Progression and Spacing of Heterocyst Differentiation

Michigan State University, East Lansing MI

Investigators

Abstract

In the presence of combined nitrogen, filaments of the photosynthetic bacterium Anabaena are comprised wholly of vegetative cells. When deprived of combined nitrogen, 5 to 10% of the cells, at semi-regular intervals, differentiate into N2-fixing heterocysts. Anaebena offers a rare opportunity to study how a prokaryote forms a multicellular pattern (the spacing of heterocysts), and an infrequent opportunity to analyze how prokaryotic cells differentiate. The following mechanism of pattern formation is generally agreed on: mature and developing heterocysts inhibit the differentiation of nearby cells into heterocysts by elaborating a differentiation-inhibiting substance that moves outward along a filament. To date, few of the genes and processes that contribute to spaced heterocyst differentiation are known. The genomic sequence of Anabaena PCC 7120, nearly finished, provides a tool of great value to help elucidate the detailed mechanisms, and overall strategy, that regulate differentiation. That tool will be used to try to identify the estimated 100-200 genes that are required specifically for pattern formation and cellular differentiation. Analysis of these genes, once they are identified, will be crucial for understanding the mechanisms that underlie differentiation. Mutagenesis and global analysis of transcript abundance by hybridization to DNA arrays constitute complementary approaches to identifying the desired set of genes. Hybridization has the potential to show increases or decreases in a great number of RNA transcripts simultaneously. However, because hybridization is correlative, it requires mutagenesis to demonstrate which genes are necessary for development. Also, mutagenesis can, but hybridization cannot, recognize the developmental importance of constitutively expressed genes that are required specifically for development. Mutation of genes whose products participate in the intercellular inhibition of differentiation could lead all cells to initiate differentiation. Because such mutations might prevent vegetative growth, they are being sought separately by conditional mutagenesis. Heterocysts, normally 5-10% of total cells, presumably contribute a like percentage of total mRNA. Measurements of hybridization normally resolve differences of over 2-fold in transcript abundance between two conditions. Therefore, unless developing heterocysts can be isolated without loss of mRNAs, they must produce more than 20-fold more of an mRNA than does an average vegetative cell for the difference to be detectable by hybridization. For these reasons, our primary approach will be by mutagenesis, but hybridization analysis will also be performed. This project builds on the small number of development-specific genes already identified. In particular, available mutants bracket developmental stages. Focus will be on hybridization analysis on genes that are expressed specifically during, and so may be specifically required for, these stages. For example, whereas genes activated in a hetR mutant are candidates for involvement in non-developmental responses to nitrogen deprivation, genes activated in a hetC mutant but not in a hetR mutant are candidates for genes involved in pattern formation and the initiation of differentiation. Similarly, genes activated in hepK and devA mutants and in mutant a71 but not in a hetC mutant are candidates for involvement in all but the earliest stages of morphological differentiation. Derivatives of transposon Tn5 mutagenize Anabaena highly effectively, and without heretofore discerned site-specificity. Such transposons will be used to isolate mutants that can grow on combined nitrogen but not on N2, map them by sequencing, and test their sites of insertion for developmental relevance by complementing the mutations with mapped clones. The developmental roles of the genes identified will be discerned and integrated.