Cupriavidus pinatubonensis JMP134 Alleviates Sulfane Sulfur Toxicity after the Loss of Sulfane Dehydrogenase through Oxidation by Persulfide Dioxygenase and Hydrogen Sulfide Release
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains, Primers and Media
2.2. Thiosulfate Oxidation by Whole-Cell and Product Detection
2.3. Real-Time Quantitative Reverse Transcription PCR (RT–qPCR)
2.4. Bioinformatics Analysis
2.5. Statistical Analysis
3. Results
3.1. Thiosulfate Oxidation by Wild-Type and Mutant Strains of C. pinatubonensis JMP134
3.2. Changes in pH Values When Bacteria Oxidize Thiosulfate
3.3. GSSG Detection in the Cell Lysates
3.4. Gene Expression in the Wild-Type and Mutant Strains
3.5. Distribution and Phylogenetic Analysis of SoxC, PDO and DsrAB
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Strains | Characteristics | Source |
---|---|---|
Wild-type strains | ||
C. pinatubonensis JMP134 | Wild type | Our lab |
E. coli S17-1 | recA pro thi hsdS, RP4 tra functions, supE44 | Invitrogen |
Deletion strains | ||
ΔsoxCD | soxC and soxD deleted in JMP134 | This study |
Δpdo12soxCD | pdo1, pdo2, soxC and soxD deleted in JMP134 | This study |
Δpdo12 | pdo1 and pdo2 deleted in JMP134 | [3] |
Δpdo1soxCD | pdo1, soxC and soxD deleted in JMP134 | This study |
Δpdo2soxCD | pdo2, soxC and soxD deleted in JMP134 | This study |
Complementation strains | ||
Δpdo12soxCD::pdo1 | Δpdo12soxCD with plasmid pBBR-pdo1 | This study |
ΔsoxCD:soxCD | ΔsoxCD with plasmid pBBR-soxCD | This study |
Plasmids | ||
pBBR1MCS2 | Kanamycin resistance, mob+, pBBR1 replicon, cloning vector | Qi qingsheng |
pK18mobsacB | Widely used gene knockout vector, kanamycin resistance | Our lab |
pBBR-pdo1 | pBBR1MCS2 containing pdo1 | This study |
pBBR-soxCD | pBBR1MCS2 containing soxCD | This study |
Target Gene | Primers | Sequence (5′-3′) d |
---|---|---|
Deletion | ||
soxC and soxD | a Up-f | CAGGAAACAGCTATGACATGATTACGAATTCACCGCCGGGTTTCTGTTG |
Up-r | CCTACCATCGGTTCCTGCAATGCCGTCTCCT | |
b Down-f | TTGCAGGAACCGATGGTAGGGTGGATTCTTGAG | |
Down-r | TTCAGGATCCCCGGGTACCGAGCTCGAATTCTGCCATTGCTCTCTCCTGTTG | |
c V-f | GGTGTTCGGCTACACCATGT | |
V-r | AGCTTTGCTCTCCGGCTAC | |
pdo1 | Up-f | CAGGAAACAGCTATGACATGATTACGAATTAGACGATTACCTGGTCTACACCTTC |
Up-r | CAGCTGTTCGTACAGGCGCGTCAAATCCTTCTAT | |
Down-f | CGCGCCTGTACGAACAGCTGATAGAAGGTTTGCAT | |
Down-r | TTCAGGATCCCCGGGTACCGAGCTCGAATTGGCTGATGATGGAGAACGAAC | |
V-f | TATTGGCTGCCATCTGCT | |
V-r | GCTCTACAAGCTCAATGCG | |
pdo2 | Up-f | CAGGAAACAGCTATGACATGATTACGAATTCGAGGTCGTAGCGGTAGTTG |
Up-r | ACACACATGAGCTATCTGAAGATTCCCCTCAAC | |
Down-f | TTCAGATAGCTCATGTGTGTCTATCCGTGGTTAGC | |
Down-r | TTCAGGATCCCCGGGTACCGAGCTCGAATTCCATTTCATCGAGGAATAGCGT | |
V-f | ATGGCGTCCCAATCCAGCTT | |
V-r | TTGCCTGGAGAGTGGCTTTG | |
Complementation | ||
soxC and soxD | Forward | CACACAGGAAACAGCTATGCAGGAACGCACACC e |
Reverse | TTCCATTCGCCATTCACTATTTTGCCTCAAGAATCCA e | |
pdo1 | Forward | CACACAGGAAACAGCTATGACACCGACCATGCCAAG e |
Reverse | TTCCATTCGCCATTCATCAGAGGGCGTTGAGGGG e | |
Linearization | ||
pBBR1MCS2 | Forward | TGAATGGCGAATGGAAATTGTAAG |
Reverse | AGCTGTTTCCTGTGTGAAATTGTTATC | |
RT‒PCR | ||
soxY | Forward | GAGTGGAACAAGACCGCTTT |
Reverse | ATCGCGATCTGCTCGGTATC | |
pdo1 | Forward | CACGCTCTACCGTTCCATCA |
Reverse | CACGTGGATGTTGTTCTCGC | |
pdo2 | Forward | CATGCCCATGCCGACCACATC |
Reverse | CGTGCCGAAGGTGAGCGTATC | |
sqr | Forward | GCGTGGTGAAGTACGAACAA |
Reverse | AGGTCGTAGCGGTAGTTGGA | |
soxF | Forward | GCGTGAGTGGAGCGGACATG |
Reverse | GATTGGACAGCGGACACGAGAC | |
yedE | Forward | TTGCACGAAGACGGTCAGGAAAC |
Reverse | CACGGCGTGTGCGGAATCTC | |
yeeE | Forward | GGAAAGCCACCAGCCCGATG |
Reverse | CCGCCAAGGTGCAGGGATTC |
Phylum | Class | Complete Sox System | Incomplete Sox System | ||||
---|---|---|---|---|---|---|---|
Total | PDO | DsrAB | Total | PDO | DsrAB | ||
Aquificae | Aquificae | 4 | 0 | 0 | 3 | 0 | 0 |
Chlorobi | Chlorobia | 0 | 0 | 0 | 7 | 0 | 7 |
Deinococcus-Thermus | Deinococci | 8 | 8 | 0 | 0 | 0 | 0 |
Proteobacteria | - | 288 | 230 | 8 | 52 | 36 | 27 |
Acidithiobacillia | 0 | 0 | 0 | 2 | 2 | 0 | |
Alphaproteobacteria | 146 | 140 | 2 | 8 | 5 | 3 | |
Betaproteobacteria | 75 | 63 | 1 | 20 | 16 | 5 | |
Deltaproteobacteria | 2 | 0 | 0 | 0 | 0 | 0 | |
Epsilonproteobacteria | 35 | 0 | 0 | 1 | 0 | 0 | |
Gammaproteobacteria | 29 | 27 | 5 | 21 | 13 | 19 | |
Hydrogenophilalia | 1 | 0 | 0 | 0 | 0 | 0 | |
Total | 300 | 238 | 8 | 62 | 36 | 34 |
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Xin, Y.; Wang, Y.; Zhang, H.; Wu, Y.; Xia, Y.; Li, H.; Qu, X. Cupriavidus pinatubonensis JMP134 Alleviates Sulfane Sulfur Toxicity after the Loss of Sulfane Dehydrogenase through Oxidation by Persulfide Dioxygenase and Hydrogen Sulfide Release. Metabolites 2023, 13, 218. https://doi.org/10.3390/metabo13020218
Xin Y, Wang Y, Zhang H, Wu Y, Xia Y, Li H, Qu X. Cupriavidus pinatubonensis JMP134 Alleviates Sulfane Sulfur Toxicity after the Loss of Sulfane Dehydrogenase through Oxidation by Persulfide Dioxygenase and Hydrogen Sulfide Release. Metabolites. 2023; 13(2):218. https://doi.org/10.3390/metabo13020218
Chicago/Turabian StyleXin, Yufeng, Yaxin Wang, Honglin Zhang, Yu Wu, Yongzhen Xia, Huanjie Li, and Xiaohua Qu. 2023. "Cupriavidus pinatubonensis JMP134 Alleviates Sulfane Sulfur Toxicity after the Loss of Sulfane Dehydrogenase through Oxidation by Persulfide Dioxygenase and Hydrogen Sulfide Release" Metabolites 13, no. 2: 218. https://doi.org/10.3390/metabo13020218