Cellular homeostasis in Bacillus subtilis: The Roles of Spx, YjbH, and MrpA

Abstract: Bacteria survive adverse and changing conditions in their direct environments and respond to threats by sensing a wide variety of stresses and mount suitable responses.<br/>In this thesis, proteins in the gram-positive soil bacterium Bacillus subtilis involved in adaption to disulfide stress (formation of non-native disulfide bonds in the cytoplasm) and a membrane protein required for cation and cytoplasmic pH homeostasis were studied. The response to disulfide stress is governed by the transcriptional regulator Spx, whereas MrpA of the Mrp-antiporter has an important role in Na+ and alkali resistance. The transmembrane topology of the largest protein in the Mrp complex, MrpA was studied bioinformatically and experimentally by using a cytochrome c fusion tag approach. MrpA was shown to comprise 21 transmembrane segments. The MrpA C-terminal was shown to be essential for B. subtilis adaptation to alkaline pH in the presence of high salt. The Spx protein is subject to post-translational control involving the ClpXP protease complex. YjbH, an adaptor protein required for recognition of Spx by ClpXP was biochemically and structurally investigated. Homology modeling combined with crosslinking mass spectrometry provided evidence that YjbH has a fold similar to that of DsbA-family of oxidative folding catalysts. The X-ray crystallographic structure of YjbH in complex with Spx was determined at 1.7?Å resolution. The structure reveals a unique architecture of YjbH, where a DsbA-like fold is linked to a C-terminal domain reminiscent of the winged-helix fold typically involved in DNA binding. The interaction between YjbH and Spx involves a relatively large surface area that extends to the C-terminal part of Spx. Taken together, the work on YjbH revealed the overall architecture, and molecular basis for Spx recognition and suggests a model for how YjbH displays the C-terminus of Spx for recognition by ClpXP.<br/> Bacteria survive adverse and changing conditions in their direct environments and respond to threats by sensing a wide variety of stresses and mount suitable responses.<br/>In this thesis, proteins in the gram-positive soil bacterium Bacillus subtilis involved in adaption to disulfide stress (formation of non-native disulfide bonds in the cytoplasm) and a membrane protein required for cation and cytoplasmic pH homeostasis were studied. The response to disulfide stress is governed by the transcriptional regulator Spx, whereas MrpA of the Mrp-antiporter has an important role in Na+ and alkali resistance. The transmembrane topology of the largest protein in the Mrp complex, MrpA was studied bioinformatically and experimentally by using a cytochrome c fusion tag approach. MrpA was shown to comprise 21 transmembrane segments. The MrpA C-terminal was shown to be essential for B. subtilis adaptation to alkaline pH in the presence of high salt. The Spx protein is subject to post-translational control involving the ClpXP protease complex. YjbH, an adaptor protein required for recognition of Spx by ClpXP was biochemically and structurally investigated. Homology modeling combined with crosslinking mass spectrometry provided evidence that YjbH has a fold similar to that of DsbA-family of oxidative folding catalysts. The X-ray crystallographic structure of YjbH in complex with Spx was determined at 1.7?Å resolution. The structure reveals a unique architecture of YjbH, where a DsbA-like fold is linked to a C-terminal domain reminiscent of the winged-helix fold typically involved in DNA binding. The interaction between YjbH and Spx involves a relatively large surface area that extends to the C-terminal part of Spx. Taken together, the work on YjbH revealed the overall architecture, and molecular basis for Spx recognition and suggests a model for how YjbH displays the C-terminus of Spx for recognition by ClpXP.<br/>