A regulatory duet: sHdrR and SoxR team up for transcriptional repression of sulfur oxidation in Hyphomicrobium denitrificans
A regulatory duet: sHdrR and SoxR team up for transcriptional repression of sulfur oxidation in Hyphomicrobium denitrificans
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DissertationPrüfungsdatum
24.06.2025Datum der Veröffentlichung
04.08.2025Erstgutachter
Dahl, ChristianeZweitgutachter
Deppenmeier, UweMetadaten
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Inhalt
The Alphaproteobacterium Hyphomicrobium denitrificans XT, an obligately chemoorganoheterotrophic methylotroph, uses C1 compounds like methanol and also C2 compounds for two purposes: oxidizing them to CO2 for energy conservation and assimilating them for biomass production. When present, thiosulfate is used as an auxiliary electron donor. Its oxidation is initiated outside of the cytoplasm, i.e. in the periplasm. Further oxidative steps occur in the cytoplasm.
The presence of thiosulfate causes growth retardation on methanol but not on formate. I set out to explain this puzzling observation and found that methanol must be oxidized to formate and re-reduced for assimilation via the serine cycle. Thiosulfate oxidation produces sulfite, which forms adducts with pyrroloquinoline quinone (PQQ), thereby inhibiting periplasmic methanol dehydrogenase and thus methanol degradation. Formate metabolism remains unaffected as it occurs in the cytoplasm.
In the next step, the transcriptional regulation of genes encoding enzymes involved in sulfur oxidation in H. denitrificans was analyzed. Understanding the transcriptional regulation of sulfur oxidation to adapt metabolic flux to environmental conditions is necessary. In H. denitrificans, there are two homologous sulfane-sulfur-responsive ArsR-type transcriptional repressors, sHdrR and SoxR, that are responsible for the transcriptional regulation of genes encoding Sox, sHdr and associated proteins. Phenotypic analysis of knockout strains demonstrated the importance of these regulators in vivo. Site-directed mutagenesis, mass spectrometry, and gel shift assays in vitro revealed that regulatory proteins undergo conformational changes prior to detaching from the target DNA. DNA binding sites and transcriptional regulatory activity were also analyzed. The combined regulatory role of both repressors was confirmed in vitro by EMSA experiments. EMSA was also used to map common binding regions. These overlap the putative -35 and -10 RNA polymerase binding sites upstream of the divergently transcribed soxY and soxA, and soxT1A and shdrR gene sets.
Genes for two potential sulfur compound transporters, SoxT1A and SoxT1B, which resemble YeeE/YedE-family thiosulfate transporters, are located in the same genetic island as those involved in sulfur oxidation (sox and shdr). SoxT1A was identified as being crucial for delivery of sulfur to the cytoplasm for oxidation, while SoxT1B plays a role in signal transduction for the transcriptional repressor SoxR. Mutants lacking these transporters exhibit disrupted sulfur oxidation, underscoring their distinct but essential roles.
Target genes regulated by the repressors sHdrR and SoxR were identified through RNA-Seq analysis of deletion mutants. SoxR regulates the sox genes for the enzymes of thiosulfate oxidation in the periplasm and the lip-shdrR-lbpA genes encoding proteins responsible for sulfite formation in the cytoplasm, while sHdrR affects only a subset of these genes, excluding the sox genes. Both repressors cooperate, potentially forming heterodimers, and interact with other transcriptional regulators. Their regulatory effect extends far beyond sulfur oxidation, significantly impacting anaerobic metabolism, particularly denitrification in H. denitrificans. Whether the interaction between the two repressors is direct or indirect in vivo is an important question for future research.
The presence of thiosulfate causes growth retardation on methanol but not on formate. I set out to explain this puzzling observation and found that methanol must be oxidized to formate and re-reduced for assimilation via the serine cycle. Thiosulfate oxidation produces sulfite, which forms adducts with pyrroloquinoline quinone (PQQ), thereby inhibiting periplasmic methanol dehydrogenase and thus methanol degradation. Formate metabolism remains unaffected as it occurs in the cytoplasm.
In the next step, the transcriptional regulation of genes encoding enzymes involved in sulfur oxidation in H. denitrificans was analyzed. Understanding the transcriptional regulation of sulfur oxidation to adapt metabolic flux to environmental conditions is necessary. In H. denitrificans, there are two homologous sulfane-sulfur-responsive ArsR-type transcriptional repressors, sHdrR and SoxR, that are responsible for the transcriptional regulation of genes encoding Sox, sHdr and associated proteins. Phenotypic analysis of knockout strains demonstrated the importance of these regulators in vivo. Site-directed mutagenesis, mass spectrometry, and gel shift assays in vitro revealed that regulatory proteins undergo conformational changes prior to detaching from the target DNA. DNA binding sites and transcriptional regulatory activity were also analyzed. The combined regulatory role of both repressors was confirmed in vitro by EMSA experiments. EMSA was also used to map common binding regions. These overlap the putative -35 and -10 RNA polymerase binding sites upstream of the divergently transcribed soxY and soxA, and soxT1A and shdrR gene sets.
Genes for two potential sulfur compound transporters, SoxT1A and SoxT1B, which resemble YeeE/YedE-family thiosulfate transporters, are located in the same genetic island as those involved in sulfur oxidation (sox and shdr). SoxT1A was identified as being crucial for delivery of sulfur to the cytoplasm for oxidation, while SoxT1B plays a role in signal transduction for the transcriptional repressor SoxR. Mutants lacking these transporters exhibit disrupted sulfur oxidation, underscoring their distinct but essential roles.
Target genes regulated by the repressors sHdrR and SoxR were identified through RNA-Seq analysis of deletion mutants. SoxR regulates the sox genes for the enzymes of thiosulfate oxidation in the periplasm and the lip-shdrR-lbpA genes encoding proteins responsible for sulfite formation in the cytoplasm, while sHdrR affects only a subset of these genes, excluding the sox genes. Both repressors cooperate, potentially forming heterodimers, and interact with other transcriptional regulators. Their regulatory effect extends far beyond sulfur oxidation, significantly impacting anaerobic metabolism, particularly denitrification in H. denitrificans. Whether the interaction between the two repressors is direct or indirect in vivo is an important question for future research.
Schlagwörter
Hyphomicrobium denitrifican, ArsR family regulators, sHdr system, sHdrR, SoxR, YeeE/YedE-family sulfur transporter, thiosulfate oxidation
Klassifikation (DDC)
570 Biowissenschaften, BiologieLi, Jingjing: A regulatory duet: sHdrR and SoxR team up for transcriptional repression of sulfur oxidation in Hyphomicrobium denitrificans. - Bonn, 2025. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-83623
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-83623
@phdthesis{handle:20.500.11811/13303,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-83623,
doi: https://doi.org/10.48565/bonndoc-625,
author = {{Jingjing Li}},
title = {A regulatory duet: sHdrR and SoxR team up for transcriptional repression of sulfur oxidation in Hyphomicrobium denitrificans},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2025,
month = aug,
note = {The Alphaproteobacterium Hyphomicrobium denitrificans XT, an obligately chemoorganoheterotrophic methylotroph, uses C1 compounds like methanol and also C2 compounds for two purposes: oxidizing them to CO2 for energy conservation and assimilating them for biomass production. When present, thiosulfate is used as an auxiliary electron donor. Its oxidation is initiated outside of the cytoplasm, i.e. in the periplasm. Further oxidative steps occur in the cytoplasm.
The presence of thiosulfate causes growth retardation on methanol but not on formate. I set out to explain this puzzling observation and found that methanol must be oxidized to formate and re-reduced for assimilation via the serine cycle. Thiosulfate oxidation produces sulfite, which forms adducts with pyrroloquinoline quinone (PQQ), thereby inhibiting periplasmic methanol dehydrogenase and thus methanol degradation. Formate metabolism remains unaffected as it occurs in the cytoplasm.
In the next step, the transcriptional regulation of genes encoding enzymes involved in sulfur oxidation in H. denitrificans was analyzed. Understanding the transcriptional regulation of sulfur oxidation to adapt metabolic flux to environmental conditions is necessary. In H. denitrificans, there are two homologous sulfane-sulfur-responsive ArsR-type transcriptional repressors, sHdrR and SoxR, that are responsible for the transcriptional regulation of genes encoding Sox, sHdr and associated proteins. Phenotypic analysis of knockout strains demonstrated the importance of these regulators in vivo. Site-directed mutagenesis, mass spectrometry, and gel shift assays in vitro revealed that regulatory proteins undergo conformational changes prior to detaching from the target DNA. DNA binding sites and transcriptional regulatory activity were also analyzed. The combined regulatory role of both repressors was confirmed in vitro by EMSA experiments. EMSA was also used to map common binding regions. These overlap the putative -35 and -10 RNA polymerase binding sites upstream of the divergently transcribed soxY and soxA, and soxT1A and shdrR gene sets.
Genes for two potential sulfur compound transporters, SoxT1A and SoxT1B, which resemble YeeE/YedE-family thiosulfate transporters, are located in the same genetic island as those involved in sulfur oxidation (sox and shdr). SoxT1A was identified as being crucial for delivery of sulfur to the cytoplasm for oxidation, while SoxT1B plays a role in signal transduction for the transcriptional repressor SoxR. Mutants lacking these transporters exhibit disrupted sulfur oxidation, underscoring their distinct but essential roles.
Target genes regulated by the repressors sHdrR and SoxR were identified through RNA-Seq analysis of deletion mutants. SoxR regulates the sox genes for the enzymes of thiosulfate oxidation in the periplasm and the lip-shdrR-lbpA genes encoding proteins responsible for sulfite formation in the cytoplasm, while sHdrR affects only a subset of these genes, excluding the sox genes. Both repressors cooperate, potentially forming heterodimers, and interact with other transcriptional regulators. Their regulatory effect extends far beyond sulfur oxidation, significantly impacting anaerobic metabolism, particularly denitrification in H. denitrificans. Whether the interaction between the two repressors is direct or indirect in vivo is an important question for future research.},
url = {https://hdl.handle.net/20.500.11811/13303}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-83623,
doi: https://doi.org/10.48565/bonndoc-625,
author = {{Jingjing Li}},
title = {A regulatory duet: sHdrR and SoxR team up for transcriptional repression of sulfur oxidation in Hyphomicrobium denitrificans},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2025,
month = aug,
note = {The Alphaproteobacterium Hyphomicrobium denitrificans XT, an obligately chemoorganoheterotrophic methylotroph, uses C1 compounds like methanol and also C2 compounds for two purposes: oxidizing them to CO2 for energy conservation and assimilating them for biomass production. When present, thiosulfate is used as an auxiliary electron donor. Its oxidation is initiated outside of the cytoplasm, i.e. in the periplasm. Further oxidative steps occur in the cytoplasm.
The presence of thiosulfate causes growth retardation on methanol but not on formate. I set out to explain this puzzling observation and found that methanol must be oxidized to formate and re-reduced for assimilation via the serine cycle. Thiosulfate oxidation produces sulfite, which forms adducts with pyrroloquinoline quinone (PQQ), thereby inhibiting periplasmic methanol dehydrogenase and thus methanol degradation. Formate metabolism remains unaffected as it occurs in the cytoplasm.
In the next step, the transcriptional regulation of genes encoding enzymes involved in sulfur oxidation in H. denitrificans was analyzed. Understanding the transcriptional regulation of sulfur oxidation to adapt metabolic flux to environmental conditions is necessary. In H. denitrificans, there are two homologous sulfane-sulfur-responsive ArsR-type transcriptional repressors, sHdrR and SoxR, that are responsible for the transcriptional regulation of genes encoding Sox, sHdr and associated proteins. Phenotypic analysis of knockout strains demonstrated the importance of these regulators in vivo. Site-directed mutagenesis, mass spectrometry, and gel shift assays in vitro revealed that regulatory proteins undergo conformational changes prior to detaching from the target DNA. DNA binding sites and transcriptional regulatory activity were also analyzed. The combined regulatory role of both repressors was confirmed in vitro by EMSA experiments. EMSA was also used to map common binding regions. These overlap the putative -35 and -10 RNA polymerase binding sites upstream of the divergently transcribed soxY and soxA, and soxT1A and shdrR gene sets.
Genes for two potential sulfur compound transporters, SoxT1A and SoxT1B, which resemble YeeE/YedE-family thiosulfate transporters, are located in the same genetic island as those involved in sulfur oxidation (sox and shdr). SoxT1A was identified as being crucial for delivery of sulfur to the cytoplasm for oxidation, while SoxT1B plays a role in signal transduction for the transcriptional repressor SoxR. Mutants lacking these transporters exhibit disrupted sulfur oxidation, underscoring their distinct but essential roles.
Target genes regulated by the repressors sHdrR and SoxR were identified through RNA-Seq analysis of deletion mutants. SoxR regulates the sox genes for the enzymes of thiosulfate oxidation in the periplasm and the lip-shdrR-lbpA genes encoding proteins responsible for sulfite formation in the cytoplasm, while sHdrR affects only a subset of these genes, excluding the sox genes. Both repressors cooperate, potentially forming heterodimers, and interact with other transcriptional regulators. Their regulatory effect extends far beyond sulfur oxidation, significantly impacting anaerobic metabolism, particularly denitrification in H. denitrificans. Whether the interaction between the two repressors is direct or indirect in vivo is an important question for future research.},
url = {https://hdl.handle.net/20.500.11811/13303}
}
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