Publicação
Regulation of the KtrAB transporter by c-di-AMP in B. subtilis
| Resumo: | Potassium is the major cation present in all living cells and has a crucial role in bacteria where it is involved in several processes such as osmoregulation, pH homeostasis and membrane potential adjustment. The KtrAB protein complex is the principal potassium transporter present in Bacillus subtilis and is composed by a regulatory subunit, KtrA, and a membrane protein, KtrB. This transporter can be regulated by an exchange of ATP/ADP and by c-di-AMP, a secondary messenger recently discovered. It has been postulated that the regulation of KtrAB by c-di-AMP occurs at two levels, by direct interaction with the regulatory subunit, KtrA, leading to an inhibition of potassium transport, and by interaction with the ydaO riboswitch, leading to an inhibition of KtrAB expression. However, studies performed until now are very preliminary and an extensive study is necessary. In this project the regulation of KtrAB transporter by c-di-AMP was characterized in the context of Bacillus subtilis. Using genetic techniques, single point mutations were inserted in the binding sites for c-di-AMP in KtrA and in ydaO riboswitch and multiple bacterial strains were constructed. The phenotype of these strains was evaluated using several functional tests to assess the impact of c-di-AMP in KtrAB regulation. Strikingly, none of the mutations inserted had an impact at a phenotypical level. However, biochemical tests demonstrate that these mutations alter c-di- AMP binding, particularly in KtrA. In agreement with the results obtained it is possible to conclude that c-di-AMP does not have an important effect in KtrAB regulation, contrary to what has been reported. In fact, it seems that Bacillus subtilis have other compensatory mechanisms that allow it to overcome the effects of mutations inserted. |
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| Autores principais: | Fernandes, Ana Patrícia Carvalho |
| Assunto: | Ciências Naturais::Ciências Biológicas |
| Ano: | 2018 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Potassium is the major cation present in all living cells and has a crucial role in bacteria where it is involved in several processes such as osmoregulation, pH homeostasis and membrane potential adjustment. The KtrAB protein complex is the principal potassium transporter present in Bacillus subtilis and is composed by a regulatory subunit, KtrA, and a membrane protein, KtrB. This transporter can be regulated by an exchange of ATP/ADP and by c-di-AMP, a secondary messenger recently discovered. It has been postulated that the regulation of KtrAB by c-di-AMP occurs at two levels, by direct interaction with the regulatory subunit, KtrA, leading to an inhibition of potassium transport, and by interaction with the ydaO riboswitch, leading to an inhibition of KtrAB expression. However, studies performed until now are very preliminary and an extensive study is necessary. In this project the regulation of KtrAB transporter by c-di-AMP was characterized in the context of Bacillus subtilis. Using genetic techniques, single point mutations were inserted in the binding sites for c-di-AMP in KtrA and in ydaO riboswitch and multiple bacterial strains were constructed. The phenotype of these strains was evaluated using several functional tests to assess the impact of c-di-AMP in KtrAB regulation. Strikingly, none of the mutations inserted had an impact at a phenotypical level. However, biochemical tests demonstrate that these mutations alter c-di- AMP binding, particularly in KtrA. In agreement with the results obtained it is possible to conclude that c-di-AMP does not have an important effect in KtrAB regulation, contrary to what has been reported. In fact, it seems that Bacillus subtilis have other compensatory mechanisms that allow it to overcome the effects of mutations inserted. |
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