Dept. of Environmental and Biomolecular Systems
Oregon Health & Science University
The Spx protein of Bacillus subtilis is a global transcriptional regulator that exerts both positive and negative control in response to oxidative stress by interacting with the C-terminal domain of the RNA polymerase (RNAP) alpha subunit (Î±CTD). One target of Spx-negative control is the ComPA signal transduction system, which activates the transcription of the srf operon at the onset of competence development. Previous genetic and structural analyses have determined that an Spx-binding surface resides in and around the Î±1 region of Î±CTD. Alanine-scanning mutagenesis of B. subtilis Î±CTD uncovered residues required for Spx function and ComA-dependent srf transcriptional activation. Analysis of srf-lacZ fusion expression, DNase I footprinting, and solid-phase promoter retention experiments indicated that Spx interferes with ComA-Î±CTD interaction and that residues Y263, C265, and K267 of the a1 region lie within the overlapping ComA- and Spx-binding sites for aCTD interaction. The requirement of the oxidized Spx for Spx-dependent activation of trxA and trxB expressions was demonstrated in previous work (Nakano et al., 2005). Evidence is presented here that oxidized Spx, while enhancing interference of activator-RNAP interaction, is not essential for negative control. Spx is under proteolytic control by the ATP-dependent protease, ClpXP. Previous studies suggested that the accumulation of Spx protein upon disulfide stress is due to the derepression of spx by PerR and YodB and the down-regulation of the ClpXP activity. The effect of disulfide stress on ClpXP activity was examined using the thiol-specific oxidant, diamide. ClpXP-catalyzed degradation of either Spx or a green fluorescent protein (GFP) derivative bearing an SsrA tag recognized by ClpXP was inhibited by diamide treatment in vitro. Spx is also a substrate for MecA/ClpCP-catalyzed proteolysis in vitro, but the same concentration of diamide that inhibited ClpXP had little observable effect on MecA/ClpCP activity. The derivative of transcriptional repressor HrcA bearing an SsrA tag is another ClpXP substrate in vivo and its degradation by ClpXP was reduced in the presence of diamide. ClpX bears a Cys4 Zinc-binding domain (ZBD), which in other Zinc-binding proteins is vulnerable to thiol-reactive electrophiles. Diamide treatment caused partial release of Zn from ClpX and the formation of high molecular weight species, as observed by electrophoresis through non-reducing gels. When two of the Zn-coordinating Cys residues of the ClpX ZBD were changed to Ser, Spx proteolysis was reduced in vitro, the Spx-dependent transcriptional controls were enhanced and the Spx protein accumulated in vivo. The results are consistent with the hypothesis that inhibition of ClpXP by disulfide stress is due to structural changes to the N-terminal ZBD of ClpX. YjbH, a negative regulator of Spx, was examined in B. subtilis. Elevated Spx protein and enhanced Spx-dependent transcriptional control were observed in the cells bearing the yjbH insertion mutants. Thus, expression of spx was negatively affected by yjbH and this negative control was maintained when the spx was expressed from an IPTG-inducible promoter. The concentration of another ClpXP substrate HrcA-SsrA was not affected by YjbH in vivo, suggesting that YjbH is a specific negative regulator for Spx concentration. A mutation that changes the first cysteine residue of YjbH CXXC motif at the N terminus to alanine did not affect Spx-dependent transcriptional control and the control of Spx concentration in untreated and diamide-treated cells. Finally YjbH is proposed to post-tranlationally modulate Spx level in B. subtilis.
OGI School of Science and Engineering
Zhang, Ying, "Function of Spx and its control by proteolysis" (2007). Scholar Archive. 158.