Characterization of Two Genes Up-regulated During Heterocyst Development in the Cyanobacterium Anabaena sp. strain PCC 7120.

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Title: Characterization of Two Genes Up-regulated During Heterocyst Development in the Cyanobacterium Anabaena sp. strain PCC 7120.
Author: Thayer, Rebecca Elizabeth
Advisors: Dr. Stephanie Curtis, Committee Chair
Dr. Eric Miller, Committee Member
Dr. Jeff Thorne, Committee Member
Abstract: Anabaena sp. strain PCC 7120 is a cyanobacterium that carries out photosynthesis in a manner similar to plants and is capable of nitrogen fixation. This organism has developed a necessary spatial separation of the incompatible processes of photosynthesis and nitrogen fixation, as nitrogen fixation is sensitive to oxygen that is produced during photosynthesis. A differentiated cell type, called a heterocyst, is formed when Anabaena is in an environment lacking nitrogen, and these cells are the sites of nitrogen fixation. Heterocyst formation occurs about every tenth cell along a filament of photosynthetic vegetative cells after 24-36 hours of nitrogen starvation. A screen for sequences up-regulated at the transcript level during heterocyst development in Anabaena identified adjacent loci alr4311 and all4312. The sequence of alr4311 suggests it encodes the ATP-binding protein of an ABC transporter complex, while that of all4312 suggests it encodes the response regulator of a two-component regulatory system. Phylogenetic analysis of the predicted protein sequences of alr4311 and all4312 indicated that both of these proteins have orthologs in Nostoc punctiforme and Anabaena variabilis, two filamentous, diazotrophic cyanobacteria. Additionally, alr4311 appears to be most similar to ABC transporters involved in the import of cobalt, while all4312 was most similar to uncharacterized response regulators. The transcripts of alr4311 and all4312 are expressed at low levels in vegetative cells, and increase in abundance after nitrogen starvation and the induction of heterocyst development. Northern analysis and real-time RT-PCR showed that expression of alr4311 and all4312 are induced as early as 3 hours after initiation of differentiation, and expression levels of both genes remain elevated through the first 24 hours of development. Expression of both of these genes was blocked in an ntcA mutant, and significantly decreased in a hetR mutant. alr4311 was shown to be part of an operon with three unknown genes immediately upstream, while all4312 is not organized in a cluster of genes. The promoters of all4312 and the alr4311 operon were mapped by primer extension analysis, and a single transcript was detected for each, mapping downstream from a —10 consensus sequence characteristic of a sigma 70 promoter. Mutational analyses in which alr4311 and all4312 were individually inactivated suggested that alr4311 is required for the response to nitrogen starvation, while all4312 appears to be important in this response, but not essential. Both the alr4311 and all4312 mutants appeared to differentiate heterocysts, suggesting that the role of each of these genes may be related to heterocyst function, rather than development of the heterocyst structure.
Date: 2006-05-31
Degree: MS
Discipline: Functional Genomics

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