UBC Theses and Dissertations
CRP and Sxy regulate competence gene promoters in haemophilus influenzae Cameron, Andrew Dafydd Skye
Many bacteria have the ability to bind and take up DNA from their environment through a process called natural competence. Competent species are widely distributed across the prokaryotic phylogenetic tree and inhabit disparate niches. Most of these species are thought to tightly regulate DNA uptake, suggesting that it is an important physiological response to conditions that can arise in diverse environments. To better understand some of the signals and mechanisms that regulate competence development, I have carried out molecular studies in Haemophilus influenzae and other γ-proteobacteria. In H. influenzae, transcription of natural competence genes depends on two proteins, CRP and Sxy. In Escherichia coli, CRP is a well-characterized transcription factor that stimulates gene expression in response to sugar and energy starvation; genetic studies have shown that CRP plays a similar role in H. influenzae. Although CRP preferentially binds to DNA sites with the consensus sequence TGTGA, results presented here demonstrate that CRP targets unusual "CRP-S" sites (sequence TG C GA) in competence gene promoters. Transcription initiation at CRP-S promoters absolutely requires Sxy, unlike other CRP-regulated promoters in the cell. Results from promoter mutagenesis and in vitro binding assays support a model in which CRP cannot bind CRP-S sites unless assisted by Sxy. Bioinformatic analysis identified competence genes, Sxy orthologs, and CRP-S sites in three γ-proteobacteria families (Enterobacteriaceae, Pasteurellaceae, and Vibrionaceae ), suggesting that many bacteria use CRP and Sxy to regulate competence. Studies in H. influenzae identified an extensive secondary structure in sxy mRNA that blocks translation. Culturing cells in starvation medium improved Sxy translation while independently stimulating CRP activity at the sxy promoter; however, the addition of nucleotides to starvation medium blocked Sxy translation. Thus, sxy mRNA secondary structure is responsive to conditions where exogenous DNA can be used as a source of nucleotides, and transcription of sxy is simultaneously enhanced if CRP signals that energy supplies are limited. In conclusion, nutritional signals transduced by CRP and Sxy are integrated by CRP-S sites in competence gene promoters.
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