In previous study, MoaD-MoaE has been shown to play an irreplaceable role in the transformation of cyclic pyranopterin monophosphate (cPMP) into molybdopterin (MPT) in E. coli (Neumann et al., 2009). To confirm the importance of DRJAMM to MPT synthase, cPMP was oxidized into its stable fluorescence derivatives, compound Z (Wuebbens and Rajagopalan, 1995; Dahl et al., 2013). When the amount of cPMP was set to 100% in drJAMM mutant strain, it was not detected in wild-type R1 strain and drJAMM complementary strain (Supplementary Figure 1), indicating that DRJAMM is essential for the activation of DRMoaD-MoaE.

It has been previously revealed that DRJAMM requires Zn²⁺ to cleave the MoaD-MoaE fusion protein (Yang et al., 2018). However, we found that other metal ions could also catalyze this cleavage activity in vitro, such as Mn²⁺, Mg²⁺, Ca²⁺, and Ni²⁺ (Figure 1A). The remaining DRMoaD-MoaE fragments were quantified using ImageJ software (National Institutes of Health, United States) to demonstrate the catalytic efficiency of DRJAMM in the presence of different metal ions, as shown in Figure 1B. Unexpectedly, DRJAMM displayed the highest catalytic efficiency in the presence of Ca²⁺, about threefold higher than that of Zn²⁺ (Figure 1B). In addition, high temperatures did not inhibit the activity of JAMMs. Since the catalytic function of PfJAMM1 was the best at 100°C in Pyrococcus furiosus (Cao et al., 2017), we set a series of temperature gradients and found that the DRJAMM exhibits catalytic activities at different temperatures. Surprisingly, DRMoaD-MoaE is stably degraded by DRJAMM even under high temperatures (Figure 1C). These results suggested that the enzyme activity of DRJAMM has strong adaptability to a wide range of temperatures, even above 100°C.

To investigate the function of DRJAMM in D. radiodurans, a drJAMM (Δdr_0402) knockout mutant was constructed and the cell survival rate and cell growth curves were measured (Figures 2A–C). It was shown that the mutant ΔdrJAMM was not sensitive to UV radiation but declined significantly under H₂O₂ (0–80 mM) than the R1 (wild-type). The sensitivity is nearly disappeared after complementation with drJAMM (Δdr_0402_Cwt) in the mutant. However, the ΔdrJAMM growth curve displayed no change during a stationary phase of approximately 30 h (Figure 2C), indicating that the drJAMM mutation does not influence the growth rate of D. radiodurans but affects its response to oxidative stress.

Further analysis of oxidative stress survival with a spot-test method also demonstrated that the mutant is highly sensitive to H₂O₂, and could not endure 80 mM H₂O₂, but could be recovered after gene complementation (Figure 2D). Hence, the ROS level was measured to verify the role of drJAMM in the antioxidant process of D. radiodurans. As shown in Figure 2E, the ROS accumulation level in mutant was about 1.3-fold than that of R1 following 40 mM H₂O₂ treatment, while 1.5-fold higher after 80 mM H₂O₂ treatment. Furthermore, the ROS level in the mutant rises with the increase in H₂O₂ concentration, which was found to be restored to wild-type levels in the complementary strain. This suggests that the absence of drJAMM will cause the accumulation of ROS. Therefore, drJAMM is critical for the antioxidation process in D. radiodurans.

To test the cleavage efficiency of DRJAMM (DR0402) to DRMoaD-MoaE (DR2607) during antioxidant processes, a His-tag was fused to the C-terminal of DRJAMM and DRMoaD-MoaE in situ. The transcriptional and expressional levels of drJAMM and drMoaD-MoaE were analyzed using qRT-PCR and western blot assays following H₂O₂ treatment in the wild-type R1 strain. The mRNA levels of drJAMM and drMoaD-MoaE are increased under H₂O₂ treatment (Figure 3A), suggesting that they both may be involved in the oxidative resistance of D. radiodurans. Similarly, western blot assays showed that the expression of DRJAMM and DRMoaD-MoaE are both remarkably elevated about 1.5-fold and 3-fold, respectively, following H₂O₂ treatment, while the expression level of DRMoaE is also increased significantly about 1.5-fold (Figures 3B–E), indicating that the cleavage activity of DRJAMM might be necessary for oxidative resistance.

DMSO reductase activity is dependent on molybdenum cofactor (Moco) synthesis that requires JAMM/MPN metalloprotease (Miranda et al., 2011). Sequence alignment suggested that dr_0397 encode a molybdopterin oxidoreductase that has been shown to play a role in dimethyl sulfoxide respiration in Rhodobacter capsulatus (Solomon et al., 2000), and is homologous to E. coli DMSO reductase (Supplementary Figure 2).

To confirm whether the absence of drJAMM will affect the DMSO respiration system in D. radiodurans, the transcriptional levels of DMSO reductase were measured using qRT-PCR. Compared with wild-type R1, the mRNA levels of the DMSO reductase are more strongly elevated in ΔdrJAMM following exposure to H₂O₂, though the levels are also induced in R1 (Figure 4A), suggesting deletion of drJAMM causes a large demand for DMSO reductase in the antioxidant process. In the absence of DMSO, the growth of all strains was inhibited, while the addition of DMSO restarted growth. However, the growth of ΔdrJAMM is still in stagnation after adding DMSO (Figure 4B), indicating that drJAMM is necessary for the DMSO respiration system. In Haloferax volcanii, the JAMM/MPN⁺ metalloprotease HvJAMM1 can activate MPT synthase, and anaerobic growth using DMSO as a terminal electron acceptor can be used as a method to monitor the activation of MPT synthase by HvJAMM1 (Hepowit et al., 2012).

To further investigate the function of DRJAMM in the DMSO respiration system, the wild-type R1, drJAMM mutant strain, and drJAMM complementary strain were grown to OD₆₀₀ = 1.0 under aerobic conditions, and then incubated with DMSO under anaerobic conditions. The DMSO reductase activity was not detected in the cell lysate of drJAMM mutant strain, but could be readily detected in wild-type R1 and drJAMM complementary strain (Figure 4C), suggesting that DRJAMM is important for the maturation of DMSO reductase protein.

All strains, plasmids and primers used in this study are listed in Supplementary Tables 1, 2. E. coli strains were grown in Luria-Bertani (LB) liquid medium (1% tryptone, 0.5% yeast extract, and 1% sodium chloride) or on agar (1.5% Bacto-agar) plates supplemented at 37°C with appropriate antibiotics. All D. radiodurans strains were grown at 30°C in tryptone glucose yeast extract (TGY) liquid media or on agar plates (0.5% tryptone, 0.1% glucose, and 0.3% yeast extract) supplemented with appropriate antibiotics.

The dr_0402 and dr_2607 genes were amplified and cloned into a modified pET28a expression vector at NdeI and BamHI site (Supplementary Table 2), respectively (Li et al., 2019). Then, the constructed plasmids were transformed into E. coli BL21 (DE3), and induced in LB medium containing 50 μg/mL kanamycin and 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 30°C for 5 h. Cells were collected and resuspended in lysis buffer (20 mM Tris–HCl, pH 8.0; 500 mM NaCl; 5% (w/v) glycerol; 3 mM β-mercaptoethanol and 9 mM imidazole), followed by sonication. After centrifugation at 20,000 g for 30 min at 4°C, the supernatant was purified using a Ni-NTA column (1 mL, GE Healthcare Biosciences, United States) equilibrated with buffer A (20 mM Tris–HCl, pH 8.0; 500 mM NaCl; 5% (w/v) glycerol and 3 mM β-mercaptoethanol), and washed by buffer B (20 mM Tris–HCl, pH 8.0; 500 mM NaCl; 5% (w/v) glycerol; 3 mM β-mercaptoethanol; 500 mM imidazole). Finally, the proteins were concentrated and purified using a Superdex75 column (GE Healthcare Biosciences, United States), DRJAMM was eluted with buffer C (20 mM Tris–HCl, pH 8.0; 200 mM KCl; and 1 mM EDTA), DRMoaD-MoaE was eluted with buffer D (20 mM Tris-HCl, pH 8.0; and 200 mM KCl).

The DRJAMM (DR0402) activity assays were performed as described previously with minor modifications (Yang et al., 2018). The 40 μM of DRJAMM and 10 μM of DRMoaD-MoaE were added into the reaction buffer (100 mM KCl; 20 mM Tris-HCl, pH 8.0; and 1 mM dithiothreitol), and then 0.4 mM of different metal ions were added into samples. The reactions with 0.4 mM Ca²⁺ were incubated at 4, 16, 30, 37, 70, and 100°C for 30 min, respectively, and quenched by the addition of SDS loading buffer followed by immediate boiling. The products were identified by Tricine-SDS-PAGE (12%).

The mutant strains were constructed by a tripartite ligation method, as described previously (He et al., 2020). Briefly, the DNA fragment upstream of dr_0402 was amplified by PCR using the primers Δdr_0402-p1 and Δdr_0402-p2, which was digested with BamHI (Supplementary Table 2). The DNA fragment downstream of dr_0402 was amplified by PCR using the primers Δdr_0402-p3 and Δdr_0402-p4, which were digested with HindIII (downstream) (Supplementary Table 2). The digested fragment was connected to a streptomycin resistance gene. After the triplet ligation product was transformed into the D. radiodurans wild-type R1 strain, the mutant colonies were then selected on TGY plates containing 10 μg/ml streptomycin, and confirmed by genomic PCR using primers Δdr_0402-p5 and Δdr_0402-p6, and DNA sequencing. For complementary strain construction, the wild type dr_0402 was amplified by PCR using Δdr_0402_Cwt-F and Δdr_0402_Cwt-R, and cloned into the plasmid pRADK containing the D. radiodurans groEL promoter; and then transformed into the Δdr_0402 mutant strain to obtain the complementary strain Δdr_0402_Cwt.

Western blotting was used to confirm protein expression levels were performed as described previously (Dai et al., 2018). The 6 × His-tag was fused to the C-terminus of DRJAMM and DRMoaD-MoaE using tripartite ligation and a double-crossover recombination method. Mouse anti-6 × His tag (Proteintech, United States) was used to detect DRJAMM, DRMoaD-MoaE, and MoaE in the strains. The pre-stained marker was used as reference (Thermo Fisher, United States). The expression level of GroEL was detected using a rabbit anti-GroEL polyclonal antibody (Sigma, United States) in D. radiodurans, which was used as the internal control.

Real-time quantitative PCR (qRT-PCR) was used to measure dr_0402 and dr_2607 gene expression under oxidative stress, as described previously (Dai et al., 2020). First, D. radiodurans cells were grown to OD₆₀₀ = 1.0 and treated with 40 mM H₂O₂ for 30 min. Then, the cells were collected by centrifugation at 5,000 g for 3 min at 4°C. Total RNA was extracted from 5 mL cell cultures using TRIZOL reagent (Invitrogen, Carlsbad, CA, United States). The qRT-PCR experiments were performed using SYBR Premix Ex Taq (TaKaRa Biotechnology, Japan). The primers used in this experiment are listed in Supplementary Table 2. The data were collected and the difference in relative transcription abundance level was calculated. Glyceraldehyde 3-phosphate dehydrogenase (GADPH) encoded by the gene dr_1343 was used as the internal control.

To measure the survival curves and observe phenotypes under H₂O₂ treatment, the wild-type D. radiodurans R1 and Δdr_0402 were grown to OD₆₀₀ = 1.0, and then treated with different concentrations of H₂O₂ for 30 min. After the reaction, the residual H₂O₂ was cleared away by adding excess catalase, and then the sample was plated on TGY plates. All the experiments were repeated three times. To measure the growth curve, the wild-type D. radiodurans R1 and Δdr_0402 were cultured to OD₆₀₀ = 1.0 at 30°C and then 500 μl was transferred into 100 ml of fresh TGY medium without antibiotics. OD₆₀₀ values were measured every 1 or 2 h.

2′,7′-dichlorofluorescein diacetate (DCFH-DA) was used as a molecular probe hydrolysis to generate DCFH, ROS can oxidize DCFH to generate DCF with fluorescence, which can be measured using a fluorescence spectrometer (SpectraMax M5, United States) with an excitation wavelength of 485 nm and emission wavelength of 525 nm. D. radiodurans R1 and the mutant strains were grown to OD₆₀₀ = 1.0 and washed three times with PBS buffer. Pellets were incubated with DCFH-DA at 37°C for 30 min. After incubation, cells were washed three times with PBS buffer and resuspended in 2 mL PBS buffer, and then 1 ml sample was treated with 0, 40, and 80 mM H₂O₂ for 30 min, respectively. The accumulation of ROS was measured the manufacturer’s protocol (Beyotime Biotechnology, China).

DMSO analyses were performed as described previously (Cao et al., 2015). In brief, the strains were grown aerobically to OD₆₀₀ = 1.0. For anaerobic growth, the strains were transferred to TGY medium containing 100 mM DMSO as a terminal electron acceptor at 30°C for 4 days.

DMSO reductase activity assay was performed as described previously (Miranda et al., 2011). A total of 250 mL of TGY cultures of each strain were grown to OD₆₀₀ = 1.0, harvested by centrifugation, washed in 15 mL buffer A (50 mM Tris-HCl, pH 7.5; 1 mM EDTA, pH 8.0; 2 M NaCl), resuspended in 20 mL buffer A, and lysed by ultrahigh pressure homogenizer (Shanghailitu, China), successively. Cell lysates were clarified by centrifugation (15,000 rpm, 30 min), and protein concentrations were measured using the Bradford assay kit (Beyotime Biotechnology, China). The DMSO reductase activity was monitored at A_600nm (15 s intervals for 3.5 min). Assays (4 mL) included cell lysate (1–1.5 mg protein), 0.3 mM methyl viologen in buffer A, and the top filled with nitrogen. The mixture was titrated to 1–1.2 A_600nm units with fresh 20 mM odium dithionite (Na₂S₂O₄) in 20 mM sodium bicarbonate (NaHCO₃) prior to the addition of 10 mM DMSO. One unit (U) of enzyme activity was defined as 1-μm substrate consumed per minute at room temperature, with an extinction coefficient A_600nm of 13.6 (mM^–1⋅cm^–1) for methyl viologen.