Bacterial strains
Bacterial strains used in this study are listed in Supplementary Table 2. Cupriavidus strains were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), the Japan Collection of Microorganisms (JCM) (Ibaraki, Japan), and the NITE Biological Resource Center (NBRC) (Chiba, Japan).
Culture media
Modified minimum medium (M9) (10 mM K2HPO4, 10 mM KCl, 20 mM NH4Cl, 10 mM MgSO4 ∙ 7H2O, and 0.1% Hunter’s trace elements, pH 7.5)46, Luria-Bertani medium (LB) (1% tryptone, 0.5% yeast extract, and 0.5% NaCl), and modified Terrific Broth (TB) (2% tryptone, 2.5% yeast extract, 0.5% NaCl, and 4% glycerol) were used. The M9 components were prepared as 100-fold concentrations, sterilized using membrane filters, and added to autoclaved water with or without 1.7% agar (for plates). Pterin and lumazine solutions were neutralized to pH 7.5 using 10 M NaOH and autoclaved. The following antibiotics were used for culturing transformants: 100 µg mL−1 ampicillin sodium salt, 20 µg mL−1 (for E. coli) or 100 µg mL−1 (for LA-1) chloramphenicol, and 40 µg mL−1 kanamycin sulfate.
Bacterial cultures
Cupriavidus strains were aerobically cultured in 20 mL test tubes containing 5 mL LB at 28 °C. Each culture (1 mL) was transferred to 100 mL M9 containing 0.1% yeast extract in a 500 mL baffled flask capped with SILICOSEN® (Shin-Etsu Polymer Co., Ltd., Tokyo, Japan) and aerobically cultured at 28 °C. After a 24-h incubation, cells were centrifuged (10,000 × g at 25 °C for 5 min) and then washed with and suspended in 0.9% NaCl for an optical density at 600 nm (OD600) of 1. The suspension (1 mL) was transferred to 100 mL M9 containing 5 mM pterin or lumazine compounds in 500 mL baffled flasks and cultured under the same conditions as above. Escherichia coli used for constructing plasmids was cultured in LB at 37 °C.
Chemicals
Pterin was obtained from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan). Lumazine and 6-HP were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 7-HP, 6-HL, 7-HL, and 6,7-DHP were obtained from Schircks Laboratories (Bauma, Switzerland). 6,7-DHL was obtained from Princeton BioMolecular Research, Inc. (NJ, USA). XC was obtained from Hebei Yaocheng Pharmaceutical Technology Co., Ltd. (Shijiazhuang, China) through the NARD Institute, Ltd. (Hyogo, Japan).
Meta-analysis of 16S rRNA genes
Soil samples (200 g) were collected from agricultural fields at the Tsukuba Plant Innovation Research Center (University of Tsukuba, Ibaraki, Japan). They were transferred to sterilized 500 mL bottles capped with SILICOSEN® (Shin-Etsu Polymer) and mixed with 10 mL 200 mM sterilized lumazine or distilled water (control). After an incubation at 25 °C for 5, 10, 15, and 20 days, soil samples (1 g) were collected and stored at −80 °C. Bottles were weighed, after which the amount of the evaporated water was replenished using distilled water. DNA was extracted from soil using a DNeasy® PowerSoil® Pro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, but skim milk (40 mg g−1 soil) was added for the extraction. Gene libraries for the V3–V4 region of 16S rRNA genes were prepared by a two-step tailed PCR amplification using KOD FX Neo (Toyobo Co., Ltd., Osaka, Japan) and specific primers (Supplementary Table 3). After the libraries were purified using DNA Clean Beads (Vazyme, Nanjing, China), their quality was assessed using a 5300 Fragment Analyzer System (Agilent Technologies, CA, USA) and dsDNA 915 Reagent Kit (Agilent Technologies). High-quality libraries were sequenced using an Illumina MiSeq PE300 platform (Illumina Inc., CA, USA) and a MiSeq Reagent Kit v3 (Illumina). The reads with complete primer sequences were collected, after which their primer sequences were removed using “fastx barcode splitter” and “fastx trimmer” in the FASTX-Toolkit (ver. 0.0.14). Short sequences (≤130 bases) with a low quality score (<20) and their pairs were removed using the “sickle” tool (ver. 1.33). Reads were merged using the paired-end read-merging script “FLASH (ver. 1.2.11)” and analyzed using Qiime2 and the Silva database.
Isolation of lumazine-degrading bacteria
Soil samples (10 g) incubated with lumazine for 20 days were suspended with 20 mL 0.9% NaCl. Supernatants (10–100 µL) were spread on M9 agar plates containing 5 mM lumazine. After incubating for 3–5 days at 28 °C, colonies were collected and cultured in M9 containing 5 mM lumazine at 28 °C under aerobic conditions to evaluate cell growth and lumazine degradation. For all isolates, a partial 16S rRNA gene sequence was amplified by PCR using KOD FX Neo (TOYOBO) and primers 10F and 800 R (Supplementary Table 3). Amplified products were purified using a QIAquick® PCR Purification Kit (Qiagen) and then sequenced using an ABI Prism 3130 genetic analyzer (Applied Biosystems, CA, USA).
Bacterial genome sequencing
LA-1 genomic DNA was extracted using QIAGEN Genomic-tip 20 G (Qiagen), purified using Short Read Eliminator XS (PacBio, CA, USA), and sheared (10–20 kb) using g-TUBE (Covaris LLC., MA, USA). The DNA library constructed using the SMRTbell® Express Template Prep Kit 2.0 (PacBio) was sequenced using a Sequel® IIe system (PacBio) and the Sequel® II binding kit 2.2 (PacBio). High-fidelity (HiFi) nucleotide sequences were generated by SMRT Link (ver. 11.0.0.146107). HiFi reads ≤1000 bp were eliminated using Filtlong (ver. 0.2.1), and the remaining reads were assembled using Flye (ver. 2.9.1-b1780). Genomic circularity was confirmed by assessing the assembly graph using Bandage (ver. 0.8.1), whereas the integrity of the assembled genome was confirmed using CheckM (ver. 1.2.2). The genome was annotated using Prokka (ver. 1.14.6).
Transcriptome analysis
RNA-seq analysis was conducted at the Department of Sports Medicine under the Open Facility Network Office, University of Tsukuba. Total RNA was purified using the RNeasy® Protect Bacteria Mini Kit (Qiagen) and RNase-Free DNase Set (Qiagen). RNA libraries were constructed using the extracted RNA (500 ng each) along with the NEBNext® Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, MA, USA) and the NEBNext® rRNA Depletion Kit (New England Biolabs). Libraries were denatured (alkalinity) and neutralized before the next-generation sequencing using the NextSeq 500 System (Illumina) and NextSeq 500/550 v2.5 kits (Illumina). Sequences were checked using CLC Genomics Workbench (ver. 20.0.4) (Qiagen) and mapped to the LA-1 genome sequences using “RNA-Seq analysis” in the CLC software to obtain the transcripts per kilobase million (TPM) value for genes.
qRT-PCR
Total RNA was purified as described above, after which cDNA was synthesized from the extracted RNA (1 µg) using the QuantiTect® Reverse Transcription Kit (Qiagen). The qRT-PCR analysis was completed using cDNA as well as the Light Cycler® 96 system (Roche Diagnostics GmbH, Mannheim, Germany), THUNDERBIRD® Next SYBR™ qPCR Mix (TOYOBO), and appropriate primers (Supplementary Table 3). Expression levels (Cq values) were normalized against the 16S rRNA gene transcript level.
Determination of lumazine metabolites
Soil samples (1 g) were suspended in 2 mL 5 M NaOH and centrifuged (10,000 × g at 25 °C for 10 min). Supernatants were filtered (0.22 µm pores) before being analyzed. For bacterial cultures, the culture media were boiled for 10 min and then 10 M NaOH was added at a final concentration of 0.1 M. After centrifugation (13,000 × g at 25 °C for 10 min), supernatants were analyzed by high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity system (Agilent Technologies) equipped with a TSKgel® ODS-120H column (4.6 × 150 mm, 3-µm particle size; Tosoh Co., Tokyo, Japan) at 30 °C. Solvents A (5 mM ammonium formate, pH 9.0) and B (methanol) were used for the 10-min linear gradient (5%–95% B). Pterin extracted from insects was similarly analyzed using 0.1% formic acid instead of ammonium formate. Specifically, solvents A (0.1% formic acid) and B (methanol) were used for the 10-min linear gradient (5%–70% B). To investigate the substrate specificity of PDA, the TSKgel® SAX column (6.0 × 150 mm, 5-µm particle size; Tosoh) was used at 30 °C, with solvents A (0.1 M NaOH and 1.0 M NaCl, pH 12.5) and B (50% acetonitrile and 50% 0.1 M NaOH) creating a 15-min linear gradient (0%–40% B). Mass spectra were obtained using the LCMS-8045 mass spectrometer with a Prominence UFLC system (Shimadzu Co., Kyoto, Japan) equipped with a TSKgel® ODS-120H column. The solvent gradient was the same as that used for HPLC.
Activity staining
Proteins were resolved by native polyacrylamide gel electrophoresis (PAGE) using u-PAGEL H (UH-T420, ATTO Co., Tokyo, Japan), with a running buffer comprising 3 g L−1 Tris and 14.4 g L−1 glycine. After the electrophoresis at 10 mA, the gel was immersed in a solution consisting of 100 mM Tris-HCl (pH 8.0), 0.06 mM 1-methoxy-5-methylphenazinium methylsulfate, 0.25 mM nitroblue tetrazolium, and 1 mM substrate for an incubation at room temperature.
Enzyme purification and identification
LA-1 was cultured in M9 containing 10 mM lumazine for 48 h (for LuDH and DHLI) or 10 mM citrate for 24 h (for XOR E2). The cells were collected by centrifugation (13,000 × g at 4 °C for 10 min), washed twice, and suspended in 50 mM Tris-HCl (pH 8.0) prior to an ultrasonication (20 W for five cycles of 1 min each) using the Branson Sonifier 250 (Branson Ultrasonics Co., CT, USA). After centrifugation (13,000 × g at 4 °C for 15 min), the supernatants were filtered (0.22 µm pores). The collected CFEs were applied to a HiTrap™ DEAE FF column (5 mL; Cytiva, Uppsala, Sweden) equilibrated with 50 mM Tris-HCl (pH 8.0). The column was washed with the same buffer, and then proteins were eluted using 50 mM Tris-HCl (pH 8.0) containing 0.5 M NaCl. Ammonium sulfate was added to the eluates in a stepwise manner for 40% saturation, stirred on ice for 30 min, and centrifuged (13,000 × g at 4 °C for 20 min). Ammonium sulfate was added to the collected supernatant for 60% saturation, after which the solution was stirred on ice for 30 min. The precipitates collected by centrifugation (13,000 × g at 4 °C for 20 min) were dissolved in a solution comprising 50 mM Tris-HCl (pH 8.0) and 200 mM NaCl prior to the ultrafiltration using Amicon® Ultra-15 10 K (Merck Millipore, Darmstadt, Germany) and the same buffer to remove the ammonium sulfate. The protein solution was applied to a Superdex™ 200 10/300 GL column (Cytiva) equilibrated with a solution consisting of 50 mM Tris-HCl (pH 8.0) and 200 mM NaCl and then eluted using the same buffer. The fractions with detectable enzyme activity were collected, and their buffer was replaced with 50 mM Tris-HCl (pH 8.0) using Amicon® Ultra-4 10 K (Merck Millipore). The protein solution was applied to a Mono Q™ 5/50 GL column (Cytiva) equilibrated with 50 mM Tris-HCl (pH 8.0) and washed with the same buffer. A linear gradient (0–500 mM) of NaCl in 50 mM Tris-HCl (pH 8.0) was used to elute proteins. Fractions containing active enzymes were collected, and then the NaCl was removed by an ultrafiltration using Amicon® Ultra-0.5 10 K (Merck Millipore) and 50 mM Tris-HCl (pH 8.0). The last two chromatographic analyses were performed using an ÄKTA pure™ 25 system (Cytiva).
Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein-containing gel pieces were reduced using 0.1 M dithiothreitol, alkylated using 0.1 M iodoacetamide, and digested with trypsin at 30 °C for 12 h. Tryptic peptides were desalted using ZipTip C18 (Merck Millipore) and then analyzed using nano-LC/MS/MS systems (DiNa HPLC system, KYA TECH Co., Tokyo, Japan/QSTAR XL, Applied Biosystems). Mass fragments were analyzed using Mascot (ver. 2.2.06) and the predicted amino acid sequences of LA-1 proteins.
Preparation of recombinant LuDH and DHLI produced in LA-1
PCR amplifications of DNA were completed using KOD FX Neo (Toyobo) and specific primers (Supplementary Table 3). The broad host range vector pNSGroE47 was amplified by PCR, and the resultant pNS fragment lacking the groE gene promoter was used for protein production in LA-1. The LA1_0151-0152 gene promoter sequence was amplified by PCR using DNA extracted from LA-1 with DNeasy® Blood and Tissue kits (Qiagen), digested with SalI and NheI, and ligated to the pNS fragment that had been digested with the same restriction enzymes to generate pNS-PDHLI. The LA1_0151-0152 genes containing the promoter region were amplified by PCR, digested with NheI and HindIII, and then ligated to the pNS fragment that had been digested with the same restriction enzymes to produce pNS-DHLI. The LA1_0145-0147 genes were amplified by PCR, digested with NheI and HindIII, and cloned into pNS-PDHLI digested with the same restriction enzymes to produce pNS-LuDH. The recombinant plasmids were introduced into LA-1 cells via electroporation (1.6 kV for 5 ms) using Gene Pulser II (Bio-Rad, CA, USA). Transformants were pre-cultured in LB containing chloramphenicol, after which 1 mL aliquots were transferred to 100 mL M9 containing 10 mM lumazine, 0.1% yeast extract, and chloramphenicol in 500 mL baffled flasks capped with SILICOSEN® (cap-type; Shin-Etsu Polymer) for a 48 h incubation at 28 °C. Cells were collected by centrifugation (10,000 × g at 4 °C for 10 min), washed with 50 mM Tris-HCl (pH 7.5), and disrupted in a solution containing 50 mM Tris-HCl (pH 7.5) and 10% glycerol as described above. After centrifugation (13,000 × g at 4 °C for 15 min), supernatants were filtered (0.22 µm pores) and applied to a HisTrap™ FF column (Cytiva) equilibrated with the same buffer. The column was washed with the same buffer supplemented with 20 mM imidazole, and then proteins were eluted by increasing the imidazole concentration to 500 mM. After removing imidazole from the eluates via ultrafiltration using Amicon® Ultra-15 50 K (Merck Millipore), the remaining solutions were analyzed by gel filtration chromatography as described above. Active fractions were concentrated and desalted by ultrafiltration and used as rLuDH and rDHLI preparations.
Preparation of recombinant enzymes produced in E. coli
Supplementary Table 4 lists the expression vectors and recombinant proteins used in this study. DNA fragments for selected genes were amplified by PCR using KOD FX Neo (Toyobo) and specific primers (Supplementary Table 3), digested with NdeI or NheI and HindIII or XhoI, and cloned into pET21a(+) or pET28a(+) (Merck Millipore). Recombinant plasmids were introduced into E. coli BL21(DE3) or BL21(DE3) cells harboring pGro7 for chaperone production (TaKaRa Bio Inc., Shiga, Japan). For the co-production of LA1_0143 and DHLI, an LA1_0143 gene fragment was amplified by PCR, digested with NcoI and HindIII, and cloned into pRSFDuet™-1 (Merck Millipore). The resulting plasmid was digested with NdeI and XhoI and ligated to LA1_0151, LA1_0152, and LA1_0151-0152 gene fragments digested with the same restriction enzymes. LA1_0152 with amino acid substitutions was produced from DNA amplified by PCR using a primer designed to introduce amino acid substitutions. To produce LA1_0151 with amino acid substitutions, DNA was amplified by PCR using a primer designed to introduce amino acid substitutions and then digested with ClaI and XhoI to replace the region between ClaI and XhoI in the LA1_0151 gene in the expression vector.
Transformants harboring the respective expression vectors (Supplementary Table 4) were pre-cultured in LB containing ampicillin (for pET21a(+)), kanamycin (for pET28a(+)), and chloramphenicol (for E. coli BL21(DE3) harboring pGro7) at 28 °C, after which 1 mL aliquots were transferred to 100 mL modified TB containing each antibiotic in 500 mL baffled flasks capped with SILICOSEN® (cap-type; Shin-Etsu Polymer) and cultured at 28 °C under aerobic conditions. After the OD600 reached 0.6–0.8, isopropyl-β-ᴅ-thiogalactopyranoside (IPTG) (0.2–0.5 mM) was added to each culture. When E. coli BL21(DE3) harboring pGro7 was used, ʟ-arabinose (2 g L−1) was added to the culture along with IPTG. To prepare recombinant LA1_0144 protein, 3-methyl-2-buten-1-ol (prenol) was added to the culture (0.1 mM final concentration) along with IPTG and ʟ-arabinose. After a 12-h incubation at 16 °C, cells were collected by centrifugation (10,000 × g at 4 °C for 10 min), washed, resuspended with a solution consisting of 50 mM HEPES-NaOH (pH 7.5), 0.3 M NaCl, and 10% glycerol, and disrupted as described above. After centrifugation (13,000 × g at 4 °C for 15 min), supernatants were filtered (0.22 µm pores) and applied to a HisTrap™ FF column (Cytiva) equilibrated with the same buffer. The column was washed using the same buffer supplemented with 20 mM imidazole before proteins were eluted by increasing the imidazole concentration to 500 mM. Imidazole was removed from the protein solutions via ultrafiltration using Amicon® Ultra-15 (10 K, 30 K, or 50 K, Merck Millipore). Buffers containing 1 mM dithiothreitol were used to prepare recombinant LA1_0146 and LA1_0147.
Enzyme assay
Lumazine, 7-HL, and xanthine dehydrogenase activities were determined in a solution containing 50 µM 2,6-dichloroindophenol (DCIP), 50 µM substrate, and 50 mM Tris-HCl (pH 7.5) at 30 °C. DCIP is an electron acceptor that can serve as an alternative to physiological electron acceptors. After adding enzymes to initiate reactions, the decrease in absorption at 600 nm for DCIP (ε600 = 21 × 103 M−1 cm−1)48 was monitored. When NAD+, NADP+, 1,4-benzoquinone, and dioxygen were used as electron acceptors, lumazine dehydrogenase activity was measured on the basis of 7-HL production levels determined by HPLC. DHLI activity was measured in a solution consisting of 50 mM Tris-HCl (pH 7.5) and 0.1 mM 6,7-DHL. The reaction was initiated by adding the enzyme, after which the decrease in absorption at 360 nm for 6,7-DHL (ε360 = 6.2 × 103 M−1 cm−1, pH 7.5) at 30 °C was monitored. Notably, the absorbance at 360 nm was low for the reaction product XC. The XCDC activity was measured in a solution comprising 1 mM XC, 0.2 mM MnCl2, and 50 mM HEPES-KOH (pH 7.5) at 30 °C. The enzyme was added to initiate the reaction, which was monitored by HPLC. The PDA activity was measured in a solution containing 0.1 mM substrate and 50 mM HEPES-NaOH (pH 7.5) at 30 °C. Absorbance was measured using a Hitachi U-3900 spectrophotometer (Hitachi Co. Ltd., Japan). HPLC analysis was performed as described above.
Analysis of protein–protein interactions
Recombinant proteins with and without a His-tag were produced as described above. Protein mixtures (5 mg each) were incubated in a solution comprising 50 mM HEPES-NaOH (pH 7.5), 0.3 M NaCl, and 10% glycerol on ice for 1 h and applied to a HisTrap™ FF column (Cytiva). After an extensive wash, His-tagged proteins were eluted. The co-eluted protein was analyzed by SDS-PAGE to monitor protein interactions.
Creation of an LA-1 gene disruptant
The LuDH (E1, LA1_0145) and E2 (LA1_3155) genes were disrupted by homologous recombination (Supplementary Fig. 7). PCR amplifications of DNA were completed using KOD FX Neo (Toyobo) and specific primers (Supplementary Table 3). A gene fragment (not including lacZα) of pUC19 (Takara) was amplified by PCR. Next, the 5′- and 3′-regions of the LA1_0145 and LA1_3155 genes were amplified by PCR, digested with BamHI and XhoI (5′-region) and XhoI and HindIII (3′-region), and simultaneously ligated to the BamHI–HindIII-digested pUC19 gene fragment. The resulting plasmids were digested with XbaI and XhoI and then ligated to the chloramphenicol resistance gene (including its promoter region) amplified by PCR using pNSGroE as the template. The generated plasmids were introduced into LA-1 cells via electroporation as described above, with transformants selected on agar-solidified LB medium containing chloramphenicol in plates. Gene disruption in the transformants was confirmed by PCR.
Degradation of pterins serving as insect pigments
Stink bugs were provided by Drs Moriyama and Fukatsu from the National Institute of Advanced Industrial Science and Technology (Ibaraki, Japan). The butterflies and silkworms used in this study were specimens and frozen products, respectively, and were obtained from commercial suppliers. Wings of male butterflies (Pieris rapae, Colias erate, and Eurema hecabe) were thoroughly pulverized using stainless steel beads (5 mm) and then suspended in 50% ethanol (10 mL per butterfly). After removing residual solids using a filter (0.45 µm pores), organic matter was lyophilized and resuspended in sterilized water. The head–thorax (stink bug, Plautia stali Scott) and skin (silkworm, Bombyx mori) separated from the abdomen and organs, respectively, were washed twice using acetone and lyophilized. The head–thorax (50 stink bugs) and skin (two silkworms) were ground in water (20 mL) using a mortar and pestle. After centrifuging samples (13,000 × g at 25 °C for 15 min), the resulting supernatants were filtered (0.45 µm pores), lyophilized, and resuspended in sterilized water to obtain organic matter.
Other methods
Protein concentrations were determined using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad). Proteins were analyzed by SDS-PAGE49. Enzyme kinetic parameters were calculated using Enzyme-Kinetics Calculator50. Phylogenetic trees were constructed according to the neighbor-joining method using MEGA (ver. 11.0.11), with 1,000 bootstrap replicates. Genome maps were created using Proksee51.
Statistics and reproducibility
All experiments were independently performed at least three times. Data are presented herein in terms of the mean ± standard deviation (SD). The number of replicates and the number of samples in experiments are indicated in the experimental procedures and figure legends. A two-tailed unpaired t-test was used to determine the significance between two groups: ns (not significant), p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.