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    Regulation of competence gene homologs in Salmonella enterica serovar Typhimurium and Escherichia coil

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    Alshabib_Ebthal_200325804_PHD_BIOL_Spring2017.pdf (14.05Mb)
    Date
    2016-12
    Author
    Alshabib, Ebthal Yahya
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    URI
    http://hdl.handle.net/10294/7677
    Abstract
    Many bacteria have the ability to take up DNA from their environment through a process termed “natural competence”. Even though there are numerous bacterial species that are recognized to be naturally competent, competence is undetected or overlooked in many species such as members of the Enterobacteriaceae. Homologs of competence genes and the main regulator of natural competence, Sxy, are conserved in Salmonella and Escherichia coli; however, these genes were found to be silent under all previously-tested conditions. Thus, I carried out molecular studies to investigate genetic induction and regulation mechanisms of natural competence in Salmonella Typhimurium, and E. coli. Despite decades of work and unsuccessful attempts to induce the cryptic competence genes, our genetic studies in S. Typhimurium show that low temperatures (22 – 25 ᵒC) and growth on semi-solid media to allow for motility induce the expression of competence genes. Nutrient deprivation is hypothesized to be another inducing signal; where the expression of competence genes increased under starvation conditions when using arabinose as a carbon source. We identified Sxy and CRP as positive regulators of competence gene expression, whereas the nucleoid-associated proteins, FIS and H-NS, and the alternate sigma factor, RpoS, repress the expression of competence genes. Bioinformatic analysis of competence gene promoters suggests that DNA supercoiling regulates competence gene expression. However, experimental relaxation of DNA supercoiling did not confirm this. Several competence gene homologs have an evolutionary history of operon and promoter shuffling, nevertheless the transcription of these genes is coordinated and synchronized in different bacterial lineages through coregulated promoters. Altogether these results demonstrate that several environmental signals, transcription factors, and regulatory mechanisms are integrated to regulate competence gene expression in S. Typhimurium. In Haemophilus influenzae, the poorly characterized competence regulator, Sxy, is proposed to be stimulated by starvation for purine nucleotides. In S. Typhimurium, we found that sxy expression is stimulated by temperature, motility on semi-solid media, and nutrient deprivation signals. Further, we found that sxy expression is activated by CRP and repressed by FIS, H-NS, and RpoS. Moreover, it was previously proposed that Sxy assists CRP binding to competence gene promoters. Our phenotypic analysis in E. coli showed that mutations that alter the Sxy C-terminus abolish CRP-S transcriptional activation, and remove the toxic effect characteristic of sxy overexpression. A genome-wide analysis of the Salmonella Sxy regulon using directional RNA-seq revealed that Sxy function is not restricted to the activation of competence genes. Sxy also plays a role in regulating sugar metabolism, DNA replication, nucleotide metabolism, and expression of several small RNAs, transcription factors, and pathogenicity genes. This project is the first to study the genetics of competence in Salmonella, and it is pioneering in identifying novel inducing signals and transcription factors that regulate competence gene expression. The outcomes of this research enhance our understanding of the role of Sxy in regulating competences and other functions. The body of knowledge surrounding regulation of competence genes will greatly inform and guide future research into competence gene regulation and help deduce the ecological role of DNA uptake in gamma-proteobacteria.
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