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Regulation of Sxy in Haemophilus influenzae and other Pasteurellaceae Volar, Milica

Abstract

Natural competence is ability of bacteria to take up exogenous DNA from their surroundings. Haemophilus influenzae, a member of the Pasteurellaceae family, tightly regulates competence development, inducing uptake only when cells are starved both for sugars and nucleotides. Two proteins are involved in activation of competence gene transcription: Sxy and CRP. While CRP is a global regulator of genes involved in sugar metabolism, Sxy regulates a small regulon composed primarily of competence genes. Competence levels are intimately connected with the abundance of Sxy protein in the cell, thus revealing the regulation of the sxy gene is an imperative for understanding how and why H. influenzae becomes naturally competent. Previous studies found that strains carrying point mutations predicted to weaken sxy RNA secondary structure overproduce Sxy and are hypercompetent, while strains with mutations predicted to strengthen this structure, do not produce Sxy and are noncompetent. This suggested that the secondary structure of sxy mRNA limits the gene's expression in the wild type in competence non-inducing conditions. I used bioinformatics and biochemical techniques to investigate whether sxy is regulated via its RNA secondary structure. Nuclease mapping confirmed that sxy RNA folds into a stable secondary structure and that the point mutations in hyper- and non-competent sxy mutants weaken or strengthen this structure, respectively. I also examined whether the regulation of sxy is conserved in Pasteurellaceae by comparing the predicted secondary structures of RNA in sxy homologues. The results suggested that the RNA secondary structure is only important in regulation of sxy in H. influenzae strains and is not conserved in other Pasteurellaceae. However, all of the examined species had long intergenic regions upstream of sxy homologues suggesting that additional regulatory element(s) is present. Based on the results in the thesis, I propose two models of sxy regulation: in one, sxy mRNA secondary structure inhibits translation initiation and in the other this structure allows binding of lignad(s) and acts as a riboswitch.

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