OxyR-regulated T6SS functions in coordination with siderophore to resist oxidative stress

ABSTRACT 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. IMPORTANCE The 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.

The type VI secretion system (T6SS) is a widely distributed transmembrane nano machine that resembles inverted contractile bacteriophage tails, which functions to inject effector proteins extracellularly or into neighboring cells (9,(12)(13)(14)(15)(16). Initially, the T6SS was thought to transport effector proteins with activities against prokaryotic and eukaryotic cells, thereby providing bacteria with survival advantages in microbemicrobe interactions and microbe-host interactions (17)(18)(19)(20).T6SS is also involved in the oxidative stress response in bacterial pathogens, thereby improving their survival (9,(21)(22)(23).In addition to playing a vital role in metal ion uptake to support survival in metal-restricted environments, T6SS is implicated in resistance to other stresses and contributes to cell survival under multiple adverse environmental conditions (9,15,22).Furthermore, T6SS participates in the recruitment of bacterial outer membrane vesicles (OMVs) in a Pseudomonas quinolone signal (PQS)-or lipopolysaccharide (LPS)-depend ent manner, thus facilitating iron acquisition, interbacterial competition, and oxidative stress resistance (14,24).
Here, we identified an OxyR ortholog, Reut_A2805, in C. pinatubonensis, which positively regulated the expression of T6SS1 by directly binding to its operon promoter region.Further studies revealed that OxyR-regulated T6SS1 promoted iron acquisition in a low-iron environment, thus eliminating the hydroxyl radicals induced by oxida tive stress, a process for which the T6SS1-mediated OMV-dependent iron acquisition pathway was essential.Moreover, the T6SS1-mediated OMV-dependent and cupriabac tin-mediated siderophore iron acquisition systems are differently regulated by oxidative stress and iron concentration.Our study revealed that the T6SS1 and cupriabactin are coordinated to combat oxidative stress under low-iron conditions, providing new insights into the roles of these two iron-acquisition systems in C. pinatubonensis.

Reut_A2805 is an OxyR ortholog in C. pinatubonensis
OxyR is an oxidative stress-related regulator, but its role in C. pinatubonensis has not been characterized.Genomic analysis revealed an OxyR ortholog (Reut_A2805) in C. pinatubonensis, the amino acid sequence of which shares 95% and 75% similarity (Fig. 1A) with OxyR in C. necator and B. thailandensis (22,35), respectively.The amino acid sequence of Reut_A2805 was aligned with OxyR from other bacterial species using ClustalW, and a phylogenetic tree was generated by MEGA11.Reut_A2805 clustered with other bacterial OxyR proteins (Fig. 1B).Based on a bioinformatics analysis, we annotated the reut_A2805 gene as the C. pinatubonensis oxyR gene and the corresponding protein as OxyR.As a conserved oxidative stress regulator that controls gene expression, OxyR reportedly protects against oxidative stress in bacteria (5,9,22).To assess the role of OxyR in resistance to oxidative stress in C. pinatubonensis, we constructed an oxyR gene deletion mutant and compared its viability with that of the wild type following exposure to H 2 O 2 for 25 min.The ΔoxyR mutant was more susceptible than the WT and the complemented strain upon H 2 O 2 challenge (Fig. 1C).The C-terminal domain of OxyR in C. pinatubonensis contains two redox-active cysteine residues (C199 and C208) that are known to mediate redox-dependent conformational switching in the identified OxyR (36).We simultaneously mutated two cysteine residues and found that the OxyR lacking two redox-active cysteine residues failed to help ΔoxyR mutant to resist oxidative stress (Fig. 1C).Therefore, the results all collectively indicated that reut_A2805 encodes OxyR, which is important in oxidative stress resistance in C. pinatubonensis.

OxyR positively regulates the expression of T6SS1 in C. pinatubonensis
As an important transcription factor, OxyR has been reported to regulate T6SS expres sion, thus helping Y. pseudotuberculosis and B. thailandensis to resist oxidative stress in a manner associated with metal ion transport (9,22).We reported that Fur-regulated T6SS1 is important in the acquisition of iron from OMVs for C. pinatubonensis, which contributes to bacterial exploitative competition, horizontal gene transfer, and oxidative stress resistance (24).To gain more insight into the functions and regulatory mechanisms of T6SS1 in C. pinatubonensis, we analyzed its promoter region using the online software Virtual Footprint.The putative OxyR binding site (AATGAGCAATTCGAT) in C. pinatubonen sis was highly conserved with OxyR box identified in Escherichia coli (37,38), and its sequence has a probability score of 5.07 (max score = 7.01), which is calculated by applying the position weight matrix to a sequence (Fig. 2A and B).Incubation of the T6SS1 promoter probe [P T6SS1 , −326 to −502 bp, relative to the ATG start codon of the first open reading frame [ORF] of the T6SS1 operon] with His 6 -OxyR led to the formation of DNA-protein complexes, whereas the unrelated protein bovine serum albumin (BSA) had no effect (Fig. 2C; Fig. S1A in Supporting Information).Furthermore, replacing this 15 bp binding site in the T6SS1 promoter probe with a 15 bp DNA fragment from the reut_A1727 encoding region abolished the formation of DNA-protein complexes in the electrophoretic mobility shift assay (EMSA) (Fig. 2C), suggesting that OxyR specifically recognizes the promoter region of the T6SS1 operon.The OxyR protein was purified in the absence of high Dithiothreitol (DTT) concentration, which was predominantly regarded as the oxidized form (39). Therefore, we speculated that the oxidized form of OxyR binds to the T6SS1 promoter.As a verification, the effect of OxyR oxidation on the binding was evaluated by EMSA using His 6 -OxyR treated with various concentrations of DTT.As shown in Fig. S1B in Supporting Information, the binding of OxyR to T6SS1 promoter DNA decreased as DTT concentration increased.
To investigate the role of OxyR in the regulation of the T6SS1 operon, a single-copy P T6SS1 ::lacZ fusion reporter was introduced into the chromosomes of the WT, ΔoxyR mutant, and complemented ΔoxyR(oxyR) strains, and the LacZ activity of each strain was quantified in M9 medium with H 2 O 2 .Deletion of oxyR significantly decreased the activity of the T6SS1 promoter, and this defect was fully restored to the WT level by oxyR complementation (Fig. 2D).Next, we verified the positive regulation of T6SS1 by OxyR by quantitative real-time PCR (qRT-PCR) analysis.Deletion of oxyR significantly reduced the expression levels of tssM1, vgrG1, clpV1, and hcp1; this decrease was completely reversed by the complementation plasmid expressing the regulatory protein OxyR (Fig. 2E).In addition, the positive regulation of OxyR in T6SS1 was confirmed at the protein level.The shuttle plasmid pME6032-hcp1-vsvg was constructed and transferred in WT, ΔoxyR mutant and ΔoxyR(oxyR) strains, and the expression of Hcp1-VSV-G was induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG).The secretion of Hcp1 was decreased in the ΔoxyR mutant compared to the WT and complemented strains (Fig. 2F).Therefore, OxyR specifically activates T6SS1 expression by binding its promoter in C. pinatubonensis.

OxyR-regulated T6SS1 combats oxidative stress by importing iron under low-iron conditions
To investigate whether the expression of OxyR-regulated T6SS1 responds to oxidative stress, we determined the promoter activity of T6SS1.The expression level of T6SS1 was enhanced by the addition of 0.02 mM H 2 O 2 , 0.03 mM CHP, or 0.25 mM diamide under low-iron conditions (Fig. 3A), directly implicating T6SS1 in oxidative stress-resistance.Furthermore, we determined the effect of T6SS1 on bacterial resistance to oxidative stress by measuring viability after exposure to oxidative stress under low-iron conditions.The ΔclpV1 mutant (lacking conserved T6SS1 structural gene) was significantly more sensitive to oxidative stress (H 2 O 2 , CHP, and diamide) than the WT (Fig. 3B).Meanwhile, the survival rate of the complemented strain was almost completely restored to the WT level (Fig. 3B), supporting a role for T6SS1 in combating oxidative stress under low-iron conditions.Oxidative stress always results in the formation of ROS, which is harmful to living organisms (40).To investigate the effect of T6SS1 on ROS during oxidative stress, we assessed the intracellular ROS levels in C. pinatubonensis strains after exposure to H 2 O 2 using the fluorescent dyes 3′-(p-hydroxyphenyl) fluorescein (HPF) and 5-(and-6)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetylester (CM-H 2 DCFDA).The ΔclpV1 mutant had significantly higher ROS levels than the WT after exposure to H 2 O 2 (Fig. 3C), indicating that T6SS1 is critical for reducing ROS accumulation in C. pinatubo nensis under oxidative stress conditions.Altogether, these data demonstrated that T6SS1 is induced and involved in oxidative stress resistance under low-iron conditions.
C. pinatubonensis T6SS1 is involved in iron acquisition, as indicated by the missing major iron transport systems FeoABC and cupriabactin siderophore under low-iron conditions (24).To test whether the increased T6SS1-dependent survival under oxidative stress conditions is due to iron acquisition, we measured the total metal content in bacterial cells treated with H 2 O 2 under low-iron conditions using inductively cou pled plasma mass spectrometry (ICP-MS).Deletion of clpV1 significantly reduced the intracellular iron level and complementation of clpV1 reversed the defect (Fig. 3D).By contrast, the accumulation of other metal ions, such as sodium and magnesium, was not affected in the ΔclpV1 mutant strain (Fig. S2 in Supporting Information), suggesting that the T6SS1 of C. pinatubonensis is associated with iron acquisition under low-iron and oxidative stress conditions.
To verify that the oxidative stress resistance of T6SS1 is related to intracellular iron, a low concentration of Fe 3+ was supplied during H 2 O 2 treatment.Whereas exogenous Fe 3+ (1 µM) markedly increased the survival rate of the WT and complemented strains ΔclpV1(clpV1) under H 2 O 2 challenge, the protective effect was largely abolished in the ΔclpV1 mutant (Fig. 3E).Fe-associated proteins including catalase and superoxide dismutase were used as controls because they are crucial for mitigatingROS-related stress (41,42).As expected, exogenous Fe 3+ markedly restored the growth of C. pinatubonensis in H 2 O 2 in the presence of the antioxidant Sod2 or KatG (Fig. 3E).Additionally, exogenous Fe 3+ (1 µM) significantly decreased the intracellular ROS level in the WT and complemented ΔclpV1(clpV1) strains (Fig. 3F).These results suggested a role for T6SS1 in importing iron from the environment under low-iron conditions in the presence of oxidative stress.

T6SS1-mediated OMV-dependent iron acquisition is essential for C. pinatubo nensis survival under low-iron and oxidative conditions
C. pinatubonensis has an iron acquisition pathway consisting of T6SS1-secreted LPSbinding effector TeoL, and TonB-dependent outer membrane receptors CubA and CstR, which contributes to the acquisition of iron in ΔcubEΔfeoB (Δ2Fe) mutant back ground, which is defective in both cupriabactin and FeoABC iron transport systems (24).To explore the role of T6SS1-mediated OMV recruitment in bacterial iron acquisi tion and oxidative stress resistance, we determined the total metal contents in bac terial cells treated with H 2 O 2 using ICP-MS under low-iron conditions.The ΔteoL and ΔcubAΔcstR (Δ2R) mutants exhibited significantly reduced intracellular iron levels compared to the WT and ΔteoL(teoL), Δ2R(cubA), and Δ2R(cstR) complemented strains (Fig. 4A).The accumulation levels of other metal ions, such as sodium and magne sium, were unaffected (Fig. S3 in Supporting Information), implicating T6SS1-mediated OMV recruitment in intracellular iron accumulation under oxidative stress conditions.Furthermore, we assessed the intracellular ROS levels in C. pinatubonensis WT, ΔteoL, and Δ2R mutant strains challenged with H 2 O 2 .Like ΔclpV1, the ΔteoL and Δ2R mutants had significantly higher ROS levels than the WT after exposure to H 2 O 2 , and their respective complemented strains recovered the reduced ROS levels (Fig. 4B), indicating that reduction of intracellular ROS in H 2 O 2 -treated strains requires the T6SS1-mediated OMV-dependent iron acquisition pathway.
The above results prompted us to examine whether this pathway is involved in iron acquisition.Indeed, the ΔteoL and Δ2R mutant strains were more sensitive to H 2 O 2 than the WT and complemented strains (Fig. 4C).Importantly, exogenous Fe 3+ (1 µM) enhanced the survival rate of the WT and complemented strains exposed to H 2 O 2 , whereas the effect of exogenous Fe 3+ was largely abolished in the ΔteoL or Δ2R mutant strain (Fig. 4C).We next constructed a ΔteoLΔ2R mutant, which has deficits in TeoL production and OMV recruitment.The survival rates of ΔteoLΔ2R and the corresponding single-gene complemented strains were determined following exposure to H 2 O 2 for 25 min, in the presence of Fe 3+ , Fe 3+ +His 6 -TeoL, or Fe 3+ +OMV WT .Although His 6 -TeoL and OMVs purified from the WT significantly increased the survival rate of the WT strain, they had no effect on the ΔteoLΔ2R mutant, indicating that the ability to obtain OMVs is crucial for resisting oxidative stress (Fig. 4D).Moreover, adding OMVs purified from the WT strain substantially improved the survival rates of ΔteoLΔ2R complemented with the OMV receptor gene cubA or cstR but not teoL (Fig. 4D).The results suggested that the T6SS1-mediated OMV-dependent iron acquisition pathway is essential for C. pinatubonensis survival under oxidative stress.

Two iron acquisition systems are coupled to combat oxidative stress
In C. pinatubonensis, we identified two types of iron acquisition systems mediated by T6SS1 and the siderophore cupriabactin (24,43).To determine the antioxidant mecha nism of these two iron acquisition systems, we generated a Δ2FeΔclpV1 mutant in the background of the ΔfeoB strain defective in FeoABC iron transport systems.Although the sensitivity to H 2 O 2 of the Δ2FeΔclpV1 mutant (lacking T6SS1 and cupriabactin) was not rescued by exogenous Fe 3+ , the sensitivity of strains Δ2FeΔclpV1(clpV1) and Δ2FeΔclpV1(cubE) was rescued by exogenous Fe 3+ (Fig. 5A), suggesting that T6SS1and cupriabactin-mediated importation of Fe 3+ is crucial in oxidative stress resistance.To explore the regulation of these two iron-acquisition systems, we first determined the effect of OxyR on the expression of the cupriabactin system by measuring the transcription levels of chromosomal P cub ::lacZ fusions.The expression of cub was not significantly affected in the C. pinatubonensis ΔoxyR mutant (Fig. S4 in Supporting Information), indicating that OxyR does not directly regulate the cupriabactin sidero phore system.Next, we analyzed the expression of C. pinatubonensis cub and T6SS1 in M9 medium with or without 20 µM H 2 O 2 containing Fe 3+ (0, 1, or 5 µM), respectively.Cub expression was markedly repressed by 5 µM Fe 3+ , and H 2 O 2 intensified the inhibitory effect (Fig. 5B), suggesting that the cub system functions to combat oxidative stress is iron concentration-dependent.By contrast, the expression of T6SS1 was increased by low iron (1 and 5 µM).Moreover, H 2 O 2 further activated its expression under low-iron conditions (0, 1, and 5 µM; Fig. 5B), suggesting that the T6SS1 iron acquisition system is actively induced to cope with oxidative stress under low-iron conditions.Altogether, these data indicated that two iron acquisition systems have different mechanisms and are essential for bacterial resistance to oxidative stress under low-iron conditions.pinatubonensis strains were exposed to 0.1 mM H 2 O 2 in M9 medium containing Fe 3+ (1 μM), Fe 3+ (1 μM) + apo-TeoL protein (1 μM), or Fe 3+ (1 μM) + OMV WT (20 µg mL −1 of phospholipids) for 25 min, and the viability of the cells was determined.Data represent the mean ± SD of three biological replicates, each of which was performed with three technical replicates.*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

DISCUSSION
The T6SS1 of C. pinatubonensis was reported to be negatively regulated by Fur, an iron transport-related repressor, which participates in the acquisition of iron, enabling bacteria to combat oxidative stress (24).However, the mechanism of oxidative stress resistance was unclear.In this study, we identified Reut_A2805 as an OxyR ortholog in C. pinatubonensis, and an ΔoxyR mutant showed a significant defect in oxidative stress resistance (Fig. 1).OxyR, as a conserved oxidative stress regulator that controls gene expression, is important in protection against oxidative stress in several bacterial taxa (5,9,22).Then, the OxyR positive regulates the expression of T6SS1 (Fig. 2), suggesting that the oxidative resistance of T6SS1 is facilitated by this regulator.The expression of the OxyR-regulated T6SS1 responded to oxidative stress, relieving the intracellular ROS accumulation induced by oxidative stress (Fig. 3).Also, T6SS1-mediated iron acquisition significantly enhanced bacterial resistance to oxidative stress (Fig. 3) in a manner dependent on the OMV-dependent iron acquisition pathway (Fig. 4).Our findings reveal the mechanism of C. pinatubonensis T6SS1 in combating oxidative stress by which bacteria sense and respond to oxidative stress via its positive regulator OxyR.
Iron has been implicated in many oxidative stress-related pathways and conditions, for it is a cofactor of many antioxidant enzymes, such as catalase and superoxide dismutase.It is also a primary cause of ROS generation via the Fenton reaction (44).Maintaining intracellular iron homeostasis is particularly important for bacterial survival.Fur, as a well-documented iron-dependent repressor, controls the switch of the iron transport system.The T6SS1 and cub systems are negatively regulated by Fur, facilitat ing iron acquisition under low-iron conditions, and promoting resistance to oxidative stress (24,43).However, the mechanisms were unclear.In this study, we determined the mechanism of oxidative stress resistance and evaluated the underlying mecha nism under low-iron conditions in the presence of oxidative stress.T6SS1 expression responded to oxidative stress and was positively regulated by OxyR, thus enhancing oxidative stress resistance via iron acquisition, while cub expression was strictly iron concentration-dependent and repressed by oxidative stress (Fig. 5).We infer that the T6SS1-mediated iron acquisition system functions in oxidative resistance is driven by oxidative stress under low-iron conditions, while the antioxidative capacity of cub-medi ated iron acquisition system functions only because of the low iron concentration.
OMV, as a unique and versatile secretion system, participates in multiple biological processes by delivering varied biologically active molecules at high concentrations to distant bacterial or mammalian cells (45).The ability of OMV to resist oxidative stress is nascently demonstrated, and the mechanism is unclear.The identification of iron in P. aeruginosa and C. pinatubonensis OMVs (14,24) and catalase in Helicobacter pylori OMVs (46) supports their role in oxidative stress resistance, which is consistent with the role of vesiculation in the oxidative stress response (47).In this study, we evaluated the relationships among iron, OMV, T6SS1, and siderophore under low-iron conditions in the presence of oxidative stress.As a dominant siderophore that mainly responds to the iron uptake in C. pinatubonensis, cupriabactin still proceeded to acquire iron in response to iron deficiency under oxidative stress conditions.Thus, its strategy is to slow down the rate of iron acquisition by decreasing the activity of the cub promoter.As a nonspecific iron acquisition system, the oxidative stress-induced T6SS1 and OMV were coupled to acquire iron, as well as other cargoes, and the bacterial iron uptake rate is relatively decreased.Hence, the strategy of T6SS1 and OMVs is further to decelerate the iron acquisition, and antioxidase may function in this process.
Based on our results, we unravel a complex iron acquisition pathway involving T6SS and siderophore iron transport systems that enhances bacterial survival under low-iron and oxidative stress conditions (Fig. 6).Under low-iron conditions, the Fur-repressed T6SS1 and cub gene clusters are de-repressed, thereby promoting iron acquisition in C. pinatubonensis by recruiting OMVs and secreting cupriabactin, respectively.Under this condition, the cub operon is highly expressed and mainly responsible for iron uptake, FIG 6 Model of T6SS1-and siderophore-mediated iron acquisition under low-iron and oxidative stress conditions in C. pinatubonensis.Under low-iron conditions, the Fur-repressed T6SS1 and cub gene clusters are de-repressed, and both facilitate the iron acquisition for C. pinatubonensis by recruiting OMV and secreting siderophore cupriabactin, respectively.Under this condition, cub operon is highly expressed and mainly responsible for the iron uptake, while T6SS1 is lowly expressed and functions weakly.Upon encountering oxidative stress, bacteria suppress iron acquisition by decreasing cub promoter activity and inducing T6SS1 expression by OxyR, in which iron is acquired in a nonspecific manner, thereby promoting resistance to oxidative stress.whereas T6SS1 is expressed at a low level.Upon encountering oxidative stress, bacte ria suppress iron acquisition by decreasing cub promoter activity and inducing T6SS1 expression by OxyR, in which iron is acquired in a nonspecific manner, thereby promot ing resistance to oxidative stress.Overall, our findings not only reveal the mechanism by which T6SS1 enhances resistance to oxidative stress but also demonstrate how the T6SS1 and cub iron acquisition systems sense and respond to oxidative stress under low-iron conditions in C. pinatubonensis, providing a clear perspective for understanding the bacterial iron acquisition in multiple complicated environments.

Plasmid construction
The primers used in this study are listed in Table S2 in Supporting Information.The plasmid pK18-ΔoxyR (reut_A2805) was used to construct the ΔoxyR in-frame deletion mutant of C. pinatubonensis.The primer pairs oxyR-1F-EcoRI/oxyR-1R and oxyR-2F/ oxyR-2R-SalI were used to amplify a 756 bp upstream segment and a 910 bp downstream segment of the oxyR, respectively.The upstream and downstream PCR fragments were ligated by overlapping PCR.The resulting PCR products were digested with EcoRI/SalI and inserted into the EcoRI/SalI sites of pK18 to produce pK18-ΔoxyR.The knock-out plasmids pK18-ΔkatG (reut_A1741) and pK18-Δsod2 (reut_A0597) were constructed in a similar method by using primers list in Table S2 in Supporting Information.
To complement the ΔoxyR mutant, primers oxyR-F-KpnI/oxyR-R-SacI were used to amplify the oxyR gene from the C. pinatubonensis genome DNA.The PCR product of oxyR was digested with KpnI/SacI and inserted into the KpnI/SacI sites of pBBR1MCS-5 to produce pBBR1MCS-5-oxyR.The complementation plasmids pBBR1MCS-2-katG and pBBR1MCS-2-sod2 were constructed in a similar method by using primers list in Table S2 in Supporting Information.To express His 6 -tagged OxyR, the plasmid pET28a-oxyR was constructed.Briefly, the primers oxyR-F-EcoRI and oxyR-R-SalI were used to amplify the oxyR gene fragment from the C. pinatubonensis genome.The PCR product of oxyR was digested with EcoRI/SalI and inserted into the BamHI/SalI sites of pET28a to generate pET28a-oxyR.The shuttle plasmid pME6032-hcp1-vsvg was constructed in a similar method.
For complementation, the complementary plasmids pBBR1MCS-5-oxyR, pBBR1MCS-2-katG, and pBBR1MCS-2-sod2 were introduced into the respective mutants by electropo ration.The integrity of the insert in all constructions was confirmed by DNA sequencing.

Overexpression and purification of recombinant protein
To express and purify soluble His 6 -tagged recombinant proteins, the plasmid pET28a-oxyR was transformed into BL21(DE3).Bacteria were cultured at 37°C in LB medium to an OD 600 of 0.5, shifted to 24°C and induced with 0.2 mM IPTG, and then cultivated for an additional 12 h at 24°C.Harvested cells were disrupted by sonication, and proteins were purified with the His•Bind Ni-NTA resin (Novagen, Madison, WI) according to the manufacturer's instructions.Eluted recombinant proteins were dialyzed against buffer (50 mM Tris, 150 mM NaCl, 10% glycerol, and pH 7.5) at 4°C.The resulting proteins were stored at −80°C until use.Protein concentrations were determined using the Bradford assay according to the manufacturer's instructions (Bio-Rad, Hercules, CA) with bovine serum albumin as standard.

Electrophoretic mobility shift assay
EMSA was performed by Zhang and colleagues (49).The P T6SS1 fragment was amplified from the C. pinatubonensis genome using the primers T6SS1-EMSA-F and T6SS1-EMSA-R.Increasing concentrations of purified His 6 -OxyR (0.01, 0.05, 0.10, and 0.15 µM) were incubated with 5 ng DNA probes in EMSA buffer (20 mM Tris, pH 7.4, 4 mM MgCl 2 , 100 mM NaCl, and 10% glycerol).After incubation for 20 min at room temperature, the binding reaction mixture was subjected to electrophoresis on a 6% native polyacryla mide gel containing 5% glycerol in 0.5 × TBE (Tris-borate-EDTA) electrophoresis buffer, and the DNA probe was detected using SYBR Green.As negative controls, irrelevant protein bovine serum albumin was included in the binding assay.

Construction of chromosomal fusion reporter strain and β-galactosidase assay
The lacZ fusion reporter vector pK18-P T6SS1p ::lacZ was transformed into E. coli S17-1 λ pir and mated with C. pinatubonensis strains as described previously (43).The lacZ fusion reporter strains were grown to stationary phase in NB or M9 medium at pH 7.0 under 30°C unless otherwise specified, and β-galactosidase activity was assayed using ONPG (o-Nitrophenyl β-D-galactopyranoside) as the substrate.These assays were performed in triplicate at least three times, and the error bars represent SDs.

Quantitative real-time PCR
Bacteria cells were harvested during the mid-exponential phase, and RNA was extracted using the RNAprep Pure Cell/Bacteria Kit and treated with RNase-free DNase (TIAN GEN, Beijing, China).The purity and concentration of the RNA were determined by gel electrophoresis and spectrophotometer (NanoDrop, Thermo Scientific).First-strand cDNA was reverse transcribed from 1 µg of total RNA with the TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China).qRT-PCR was performed in CFX96 Real-Time PCR Detection System (Bio-Rad, USA) with TransStart Green qPCR SuperMix (TransGen Biotech, Beijing, China).For all primer sets (Table S2 in Supporting Information), the following cycling parameters were used: 95°C for 30 s followed by 40 cycles of 94°C for 15 s and 55°C for 30 s.For standardization of the results, the relative abundance of 16S rRNA was used as the internal standard.All samples were analyzed in triplicate, and the expression of target genes was calculated as relative fold values using the 2 -ΔΔCT method.These assays were performed in triplicate at least three times, and the error bars represent the SEM.

Protein secretion assay
The shuttle plasmid pME6032-hcp1-vsvg was constructed and transferred in relevant strains, and the expression of Hcp1-VSV-G was induced by IPTG.Secretion assay for Hcp1 was performed according to described methods (50).Briefly, strains were inoculated into 100 mL NB and incubated with continuous shaking until OD 600 reached 1.5 at 30°C. 1 mL of culture was centrifuged, and the cell pellet was resuspended in 100 µL Sodium dodecyl sulfate (SDS)-loading buffer; the whole-cell lysate sample was defined as Hcp1 cells .80 mL of the culture was centrifuged, and the supernatant was filtered through a 0.22 µm filter (Millipore, MA, USA).The secreted proteins in the supernatant were collected by filtration over a nitrocellulose filter (BA85) (Whatman, Germany) for three times.The filter was soaked in 100 µL SDS sample buffer for 15 min at 65°C to recover the proteins present, and the sample was defined as Hcp1 sup .All samples were normalized to the OD 600 of the culture and volume used in preparation.

Bacterial survival assay
Mid-exponential phase C. pinatubonensis strains grown in NB medium were collected, washed, diluted 50-fold into M9 medium, and treated with H 2 O 2 (0.1 mM), CHP (0.03 mM), or diamide (0.25 mM), respectively, at 30°C for 25 min.After treatment, the cultures were serially diluted and plated onto NB agar plates, and colonies were counted after 48 h growth at 30°C.Percentage survival was calculated by dividing the number of Colony forming units (CFU) of stressed cells by the number of CFU of cells without stress.All these assays were performed in triplicate at least three times.

OMV isolation, purification, and quantification
OMVs were isolated, purified, and quantified as described (24).Briefly, to obtain OMVs without bacterial cells, overnight batch culture was centrifuged for 20 min at 6,000 × g and 4°C.The supernatant was filtered through 0.45 and 0.22 µm vacuum filter, respec tively, to thoroughly remove the remaining bacteria.The resulting filtrate was ultracen trifuged for 1 h at 200,000 × g at 4°C using an angle rotor (70 Ti, Beckman Coulter, USA), and the pellets were washed twice with phosphate-buffered saline (PBS), which were subsequently resuspended in 50 mM HEPES-0.85% NaCl.For purification, crude OMV samples were adjusted to 1 mL of 45% (wt/vol) iodixanol (OptiPrep; Sigma-Aldrich) in HEPES-NaCl, transferred to the bottom of ultracentrifuge tubes, and layered with iodixanol-HEPES-NaCl (2 mL of 40, 35, 30, 25, and 20%).The samples were ultracentri fuged for 4 h at 150,000 × g at 4°C using a swing rotor (SW40 Ti, Beckman Coulter, USA).Then, 1 mL fractions were collected from each gradient and detected by SDS-PAGE.The fraction containing OMV was ultracentrifuged for 1 h at 200,000 × g at 4°C using an angle rotor and resuspended in HEPES-NaCl.For quantification, the protein concentration and the phospholipid concentration of the OMV were measured, respectively.

FIG 1
FIG 1 Sequence analysis of OxyR from C. pinatubonensis.(A) Alignment of Reut_A2805 with OxyR from C. necator and B. thailandensis.The multiple sequence alignment was performed using ClustalW, and the figure was produced by ESPript.The conserved sequences were colored in red.(B) A maximum-likelihood phylogenetic tree generated by the ClustalW alignment of the amino acid sequences of Reut_A2805 and OxyR from different species using MEGA11.The names of microorganisms were listed in the label, and the Protein ID was listed in the Supporting Table S3.The numbers at each node indicate the percentage of 1,000 bootstrap sample whose tree contained the indicated node.The scale bar at the lower left represents a genetic distance of 0.1.(C) OxyR is essential for C. pinatubonensis to resist oxidative stress.The viability of mid-exponential phase C. pinatubonensis strains was determined after exposure to 0.1 mM H 2 O 2 for 25 min in M9 medium.Data represent the mean ± SD of three biological replicates, each of which was performed with three technical replicates.***P < 0.001.

FIG 2
FIG 2 OxyR directly and positively regulates T6SS1 expression in C. pinatubonensis.(A) Gene organization of the T6SS1 gene cluster in C. pinatubonensis.(B) Identification of the OxyR binding site in the promoter region of T6SS1.The putative OxyR binding site was identified by the online software Virtual Footprint and indicated by shading, and its sequence has a probability score of 5.07 (max score = 7.01).The Y-axis represents relative nucleotide probability, and the X-axis represents nucleotide position.The ATG start codon of the first ORF of the T6SS1 operon was marked in bold, and the -35 and -10 elements of the T6SS1 promoter are underlined.+1 denotes the transcription start point.(C) OxyR binds the T6SS1 promoter.EMSA was performed to analyze the interaction between OxyR with the T6SS1 promoter (P T6SS1 ) and OxyR box mutant DNA (P T6SS1M ).(D-F) OxyR activates the expression of T6SS1.β-galactosidase activity of T6SS1 promoter from chromosomal lacZ fusions in relevant C. pinatubonensis strains was measured under H 2 O 2 condition (D).Cells of relevant C. pinatubonensis strains were grown to stationary phase in M9 medium with 0.02 mM H 2 O 2 , and the expression of tssM1, vgrG1, clpV1, and hcp1 (the main components of T6SS1) was measured by quantitative real-time PCR (qRT-PCR) (E).The relevant C. pinatubonensis strains expressing C-terminal VSV-G-tagged Hcp1 were grown in M9 medium with 0.02 mM H 2 O 2 to the late logarithmic phase at 30°C.Production (Total) and secretion (Secreted) of Hcp1-VSV-G were detected by immunoblotting using anti-VSV-G antibodies.Isocitrate dehydrogenase (ICDH) was used as a loading control and lysis control for the total and secreted fractions (F).Data represent the mean ± SD of three biological replicates, each of which was performed with three technical replicates.**P < 0.01; ***P < 0.001.

FIG 3
FIG 3 OxyR-regulated T6SS1 combats oxidative stress by importing iron under low-iron conditions.(A) T6SS1 expression is induced by oxidative stress under low-iron conditions.Cells of relevant C. pinatubonensis strains harboring P T6SS1 ::lacZ were grown in M9 medium containing 0.02 mM H 2 O 2 , 0.03 mM CHP, or 0.25 mM diamide, and the LacZ activity was measured.(B) T6SS1 is involved in oxidative stress resistance.The viability of C. pinatubonensis strains in the mid-exponential phase was determined after exposure to 0.1 mM H 2 O 2 , 0.2 mM CHP, or 1 mM diamide for 25 min in M9 medium.(C) Deletion of T6SS1 led to the accumulation of the intracellular ROS under oxidative stress conditions.The intracellular ROS in mid-exponential phase bacterial strains exposed to H 2 O 2 was determined with the HPF or H 2 DCFDA probe, and fluorescence signals were measured using a microplate reader with excitation/emission wavelengths of 490/515 nm (HPF) and 495/520 nm (CM-H 2 DCFDA).(D) Iron uptake requires T6SS1 under oxidative stress conditions.Stationary phase of C. pinatubonensis strains was exposed to M9 medium with or without 0.5 mM H 2 O 2 for 20 min, and the iron associated with bacterial cells was measured by inductively coupled plasma mass spectrometry (ICP-MS).(E and F) Alleviation of sensitivity and reduction of intracellular ROS in H 2 O 2 -treated C. pinatubonensis strains by exogenous Fe 3+ required T6SS1.Relevant mid-exponential phase C. pinatubonensis strains were exposed to 0.1 mM H 2 O 2 in M9 medium with or without exogenously provided Fe 3+ (1 μM) for 25 min.The viability of the cells (E) and the intracellular ROS (F) was determined, respectively.Data represent the mean ± SD of three biological replicates, each of which was performed with three technical replicates.*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

FIG 4
FIG 4 T6SS1-mediated OMV-dependent iron acquisition is essential for C. pinatubonensis survival under oxidative stress.(A) The T6SS1-mediated OMV-depend ent iron acquisition is important for the accumulation of intracellular iron under oxidative stress conditions.Stationary phase of C. pinatubonensis strains was exposed to 0.5 mM H 2 O 2 in M9 medium for 20 min, and the iron associated with bacterial cells was measured by ICP-MS.(B) Reduction of intracellular ROS in H 2 O 2 -treated C. pinatubonensis strains requires the T6SS1-mediated OMV-dependent iron acquisition.The intracellular ROS in mid-exponential phase bacterial strains exposed to H 2 O 2 was determined with the HPF or CM-H 2 DCFDA probe, and fluorescence signals were measured using a microplate reader.(C) Alleviation of the sensitivity of C. pinatubonensis strains to H 2 O 2 by exogenous Fe 3+ requires T6SS1-mediated OMV-dependent iron acquisition.Relevant mid-exponential phase C. pinatubonensis strains were exposed to 0.1 mM H 2 O 2 in M9 medium with or without exogenously provided Fe 3+ (1 μM) for 25 min, and the viability of the cells was determined.(D) The T6SS1-mediated OMVs recruitment system contributes to oxidative stress resistance.Relevant mid-exponential phase C.

FIG 5
FIG 5 Two iron acquisition systems are differently induced and coupled to combat oxidative stress.(A) Alleviation of the sensitivity of C. pinatubonensis strains to H 2 O 2 by exogenous Fe 3+ requires two iron acquisition systems mediated by T6SS1 and cupriabactin.Relevant mid-exponential phase C. pinatubonensis strains were exposed to 0.1 mM H 2 O 2 in M9 medium with or without exogenously provided Fe 3+ (1 μM) for 25 min, and the viability of the cells was determined.(B) Expression of cub and T6SS1 is affected by iron concentrations and oxidative stress, respectively.β-galactosidase analysis of cub and T6SS1 promoter activities was performed in C. pinatubonensis strains grown to stationary phase in M9 medium with or without 0.02 mM H 2 O 2 containing different concentrations of Fe 3+ (0, 1, and 5 μM).Data represent the mean ± SD of three biological replicates, each of which was performed with three technical replicates.*P < 0.05; **P < 0.01; ns, not significant.