Article

An ortholog of OxyR in Legionella pneumophila is expressed postexponentially and negatively regulates the alkyl hydroperoxide reductase (ahpC2D) operon.

Department of Pathology and Laboratory Medicine, Queen Elizabeth II Health Sciences Center, Halifax, Nova Scotia, Canada.
Journal of bacteriology (Impact Factor: 2.69). 06/2008; 190(10):3444-55. DOI: 10.1128/JB.00141-08
Source: PubMed

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) (pBADLpoxyR)-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.

1 Follower
 · 
80 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: While screening genomic library of A. baumannii DS002 isolated from organophosphate (OP) polluted soils, nine open reading frames were identified coding for Glutathione S-transferase (GSTs) like proteins. These GSTs (AbGST01 to AbGST09) have shown phylogenetic relationship to a number of well characterized GST classes found in taxonomically diverse group of organisms. Interestingly, the Abgst01 (KF151191) got upregulated when grown in presence of an OP insecticide, methyl parathion (MeP). Even its product, AbGST01 dealkylated methyl parathion to desmethyl parathion. An OxyR binding motif was identified upstream of Abgst01 gene. The Abgst-lacZ fusions constructed by omitting OxyR binding site showed drastic reduction in promoter activity. Reconfirming its role in abgst01 expression, very low β-galactosidase activity levels were observed when Abgst-lacZ fusions were mobilized into oxyR (KF151190) null mutants of A. baumannii DS002. The OxyR binding sites are not found upstream of other Abgst01 (Abgst02- Abgst09) promoters. However they contained consensus sequence motifs that possibly can serve as target sites for certain well characterised transcription factors. In support of this observation, the Abgsts have responded differently to various oxidative stress inducers. The Abgsts identified in A. baumannii DS002 are found to be highly conserved among all known genome sequences of A. baumannii strains. The versatile ecological adaptability of A. baumannii strains is apparent if sequence conservation is seen together with their involvement in detoxification process.
    Microbiology 10/2013; 160. DOI:10.1099/mic.0.070664-0 · 2.84 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Legionella pneumophila uses aquatic protozoa as replication niche and protection from harsh environments. Although L. pneumophila is not known to have a circadian clock, it encodes homologues of the KaiBC proteins of Cyanobacteria that regulate circadian gene expression. We show that L. pneumophila kaiB, kaiC and the downstream gene lpp1114, are transcribed as a unit under the control of the stress sigma factor RpoS. KaiC and KaiB of L. pneumophila do not interact as evidenced by yeast and bacterial two-hybrid analyses. Fusion of the C-terminal residues of cyanobacterial KaiB to Legionella KaiB restores their interaction. In contrast, KaiC of L. pneumophila conserved autophosphorylation activity, but KaiB does not trigger the dephosphorylation of KaiC like in Cyanobacteria. The crystal structure of L. pneumophila KaiB suggests that it is an oxidoreductase-like protein with a typical thioredoxin fold. Indeed, mutant analyses revealed that the kai operon-encoded proteins increase fitness of L. pneumophila in competitive environments, and confer higher resistance to oxidative and sodium stress. The phylogenetic analysis indicates that L. pneumophila KaiBC resemble Synechosystis KaiC2B2 and not circadian KaiB1C1. Thus, the L. pneumophila Kai proteins do not encode a circadian clock, but enhance stress resistance and adaption to changes in the environments.
    Environmental Microbiology 07/2013; DOI:10.1111/1462-2920.12223 · 6.24 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Legionella pneumophila is a water-borne pathogen that causes a severe lung infection in humans. It is able to replicate inside amoeba in the water environment, and inside lung macrophages in humans. Efficient regulation of gene expression is critical for responding to the conditions that L. pneumophila encounters and for intracellular multiplication in host cells. In the last two decades, many reports have contributed to our understanding of the critical importance of small regulatory RNAs (sRNAs) in the regulatory network of bacterial species. This report presents the current state of knowledge about the sRNAs expressed by L. pneumophila and discusses a few regulatory pathways in which sRNAs should be involved in this pathogen.
    Current topics in microbiology and immunology 08/2013; 376. DOI:10.1007/82_2013_347 · 3.47 Impact Factor