The B. subtilis protein quality control system includes chaperones like the Hsp70 (DnaKJE) and Hsp60 (GroE) system, as well as other conserved chaperone systems such as ribosome-associated chaperones (Trigger factor), Hsp90 (HtpG), small heat shock proteins and redox chaperones (Schumann et al., 2002; Moliere and Turgay, 2009), together with AAA+ protease complexes.

The AAA+ unfoldase ClpB, which together with DnaK is necessary for protein refolding and disaggregation (Glover and Lindquist, 1998; Weibezahn et al., 2004; Haslberger et al., 2007; Winkler et al., 2010, 2012a; Oguchi et al., 2012; Seyffer et al., 2012), is widely conserved in most bacterial species, but is notably absent from B. subtilis. However, it was demonstrated that B. subtilis ClpC, which is closely related to ClpB, can—together with the adaptor protein MecA or its paralog YpbH—disaggregate and refold protein aggregates in vitro when not associated with ClpP (Schlothauer et al., 2003; Haslberger et al., 2008).

In B. subtilis, the AAA+ protease complexes ClpCP, ClpEP and ClpXP are part of the protein quality control system. ClpC was identified as a stress-induced protein, the ΔclpC strain is thermosensitive and, similar to ΔclpP or ΔclpX strains, display impaired degradation of misfolded proteins (Krüger et al., 1994, 2000; Msadek et al., 1994; Gerth et al., 1998, 2004; Kock et al., 2004). ClpE expression is tightly controlled and is only induced after severe heat shock, implying that ClpEP might function as an additional protease system under other severe stress conditions (Derre et al., 1999a; Gerth et al., 2004; Miethke et al., 2006). Consistent with their function in protein homeostasis, ClpC, ClpX, ClpE, and ClpP were all observed to associate with subcellular protein aggregates, especially upon heat shock or heterologous protein synthesis (Krüger et al., 2000; Jürgen et al., 2001; Miethke et al., 2006; Kain et al., 2008; Kirstein et al., 2008; Simmons et al., 2008).

As previously demonstrated for other bacteria (Sauer and Baker, 2011), ClpXP of B. subtilis is necessary for the degradation of proteins whose translation is stalled. These unfinished polypeptides are prone to aggregation and must be eliminated. In a process called trans-translation, stalled ribosomes are rescued by the activities of the SmpB protein in conjunction with the transfer and messenger RNA (tmRNA). The tmRNA is a specialized small RNA, which aided by SmpB first acts as a tRNA and subsequently like an mRNA. This not only liberates the ribosome, but also results in the addition of a short sequence, termed an SsrA tag to the C-terminus of the unfinished protein (Keiler et al., 1996; Muto et al., 2000; Abe et al., 2008; Keiler, 2008; Ujiie et al., 2009). ClpXP recognizes the C-terminal SsrA tag, and degrades these unfinished proteins, thereby preventing their aggregation (Keiler et al., 1996; Wiegert and Schumann, 2001; Sauer and Baker, 2011).

The membrane-associated FtsH AAA+ protease is most likely also directly involved in protein quality control, since a deletion of ftsH causes pleiotropic effects, including salt, and heat sensitivity (Deuerling et al., 1995, 1997). The two B. subtilis AAA+ protease Lon paralogs, LonA and LonB, do not have a significant role in the degradation of misfolded proteins (Riethdorf et al., 1994; Schmidt et al., 1994; Krüger et al., 2000; Serrano et al., 2001; Simmons et al., 2008). Only very little is known about the possible in vivo role of the B. subtilis ClpYQ (CodWX) AAA+ protease complex (Slack et al., 1995; Kang et al., 2003; Simmons et al., 2008; Figure 1).

An interesting feedback mechanism was observed for the regulation of chaperone synthesis in B. subtilis. The transcription of the dnaK and groE operon is controlled by the repressor HrcA, which is also encoded as the first gene of the dnaK operon. The GroEL chaperone is necessary for maintaining the repressor activity of HrcA. However, when GroEL interacts with unfolded proteins, HrcA repressor activity cannot be maintained and the synthesis of GroEL and DnaK is induced. The elevated levels of chaperones help to protect and repair the proteome. This subsequently restores the repressor activity of HrcA, thereby terminating the transcriptional induction of chaperones (Mogk et al., 1997; Schumann et al., 2002).

The same AAA+ protease complexes can be involved in general proteolysis for protein quality control and in regulatory proteolysis to control the activity of transcription factors and other key regulatory proteins. In B. subtilis, not only chaperones like GroEL are involved in sensing protein folding stress, but the AAA+ protease complexes ClpCP or ClpXP with their adaptor proteins McsB and YjbH are involved in sensing various stresses and are also involved in the regulation of their own synthesis by controlling e.g., CtsR or Spx stability (Zuber, 2004; Kirstein et al., 2009b; Rochat et al., 2012; Runde et al., 2014; Engman and von Wachenfeldt, 2015; Mijakovic et al., 2016).

As mentioned above, McsB can act as an adaptor for the AAA+ protein ClpC. It has been shown that this activity depends on the ability of McsB to function as a protein kinase (Kirstein et al., 2005, 2007). Only when active as a kinase McsB can stimulate ClpC activity, and this specific activation depends on site-specific phosphorylation of ClpC by McsB (Elsholz et al., 2010b). The kinase activity of McsB has also shown to be required for the degradation of specific substrates by the ClpCP protease. However, McsB might be involved in regulatory proteolysis of not only transcription factors such as CtsR, but also other proteins. There are strong indications that the ClpC adaptor proteins McsB like MecA or YpbH play an important role together with ClpCP not only in regulatory proteolysis of CtsR, but also in general proteolysis and protein quality control (Kirstein et al., 2008).

When grown into stationary phase, a subpopulation of B. subtilis cells develop the ability to actively take up extracellular DNA. ComK is the transcription factor necessary and sufficient to induce the transcription of the competence state (K-state) regulon. ComK induces the transcription of competence genes, which encode the proteins necessary to form the DNA receptors that recognize and transport extracellular DNA into the cell. Concurrently, DNA repair and recombination systems are upregulated, whereas general transcription, translation, cell division and growth are impaired (van Sinderen et al., 1995; Haijema et al., 2001; Berka et al., 2002; Hamoen et al., 2003; Chen et al., 2005; Hahn et al., 2005, 2015). Thus, the K-state cells are not only able to take up DNA, but also exhibit properties such as growth inhibition that are characteristic of persister-like cellular states (Hahn et al., 2015), and which can confer a survival advantage in the face of antibiotics or other stressors (Yüksel et al., 2016).

In exponentially growing B. subtilis cells, ComK is constantly antagonized by the adaptor protein MecA. MecA not only targets ComK for degradation by ClpCP, but also directly inhibits ComK activity (Dubnau and Roggiani, 1990; Kong and Dubnau, 1994; Turgay et al., 1997, 1998; Persuh et al., 1999). At higher cell density in post-exponential cells, signaling via a quorum sensing system causes the stable phosphorylation of the response regulator ComA, which results in the synthesis of the small protein ComS (D'Souza et al., 1994; Hamoen et al., 1995). ComS competes with ComK for binding to MecA (Prepiak and Dubnau, 2007), which results in the release of ComK from MecA-mediated inhibition and degradation (Turgay et al., 1997, 1998). Since ComK is a positive autoregulatory transcription factor, this release results in the exponential synthesis of ComK in the subpopulation of competence-developing B. subtilis cells. The MecA-dependent retargeting of the abundant ComK protein for ClpCP degradation is essential for the escape from competence (Turgay et al., 1998).

This post-translational regulatory mechanism—where the activity of an adaptor protein is controlled by the signal-induced synthesis of a small protein that acts like an anti-adaptor protein—was also observed in E. coli for the proteolytic control of the general stress sigma factor σ^S by the adaptor protein RssB-P (Becker et al., 1999; Bougdour et al., 2006; Hengge, 2009; Battesti and Gottesman, 2013; Battesti et al., 2013; Micevski et al., 2015).

Endospore formation is a terminal cellular developmental process leading to two different types of cells in a structure termed the sporangium. This event begins with asymmetric cell division, after which the larger mother cell encloses the smaller forespore cell and supports its development into an endospore. This concerted cellular developmental process culminates in the release of the endospore from the lysing mother cell (Rudner and Losick, 2001; Higgins and Dworkin, 2012). The endospore is metabolically inactive and highly resistant to most stressors and environmental extremes (Piggot and Hilbert, 2004). Once the cell has committed to this developmental process, it is irreversible (Dworkin and Losick, 2005). Consequently, half of the progeny will transform into an endospore, whereas the other half will die. It is therefore critical that this process is tightly regulated. Indeed, the decision whether or not to commit to this complex developmental process is controlled by multiple regulatory circuits that integrate several distinct signals (Higgins and Dworkin, 2012). Interestingly, AAA+ protease complexes have several important roles at various stages of this complex decision-making process. The roles of ClpCP, ClpXP and FtsH sporulation have been elucidated in detail (Pan et al., 2001; Bradshaw and Losick, 2015; Tan et al., 2015).

One of the interesting aspects of sporulation is an asymmetric cell division that results in two unequally sized daughter cells a smaller forespore and a larger mother cell. Upon asymmetric division, both cells engage specific and distinct gene expression programs that ultimately determine their markedly different fates (Piggot and Hilbert, 2004). The first cell type-specific genetic program is the activation of the alternative sigma factor F in the forespore, which depends on both a partner-switching mechanism involving the anti-sigma factor SpoIIAB and the anti-anti-sigma factor SpoIIAA, and also on the activity of the PP2C phosphatase SpoIIE (Stragier and Losick, 1996).

Sigma F and all factors required for its activation are produced at the onset of sporulation and thus are present in both cell compartments (Gholamhoseinian and Piggot, 1989). For over two decades it was not understood how Sigma F is activated exclusively in the forespore. SpoIIE is the critical controller of the activation of Sigma F: it de-phosphorylates SpoIIAA, which can then activate Sigma F (Stragier and Losick, 1996). Intriguingly, SpoIIE is expressed in both compartments but the protein is found only in the forespore (Gholamhoseinian and Piggot, 1989). Bradshaw and Losick recently implicated the AAA+ protease FtsH in the compartment specific regulation of SpoIIE stability during the early stages of sporulation (Bradshaw and Losick, 2015).

They showed that SpoIIE is subject to FtsH-dependent degradation in the mother cell, but is protected from proteolysis in the forespore. This specific stabilization results in the accumulation of active SpoIIE proteins in the forespore that lead to the forespore-specific activation of Sigma F (Figure 4A). The stabilization of SpoIIE in the forespore is not linked to differences in FtsH expression or activity in the different compartments.

Normally, SpoIIE is degraded by FtsH upon recognition of an N-terminal degradation tag. However, relocation of SpoIIE from the polar divisome to the cell pole results in stabilization of SpoIIE by a mechanism that is not yet fully understood but seems to involve SpoIIE oligomerization (Bradshaw and Losick, 2015; Figure 4A). Nonetheless, the local control of SpoIIE degradation is a great example of how proteolysis can be a crucial regulatory mechanism in the control of cell polarity.

Interestingly, FtsH is not the only AAA+ protease that is involved in the control of SigmaF activity. It has been shown that the ClpCP protease is responsible for the degradation of the anti-sigma factor SpoIIAB (Pan et al., 2001). Under normal growth conditions, SpoIIAB interacts and thus inactivates Sigma F (Duncan and Losick, 1993). This interaction is also thought to stabilize SpoIIAB. Upon de-phosphorylation of the anti-anti-sigma factor SpoIIAA by SpoIIE, SigmaF is liberated (Stragier and Losick, 1996) and SpoIIAB is subject to ClpCP-dependent degradation (Pan et al., 2001). Although, this proteolytic mechanism is not directly involved in the activation of SigmaF, it is required to maintain the stability of free Sigma F (Pan et al., 2001). Targeting of SpoIIAB for ClpCP-dependent degradation is enabled by the presence of the C-terminal amino acid sequence LCN (Pan et al., 2001; Pan and Losick, 2003). Interestingly, none of the described ClpC adaptors are involved in the proteolysis of SpoIIAB, which implicates a hitherto unidentified adaptor or molecular mechanism in this process (Kirstein et al., 2009b). Since artificially LCN-tagged proteins are also subject to degradation during exponential growth (Pan and Losick, 2003), it is unlikely that this process depends on a sporulation-specific adaptor protein (Figure 4B).

Regulated proteolysis is also involved in the control mechanisms ensuring proper spore formation. The ClpXP protease together with the adaptor protein CmpA are involved in the quality control of the spore envelope. In cells that produce spores with a proper spore envelope, CmpA is degraded through ClpXP-dependent proteolysis and sporulation continues. However, in cells that display defects in the spore envelope maturation, CmpA is stabilized and mediates ClpXP-dependent degradation of the coat morphogenetic protein SpoIVA. This proteolytic event causes instability and subsequent lysis of the spore, thereby ensuring that only properly assembled spores are produced within the population. The presence of ClpXP and CmpA is required but not sufficient for degradation of SpoIVA and also of CmpA itself. The proteolytic activity of this regulatory circuit depends on the presence of a specific signal or component that is under the control of the cell type-specific Sigma K. However, the nature of this signal or component is unclear and requires further investigation (Tan et al., 2015; Figure 4C).

The three mechanisms described above are examples of how regulated protein degradation is involved in the control of sporulation. In addition, evidence exists that AAA+ proteases and their associated proteolytic events play even more roles in the control of sporulation. A recent global high-throughput genetic screen highlighted the pleiotropic function of ClpC in the control of sporulation. Meeske and colleagues showed that cells lacking clpC had a dramatic defect in sporulation efficiency and displayed different phenotypes, such as delayed entry, asymmetric engulfment, reduced or no Sigma G activity and a concomitant small forespore phenotype (Meeske et al., 2016). This observation suggests that ClpC is specifically involved in the control of distinct but yet unknown regulatory events during sporulation.

A first analysis of B. subtilis strains with clpC, clpX, or clpP mutations suggested that these genes are important for swimming motility (Rashid et al., 1996; Liu and Zuber, 1998; Msadek et al., 1998). It was demonstrated that ClpCP and ClpXP enable motility via regulatory proteolysis of the transcription factors ComK, DegU and Spx, which directly or indirectly influence the transcription of flagellar genes (Liu and Zuber, 1998; Ogura and Tsukahara, 2010; Molière et al., 2016).

Interestingly, B. subtilis cells can switch from swimming to swarming motility on surfaces, which is accompanied by a hyperflagellation of the swarming cells (Kearns, 2010). The transcriptional activator SwrA determines the number of flagella in B. subtilis cells (Mukherjee and Kearns, 2014). This transition is controlled by regulated proteolysis of SwrA, which in swimming cells is targeted by the adaptor protein SmiA for LonA-dependent degradation (Mukherjee et al., 2015).

The transformation of B. subtilis cells from the motile to the sessile state depends on the presence of the SlrR regulatory protein. In the SlrR low state, motility and autolysis genes are expressed. In contrast, in the SlrR high state SlrR together with SinR repress motility and autolysis genes, resulting in long chains of sessile cell and biofilm formation. The induction of SlrR expression is well understood and depends on a complex three-protein regulatory circuit (Chai et al., 2010b; Norman et al., 2013). Interestingly, the switch from the SlrR high state to the motile, SlrR low depends on the controlled degradation of SlrR. It is not clear how SlrR is degraded, but it is known that an LexA-like auto-cleavage of SlrR is involved in SlrR stability. Interestingly, it was shown that the AAA+ protease ClpCP influences the stability of SlrR, but the precise molecular mechanisms have not yet been described (Chai et al., 2010a).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.