Wolk 9723193 Anabaena, a filamentous cyanobacterium, carries out oxygenic photosynthesis and aerobic N2-fixation in two types of cells, vegetative cells and heterocysts, respectively. Comprised wholly of vegetative cells in the presence of fixed nitrogen, filaments respond to nitrogen deprivation by having 5 to 10% of their cells, at semi-regular intervals, differentiate into heterocysts. This organism offers one of the few opportunities to study the ways in which a prokaryote regulates structural cellular differentiation, and a nearly unique opportunity to study prokaryotic formation of multicellular patterns. A number of developmental regulatory genes and genes involved in synthesis of heterocyst-specific structures have been identified, and certain of their interdependencies identified. However, the detailed mechanisms that regulate the progression of the differentiation process once that process has been initiated remain largely unknown, and no overall regulatory strategy has been discerned. hepA is a gene that is activated specifically in immature heterocysts starting several hours after nitrogen stepdown and whose activity is required for normal formation of a principal structural element of heterocysts, the outer, polysaccharide layer of their envelope. Aspects of the regulation of hepA will be investigated. Experimental evidence indicates that the pattern of spaced heterocysts results from an inhibition, by mature and developing heterocysts, of the differentiation of nearby cells into heterocysts, and suggests that the inhibition is mediated by the elaboration of some differentiation-inhibiting substance that moves outward along a filament. Because the differentiation of heterocysts is organized by inhibition, it appears appropriate for Anabaena to have genes whose products inhibit the initiation or progression of differentiation. However, a mutation in such a gene could lead all vegetative cells to start to differentiate into heterocysts, a result that might heretofore have been recog nized only as a lethal mutation. Analysis of a restricted class of conditional mutations will show whether there are such genes that control differentiation in Anabaena. Results obtained with these conditional mutations can elucidate mechanisms that underlie cellular differentiation and (or) multicellular pattern formation. The oxygen in the earth's atmosphere came initially from the growth of oxygen-producing cyanobacteria. This oxygen prevented the assimilation of nitrogen gas. Certain filamentous cyanobacteria overcame that impediment by inventing multicellular division of labor: spaced cells in a filament became heterocysts, cells unable to photosynthesize and grow, but able to fix nitrogen gas in an oxygen-containing environment. Thus arose what may have been the first multicellular pattern, and Anabaena, still an outstanding example of bacterial multicellularity. Heterocyst formation is being analyzed because it provides an example of bacterial differentiation that may be regulated in a unique fashion, and because of the important contribution of heterocysts to global nitrogen fixation. Analysis of the regulation of hepA and the use of conditional mutants represent approaches to understanding how differentiation progresses. Conditional mutation may also assist mechanistic analysis of pattern formation.
