Characterization of an OxyR-regulated alkyl hydroperoxide reductase (ahpC2D) operon in Legionella pneumophila.
Date
2007
Authors
LeBlanc, Jason J.
Journal Title
Journal ISSN
Volume Title
Publisher
Dalhousie University
Abstract
Description
Legionella pneumophila expresses two catalase-peroxidase enzymes that exhibit only weak hydrogen peroxide (H2O2)-scavenging activity, suggesting that other enzymes might decompose H2O 2. Likely candidates included two alkyl hydroperoxide reductases (AhpC) that were identified in the L. pneumophila genome. AhpC1 and AhpC2/AhpD (AhpC2D) show similarity to the peroxide scavenging systems found in Helicobacter pylori and Mycobacterium tuberculosis , respectively. Our results indicated that: (i) expression of L. pneumophila ahpC1 and ahpC2 restores H2 O2 resistance in a catalase/peroxidase-deficient mutant of Escherichia coli; (ii) both L. pneumophila ahpC1::km and ahpC2D::km mutants are more sensitive to various peroxides or reactive oxygen intermediate (ROI)-producing compounds than wild-type, a phenotype that could be alleviated by complementation; (iii) L. pneumophila ahpC mutants were not affected in their intracellular growth in macrophage-like cells; (iv) expression of ahpC1 appeared post-exponentially in broth culture, whereas ahpC2 was expressed during early exponential phase; (v) ahpC1 mRNA levels were consistently higher than those of ahpC2D; (vi) ahpC2D expression is significantly increased upon loss of AhpC1 function. To address whether the L. pneumophila OxyR homologue (LpOxyR) could function as a regulator of the oxidative stress response as seen in other organisms, experiments were performed to demonstrate that: (i) reduced and oxidized forms of LpOxyR can bind the promoter region of ahpC2D (Pahpc2); (ii) defective LpOxyR binding resulted in loss of ahpC2 transcriptional activity; (iii) reduced LpOxyR displayed an extended DNA footprint that overlaps with the putative -35 region of ahpC2 which was fully accessible to RNA polymerase with oxidized LpOxyR; (iv) expression of LpOxyR was partially able to restore peroxide resistance in an E. coli oxyR::km mutant. However, unlike E. coli OxyR, LpOxyR was unable to bind to its own promoter. Since LpOxyR expression was growth phase-dependent, attempts were also made to determine possible regulators of oxyR expression. An acrylamide capture of DNA-bound complexes technique was used in an attempt to identify transcriptional regulators with no success. In summary, this study reports that AhpC1 or AhpC2D provide an essential peroxide-scavenging function to L. pneumophila and that LpOxyR functions as a peroxide sensor/transcriptional regulator capable of activating transcription of ahpC2D in response to oxidative stress.
Thesis (Ph.D.)--Dalhousie University (Canada), 2007.
Thesis (Ph.D.)--Dalhousie University (Canada), 2007.
Keywords
Biology, Microbiology.