H-080. Promoter Analysis of Aminopeptidase Gene in Nostoc punctiforme

R. J. Ireland, III, M. L. Summers;
California State Univ., Northridge, CA.

Background: Nostoc punctiforme is a filamentous cyanobacterium with the capacity for differentiation into a spore-like akinete due to phosphate limitation or low light. Akinetes can survive long-term exposure to stresses such as cold and desiccation. Differential display and microarray analysis identified several genes in Nostoc punctiforme with possible involvement in akinete formation. Two genes, a putative aminopeptidase of the M28 family and a dnaK heat shock gene share the same intergenic region, though transcribed in opposite directions. It is hypothesized that these two genes are akinete-specific and are both regulated by a repressor protein. In addition, highly conserved regions identified between closely related cyanobacterial species are hypothesized to be critical to promoter function and regulation. Methods: Rapid amplification of cDNA ends (RACE) was used to find the +1 start sites for both genes. PCR fragments containing various combinations of conserved motifs were cloned into a promoterless green fluorescent protein (GFP) transcriptional reporter plasmid plasmid vector. Promoter regulation within cells of a filament, as assessed by GFP expression by epifluorescence microscopy, analysis was used to map promoter regulatory elements. Results: Epifluorescence microscopy of promoter-reporter deletion strains showed that cell-type specific expression of the aminopeptidase gene involves repressor binding site in the un-translated leader sequence. Constitutive expression in all cell types was observed when this region was eliminated. Conclusions: Bioinformatic analysis of the promoter region has identified conserved regions of the promoter found in orthologous genes of closely related Anabaena variabilis and Anabaena 7120. Transcriptional reporter analysis demonstrated importance of conserved regions lying between the startsite and coding region of the aminopeptidase gene.