Date Thesis Awarded

5-2018

Access Type

Honors Thesis -- Access Restricted On-Campus Only

Degree Name

Bachelors of Science (BS)

Department

Biology

Advisor

Mark Forsyth

Committee Members

Margaret Saha

Lisa Landino

Oliver Kerscher

Abstract

Two component signal transduction systems in bacteria are key for environmental adaptation. Signaling via these systems is traditionally considered to be relatively simple, only involving interactions between the sensory protein and its cognate response regulator. The ArsRS two component system is vital for the acid response in Helicobacter pylori. This study investigates the mechanism by which ArsRS responds to acid in H pylori. Recent studies in our lab have challenged the the classic model in which the response regulator ArsR is activated by the phosphorylation of a conserved aspartic acid by the transfer of a phosphoryl group from a histidine within the acid sensing histidine kinase ArsS. We show that only phosphomimetic substitutions for the aspartic acid at the 52nd position on ArsR (D52E and D52N) yield viable H. pylori mutants, while substitutions of alanine, glycine or serine at this position fail to yield viable mutants. As deletion of arsR is a lethal mutation, the recovery of phosphomimetic amino acid substitutions and failure to recover non-phosphomimics suggests that the crucial activity of ArsR depends upon phosphorylation at D52 and that ArsR D52E and D52N must be at least partially active. We thus examined the extent to which these phosphomimetic mutations affect the expression of two of the genes in the ArsRS regulon, the acid-repressed outer membrane adhesion protein, sabA and the acid-induced urease subunit, ureA, under acidic conditions. Our findings show a slight but significant decrease in sabA transcription in H. pylori mutants with the phosphomimetic forms of ArsR but did not show a phenotype of constitutive acid regulation of ureA as expected of phosphomimetic response regulators. These mutants do, however, show full acid repression of sabA and acid induction of ureA at pH 5, indicating another step of activation in the acid response besides phosphorylation of ArsR D52. Our investigation into the role of ArsS in the Acid Response System (ARS), found that the histidine-kinase activity of ArsS is necessary for both acid-induced repression of sabA and acid-induced activation of ureA in both wild type H. pylori and H. pylori with phosphomimetic ArsR D52E. Thus, a functional ARS is lost without phosphorylation by ArsS. Furthermore, a mutant H. pylori strain with a loss of ArsS kinase ability and a phosphomimetic ArsR failed to show acid-induction of ureA, indicating that the acid induction of ureA seen in the ArsR D52E mutant does not come from an additional activation of ArsR, but from phosphorylation by ArsS of a protein other than ArsR. Lastly, we explore the role of cross talk between ArsRS and CrdRS or FlgR in the acid regulation of ureA and sabA via genetic deletions in the response regulators CrdR and FlgR and the non-cognate histidine kinase CrdS. Our results indicate that cross-talk with these non-cognate TCS proteins is not involved in the acid regulation of sabA and ureA. Our findings challenge the classic model of two component systems and suggest a much more complex interaction between sensory proteins and response regulator proteins in order to regulate gene expression.

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