Abstract
Cupriavidus metallidurans strains are bacteria that belong to the Burkholderiaceae family and characterized by their resistance to multiple metals. Most have been isolated from industrial sites linked to mining, metallurgic and chemical industries. Although many metal resistance determinants have been extensively studied, the rapid evolution of C. metallidurans strains towards significantly increased metal resistance is not and is the subject of this thesis.
As C. metallidurans NA4, isolated from drinking water aboard the International Space Station, was used as a model in this study, a first step was to characterize its genome in detail. Strain NA4 was sequenced using Nanopore® technology and a hybrid assembly with existing Illumina data resulted in the closure of its six replicons and proper gene annotation. A distinctive pattern in the location of metal resistance determinants was observed, with the chromid playing a pivotal role in contrast to type strain CH34, for which the megaplasmids pMOL28 and pMOL30 are the main actors.
Next, C. metallidurans NA4 was used to perform adaptive laboratory evolution experiments with different precious metals, namely silver and platinum.
For silver, the canonical resistance mechanisms did surprisingly not participate in the adaptive evolution of C. metallidurans NA4 to silver. In contrast, a novel and unique resistance mechanism, with a pivotal role for the previously uncharacterized small periplasmic proteins (PrsQ1 and PrsQ2) that are partially controlled by two-component regulatory system, was discovered in the adapted strain NA4S. These small proteins belong to a group of ca. 20 homologous proteins, whose members can only be found in the related genera Cupriavidus and Ralstonia, distributed over the C. metallidurans genome. They do not contain conserved histidine and methionine residues typical for proteins binding metals and structure prediction indicated that they could be intrinsically disordered. As the increased silver resistance in NA4S is not mediated by efflux and the formation of silver nanoparticles was observed, we hypothesize that this formation prevents ionic silver to exert its action in the cytoplasm and provides the ability to withstand much higher silver concentrations than efflux-mediated resistance. In a next step, the underlying regulatory mechanism was further studied. Specifically, the two-component regulatory system AgrRS (agrS is mutated in the adapted strain NA4S) was scrutinized, together with the two-component regulatory system CzcR2S2 (increased transcription in NA4S), in order to examine binding and interaction of the response regulators AgrR and CzcR2 with target sites. Interestingly, mutants of AgrR and CzcR2 unable to be phosphorylated were found to be the active form in electrophoretic mobility shift assays and showed binding to the promoter regions of agrR (response regulator), copR2 (response regulator), prsQ2 (small periplasmic protein) and czcR2 (response regulator). The binding and interaction with specific target sites was further studied with various enzymatic and chemical footprinting and premodification binding interference techniques, resulting in a high-resolution contact map of AgrR binding to its own promoter region and of CzcR2 binding to the copR2 promoter region. Furthermore, the identified binding regions showed that the response regulators might be substituting the absence of a good -35 element by acting as class II transcriptional activators. This led to a general scheme for the regulation of the observed increase in silver resistance in which AgrS inactivation (either completely or its phosphotransfer capacity) results mainly in unphosphorylated AgrR, which subsequently activates target genes, including the pivotal prsQ2 gene. Furthermore, our results show that there is crosstalk between the different two-component regulatory systems and redundancy in the adaptation to silver.
In addition, an evolved strain, NA4Pt, with an increased resistance to platinum was also obtained. Since no canonical resistance mechanisms to platinum are known, NA4Pt was extensively studied using high-throughput genomic and transcriptomic sequencing. The latter showed pleiotropic alterations in membrane-related processes, such as pili, peptidoglycan turnover and electron transfer.
This research work has investigated different adaptation and resistance mechanisms, and in particular a novel silver resistance mechanism. Although, they still need to be further studied to decipher them completely, these findings showed the functionality of a novel protein family, an unanticipated mechanism of resistance, and the potential of C. metallidurans to adapt to toxic metal concentrations. Furthermore, the role of two-component regulatory systems in regulating the silver resistance revealed the importance of unphosphorylated response regulators.
As C. metallidurans NA4, isolated from drinking water aboard the International Space Station, was used as a model in this study, a first step was to characterize its genome in detail. Strain NA4 was sequenced using Nanopore® technology and a hybrid assembly with existing Illumina data resulted in the closure of its six replicons and proper gene annotation. A distinctive pattern in the location of metal resistance determinants was observed, with the chromid playing a pivotal role in contrast to type strain CH34, for which the megaplasmids pMOL28 and pMOL30 are the main actors.
Next, C. metallidurans NA4 was used to perform adaptive laboratory evolution experiments with different precious metals, namely silver and platinum.
For silver, the canonical resistance mechanisms did surprisingly not participate in the adaptive evolution of C. metallidurans NA4 to silver. In contrast, a novel and unique resistance mechanism, with a pivotal role for the previously uncharacterized small periplasmic proteins (PrsQ1 and PrsQ2) that are partially controlled by two-component regulatory system, was discovered in the adapted strain NA4S. These small proteins belong to a group of ca. 20 homologous proteins, whose members can only be found in the related genera Cupriavidus and Ralstonia, distributed over the C. metallidurans genome. They do not contain conserved histidine and methionine residues typical for proteins binding metals and structure prediction indicated that they could be intrinsically disordered. As the increased silver resistance in NA4S is not mediated by efflux and the formation of silver nanoparticles was observed, we hypothesize that this formation prevents ionic silver to exert its action in the cytoplasm and provides the ability to withstand much higher silver concentrations than efflux-mediated resistance. In a next step, the underlying regulatory mechanism was further studied. Specifically, the two-component regulatory system AgrRS (agrS is mutated in the adapted strain NA4S) was scrutinized, together with the two-component regulatory system CzcR2S2 (increased transcription in NA4S), in order to examine binding and interaction of the response regulators AgrR and CzcR2 with target sites. Interestingly, mutants of AgrR and CzcR2 unable to be phosphorylated were found to be the active form in electrophoretic mobility shift assays and showed binding to the promoter regions of agrR (response regulator), copR2 (response regulator), prsQ2 (small periplasmic protein) and czcR2 (response regulator). The binding and interaction with specific target sites was further studied with various enzymatic and chemical footprinting and premodification binding interference techniques, resulting in a high-resolution contact map of AgrR binding to its own promoter region and of CzcR2 binding to the copR2 promoter region. Furthermore, the identified binding regions showed that the response regulators might be substituting the absence of a good -35 element by acting as class II transcriptional activators. This led to a general scheme for the regulation of the observed increase in silver resistance in which AgrS inactivation (either completely or its phosphotransfer capacity) results mainly in unphosphorylated AgrR, which subsequently activates target genes, including the pivotal prsQ2 gene. Furthermore, our results show that there is crosstalk between the different two-component regulatory systems and redundancy in the adaptation to silver.
In addition, an evolved strain, NA4Pt, with an increased resistance to platinum was also obtained. Since no canonical resistance mechanisms to platinum are known, NA4Pt was extensively studied using high-throughput genomic and transcriptomic sequencing. The latter showed pleiotropic alterations in membrane-related processes, such as pili, peptidoglycan turnover and electron transfer.
This research work has investigated different adaptation and resistance mechanisms, and in particular a novel silver resistance mechanism. Although, they still need to be further studied to decipher them completely, these findings showed the functionality of a novel protein family, an unanticipated mechanism of resistance, and the potential of C. metallidurans to adapt to toxic metal concentrations. Furthermore, the role of two-component regulatory systems in regulating the silver resistance revealed the importance of unphosphorylated response regulators.
| Original language | English |
|---|---|
| Qualification | Other |
| Awarding Institution |
|
| Supervisors/Advisors |
|
| Date of Award | 18 Nov 2019 |
| State | Published - 18 Nov 2019 |