Genetic evidence that multiple proteases are involved in modulation of ...

FEMS Microbiology Letters, 364, 2017, fnx054 doi: 10.1093/femsle/fnx054 Advance Access Publication Date: 8 March 2017 Research Letter

R E S E A R C H L E T T E R – Physiology & Biochemistry

Genetic evidence that multiple proteases are involved in modulation of heat-induced activation of the sigma factor SigI in Bacillus subtilis Tai-Yen Liu, Shu-Hung Chu, Yi-Nei Hu, Jyun-Jhih Wang and Gwo-Chyuan Shaw∗ Institute of Biochemistry and Molecular Biology, School of Life Sciences, National Yang-Ming University, Taipei 112, Taiwan, Republic of China ∗

Corresponding author: Institute of Biochemistry and Molecular Biology, School of Life Sciences, National Yang-Ming University, Taipei 112, Taiwan. Tel: +886-2-2826-7127; Fax: +886-2-2826-4843; E-mail: [email protected] One sentence summary: Multiple proteases are involved in SigB-relevant and SigB-irrelevant modulation of heat-induced activation of the sigma factor SigI in Bacillus subtilis. Editor: Andre Klier

ABSTRACT The Bacillus subtilis sigI-rsgI operon encodes the heat-inducible sigma factor SigI and its cognate anti-sigma factor RsgI. The heat-activated SigI positively regulates expression of sigI itself and genes involved in cell wall homeostasis and heat resistance. It remains unknown which protease(s) may contribute to degradation of RsgI and heat-induced activation of SigI. In this study, we found that transcription of sigI from its σ I -dependent promoter under heat stress was downregulated in a strain lacking the heat-inducible sigma factor SigB. Deletion of protease-relevant clpP, clpC or rasP severely impaired sigI expression during heat stress, whereas deletion of clpE partially impaired sigI expression. Complementation of mutations with corresponding intact genes restored sigI expression. In a null mutant of rsgI, SigI was activated and sigI expression was strongly upregulated during normal growth and under heat stress. In this rsgI mutant, further inactivation of rasP or clpE did not affect sigI expression, whereas further inactivation of clpP or clpC severely or partially impaired sigI expression. Spx negatively influenced sigI expression during heat stress. Possible implications are discussed. Given that clpC, clpP and spx are directly regulated by SigB, SigB appears to control sigI expression under heat stress via ClpC, ClpP and Spx. Keywords: regulated proteolysis; heat stress response; regulatory network

INTRODUCTION The soil-dwelling bacterium Bacillus subtilis is liable to be exposed to environmental heat stress. The heat-sensitive sensors can sense the heat signal and transduce it via a variety of mediators to evoke responses for helping cells adapt to and survive the heat stress. SigB is a general stress sigma factor that can be activated by a drop in the intracellular level of ATP during stationary phase or by exposure to environmental stresses such as

heat stress. The large SigB regulon consists of more than 150 genes that are involved in general stress adaptation and resistance (Hoper, Volker and Hecker 2005; Hecker, Pane-Farre and Volker 2007). The sigma factor SigW of B. subtilis is one of the seven extracytoplasmic function (ECF) sigma factors that are induced in response to various cell envelope stresses (Helmann 2002). sigW is co-transcribed with its downstream gene rsiW, which encodes the cognate transmembrane anti-sigma factor.

Received: 1 December 2016; Accepted: 7 March 2017 C FEMS 2017. All rights reserved. For permissions, please e-mail: [email protected]

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Interaction of SigW with the N-terminal region of RsiW sequesters and inactivates SigW on the inner surface of the cytoplasmic membrane (Yoshimura et al. 2004). In the presence of a stress signal, the membrane proteases PrsW (YpdC) and RasP (YluC) cleave RsiW at site 1 and site 2, respectively, for the release of the soluble N-terminal region of RsiW (Schobel et al. 2004; Ellermeier and Losick 2006; Heinrich and Wiegert 2006). It has been proposed that other membrane proteases might also be involved in trimming of RsiW (Heinrich, Hein and Wiegert 2009; Ho and Ellermeier 2012). Recently, it was reported that RsiV, which is the cognate anti-sigma factor of the ECF sigma factor SigV, can be cleaved by RasP but not by PrsW (Hastie, Williams and Ellermeier 2013). Bacillus subtilis contains three different HSP100 Clp ATPases (ClpC, ClpE and ClpX). Each of them can interact with the ClpP protease to form large hetero-oligomeric complexes (Gerth et al. 1996, 1998; Derre et al. 1999). clpC, clpE, clpP and clpX are members of the heat shock stimulon (Schumann 2003). Expressions of clpC, clpE and clpP are under negative regulation of the transcriptional repressor CtsR. The DNA-binding activity of CtsR is intrinsically inactivated during heat stress, leading to de-repression of clpC, clpE and clpP (Elsholz et al. 2010). The inactivated CtsR is further modified by McsB through arginine phosphorylation for proteolysis by ClpCP (Fuhrmann et al. 2009). It is also known that the cleaved N-terminal region of RsiW can be further degraded by ClpXP and ClpEP to release and activate SigW (Zellmeier, Schumann and Wiegert 2006). The global transcriptional regulator Spx can be degraded by both ClpXP and ClpCP (Nakano et al. 2002). spx is a member of the SigB regulon (Hoper, Volker and Hecker 2005) and belongs to the heat shock stimulon (Helmann et al. 2001). Spx can interact with RNA polymerase to stimulate transcription in response to oxidative stress and heat stress (Leelakriangsak and Zuber 2007; Lin and Zuber 2012; Runde et al. 2014) or inhibits activator-stimulated transcription (Nakano et al. 2003). The heat-inducible sigI–rsgI operon encodes the alternative sigma factor SigI (not an ECF sigma factor) and its cognate anti-sigma factor RsgI (Zuber, Drzewiecki and Hecker 2001; Asai et al. 2007). Interaction of the cytoplasmic N-terminal region of the transmembrane protein RsgI with SigI sequesters and inactivates SigI on the inner surface of the cytoplasmic membrane (Asai et al. 2007). Heat-activated SigI can stimulate expression of sigI itself and genes involved in cell wall homeostasis and heat resistance (Asai et al. 2007; Tseng and Shaw 2008; Tseng et al. 2011). The response regulator WalR is essential for cell growth and plays a key role in coordinating cell wall metabolism with cell division (Bisicchia et al. 2007; Dubrac et al. 2008; Fukushima et al. 2008). The heat-inducible WalR can directly and positively regulate sigI transcription under heat stress through a binding site located upstream of the σ I promoter of sigI (Huang et al. 2013). To date, it remains unknown which protease(s) contribute to degradation of RsgI and heat-induced activation of SigI. In this study, we aimed to identify genes possibly involved.

as a host for cloning purposes, and B. subtilis cells were grown in Luria−Bertani (LB) medium containing 10 g/l tryptone, 5 g/l yeast extract and 5 g/l NaCl. Antibiotics were used at the following concentrations (μg/ml): ampicillin, 100; chloramphenicol, 5; erythromycin, 1; spectinomycin, 100.

Construction of plasmids To construct plasmids pGS2780, pGS2784, pGS2793, pGS2794 and pGS2747 for the disruption of the chromosomal clpP, rasP, clpC, clpE and comK, respectively, DNA fragments were amplified by PCR with primer pairs B500 plus B501, B192 plus B193, B532 plus B533, B535 plus B536 and B412 plus B413, respectively, and then cloned individually between HindIII and BamHI sites of the thermosensitive plasmid pRN5101 (Fedhila et al. 2002). To construct plasmid pGS1753 or pGS1762, which carries a transcriptional fusion of the σ I -dependent promoter region of sigI or the σ A -dependent promoter region of sigI with bgaB (encoding a thermostable β-galactosidase), a DNA fragment was amplified by PCR with the primer pair T344 plus T355 or T383 plus T384, cleaved with restriction enzymes, and cloned between EcoRI and BamHI sites of the integrative promoter probe vector pDL (Yuan and Wong 1995). To construct plasmid pGS2776 or pGS2786, which carries the ctsR promoter plus the clpC coding region or the ctsR promoter plus the rasP coding region, two DNA fragments were amplified by PCR with primer pairs B494 plus B495 and B496 plus B497 or with primer pairs B494 plus B495 and B486 plus B487, cleaved with restriction enzymes, and cloned between KpnI and EcoRI or between KpnI and HindIII sites of pSG1154 (Lewis and Marston 1999).

Construction of strains with a bgaB fusion or an ectopically expressed gene integrated at the amyE locus Competent cells of B. subtilis were prepared and transformed by pDL- or pSG1154-derived integrative plasmids as described previously (Contente and Dubnau 1979). Transformants were selected by growth on chloramphenicol-containing (for pDLderived plasmids) or spectinomycin-containing (for pSG1154derived plasmids) LB agar plates. Correct integrants were screened by the method of O’Kane, Stephens and McConnell (1986).

Construction of disruption mutants of B. subtilis Disruption of chromosomal genes with pRN5101-derived plasmids through a single-crossover recombination was performed as described previously (Fedhila et al. 2002). The integrative plasmid was introduced into B. subtilis cells by the method of Contente and Dubnau (1979). The correctness of integrants was verified by PCR.

Ectopic complementation of the clpC or rasP mutation

MATERIALS AND METHODS Strains, plasmids, oligonucleotides, media and growth conditions The bacterial strains and plasmids used in this study are listed in Supplementary Table S1. The oligonucleotide primers are listed in Supplementary Table S2. Escherichia coli DH5α, which was used

Plasmid pGS2776 or pGS2786, which carries the σ B -dependent promoter of ctsR plus the clpC or rasP coding region, was integrated at the amyE locus of the clpC or rasP mutant. The integrative plasmid pGS1753, which carries the σ I -dependent promoter region of sigI transcriptionally fused with bgaB, was integrated at the σ I -dependent promoter region of the chromosomal sigI gene through a homologous recombination. The correct integration was verified by PCR.

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Assays of BgaB activities Activities of the thermostable β-galactosidase BgaB were measured as described previously (Schrogel and Allmansberger 1997), but with modifications (Tseng and Shaw 2008).

RESULTS AND DISCUSSION SigB positively influenced expressions of mreBH and sigI during heat stress in B. subtilis The B. subtilis mreBH gene, which encodes an actin homolog, was previously shown to be positively regulated by the sigma factor SigI (Tseng and Shaw 2008) and the response regulator WalR (Huang et al. 2013). During the course of the study, we serendipitously found that expression of a transcriptional fusion of the regulatory region of mreBH with the thermostable β-galactosidase reporter gene bgaB integrated at the amyE locus was downregulated in a null mutant of sigB under heat stress (Fig. 1a). However, sequence analysis of the regulatory region of mreBH revealed no potential σ B -dependent promoter sequence. To examine whether the influence of mreBH expression by SigB might be mediated through SigI or WalR, we constructed a transcriptional fusion of the regulatory region of sigI or walR with bgaB. The results showed that expression of the walR–bgaB fusion was not affected by the null mutation of sigB (Fig. 1b), whereas expression of the sigI–bgaB fusion was downregulated in the null mutant of sigB under heat stress (Fig. 1c). It is known that sigI transcription is driven by a σ A -dependent promoter (Salzberg et al. 2013) and a σ I -dependent promoter (Tseng and Shaw 2008). To determine which one is relevant with the observed effect, these two promoters were separately fused with bgaB. The results showed that expression from the σ A -dependent promoter–bgaB fusion was not affected by sigB mutation (Fig. 1d), whereas expression from the σ I -dependent promoter–bgaB fusion was downregulated in the sigB mutant under heat stress (Fig. 1e). Since no potential σ B -dependent promoter sequence is present in the regulatory region of sigI, SigB appears to influence sigI expression in an indirect manner. We then turned to seek which SigB-regulated genes might be relevant with heat induction of sigI.

Deletion of SigB-regulated clpC or clpP severely impaired sigI expression from the σ I -dependent promoter The clpC operon, which is transcribed from a σ A -dependent promoter and a σ B -dependent promoter (Kruger, Msadek and Hecker 1996), is under the repression of the repressor CtsR to prevent unstressed transcription (Kruger and Hecker 1998). When the DNA-binding activity of CtsR is intrinsically inactivated by heat stress (Elsholz et al. 2010), the heat-activated sigma factor SigB may stimulate the σ B -dependent promoter to contribute to clpC transcription. This possibility led us to test whether SigB might influence heat induction of sigI via clpC. sigI expression from the σ I -dependent promoter was then examined in the wild-type B. subtilis and the clpC mutant. The result showed that deletion of clpC severely impaired sigI expression from the σ I -dependent promoter under heat stress (Fig. 2a). To rule out the possibility of a polar effect of clpC deletion on downstream genes within the same operon, we carried out a complementation experiment. Ectopic complementation of the clpC mutation with an intact clpC gene expressed from the σ B dependent promoter of ctsR restored sigI expression from the

Figure 1. Effects of deletion of sigB on expression of transcriptional fusions of various promoters with bgaB in B. subtilis. Cells were grown in LB medium at 37◦ C to an OD600 of 0.3, and then the culture was split into two fractions. One fraction was maintained at 37◦ C (circles), and the other was shifted to 51◦ C (squares) at time zero. Cells continued to grow for various times as indicated before harvesting for BgaB activity assays. Solid symbols, no deletion; open symbols, sigB deletion. (a) Expression of the mreBH promoter region–bgaB fusion (pGS1722) in the wild-type B. subtilis (BM1140) or the sigB mutant (BM2292). (b) Expression of the walR promoter region–bgaB fusion (pGS1831) in the wild-type B. subtilis (BM1304) or the sigB mutant (BM2294). (c) Expression of the σ A -dependent promoter plus the σ I -dependent promoter of the sigI–bgaB fusion (pGS2221) in the wild-type B. subtilis (BM1827) or the sigB mutant (BM2308). (d) Expression of the σ A -dependent promoter of the sigI–bgaB fusion (pGS1762) in the wild-type B. subtilis (BM1187) or the sigB mutant (BM2622). (e) Expression of the σ I -dependent promoter of the sigI–bgaB fusion (pGS1753) in the wild-type B. subtilis (BM1172) or the sigB mutant (BM2623). Each value is the mean ± SD of three independent experiments.

σ I -dependent promoter under heat stress (Fig. 3a). The clpP gene is organized as a monocistronic operon and is also transcribed from a σ A -dependent promoter and a σ B -dependent promoter (Gerth et al. 1998). Deletion of clpP almost abolished sigI expression from the σ I -dependent promoter under heat stress (Fig. 2b). These results indicate that clpC and clpP are involved in modulation of heat induction of sigI expression.

Deletion of rasP severely impaired sigI expression from the σ I -dependent promoter The membrane protease gene rasP is expressed from a putative σ A -dependent promoter preceding the uppS operon (Hastie, Williams and Ellermeier 2013). Although rasP does not belong to the SigB regulon, we still attempted to examine the effect of deletion of rasP on sigI expression. The result showed that deletion of rasP severely impaired sigI expression from the σ I -dependent promoter under heat stress (Fig. 2c). Ectopic

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Figure 2. Expression of the σ I -dependent promoter of the sigI–bgaB fusion in various B. subtilis strains. Cells were grown in the same way as described in Fig. 1. Solid symbols, in the wild-type; open symbols, in mutants; circles, 37◦ C; squares, 51◦ C. (a) In the wild-type B. subtilis (BM1172) or the clpC mutant (BM2608). (b) In the wild-type B. subtilis (BM1172) or the clpP mutant (BM2609). (c) In the wildtype B. subtilis (BM1172) or the rasP mutant (BM2624). (d) In the wild-type B. subtilis (BM1172) or the clpE mutant (BM2610). Each value is the mean ± SD of three independent experiments.

Figure 4. Effects of inactivations of various genes in the rsgI mutant on expression of the σ I -dependent promoter of the sigI–bgaB fusion. Cells were grown in the same way as described in Fig. 1. Solid symbols in (a) to (d), without inactivation (BM2653); open symbols, with inactivation of (a) rasP (BM2671), (b) clpE (BM2675), (c) clpP (BM2668) or (d) clpC, (BM2676); circles, 37◦ C; squares, 51◦ C. Each value is the mean ± SD of three independent experiments.

The effect of deletion of clpE on sigI expression was also investigated. The results showed that deletion of clpE caused a partial impairment of expression of sigI from the σ I -dependent promoter under heat stress (Fig. 2d).

Further inactivation of rasP or clpE in a null mutant of rsgI did not affect sigI expression

Figure 3. Effect of ectopic complementation of the clpC or rasP mutation on expression of the σ I -dependent promoter of the sigI–bgaB fusion. Cells were grown in the same way as described in Fig. 1. Solid symbols, with complementation; open symbols, without complementation; circles, 37◦ C; squares, 51◦ C. (a) Expression of the bgaB fusion in the clpC mutant (BM2670) or the clpC mutant complemented with the clpC gene (BM2669). (b) Expression of the bgaB fusion in the rasP mutant (BM2658) or the rasP mutant complemented with the rasP gene (BM2673). Each value is the mean ± SD of three independent experiments.

complementation of the rasP mutation with an intact rasP gene expressed from the ctsR promoter restored sigI expression from the σ I -dependent promoter under heat stress (Fig. 3b), thus excluding the possibility of a polar effect of rasP deletion on downstream genes within the same operon. These results indicate that rasP is also involved in modulation of heat induction of sigI expression. Inspection of the nucleotide sequence of the regulatory region of the monocistronic prsW gene revealed only a putative σ A -dependent promoter (data not shown). We also investigated the effect of deletion of prsW on sigI expression, but no significant effect was observed (data not shown).

Deletion of clpE caused a partial impairment of heat induction of sigI expression The clpE gene is organized as a monocistronic operon and is transcribed from two σ A -dependent promoters (Derre et al. 1999).

We next analyzed the possible connection between the antisigma factor RsgI and these proteases in heat induction of sigI expression. The effect of further inactivation of clpC, clpE, clpP, clpX or rasP in an rsgI deletion mutant on sigI expression was examined. As expected, sigI expression in the null mutant of rsgI was strongly upregulated during normal growth at 37◦ C and under heat stress at 51◦ C, most likely due to the activation of SigI (Fig. 4a). Further inactivation of rasP or clpE in the rsgI mutant did not affect sigI expression (Fig. 4a and b). These results are in agreement with the notion that RasP and ClpE are likely involved in the degradation of the anti-sigma factor RsgI. In the absence of RsgI, there is no requirement of these two proteases for activation of SigI.

Further inactivation of clpP or clpC in the null mutant of rsgI severely or partially impaired sigI expression In contrast, further inactivation of clpP in the rsgI mutant almost abolished sigI expression during normal growth and under heat stress (Fig. 4c). Unexpectedly, further inactivation of clpC in the rsgI mutant did not affect sigI expression during normal growth at 37◦ C but partially impaired sigI expression under heat stress at 51◦ C (Fig. 4d). The transcriptional regulator ComK is a substrate of ClpCP (Turgay et al. 1998). We then investigated the possibility of whether the impaired sigI expression in the clpC mutant might be caused by an increased level of ComK, which led to repression of sigI expression. The result showed that inactivation of comK slightly reduced rather than enhanced sigI expression from the σ I -dependent promoter (Supplementary Fig. S1),

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thus making this possibility unlikely. Spx is also a substrate of ClpCP (Nakano et al. 2002). spx expression is heat-inducible. The Spx protein is barely detectable during normal growth (Nakano et al. 2001). These features raised the possibility that ClpC might control sigI expression through degradation of Spx during heat stress (see below).

Spx negatively influenced sigI expression during heat stress

Figure 5. Effect of deletion of clpX or spx on expression of the σ I -dependent promoter of the sigI–bgaB fusion. Cells were grown in the same way as described in Fig. 1 except for heat stress treatment at 45◦ C. Circles, 37◦ C; squares, 45◦ C. (a) Solid symbols, in the wild-type B. subtilis (BM1172); open symbols, in the clpX mutant (BM2714). (b) Solid symbols, in the wild-type B. subtilis (BM1172); open symbols, in the spx mutant (BM2719). (c) Solid symbols, in the clpX mutant (BM2714); open symbols, in the spx clpX double mutant (BM2713). Each value is the mean ± SD of three independent experiments.

Due to the observation that the spx mutant could not grow at 51◦ C, the following spx-relevant experiments during heat stress were carried out at 45◦ C. The heat-inducible clpX gene is organized as a monocistronic operon and is transcribed from an extended –10 σ A -dependent promoter (Gerth et al. 1996). We first examined the effect of deletion of clpX on sigI expression from the σ I -dependent promoter. Unexpectedly, the result showed that deletion of clpX enhanced rather than reduced sigI expression during heat stress at 45◦ C (Fig. 5a). Given that Spx is a substrate of ClpXP, we then investigated whether the observed effect of clpX deletion might involve Spx. The result showed that deletion of spx in the wild-type cells enhanced sigI expression from the σ I -dependent promoter during heat stress (Fig. 5b). Deletion of spx in the clpX mutant also enhanced sigI expression but to a lesser extent (Fig. 5c). These results support the notion that Spx negatively influences sigI expression under heat stress. Future in vitro experiments will clarify whether Spx influences sigI expression directly or indirectly. An interesting question is why deletion of clpX caused an enhancement of sigI expression. In addition to degrading Spx, ClpXP may possibly be also capable

Figure 6. A model for the intricate regulatory network of SigB-relevant and SigB-irrelevant modulation of heat-induced activation of the sigma factor SigI by multiple proteases in B. subtilis. When the DNA-binding activity of the repressor CtsR is intrinsically inactivated by heat stress (Elsholz et al. 2010), clpC, clpE and clpP are derepressed. The heat-activated sigma factor SigB then stimulates the σ B -dependent promoters for transcription of clpC and clpP, thus contributing to sigI transcription during heat stress through degradation of Spx. Meanwhile, ClpE and RasP contribute to SigB-irrelevant modulation of activation of SigI. Spx, whose synthesis is also under the positive control of SigB, negatively influences sigI expression under heat stress.

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of degrading the cytosolic SigI. Further biochemical characterization will clarify this possibility. Given that Spx negatively influences sigI expression during heat stress and that Spx is also a substrate of ClpCP, it could suggest that ClpCP contributes to sigI expression under heat stress through degradation of Spx (Fig. 6). As mentioned above, clpC and clpP are subject to the repression of CtsR under unstressed conditions (Kruger and Hecker 1998). When the DNA-binding activity of CtsR is intrinsically inactivated by heat stress (Elsholz et al. 2010), the heat-activated sigma factor SigB then can stimulate the σ B -dependent promoters for transcription of clpC and clpP. Thus, SigB can contribute, at least in part, to heat induction of sigI via clpC and clpP (Fig. 6). Heat induction of sigI expression appears to occur at least at two levels: (i) the heat-inducible WalR and SigI can stimulate sigI transcription, thus contributing to heat induction of sigI expression at the transcriptional level (Fig. 6); and (ii) SigB-relevant and SigB-irrelevant heat induction of multiple proteases can modulate SigI activity at the post-translational level (Fig. 6). Some other important work will need to be done in the future: (i) identification of other protease(s) that may be involved in degradation of RsgI, and (ii) biochemical identification and characterization of multiple cleaved forms of RsgI due to degradation by various proteases at distinct sites. In summary, we present genetic evidence that multiple proteases including ClpCP, ClpEP, ClpXP and RasP are involved in modulation of heat-induced activation of the sigma factor SigI in Bacillus subtilis. We have also shown that Spx negatively influences sigI expression during heat stress. ClpCP apparently contributes to sigI expression under heat stress through degradation of Spx. Given that clpC, clpP and spx are known to be directly regulated by SigB, SigB appears to control sigI expression during heat stress via ClpC, ClpP and Spx.

SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online.

ACKNOWLEDGEMENTS We thank Kurs ¨ ¸ ad Turgay, Jeff Errington, Marc Bramkamp and Thomas Wiegert for kindly providing strains. We are also grateful to the reviewers for valuable comments and suggestions.

FUNDING This work was supported by the Ministry of Science and Technology of the Republic of China (MOST 104-2311-B-010-006MY2). Conflict of interest. None declared.

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