OxyR, a LysR family transcriptional regulator, plays vital roles in bacterial oxidative stress response. In this study, we found that the deletion of oxyR not only inhibited the antioxidant capacity of S. marcescens FS14, but also decreased the production of prodigiosin. Further study revealed that OxyR activated the prodigiosin biosynthesis at the transcriptional level. Complementary results showed that not only the wild-type OxyR but also the reduced form OxyRC199S could activate the prodigiosin biosynthesis. We further demonstrated that reduced form of wild type OxyR could bind to the promoter of pig gene cluster, and identified the binding sites which is different from oxidized OxyR binding sites in E. coli. Our results demonstrated that OxyR in FS14 uses oxidized form to regulate the expression of the antioxidant related genes and utilizes reduced form to activate prodigiosin production. Further in silico analysis suggested that the activation of prodigiosin biosynthesis by reduced OxyR should be general in S. marcesencs. To our knowledge, this is the first report to show that OxyR uses the reduced form to activate the gene\'s expression, therefore, our results provide a novel regulation mechanism of OxyR.

The formation of reactive oxygen species is harmful and can destroy intracellular macromolecules such as lipids, proteins, and DNA, even leading to bacterial death. To cope with this situation, microbes have evolved a variety of sophisticated mechanisms, including antioxidant enzymes, siderophores, and the type VI secretion system (T6SS). However, the mechanism of oxidative stress resistance in Cupriavidus pinatubonensis is unclear. In this study, we identified Reut_A2805 as an OxyR ortholog in C. pinatubonensis, which positively regulated the expression of T6SS1 by directly binding to its operon promoter region. The study revealed that OxyR-regulated T6SS1 combats oxidative stress by importing iron into bacterial cells. Moreover, the T6SS1-mediated outer membrane vesicles-dependent iron acquisition pathway played a crucial role in the oxidative stress resistance process. Finally, our study demonstrated that the T6SS1 and siderophore systems in C. pinatubonensis exhibit different responses in combating oxidative stress under low-iron conditions, providing a comprehensive understanding of how bacterial iron acquisition systems function in diverse conditions.IMPORTANCEThe ability to eliminate reactive oxygen species is crucial for bacterial survival. Continuous formation of hydroperoxides can damage metalloenzymes, disrupt DNA integrity, and even result in cell death. While various mechanisms have been identified in other bacterial species to combat oxidative stress, the specific mechanism of oxidative stress resistance in C. pinatubonensis remains unclear. The importance of this study is that we elucidate the mechanism that OxyR-regulated T6SS1 combats oxidative stress by importing iron with the help of bacterial outer membrane vesicle. Moreover, the study highlights the contrasting responses of T6SS1- and siderophore-mediated iron acquisition systems to oxidative stress. This study provides a comprehensive understanding of bacterial iron acquisition and its role in oxidative stress resistance in C. pinatubonensis under low-iron conditions.

Sulfane sulfur, a collective term for hydrogen polysulfide and organic persulfide, often damages cells at high concentrations. Cells can regulate intracellular sulfane sulfur levels through specific mechanisms, but these mechanisms are unclear in Corynebacterium glutamicum. OxyR is a transcription factor capable of sensing oxidative stress and is also responsive to sulfane sulfur. In this study, we found that OxyR functioned directly in regulating sulfane sulfur in C. glutamicum. OxyR binds to the promoter of katA and nrdH and regulates its expression, as revealed via in vitro electrophoretic mobility shift assay analysis, real-time quantitative PCR, and reporting systems. Overexpression of katA and nrdH reduced intracellular sulfane sulfur levels by over 30% and 20% in C. glutamicum, respectively. RNA-sequencing analysis showed that the lack of OxyR downregulated the expression of sulfur assimilation pathway genes and/or sulfur transcription factors, which may reduce the rate of sulfur assimilation. In addition, OxyR also affected the biosynthesis of L-cysteine in C. glutamicum. OxyR overexpression strain Cg-2 accumulated 183 mg/L of L-cysteine, increased by approximately 30% compared with the control (142 mg/L). In summary, OxyR not only regulated sulfane sulfur levels by controlling the expression of katA and nrdH in C. glutamicum but also facilitated the sulfur assimilation and L-cysteine synthesis pathways, providing a potential target for constructing robust cell factories of sulfur-containing amino acids and their derivatives. IMPORTANCE C. glutamicum is an important industrial microorganism used to produce various amino acids. In the production of sulfur-containing amino acids, cells inevitably accumulate a large amount of sulfane sulfur. However, few studies have focused on sulfane sulfur removal in C. glutamicum. In this study, we not only revealed the regulatory mechanism of OxyR on intracellular sulfane sulfur removal but also explored the effects of OxyR on the sulfur assimilation and L-cysteine synthesis pathways in C. glutamicum. This is the first study on the removal of sulfane sulfur in C. glutamicum. These results contribute to the understanding of sulfur regulatory mechanisms and may aid in the future optimization of C. glutamicum for biosynthesis of sulfur-containing amino acids.

Regulation of the cell cycle process is an effective measure to ensure the stability and fidelity of genetic material during the reproduction of bacteria under different stresses. The small RNA DsrA helps bacteria adapt to environments by binding to multiple targets, but its association with the cell cycle remains unclear. Detection by flow cytometry, we first found that the knockout of dsrA promoted replication initiation, and corresponding overexpression of DsrA inhibited replication initiation in Escherichia coli. The absence of the chaperone protein Hfq, the DNA replication negative regulator protein Dps, or the transcription factor OxyR, was found to cause DsrA to no longer inhibit replication initiation. Excess DsrA promotes expression of the oxyR and dps gene, whereas β-galactosidase activity assay showed that deleting oxyR limited the enhancement of dps promoter transcriptional activity by DsrA. OxyR is a known positive regulator of Dps. Our data suggests that the effect of DsrA on replication initiation requires Hfq and that the upregulation of Dps expression by OxyR in response to DsrA levels may be a potential regulatory pathway for the negative regulation of DNA replication initiation.

Reactive oxygen species (ROS) are byproducts of oxygen metabolism produced by virtually all organisms living in an oxic environment. ROS are also produced by phagocytic cells in response to microorganism invasion. These highly reactive molecules can damage cellular constituents (proteins, DNA, and lipids) and exhibit antimicrobial activities when present in sufficient amount. Consequently, microorganisms have evolved defense mechanisms to counteract ROS-induced oxidative damage. Leptospira are diderm bacteria form the Spirochaetes phylum. This genus is diverse, encompassing both free-living non-pathogenic bacteria as well as pathogenic species responsible for leptospirosis, a widespread zoonotic disease. All leptospires are exposed to ROS in the environment, but only pathogenic species are well-equipped to sustain the oxidative stress encountered inside their hosts during infection. Importantly, this ability plays a pivotal role in Leptospira virulence. In this review, we describe the ROS encountered by Leptospira in their different ecological niches and outline the repertoire of defense mechanisms identified so far in these bacteria to scavenge deadly ROS. We also review the mechanisms controlling the expression of these antioxidants systems and recent advances in understanding the contribution of Peroxide Stress Regulators in Leptospira adaptation to oxidative stress.

Genome minimization is an effective way for industrial chassis development. In this study, Zymomonas mobilis ZMNP, a plasmid-free mutant strain of Z. mobilis ZM4 with four native plasmids deleted, was constructed using native type I-F CRISPR-Cas system. Cell growth of ZMNP under different temperatures and industrial effluent of xylose mother liquor were examined to investigate the impact of native plasmid removal. Despite ZMNP grew similarly as ZM4 under different temperatures, ZMNP had better xylose mother liquor utilization than ZM4. In addition, genomic, transcriptomic, and proteomic analyses were applied to unravel the molecular changes between ZM4 and ZMNP. Whole-genome resequencing result indicated that an S267P mutation in the C-terminal of OxyR, a peroxide-sensing transcriptional regulator, probably alters the transcription initiation of antioxidant genes for stress responses. Transcriptomic and proteomic studies illustrated that the reason that ZMNP utilized the toxic xylose mother liquor better than ZM4 was probably due to the upregulation of genes in ZMNP involving in stress responses as well as cysteine biosynthesis to accelerate the intracellular ROS detoxification and nucleic acid damage repair. This was further confirmed by lower ROS levels in ZMNP compared to ZM4 in different media supplemented with furfural or ethanol. The upregulation of stress response genes due to the OxyR mutation to accelerate ROS detoxification and DNA/RNA repair not only illustrates the underlying mechanism of the robustness of ZMNP in the toxic xylose mother liquor, but also provides an idea for the rational design of synthetic inhibitor-tolerant microorganisms for economic lignocellulosic biochemical production.

OBJECTIVE: Bacteroides fragilis is an anaerobic bacterium that is commonly found in the human gut microbiota and an opportunistic pathogen in extra-intestinal infections. B. fragilis displays a robust response to oxidative stress which allows for survival in oxygenated tissues such as the peritoneal cavity and lead to the formation of abscesses. In this study, we investigated the synergy of the oxidative stress response regulators OxyR and BmoR in the ability of B. fragilis to resist oxidative damage and to survive in extra-intestinal infection.
METHODS: A ΔbmoR ΔoxyR double mutant B. fragilis strain was constructed, and its oxidative stress response was compared to parental and single mutant strains in phenotypical assays and gene expression analysis. The pathogenic potential in an in vivo mouse model of abscess formation was also evaluated.
RESULTS: Expression analysis showed a coordinated control of thioredoxin C (trxC) gene expression by BmoR and OxyR during oxygen exposure, with upregulation of trxC in the bmoR mutant strain (4.9-fold increase), downregulation in the oxyR mutant (2.5-fold decrease), and an intermediate level of deregulation (2-fold increase) in the double mutant strain compared to the parent strain. Expression analysis during oxidative stress conditions also showed that BmoR is a major repressor of the CoA-disulfide reductase gene (upregulated 47-fold in the bmoR mutant) while OxyR plays a minor repression role in this gene (upregulated 2.5-fold in the oxyR mutant). Exposure to atmospheric oxygen for up to 72 h revealed that the deletion of bmoR alone had no significant effect in in vitro survival phenotype assays, though it partially abolishes the OxyR sensitivity phenotype in the bmoR/oxyR double mutant strain compared to oxyR mutant. In vivo assays showed that bmoR and oxyR mutants were significantly impaired in the formation and development of abscesses compared to the parent strain in an experimental intra-abdominal infection mouse model.
CONCLUSIONS: Although the full extent of genes whose expression are modulated by BmoR and OxyR is yet to be defined, we present evidence that these regulators have overlapping functions in B. fragilis response to oxidative stress and ability to form abscess in extra-intestinal tissues.

In many bacteria, OxyR acts as a transcriptional regulator that facilitates infection via degrading hydrogen peroxide (H2O2) generated by the host defense response. Previous studies showed that OxyR also plays an important role in regulating biofilm formation, cell motility, pili relate-genes expression, and surface polysaccharide production. However, the role of OxyR has not been determined in Acidovorax citrulli strain xjl12. In the current study, the qRT-PCR and western blot assays revealed that the expression level of oxyR was significantly induced by H2O2. The oxyR deletion mutant of A. citrulli was significantly impaired bacterial tolerance to oxidative stress and reduced catalase (CAT) activity. In addition, oxyR mutant resulted in reduced swimming motility, twitching motility, biofilm formation, virulence, and bacterial growth in planta by significantly affecting flagellin and type IV pili-related gene (fliC and pilA) expression. The qRT-PCR assays and western blot revealed that OxyR positively regulated the expression of fliC and pilA. Furthermore, bacterial one-hybrid assay demonstrated that OxyR directly affected pilA and fliC promoter. Through bacterial two-hybrid assay, it was found that OxyR can directly interact with PilA and FliC. These results suggest that OxyR plays a major role in the regulating of a variety of virulence traits, and provide a foundation for future research on the global effects of OxyR in A. citrulli.

Plant secondary metabolites perform numerous functions in the interactions between plants and pathogens. However, little is known about the precise mechanisms underlying their contribution to the direct inhibition of pathogen growth and virulence in planta. Here, we show that the secondary metabolite sulforaphane (SFN) in crucifers inhibits the growth, virulence, and ability of Xanthomonas species to adapt to oxidative stress, which is essential for the successful infection of host plants by phytopathogens. The transcription of oxidative stress detoxification-related genes (catalase [katA and katG] and alkylhydroperoxide-NADPH oxidoreductase subunit C [ahpC]) was substantially inhibited by SFN in Xanthomonas campestris pv. campestris (Xcc), and this phenomenon was most obvious in sax gene mutants sensitive to SFN. By performing microscale thermophoresis (MST) and electrophoretic mobility shift assay (EMSA), we observed that SFN directly bound to the virulence-related redox-sensing transcription factor OxyR and weakened the ability of OxyR to bind to the promoters of oxidative stress detoxification-related genes. Collectively, these results illustrate that SFN directly targets OxyR to inhibit the bacterial adaptation to oxidative stress, thereby decreasing bacterial virulence. Interestingly, this phenomenon occurs in multiple Xanthomonas species. This study provides novel insights into the molecular mechanisms by which SFN limits Xanthomonas adaptation to oxidative stress and virulence, and the findings will facilitate future studies on the use of SFN as a biopesticide to control Xanthomonas.

The detection of reactive oxygen species (ROS) and the analysis of oxidative stress are frequent applications of functional flow cytometry. Identifying and quantifying the ROS species generated during oxidative stress are crucial steps for the investigation of molecular mechanisms underlying stress responses. Currently, there is a wide availability of fluorogenic substrates for such purposes, but limitations in their specificity and sensitivity may affect the accuracy of the analysis. The aim of our work was to validate a new experimental model based in different strains of Escherichia coli B deficient in key genes for antioxidant defense, namely oxyR, sodA and sodB. We applied this model to systematically assess issues of specificity in fluorescent probes and the involvement of different ROS in a bacterial model of oxidative stress, as the probes can react with a variety of oxidants and free radical species. Our results confirm the higher sensitivity and specificity of the fluorescent probe mitochondrial peroxy yellow 1 (MitoPY1) for the detection of H2O2, and its very low capacity for organic hydroperoxides, thus extending MitoPY1\'s specificity for H2O2 in mammalian cells to a bacterial model. On the contrary, the fluorescent probe 2\',7\'-dichlorodihydrofluorescein diacetate (H2DCF-DA) is more sensitive to organic peroxides than to H2O2, confirming the lack of selectivity of H2DCF-DA to H2O2. Treatment with organic peroxides and H2O2 suggests a superoxide-independent oxidation of the fluorescent probe Hydroethidine (HE). We found a positive correlation between the lipophilicity of the peroxides and their toxicity to E. coli, suggesting greater quantitative importance of the peroxidative effects on the bacterial membrane and/or greater efficiency of the protection systems against the intracellular effects of H2O2 than against the membrane oxidative stress induced by organic peroxides. Altogether, our results may aid in preventing or minimizing experimental errors and providing recommendations for the proper design of cytometric studies of oxidative stress, in accordance with current recommendations and guidelines.