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Transcriptional Profiling and Molecular Characterization of the yccT Mutant Link: A Novel STY1099 Protein With the Peroxide Stress Response and Cell Division of Salmonella enterica Serovar Enteritidis

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Transcriptional Profiling and Molecular Characterization of the yccT Mutant Link: A Novel STY1099 Protein With the Peroxide Stress Response and Cell Division of Salmonella enterica Serovar Enteritidis

Sinisa Vidovic et al. Biology (Basel).

Abstract

Uncharacterized protein STY1099, encoded by the yccT gene, was previously identified as the most altered (i.e., upregulated) protein among the ZnO nanoparticle (NP) stimulon of Salmonella enterica serovar Enteritidis. Here we combined various stress response-related assays with functional genetics, global transcriptomic and proteomic analyses to characterize the yccT gene and its STY1099 product. Exposure of S. enterica Enteritidis to H2O2 (i.e., hydrogen peroxide) resulted in a significant (p < 0.0001) upregulation of the yccT gene, whereas exposure to paraquat (i.e., superoxide) did not alter the expression of the yccT gene. The ∆yccT mutant of S. enterica Enteritidis exposed to 0.75 mM H2O2, showed significantly reduced (p < 0.05) viability compared to the wild type strain. Further, comparative transcriptome analyses supported by Co-immunoprecipitation (Co-IP) assay revealed that STY1099 protein plays a role in redox homeostasis during the peroxide stress assault via involvement in the processes of respiratory nitrate reductase, oxidoreductase activities, cellular uptake and stress response. In addition, we found that the STY1099 protein has the monopolar subcellular location and that it interacts with key cell division proteins, MinD, and FtsH, as well as with a rod shape-determining protein MerB.

Keywords: STY1099 protein; Salmonella enterica serovar Enteritidis; cell division; nitrate reductase; peroxide stress response.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characterization of the yccT gene and its STY1099 protein sequence. (A) Phylogeny of the yccT gene sequence using seven bacterial species from Enterobacteriaceae, Vibrionaceae, and Pasteurellaceae families. (B) Sequence alignment of STY1099 protein from Salmonella enterica, Escherichia coli, Shigella flexneri, Citrobacter freundi, Enterobacter cloacae, Vibrio cholerae, Haemophilus influenzae and Serratia spp. Color gradient depicts highly conserved sequences (dark blue), moderate conserved (light blue) and no conserved sequences (white). Binding sites for SO4 (red), Na+ (pink), Zn (dark green) and OH (light green) have been highlighted by rectangles above the STY1099 sequence. (C) A predicted model of the STY1099 tertiary structure was generated using a publicly available RaptorX platform at www.raptorx.uchicago.edu/StructurePrediction/documentation.
Figure 2
Figure 2
Messenger RNA (mRNA) expression levels of yccT (STY1099 protein), gutM, napD (interactomes of STY1099) and oxyR, sodA (positive controls for oxidative stress) in the wild type S. enterica serovar Enteritidis strain during its exponential growth exposed to 3 mM of H2O2. Values on the y axis are relative expression levels (fold change) normalized to wild type during the oxidative treatment. The data correspond to the mean value of three biological replications. Error bars correspond to the standard deviation.
Figure 3
Figure 3
mRNA expression levels of yccT in the wild type strain exposed to 0.75 mM, 1 mM and 3 mM concentrations of H2O2, respectively, during the exponential growth phase. The data correspond to the mean value of three biological replications.
Figure 4
Figure 4
Hydrogen peroxide killing assay. (A) Evaluation of the effect of H2O2 on the ∆yccT, ∆gutM, ∆napD mutant strains compared to that of their parental wild type strain during their exponential growth phases. (B) Validation of the effect of the yccT gene deletion on the survivability of S. Enteritidis during peroxide treatment.
Figure 5
Figure 5
Subcellular localization of STY1099 protein. Single confocal section of deconvolved three D acquisitions are shown. (A) GFP expressing bacteria. (B,C) STY1099-GFP fusions. Panels (A,B) share the same lookup table, panel C shows the STY1099-GFP fusion with a lookup table intended to show the bacterial cell outline. Scale bar 2 µm.
Figure 6
Figure 6
Gene ontology (GO) enrichment analysis portraying the most important biological processes and molecular functions of S. enterica serovar Enteritidis during the exponential growth phase that were affected by ∆yccT mutation. Included in analysis were genes that were more than two-fold up- or downregulated with FDR (p < 0.05) and high reproducibility across the biological replicates.
Figure 7
Figure 7
Validation of RNA-sequencing (RNA-seq) data by qRT-PCR analysis. Data represent fold changes in expression of selected seven genes in the wild type and the ∆yccT mutant treated with 3 mM H2O2. Genes differently expressed between the wild type and ∆yccT mutant during H2O2 treatment represent the mean value of three biological replications.
Figure 8
Figure 8
Validation of STY1099 expression of the yccT-flag gene. Western blot analysis shows the results for first antibody: anti His (lanes 1–4) and second antibody: anti-FLAG (lanes 5–10). Negative controls lanes 5 and 6.

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