RESULTS ············································································································

4.2.1 Spx-dependent transcriptional control is enhanced in the absence of yjbH

In B. subtilis, Spx is a global transcriptional regulator that is active and abundant in cells suffering from disulfide stress. The trxB gene encoding thioredoxin reductase, which is activated by Spx (Nakano et al., 2003a; Nakano et al., 2005), was highly induced in both yjbH::tetF and yjbH::tetR insertion mutants (forward and reverse orientation of a Tetracycline-resistance gene cassette, respectively) (Fig. 4.2A and B). Spx negatively controls expression of the srf operon by interfering with ComA-dependent transcriptional activation of the srf promoter (Nakano et al., 2003a; Nakano et al., 2003b; Zhang et al., 2006). The transcription of srfA was repressed in the yjbH insertion mutant strains (Fig. 4.2C and D). To determine if yjbH causes activation of trxB transcription and

108 repression of srfA through the regulation of Spx, the level of Spx protein in wild-type and yjbH mutant cells was examined.

4.2.2 Spx accumulated in the yjbH mutant strain.

In normal growing cells, Spx concentration is kept at a low level due to transcriptional repression exerted by PerR and YodB (Leelakriangsak et al., 2007) and post-translational, proteolytic control by ClpXP. High levels of Spx accumulate in clpX and clpP mutant cells and the ATP-dependent protease ClpXP degrades Spx in vivo and in vitro (Nakano et al., 2001; Nakano et al., 2003a; Nakano et al., 2003b). The in vivo effect of yjbH was examined to determine its contribution to the control of Spx concentration. B. subtilis cells of wild-type JH642 and the yjbH insertion mutants were grown in DSM medium until mid-log phase. One culture was treated with diamide and the other was left untreated for 30 min. Cell extracts were obtained by lysozyme treatment in protoplast buffer and were applied to an SDS-polyacrylamide gel for electrophoresis. Western blot analysis was performed using anti-Spx antiserum. Spx protein could be detected by western blot in cells of both yjbH strains but not wild-type cells (Fig. 4.2, lanes 1 and 3). This indicates that in vivo, yjbH mutation can cause up- regulation of Spx concentration.

4.2.3 YjbH controls Spx at the post-transcriptional level.

To determine which stage of spx expression was affected by YjbH, the spx gene promoter was replaced by an IPTG-inducible Phyperspank promoter to eliminate PerR/YodB negative transcriptional control of spx. The trxB promoter was fused with lacZ report gene, and the resulting construct was inserted at the thrC locus. The expression of trxB-lacZ was up-regulated when Spx was induced by IPTG (Fig. 4.4), but much higher activity was observed in yjbH mutation cells, indicating that YjbH exerts negative control on Spx at the post-transcriptional level.

4.2.4 An IPTG-inducible YjbH could complement loss of yjbH-dependent negative control of Spx.

The IPTG-inducible alleles encoding the wild YjbH proteins were introduced into the amyE locus of the yjbH insertion mutant, bearing either a trxB-lacZ fusion or a srfA- lacZ fusion, to determine if the alleles could complement yjbH with respect to Spx activity. The induction of an IPTG-controlled wild-type allele of yjbH resulted in reduced trxB-lacZ expression and increased srfA-lacZ relative to that of the yjbH mutant and non- induced control group and the promoter activities of srfA and trxB showed the same level of activity as observe in the wild-type background (Fig. 4.2 A, B, C, D). Furthermore, Spx protein concentration was reduced when the expression of the complementing allele of yjbH was induced (Fig. 4.3, lanes 1, 3, 5 and 7). The induction of the wild-type allele of yjbH from an ectopic position (the amyE locus) within the yjbH mutant genome can complement the loss of YjbH-dependent negative control of Spx.

4.2.5 Diamide abolishes negative control of YjbH on Spx.

The thiol-specific oxidant diamide causes high level Spx to accumulate to high levels [result in Fig. 4.3, lanes 1 and 2 (Nakano et al., 2003a)] due to derepression of spx transcription from PerR and YodB (Leelakriangsak et al., 2007) and decreased ClpXP proteolytic control. Microarray analysis showed that yjbH expression is induced 5-fold upon diamide treatment (Leichert et al., 2003) and that induction of an spx allele encoding a protease-resistant form of Spx caused induction of yjbIH operon (Nakano et al., 2003a). Upon diamide treatment though yjbH expression is increased, it failed to down-regulate Spx in wild-type cell (Fig. 4.3, lanes 1 and 2). When YjbH was overexpressed through an IPTG-controlled promoter, the negative effect on Spx was still abolished by diamide treatment (Fig. 4.3, lanes 7 and 8). This result indicated diamide- induced accumulation of Spx is not due to transcriptional regulation of yjbH.

4.2.6 Amino acid substitutions in the CXXC motif of YjbH do not significantly affect the negative control of Spx by YjbH.

The CXXC motif of YjbH is a likely target for oxidant-dependent inactivation of its negative control of Spx. To determine the role of the CXXC motif, an amino acid

110 substitution in the CXXC motif was generated by in vitro PCR mutagenesis and the product of the resulting allele was tested for activity in vivo. A cysteine to alanine substitution was created at position 31 of the first Cys of the YjbH CXXC motif. The IPTG-inducible alleles encoding the mutant YjbH proteins were introduced into the amyE locus of the yjbH insertion mutant, bearing either a trxB-lacZ fusion or a srfA-lacZ fusion, to determine if the alleles could complement yjbH with respect to Spx activity. The expression of the C31A allele of yjbH in the yjbH, trxB-lacZ strain resulted in reduced levels of expression, similar to that observed in the wild-type complemented strain (Fig. 4.2 A and B), indicating Cys31 of YjbH is not necessary for negative control of Spx. The srf-lacZ fusion expression which was repressed in both yjbH insertion mutants can be complemented by IPTG-induced wild-type or C31A mutant alleles of the yjbH gene, as shown by the increase in srf-lacZ expression (Fig. 4.2 C and D). The induction of C31A alleles of yjbH from the amyE locus within the yjbH mutant genome can increase srf-lacZ expression, due to the reduced Spx concentration. These results were confirmed by the western blot analysis of the yjbH insertion mutant strain bearing either C31A mutant inducible YjbH in Fig. 4.3, lanes 9 and 11.

The CXXC motif is sometimes involved in redox control. Western analysis also indicated accumulation of Spx upon diamide treatment is not affected by C31A mutant YjbH (Fig. 4.3, lane 12). So C31 of CXXC motif of YjbH might not be involved in the sensing of disulfide stress.

4.2.7 YjbH is not involved in negative control of other ClpXP substrate.

Besides Spx, the SsrA-tagged derivative of HrcA [HrcA-ssrA(AA)] is another substrate for ClpXP in vivo (Wiegert & Schumann, 2001). It is encoded by the hrcA-ssrA (AA) allele that is transcribed from a constitutively active promoter of the dnaK gene. Western-blot analysis showed that HrcA-ssrA(AA) (the high molecular weight band) is only detectable in clpX mutant cells [Fig. 4.5, lanes 3 and 7 and (Wiegert & Schumann, 2001)], but not in yjbH::tetF and yjbH::tetR strains (Fig. 4.5, lanes 9 and 11) before diamide treatment. HrcA-ssrA(DD) is resistant to proteolysis by ClpXP, and is present in high levels in untreated and diamide-treated cells (Fig. 4.5, lane 5). Diamide induces

endogenous hrcA expression (the low molecular weight band) from its own promoter and (Fig. 4.5, lanes 2, 4, 8 and 12).