Fluorescence in situ Localization of Gene Expression Using a lacZ Reporter in the Heterocyst-forming Cyanobacterium Anabaena variabilis

Anabaena variabilis  ATCC 29413 has one Mo nitrogenase that is made under oxic growth conditions in specialized cells called heterocysts and a second Mo nitrogenase that is made only under anoxic conditions in vegetative cells. The two large  nif  gene clusters responsible for these two nitrogenases are under the control of the promoter of the first gene in the operon,  nifB1  or  nifB2 . Despite differences in the expression patterns of  nifB1  and  nifB2 , related to oxygen and cell type, the regions upstream of their transcription start sites (tss) show striking homology, including three highly conserved sequences (CS). CS1, CS2, and the region just upstream from the tss were required for optimal expression from the  nifB1  promoter, but CS3 and the 5′ untranslated region (UTR) were not. Hybrid fusions of the  nifB1  and  nifB2  upstream regions revealed that the region including CS1, CS2, and CS3 of  nifB2  could substitute for the similar region of  nifB1 ; however, the converse was not true. Expression from the  nifB2  promoter region required the CS1, CS2, and CS3 regions of  nifB2  and also required the  nifB2  5′ UTR. A hybrid promoter that was mostly  nifB2  but that had the region from about position −40 to the tss of  nifB1  was expressed in heterocysts and in anoxic vegetative cells. Thus, addition of the  nifB1  promoter region (from about position −40 to the tss of  nifB1 ) in the  nifB  hybrid promoter supported expression in heterocysts but did not prevent the mostly  nifB2  promoter from also functioning in anoxic vegetative cells.

IMPORTANCE  In the filamentous cyanobacterium  Anabaena variabilis , two Mo nitrogenase gene clusters,  nif1  and  nif2 , function under different environmental conditions in different cell types. Little is known about the regulation of transcription from the promoter upstream of the first gene of the cluster, which drives transcription of each of these two large operons. The similarity in the sequences upstream of the primary promoters for the two  nif  gene clusters belies the differences in their expression patterns. Analysis of these  nif  promoters in strains with mutations in the conserved sequences and in strains with hybrid promoters, comprising parts from  nif1  and  nif2 , provides strong evidence that each promoter has key elements required for cell-type-specific expression of the  nif1  and  nif2  gene clusters.