H-047. Cell-Type-Specific Patterned Transcriptional Expression in Nostoc punctiforme

J. B. Polin, M. L. Summers;
California State Univ., Northridge, CA.

Key regulatory genes are hypothesized to control differentiation into akinetes in Nostoc punctiforme, a filamentous cyanobacterium capable of differentiation into nitrogen-fixing heterocysts, motile hormogonia, or spore-like akinetes. Previous microarray analysis using a zwf mutant strain that synchronously differentiates into akinetes upon dark incubation in the presence of fructose indicated up-regulation of 1300 genes during akinete formation. These include four putative regulatory genes encoding homologues of PhoH-like protein, a transcriptional regulator in the TetR family, a two-component hybrid sensor and regulator, and a DNA/RNA helicase. Rapid amplification of cDNA ends (RACE) was used to identify transcriptional start sites of each gene. Multiple sequence alignment with intergenic regions of orthologous genes from other akinete-forming bacteria identified conserved DNA sequences with likely involvement in transcriptional regulation. Reporter vectors were constructed by insertion of PCR fragments containing each promoter region in front of a promoterless GFP gene in a pSUN 119 plasmid and transformed into wild-type N. punctiforme by electroporation to create reporter strains. Heterocysts and akinetes were then induced sequentially by first nitrogen limitation and then phosphate limitation. Epifluorescence microscopy of differentiating filaments confirmed cell-type-specific patterned transcriptional expression as predicted from the microarray results. Putative deletional mutants have been created and will be used for physiological studies.