Transcriptome and physiological responses to hydrogen peroxide of the facultatively phototrophic bacterium Rhodobacter sphaeroides

Abstract

The transcriptome responses to hydrogen peroxide, H2O2, of the facultatively phototrophic bacterium Rhodobacter sphaeroides grown under semiaerobic conditions were investigated. At 7 min after the addition of 1 mM H2O2, the expression of approximately 9% of all genes (total, 394) was changed reliably by at least twofold. At 30 min, the number of genes (total, 88) and the magnitude of expression changes were much lower, indicating rapid recovery from stress. Two types of responses were observed: (i) an H2O2 stress response per se and (ii) a shift to high-oxygen metabolism. The former response involved the upregulation of genes for H2O2 detoxification, protein folding and proteolysis, DNA damage repair, iron transport and storage, iron-sulfur cluster repair, and the downregulation of genes for protein translation, motility, and cell wall and lipopolysaccharide synthesis. The shift to high-oxygen metabolism was evident from the differential regulation of genes for aerobic electron transport chain components and the downregulation of tetrapyrrole biosynthesis and photosystem genes. The abundance of photosynthetic complexes was decreased upon prolonged exposure of R. sphaeroides to H2O2, thus confirming the physiological significance of the transcriptome data. The regulatory pathways mediating the shift to high-oxygen metabolism were investigated. They involved the anaerobic activator FnrL and the antirepressor-repressor AppA-PpsR system. The transcription of FnrL-dependent genes was down at 7 min, apparently due to the transient inactivation by H2O2 of the iron-sulfur cluster of FnrL. The transcription of the AppA-PpsR-dependent genes was down at 30 min, apparently due to the significant decrease in appA mRNA.

FIG. 1.

Electron transport chains involved in energy generation under semiaerobic conditions in R. sphaeroides 7 min (A) and 30 min (B) after the addition of 1 mM H₂O₂. Arrows indicate electron flow. Thicker lines correspond to the anticipated higher electron flow. Known sites for generation of proton-motive force are shown. Expression of genes under no-stress conditions is set as background for comparisons (uncolored). Lack of color corresponds to expression that is not significantly changed from expression under no-stress conditions. Blue color corresponds to decreased expression (light blue, 0.33-0.5; bright blue, <0.33), and pink corresponds to increased expression (light pink, 2-3; pink, >3) of the corresponding genes. Proteins whose genes are expressed below reliable detection are not shown. DH, dehydrogenase; Q, quinone/quinol pool; LH, ligh-harvesting complex; RC, reaction center complex; cyt., cytochrome. The RC, LHI, and LHII complexes comprise the R. sphaeroides PS.

FIG. 2.

Upper panel shows photosystem gene cluster and protoporphyrin IX biosynthesis genes regulated by FnrL and AppA/PpsR. Each gene is represented by a box colored according to its function. bch genes, green; carotenoid crt genes, red; genes encoding structural polypeptides of photocomplexes, blue; genes encoding assembly factors or proteins of unknown function, gray; genes encoding regulatory factors, orange; genes encoding enzymes common to Bchl and ubiquinone biosynthesis, pink; protoporphyrin IX biosynthesis genes, magenta. Putative transcripts are shown as black horizontal arrows. Lower panels show relative expression levels at 7 and 30 min after exposure to H₂O₂ compared to no-stress conditions. Expression of every gene under the no-stress conditions is assigned 1 (not shown). Expression levels are according to the presented color scheme.

FIG. 3.

Relative change in expression compared to no-stress (0 min) conditions measured by qPCR. The average values of two independent qPCR amplifications performed in triplicate are presented. Gray bars, 7 min; black bars, 30 min.

FIG. 4.

Effect of the periodic (every 20 min) addition of H₂O₂ (0.45 mM, final concentration) on the abundance of R. sphaeroides photosynthetic complexes. Solid traces, control cultures; dashed traces, H₂O₂-exposed cultures. Under these conditions, H₂O₂ did not affect growth rates. Note that the abundance of photosynthetic complexes in the control culture increases with time due to oxygen consumption by the growing culture.