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. 2015 Feb 18:15:34.
doi: 10.1186/s12866-015-0357-0.

An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

Jingcheng Dai  1   2 , Hehong Wei  3   4 , Chunyuan Tian  5 , Fredrick Heath Damron  6 , Jizhong Zhou  7 , Dongru Qiu  8   9
Affiliations

An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

Jingcheng Dai et al. BMC Microbiol. .

Abstract

Background: Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR.

Results: RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essential for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains.

Conclusion: In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.

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Figures

Figure 1
Figure 1
The gene clusters of rpoE-rseA-rseB-rseC and rpoE2-ChrR and the flanking loci on the chromosome of the S. oneidensis MR-1 strains. The conserved gene cluster rpoE-rseA-rseB-rseC and the flanking genes are also found in the genomes of Escherichia coli and Pseudomonas aeruginosa.
Figure 2
Figure 2
The rpoE mutant had growth defects when cultured in minimal media, high salinity, and high or low temperature. Bacterial growth, as measured by OD600, are show for the strains growing in various conditions: A) Rich medium (LB broth); B) Nutrient-poor environment (the modified M1 minimal medium); C) Higher temperature (at 33°C and in the LB medium), D) Low temperature (at 4°C and in the LB medium); E) High salt stress (LB medium supplemented with 3% of sodium chloride, w/v).
Figure 3
Figure 3
Expression of the rpoE in the Shewanella oneidensis MR-1 activates expression of rpoE , rseA , and degQ . Strain MR-1 carrying the pHERD30T-rpoE plasmid was grown in the presense of L-arabinose (0.05% w/v). The MR-1 strain carrying only empty pHERD30T (with the pBAD promoter) vector was used as control. Note that the transcripts of rpoE in the treatment (the right lanes) include the in trans expression of plasmid-borne rpoE gene, which further enhances the expression of chromosomal loci of rpoE (SO_1342), rseA (SO_1343), and degQ (SO_3942). The transcription of the chromosomal rpoE gene and down-stream cognate anti-sigma factor gene rseA is driven by the same promoter, and an RpoE-recognized promoter for autoregulation has been identified upstream of the rpoE-rseA-rseB-rseC operon. The cells were collected for RNA extraction after 1 hour of induction. A) Transcription of the genes was examined by using semi-quantitative RT-PCR; 16S rRNA gene expression was analyzed and used as the loading control. B) Trace quantity plotting of Figure 3A using ‘Quantity One’ software. The assays were performed in triplicates.
Figure 4
Figure 4
DegQ is required for optimal growth of strain MR-1 under high temperature. Genetic complementation by plasmid-borne degQ rescued bacterial growth of the degQ mutant. The MR-1ΔdegQ strains carrying empty vector (labeled as MR-1ΔdegQ) and pHERD30T-degQ plasmid (MR-1ΔdegQ+degQ) and the pHERD30T-carrying wild type MR-1 (MR-1) strains were grown in LB broth supplemented with 15 μg/ml of gentamycin. Bacterial strains were grown at 35°C.
Figure 5
Figure 5
Effects of paraquat and hydrogen peroxide (H 2 O 2 ) on the bacterial growth of the Shewanella oneidensis strains. MR-1 wild type strain, the rpoE2, pgpD (SO_3349) and cgpD (SO_1563) in-frame deletion mutants and the pgpD-cgpD double mutant strains were grown in the LB broth containing A) 0, 0.5, 1, 2, 3, and 4 mM of paraquat or B) 0, 0.1, 0.3, 0.5, 0.7 and 1 mM of hydrogen peroxide and incubated at 28°C for 18 hrs.
Figure 6
Figure 6
Induced transcription of the member genes of RpoE2-regulated operons in the rpoE 2 null in-frame deletion mutant (MR-1ΔrpoE2) carrying the plasmid-borne rpoE2 gene. The strain carrying pHERD30T empty vector was used as control and 0.01% (w/v) of L-arabinose was added to the bacterial cultures of both control (carrying pHERD30T vector) and treatment (carrying pHERD30T-rpoE2) during late exponential phase (OD600 > 0.8). The cells were collected for RNA extract after 1 hour of induction. A) Transcription of the genes was examined by using semi-quantitative RT-PCR; 16S rRNA gene exp ression was analyzed and used as the loading control. B) Trace quantity plotting of figure 6A using ‘Quantity One’ software.The quantitative data represents three times of assays in duplicates.
Figure 7
Figure 7
Glutathione (GSH) peroxidase (GPx) activity assays of PgpD and CgpD. A) Histidine-tagged CgpD and PgpD proteins were purified. B) Glutathione was added and the peroxidase activity is defined as the amount of hydrogen peroxide (mg) broken down in one minute (min) by one mg of purified enzymes (mg H2O2/min/mg) under the assay conditions described.
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