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An RNA secondary structure regulates sxy expression and competence development in haemophilus influenzae Bannister, Laura Anne

Abstract

The process of natural transformation is the ability of cells to take up DNA from their environment and recombine this DNA into their genome to yield stable inheritance of an altered genotype. In Haemophilus influenzae, competence for transformation is tightly regulated, and is induced to maximal levels when cells undergo nutrient limitation. One signal for competence development is a rise in concentration of intracellular cAMP. A second proposed signal is an increase in the concentration of Sxy, a protein that appears to activate competence by regulating the expression of other competence genes. In this thesis, I examined the evolution and expression of sxy, focusing on whether the function of Sxy is specific to competence development, and whether the pattern of expression of sxy is consistent with competence being controlled by levels of Sxy within the cell. I found potential Sxy homologs in both transformable and nontransformable bacteria, indicating that Sxy may have a function that is unrelated to competence development. Neither Sxy nor any of its homologs contain amino acid motifs that share similarity with those of known transcription factors; therefore, my results do not confirm the hypothesis that Sxy directly activates transcription of competence genes. Sxyv.lacZ reporter gene analysis indicates that sxy expression is increased within cells well before maximum competence development in rich medium, suggesting that an increase in Sxy concentration is not the limiting step of competence development. Reporter gene analysis also indicates that maximal amounts of Sxy are not any higher in starvation medium than in rich medium, suggesting that an increase in Sxy concentration is not the second, cAMP-independent signal that increases the competence of cells in starvation medium relative to those in rich medium. Finally, I suggest that sxy may be regulated at the translational level, perhaps by the inhibition of translation by an mRNA secondary structure that had been previously proposed. I confirmed the existence of this secondary structure, using both in vivo and in vitro experiments. In this thesis, I also discuss attempts to find repressors of competence following H. influenzae mutagenesis with Tn926. Although I did not recover any such mutants, I did recover mutants in the fis operon of H. influenzae that have delayed spontaneous competence, and further experiments showed that fis null mutants have transformation frequencies in rich medium that are reduced - 500-fold relative to wild type cells. I suggest some ways in which Fis may be involved in natural transformation.

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