factors interacted with two or more sigma factors in the BacTH analysis, but no interactions were found with group 3 sigma factors or the highly divergent sigma factor SigI. Twenty-three positive interactions were identified using the BacTH system, which were then quantified by using beta-galactosidase assays. We then tried to confirm twelve of the interactions showing the highest beta-galactosidase activity by using GST-pulldown assays and Western blotting. Two novel sigma/anti-sigma interacting pairs were identified by this study. The sigma factor SigB2 was shown to interact with the anti-sigma factor SapB2 and the sigma factor SigD was shown to interact with the anti-sigma factor SapD. These findings will help increase our understanding of complex regulatory processes that occur at the transcriptional level during cellular differentiation, and following stresses associated with production of high-value compounds in N. punctiforme.

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