Construction And Use Of Gfp Reporter Vectors For Analysis Of Cell-Type-Specific Gene Expression In Nostoc Punctiforme

Two transcriptional reporter shuttle vectors were constructed for the filamentous cyanobacterium Nostoc punctiforme using the green fluorescence protein (GFP) reporter. Both the ampicillin- and kanamycin-resistant versions of the plasmid allow promoters to be directionally cloned into a multiple cloning site preceding a promoterless gfp gene using an Escherichia coli host. The ability of the self-replicating shuttle plasmids to report cell-type-specific gene expression in N. punctiforme was tested by cloning promoters expressed in normal vegetative cells, nitrogen-fixing heterocysts and spore-like akinetes. A PpsaC reporter gene fusion was expressed in vegetative cells and not in heterocysts, whereas GFP driven from PhetR was found highly expressed in heterocysts. GFP expression driven by the promoter for the N. punctiforme homologue of the akinete-specific gene avaK was expressed in developing akinetes. Decreased expression of GFP from the PpsaC reporter in hormogonia was also observed. The results demonstrate the utility of these GFP vectors to study cell-type-specific gene expression in differentiating filamentous cyanobacteria.