The rearrangement of nitrogen-fixation (nif) genes during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 offers an example of an environmentally induced cellular differentiation involving programmed genome rearrangement. under nitrogen limiting conditions, Anabaena grows as a simple multicellular organism composed of two interdependent cell types; vegetative cells and heterocysts. Approximately 10% of the vegetative cells evenly spaced along the filament terminally differentiate into heterocysts, which provide fixed nitrogen to neighboring vegetative cells. Heterocycyst development involves many morphological and biochemical changes, including activation of the nif genes and repression of genes required for carbon fixation and the oxygen-evolving photosystem II. Three site-specific programmed genome rearrangements occur during the late stages of heterocyst differentiation: the excision of an 11 -kb element form with the nifD gene, the excision of a 55--kb element from within the fdxN gene, and [the excision of a 10.5-kb element from within the hupL gene]. The long-term objective of this project is to understand [the genetics of microbial development and terminal differentiation by studying] gene regulation during heterocyst differentiation, with an emphasis on the developmental period prior to genome rearrangement when a cell becomes committed to differentiate. The project is focused on the coordinated regulation of the DNA rearrangements and the [identification of genes that are required for late developmental events].
The specific aims of the proposed research are as follows; 1. [We will test the hypothesis that the xisC gene is necessary and sufficient for the hupL rearrangement by producing an xisC null mutation and by over expression of xisC. These experiments will also be used to assess the role of the rearrangement in heterocyst development and function. Reporter fusions will be used to monitor hupl expression during heterocyst differentiation in the mutant strains. The regulation of the rearrangement will be addressed as stated in aims (2) and (4).] 2. The hypothesis that the genome rearrangements are coordinately regulated by a common set of trans-acting DNA-binding proteins will be tested. The role of the DNA-binding proteins NtcA (BifA) and Factor-2 on the xisA, xisF, [and xisC] recombinase genes will be determined using both genetic and biochemical approaches. Upstream sequences of xisF and [xisC] will be used to identify new regulatory factors. 3. Sequences on the fdxN element just downstream of the xisF recombinase gene appear to be required for the cell-type specificity of rearrangement. Genetic and biochemical approaches will be used to identify the factors involved an to determine the mechanism of cell-type specificity. 4. [To determine the genetic basis of terminal differentiation, we will identify regulatory genes required for the DNA rearrangements.} Cells become committed to form mature heterocysts prior to genome rearrangement. The genes required during this crucial developmental period will be identified by transposon mutagenesis. Two complementary strategies will be used for the identification of mutants.
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