Electronic Thesis and Dissertation Repository


Doctor of Philosophy




David Haniford, Ph.D.


Transposons are integrally tied to the spread of antibiotic resistance among bacterial species, and cellular stress can cause increased transposition (which might benefit the bacteria as a species by increasing genetic plasticity). It is therefore important to understand how transposons have become integrated into bacterial regulatory networks. The goals of this thesis are to further investigate 1) new host-factors that regulate transposition, 2) post-transcriptional regulation as a means of regulating transposase expression, and 3) how cellular stress-response is tied to transposon mobility. I use the well studied Tn10/IS10 and Tn5/IS50 transposons in Escherichia coli as model systems. I show that Tn10 transposition is strongly repressed at the level of transposase translation by the global post-transcriptional regulator, Hfq—an RNA chaperone that enhances the base-pairing interactions of trans-encoded small regulatory RNAs (sRNAs) with their target mRNAs. As translation of the IS10 transposase mRNA (RNA-IN) is strongly repressed by an antisense regulator (RNA-OUT), I investigated whether Hfq was enhancing this negative regulation. Further evidence shows that Hfq down-regulates IS10 transposase translation by two pathways, one of which involves RNA-OUT. Hfq had not previously been shown to function in regulation by a cis-antisense RNA. I show that Hfq binds RNA-IN and RNA-OUT via known mRNA- and sRNA-binding sites, and that it enhances the rate of RNA-IN/OUT pairing in vitro. This ability was lost, along with in vivo regulation of transposition, to Hfq mutants lacking RNA-binding specificity at these surfaces. Evidence is presented that Hfq alters the secondary structures of both RNA-IN and RNA-OUT such that inter-molecular base pairing would be facilitated. I also show that Hfq strongly down-regulates Tn5 transposition. Unlike Tn10, this regulation is exercised at the level of transposase transcription. Evidence is presented that Hfq and the global transcription factor Crp work in the same regulatory network to limit Tn5 transposition. Finally, I demonstrate that Tn5 transposition is induced by nutrient starvation. Taken together, this work implicates Hfq as a component of a cellular defense mechanism against transposons and shows that Tn5 is able to respond to environmental conditions.

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