LeBlanc, Jason J.;
Brassinga, Ann Karen C.;
Ewann, Fanny;
Davidson, Ross J.;
Hoffman, Paul S.
An Ortholog of OxyR in Legionella pneumophila Is Expressed Postexponentially and Negatively Regulates the Alkyl Hydroperoxide Reductase ( ahpC2D ) Operon
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Medientyp:
E-Artikel
Titel:
An Ortholog of OxyR in Legionella pneumophila Is Expressed Postexponentially and Negatively Regulates the Alkyl Hydroperoxide Reductase ( ahpC2D ) Operon
Beteiligte:
LeBlanc, Jason J.;
Brassinga, Ann Karen C.;
Ewann, Fanny;
Davidson, Ross J.;
Hoffman, Paul S.
Erschienen:
American Society for Microbiology, 2008
Erschienen in:
Journal of Bacteriology, 190 (2008) 10, Seite 3444-3455
Sprache:
Englisch
DOI:
10.1128/jb.00141-08
ISSN:
1098-5530;
0021-9193
Entstehung:
Anmerkungen:
Beschreibung:
ABSTRACT Legionella pneumophila expresses two peroxide-scavenging alkyl hydroperoxide reductase systems (AhpC1 and AhpC2D) that are expressed differentially during the bacterial growth cycle. Functional loss of the postexponentially expressed AhpC1 system is compensated for by increased expression of the exponentially expressed AhpC2D system. In this study, we used an acrylamide capture of DNA-bound complexes (ACDC) technique and mass spectrometry to identify proteins that bind to the promoter region of the ahpC2D operon. The major protein captured was an ortholog of OxyR (OxyR Lp ). Genetic studies indicated that oxyR Lp was an essential gene expressed postexponentially and only partially complemented an Escherichia coli oxyR mutant (GS077). Gel shift assays confirmed specific binding of OxyR Lp to ahpC2D promoter sequences, but not to promoters of ahpC1 or oxyR Lp ; however, OxyR Lp weakly bound to E. coli OxyR-regulated promoters ( katG , oxyR , and ahpCF ). DNase I protection studies showed that the OxyR Lp binding motif spanned the promoter and transcriptional start sequences of ahpC2 and that the protected region was unchanged by treatments with reducing agents or hydrogen peroxide (H 2 O 2 ). Moreover, the OxyR Lp (pBADLp oxyR )-mediated repression of an ahpC2 - gfp reporter construct in E. coli GS077 (the oxyR mutant) was not reversed by H 2 O 2 challenge. Alignments with other OxyR proteins revealed several amino acid substitutions predicted to ablate thiol oxidation or conformational changes required for activation. We suggest these mutations have locked OxyR Lp in an active DNA-binding conformation, which has permitted a divergence of function from a regulator of oxidative stress to a cell cycle regulator, perhaps controlling gene expression during postexponential differentiation.