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Chaperone proteins are essential components in the maintenance and turnover of the proteome. Many chaperones play integral functions in the folding and unfolding of cellular substrates under many conditions, including heat stress. Most chaperones can be characterized into two categories; the typical ATP dependent chaperones and the ATP independent chaperones. One ATP independent chaperone class it the Small Heat Shock Proteins (sHSPs), which as molecular life vests and are thought to protect misfolding proteins from irreversible aggregation. One such organism, the cyanobacterium Synechocystis sp. PCC 6803, is an excellent model for the study and understanding of these proteins and their functions in vivo. The genome of Synechocystis encodes only one sHSP, Hsp16.6, and it has be shown to be essential for acquired thermotolerance. Two mutant derivatives of Hsp16.6 with single amino acid substitutions in the N-terminal arm (L9P and E25K) have loss-of-function phenotypes similar to knock out strains, but each has very different biochemical properties. The mutant L9P has an inability to interact with putative substrates during heat stress in vivo, while the mutant E25K appears unable to release substrates. Using a directed evolution approach, suppressors have been isolated that recover the lost thermotolerance of their respective parent strains, either L9P (16 suppressors) or E25K (10 suppressors). Illumina sequencing and comparative genomics have been used to identify alterations in the genomes of the suppressor strains in order to define genetic circuits involved in thermotolerance.

Type IV pili (T4P) systems are widely utilized among bacteria to power and direct surface motility. The production and secretion of a viscous polysaccharide to provide friction and resistance to the extended pilus structure is seen in several species of cyanobacteria including Nostoc punctiforme. The complex coregulation of polysaccharide secretion and T4P motor activity is not fully understood, although studies indicate a consistent relationship between functional motility and intact pathways of polysaccharide secretion and pilus extension in cyanobacteria. Using a combination of protein-protein interaction analysis, cytological studies, and comparative genomics this study proposes a theoretical mechanism for T4P motor influenced regulation of hormogonium polysaccharide secretion by a heat-shock protein (HSP) DnaK-type chaperone system in N. punctiforme. The results of this study indicate a tripartite HSP system consisting of DnaK1, DnaJ3, and coregulator GrpE is influenced by the activation of certain motor proteins in the T4P complex and are required for the production and secretion of hormogonium polysaccharide. Conservation of this system in Synechocystis sp. also implies a potential system that is conserved among all motile cyanobacteria for regulation of T4P.

Nostoc commune UTEX 584 is a photosynthetic desiccation-tolerant cyanobacterium capable of fixing nitrogen. Biotinylated nifH and nifD gene probes from Azotobacter vinelandii (a gift from Dr. Dennis Dean) hybridized to nifH and nifD sequences isolated from Nostoc commune UTEX 584. This result supports the view that the nitrogenase structural proteins and their genes are highly conserved in nitrogen-fixing organisms (Rice et al., 1980). Southern transfers of genomic DNA prepared from N- commune UTEX 584 were digested with Hind III and hybridized with nifH-specific and nifD-specific probes. Multiple copies of nifH (three) and nifD (two) were detected. Using colony hybridization, a recombinant N- commune UTEX 584 genomic DNA-pBR322 plasmid library was screened with the biotinylated nifH-specific probe and positive hybridizing nifH clones were isolated. A restriction map of the 3.5 kb Hind III insert of the recombinant plasmid (pND001) was produced. From partial sequencing data it was possible to determine the positions of the N. commune UTEX 584 nifH and nifD genes within the cloned fragment and compare the partial nucleotide sequence and deduced amino acid sequences of the E- commune UTEX 584 nifHD genes with other organisms. Isolation of the nifH and nifD genes from Nostoc commune UTEX 584 now permits a more detailed study of nif gene expression in this desiccation-tolerant photosynthetic microorganism. / M.S.