DegS and RseP Homologous Proteases Are Involved in Singlet Oxygen Dependent Activation of RpoE in Rhodobacter sphaeroides

et al. (2013) DegS and RseP Homologous Proteases Are Involved in Singlet Oxygen Dependent Activation of RpoE in Rhodobacter sphaeroides. PLoS ONE 8(11): e79520. doi:10.1371/journal.pone.0079520 Editor: Rajeev Misra DegS and RseP Homologous Proteases Are Involved in Singlet Oxygen Dependent Activation of RpoE in Rhodobacter sphaeroides Aaron M. Nuss 0 Fazal Adnan 0 Lennart Weber 0 Bork A. Berghoff 0 Jens Glaeser 0 Gabriele Klug 0 0 1 Institute of Microbiology and Molecular Biology, Giessen University , Giessen, Germany , 2 Department of Molecular Infection Biology, Helmholtz Centre for Infection Research , Braunschweig, Germany , 3 Department of Cell and Molecular Biology, Biomedical Center, Uppsala University , Uppsala , Sweden Singlet oxygen (1O2) is the main agent of photooxidative stress and is generated by photosensitizers as (bacterio)chlorophylls. It leads to the damage of cellular macromolecules and therefore photosynthetic organisms have to mount an adaptive response to 1O2 formation. A major player of the photooxidative stress response in Rhodobacter sphaeroides is the alternative sigma factor RpoE, which is inactivated under non-stress conditions by its cognate anti-sigma factor ChrR. By using random mutagenesis we identified RSP_1090 to be required for full activation of the RpoE response under 1O2 stress, but not under organic peroxide stress. In this study we show that both RSP_1090 and RSP_1091 are required for full resistance towards 1O2. Moreover, we revealed that the DegS and RseP homologs RSP_3242 and RSP_2710 contribute to 1O2 resistance and promote ChrR proteolysis. The RpoE signaling pathway in R. sphaeroides is therefore highly similar to that of Escherichia coli, although very different anti-sigma factors control RpoE activity. Based on the acquired results, the current model for RpoE activation in response to 1O2 exposure in R. sphaeroides was extended. - Light and oxygen in combination with a photosensitizer lead to the formation of toxic singlet oxygen (1O2). The photosensitizer absorbs light and transfers energy to molecular oxygen, causing a spin conversion of an electron, thereby forming the highly reactive 1O2 [1]. Excess of 1O2 is toxic for the cell, as it can react with macromolecules like proteins, lipids and nucleic acids [2,3]. The cell needs to respond to this so called photooxidative stress to prevent cellular damages which consequently would lead to cell death. Facultative photosynthetic -proteobacteria like Rhodobacter sphaeroides induce the formation of the photosynthetic apparatus when the oxygen tension in the environment decreases. The synthesized bacteriochlorophyll molecules and their precursors can act as potent cellular photosensitizers. Nevertheless, even when photosynthetic pigments are highly abundant in the cell, R. sphaeroides grows well in the presence of light and oxygen. The presence of carotenoids protects against 1O2 caused damages and in addition, R. sphaeroides mounts a molecular response to 1O2 exposure, which is independent of carotenoids [4,5]. This response partly depends on the alternative group IV sigma factor RpoE. RpoE is inactivated by forming a stable complex with its cognate antisigma factor ChrR in a 1:1 stoichiometry [6,7]. When R. sphaeroides cells are exposed to 1O2, the RpoE:ChrR complex dissociates, RpoE binds to the RNA polymerase and induces the expression of target genes [4,6]. When the crystal structure of the RpoE:ChrR complex was solved it was shown that the zinc containing anti-sigma domain (ASD) of ChrR is necessary for the interaction with RpoE [7]. The ASD is conserved in many bacterial anti-sigma factors [7]. A second zinc containing ChrR domain, the cupin like domain (CLD), is necessary for activation of RpoE by 1O2. It was proposed that amino acid side chains or a ligand in the ChrR-CLD are targets of unknown chemical modification by 1O2 that lead to dissociation of the RpoE:ChrR complex [7]. The CLD could also play a role in promoting an association of the RpoE:ChrR complex with the photosynthetic membrane, the main source of 1O2 generation [8]. In bacteria one mechanism of sigma factor activation is the proteolysis of the cognate anti-sigma factor. In the Gram Figure 1. Genetic organization of the RSP_1091-1087 and rpoEchrR operons on the R. sphaeroides chromosome 1. The insertion site of Tn5 which resulted in reduced RpoE activity is indicated. The Tn5 inserted 683 bp downstream of the start codon of the RSP_1090 gene. RSP_1090 located in a putative operon with RSP_1091, RSP_1089, RSP_1088 and RSP_1087. Both operons are preceded by an RpoE dependent promoter. Annotated protein functions are depicted below the locus tag numbers. doi: 10.1371/journal.pone.0079520.g001 negative bacterium Escherichia coli, the alternative sigma factor E (also known as RpoE) is inactivated by the binding of its cognate anti-sigma factor RseA, which is membrane localized. Under cell envelope stress conditions, RseA is stepwise proteolyzed, thus RpoE is released and can bind to the RNA polymerase [9]. Interestingly, the N-terminal ASD of ChrR and RseA are similar in structure, but not in amino acid sequence [7,10]. Homologs of the RpoE:ChrR complex can be found in many -, - and -proteobacteria [11]. In the -proteobacterium Caulobacter crescentus RpoE activity is not only induced by 1O2, but also by exposure to organic peroxide (tert-butylhydroperoxide, tBOOH), cadmium and UV-A irradiation [12]. Specific amino acid residues in the anti-sigma factor ChrR may be required for the specific response to either 1O2, organic peroxide and UV-A irradiation or cadmium [12]. The R. sphaeroides RpoE regulon is well defined, but the exact mechanism of RpoE:ChrR dissociation is still unknown. Recent work reported that the anti-sigma factor ChrR is degraded in the presence of 1O2 and tBOOH [13,14], but the proteases involved in ChrR proteolysis are yet unknown. This motivated us to search for factors that are involved in RpoE activation under photooxidative stress. A Tn5 mutagenesis of the R. sphaeroides wild type revealed that insertion of Tn5 into the RSP_1090 generated a strain highly sensitive to 1O2. Consequently, we investigated the impact of genes encoded in the RSP_1091-1087 operon in the photooxidative stress response and showed that RSP_1090 affects the stability of ChrR. In E. coli the proteases DegS and RseP are involved in proteolysis of the RpoE anti-sigma factor RseA. Because the Tn5 mutagenesis did not reveal 1O2 sensitive protease-mutants in R. sphaeroides and the RSP_1090 product has no homology to proteases, the DegS and RseP homologs RSP_3242 and RSP_2710 were deleted in R. sphaeroides in order to elucidate if these proteases are involved in ChrR degradation and RpoE activation. Our results support a function of these proteases in singlet oxygen-dependent proteolysis of ChrR. Therefore, central factors involved in RpoE activation are shared between R. sphaeroides and E. coli despite the (...truncated)