Persistent environmental stress conditions, such as high temperature, pH, and nutritional limitations can lead to the accumulation of unfolded and damaged protein aggregates in the cell that may have a derogatory effect on cell viability (Oldfield & Dunker 2014)). Bacteria have developed a wide range of mechanisms to sense and maintain protein homeostasis, employing a network of molecular chaperones and proteases. Regulatory proteolysis, one of the ways of altering the protein content of the bacterial cell in response to stress, is predominantly characterized in bacteria by the activity of a large and diverse superfamily of ATP-dependent proteolytic complexes, AAA+ (ATPases Associated with diverse cellular Activities) proteases (Sauer & Baker 2011) (Matyskiela & Martin 2013). Mode of action of a characteristic AAA+ protease involves recognition and ATP-dependent unfolding of specific substrates by AAA+ unfoldase, followed by translocation of the unfolded protein into the degradation chamber of the associated peptidase.
Depending on the genome size, bacteria may have up to five active AAA+ proteases that play diverse roles in bacterial regulatory and quality control circuits (Sauer & Baker 2011) . In this review we will discuss molecular structure of AAA+ proteases, their diversity, as well as the major functions of these proteases in bacterial cells, namely, degradation of aggregated and misfolded proteins, reversing protein aggregation, and disassembly of stable protein complexes.
Characteristic structural features of AAA+ proteases
Typical AAA+ protease possess a core αβα nucleotide-binding domain with two major nucleotide binding and hydrolysis motifs referred to as Walker A and Walker B (Walker et al. 1982) (Neuwald et al. 1999) (Ogura & Wilkinson 2001) . A defining region of 200-250 amino acids, the AAA+ module, is comprised of a core αβα nucleotide binding domain and a smaller α-helical domain that is highly conserved structurally and serves as a defining characteristic of all AAA+ superfamily members (Neuwald, Aravind et al. 1999) (Ogura & Wilkinson 2001) (Ammelburg et al. 2006) . Different members of the AAA+ protein superfamily often display variability in the structure of their core AAA+ modules, which possibly play a role in directing these proteins towards specific functions, and would explain remarkable diversity of mechanisms of action of different AAA+ proteases in bacterial cells.
In their biologically active form, AAA+ proteases assemble into oligomers (Iyer et al. 2004) . The hexameric configuration allows the positioning of the ATP-binding sites at the interface between subunits of the holoenzyme, linking the nucleotide-binding sites of adjacent subunits (Siddiqui et al. 2004) (Lum et al. 2004) (Schlieker et al. 2004). All hexameric AAA+ proteins also have a central cavity or pore that is used by numerous AAA+ proteins to thread their substrates through (Ogura & Wilkinson 2001). The size of the pore may vary with translocation of different substrates (Alexopoulos et al. 2012) , and is also affected by ATP binding and hydrolysis by AAA+ module (Wang 2004) (Wang et al. 2001) . During the hydrolysis process, the movement of N-terminal and C-terminal domains of the AAA+ module generates a mechanical force that can be used to affect remodeling events in associated molecules (Ogura & Wilkinson 2001).
Proteolytic activity of AAA+ proteins: unfolding and degradation of protein aggregates
AAA+ proteins play a crucial role in bacterial proteolysis by unfolding protein substrates and delivering them to the degradative chamber of peptidase (Pickart & Cohen 2004). These proteolytic functions in bacteria are carried out by several different AAA+ proteases, ClpAP, ClpXP, HslUV, Lon, FtsH, and Mpa•20S, that can be divided into two major groups: unfoldase/protease complexes (ClpXP, ClpAP , Mpa•20S and HslUV), with unfoldase and peptidase components on separate polypeptides, and those that contain both components on a single polypeptide. Regradless of the group, all bacterial AAA+ proteases share the same three-step mechanism of substrate degradation. During the first step, the substrate is recognised by the unfoldase. Following recognition, the substrate is then unfolded using the energy of ATP binding and hydrolysis, and the unfolded substrate is then translocated into the associated peptidase, where the polypeptide chain is hydrolysed into 3–8 amino acids long peptide fragments (Gribun et al. 2005; Sprangers et al. 2005) .
Two-component AAA+ proteases
ClpXP and ClpAP proteases: a paradigm of bacterial AAA+ proteolytic machinery
Two-component AAA+ Clp-containing proteases play a critical role in controlling stress response and regulating bacterial virulence (Barchinger & Ades 2013; Frees et al. 2013; Micevski & Dougan 2013). All Clp-containing systems form barrel-shaped complexes in which the hexameric ring-shaped oligomers of AAA+ unfoldase is concentrically aligned with two heptameric rings of ClpP protease (Wang et al. 1997; Bochtler et al. 2000; Sousa et al. 2000). ClpP is a serine protease with a chymotrypsin-like activity (Arribas & Castano 1993; Thompson & Maurizi 1994). Two heptameric rings of the enzyme assemble a barrel-like degradation chamber (Wang, Hartling et al. 1997), while The N-terminal peptides of ClpP form a narrow axial entry portal at the entrance to the catalytic chamber, that prevent entry of the folded proteins. Unfolded proteins and small peptides, on the other hand, can be degraded by ClpP even in the absence of ATP hydrolysis (Jennings et al. 2008b).This process is significantly accelerated by the activity of ClpP-associated unfoldases, such as ClpX and ClpA, that actively unfold and translocate substrate proteins through the axial entry portal into the ClpP barrel (Weber-Ban et al. 1999). Recent studies show that binding of unfoldases also leads to conformational changes in the N-terminal region of ClpP that allow entrance and degradation of small peptides (Jennings et al. 2008a).
The affinity of the unfoldase-ClpP interaction depends on whether or not the ATPase domain (one for ClpX and two for ClpA) of the unfoldase is bound to substrate (Joshi et al. 2004). In bacteria, substrate recognition requires presence of a specific amino acid motif, a degron (Schrader et al. 2009) that can be either intrinsic to the target protein, or added to the polypeptide as a tag by small stable 10S RNA (ssrA ) during the translation (Dougan et al. 2002a)(Karzai et al. 2000). SsrA tag is composed of 11 amino acids (AANDENYALAA), and is recognized by ClpX and ClpA unfoldases. Degradation of ssrA-tagged proteins is carried out almost exclusively by ClpXP and ClpAP complexes (Farrell et al. 2005; Lies & Maurizi 2008). Recent studies show that apart from specific binding of ssrA-tagged substrates, ClpX also recognizes five additional specific motifs near the C- and the N-terminus of target proteins (Flynn et al. 2003). Similarly, two additional recognition motifs (N-degron and C-degron) were identified for ClpA (Hoskins et al. 2000; Hoskins & Wickner 2006; Maglica et al. 2008).
Some protein substrates require additional components, adaptor proteins, to facilitate binding to unfoldase, and in some cases to activate either the substrate or the unfoldase for delivery to the protease for degradation . In E coli, adaptor proteins SspB, UmuD and RssB mediate substrate binding to ClpXP (Levchenko et al. 2000; Zhou et al. 2001; Neher et al. 2003), while adaptor protein ClpS delivers substrates to ClpAP (Dougan et al. 2002b; Zeth et al. 2002; Erbse et al. 2006; Hou et al. 2008; Roman-Hernandez et al. 2011).
Following the initial recognition step, unfoldase components of the ClpXP and ClpAP proteases use the energy released from the binding and hydrolysis of ATP to unfold the substrate and translocate it into the degradation chamber (Weber-Ban, Reid et al. 1999; Reid et al. 2001). Substrate unfolding is a rate-limiting step for proteolysis by AAA+ proteases, since although degradation of folded substrates still occurs, it proceeds much slower than degradation of unfolded substrates (Kenniston et al. 2003).
HslUV protease: a hypothetical ancestor of eukaryotic proteasome
HsIUV is another member of the two-component AAA+ protease family, where HslU hexamer serves as the AAA+ protein unfoldase/translocase, whereas HslV forms a
double-ring dodecamer that encloses the proteolytic compartment. Large and small AAA+ domains of HsIUV use ATP binding and hydrolysis to facilitate conformational
changes that drive substrate unfolding and translocation. These mechanical processes occur through ATP-powered movements of loops containing conserved GYVG sequence (Park et al. 2005). HslUV complex is homologues to eukaryotic proteasomes in its structure and mechanism of action (Gille et al. 2003), and like the proteasome, it requires binding of ATP in a magnesium-dependent manner for substrate binding and subsequent unfolding (Burton et al. 2005). Large AAA+ domain of HslU unfoldase contains a unique intermediate (I) domain that forms a funnel-shaped cavity above the axial pore (Kwon et al. 2003; Kwon et al. 2004). Recent study by Sundar et al demonstrated that the I domain of HslU is required for robust ATP hydrolysis, and plays an essential role in coordinating substrate binding, protein degradation, and ATP hydrolysis (Sundar et al. 2012).
Mpa/20S proteasome complex in Actinobacteria
The proteasome carries out the majority of cytosolic protein degradation inside a eukaryotic cell, while most bacteria do not contain this proteolytic machinery. However, Actinobacteria, including Mycobacteria, have acquired proteasomes by horizontal gene transfer (Tamura et al. 1995; Hu et al. 2006). In Mycobacteria, proteosomal substrate targeting is mediated by small protein Pup that is covalently attached to lysine residues of substrate proteins through its C-terminus. Similarly to the SsrA-ClpX-ClpP system, Pup-tag is then recognized by the AAA+ protein Mpa (called ARC in other actinobacteria) that binds and unfolds Pup-tagged proteins by threading them through its central pore and translocates them into the 20S proteasome for efficient degradation (Chen et al. 2009; Liao et al. 2009; Sutter et al. 2009).
One-component AAA+ proteases
FtsH: quality control of cytoplasmic and membrane proteins
FtsH, a member of the one-component AAA+ proteolytic family, is an evolutionarily conserved single-polypeptide Zn2+-dependent peptidase(Gur 2013) that forms homohexameric ring-shaped structure (Krzywda et al. 2002; Suno et al. 2006; Bieniossek et al. 2009; Langklotz et al. 2012; Okuno & Ogura 2013). Similarly to Clp-containing systems, FtsH also unfolds and directs substrates for degradation, but it contains additional domain at the amino terminus of its AAA+ module that anchors the protein to the inner membrane of the bacterial cell, and a domain at carboxyl terminus that has protease activity (Tomoyasu et al. 1993; Suno, Niwa et al. 2006). FtsH therefore plays an important role in quality control of bacterial inner-membrane environment, and degrades membrane proteins that fail to form functional membrane protein complexes (Okuno & Ogura 2013), such as SecY, an integral membrane subunit of the protein translocation machinery in the inner membrane (Akiyama et al. 1996a; Akiyama et al. 1996b). FtsH initiates ATP-dependent degradation of membrane proteins by recognizing their N- or C-terminal cytoplasmic segments (over 20 amino acid residues long)(Chiba et al. 2000; Chiba et al. 2002). In comparison to ClpXP, ClpAP, and HslUV , FtsH appears to have weaker unfolding activity (Herman et al. 1995). When FtsH encounters the fragment with structurally stable domain, the degradation process stops, leading to accumulation of stable polypeptide fragments in the inner membrane of the bacteria (Kihara et al. 1999; Chiba, Akiyama et al. 2000; Chiba, Akiyama et al. 2002), which plays an important regulatory role in a number of cellular functions.
FtsH also pays an essential role in quality control of cytoplasmic proteins such as the heat shock transcriptional factor 32 that is quickly degraded by FstH under physiological growth conditions, and this degradation is transiently inhibited during heat stress. Degradation of 32 requires the activity of the DnaK chaperone system (Tomoyasu et al. 1995): binding of DnaK and its co-chaperone DnaJ induces conformational changes within the 32 polypeptide that allow its recognition by FtsH (Rodriguez et al. 2008). While cytoplasmic AAA+ proteases such as ClpAP, ClpXP and HslUV also contribute to the degradation of 32 , it is preferentially recognized and degraded by the membrane-anchored FtsH(Herman, Thevenet et al. 1995; Tomoyasu, Gamer et al. 1995).
Lon: degradation of misfolded proteins
Lon, a second member of a bacterial one-component AAA+ proteases family, plays a pivotal role in the removal of premature translational termination products and proteins containing non-natural amino acids (Shineberg & Zipser 1973; Gottesman & Zipser 1978; Chung & Goldberg 1981; Rotanova et al. 2004; Tsilibaris et al. 2006)(Rosen et al. 2002), and is up-regulated in bacteria under numerous stress conditions (Goff et al. 1984; Goff & Goldberg 1985; Van Melderen & Aertsen 2009). Lon AAA+ protease consists of N-domain that participates in substrate recognition (Roudiak & Shrader 1998; Melnikov et al. 2008; Chir et al. 2009), a central AAA+ module, and a C-terminal peptidase domain. Lon subunits assemble into a hexamer, and substrates that are recognized, are unfolded in an ATP-dependent manner, and translocated through the axial pore in the AAA+ ring into the internal peptidase chamber for degradation (Botos et al. 2004; Park et al. 2006; Sauer & Baker 2011). Current studies demonstrate that hexamers of E. coli Lon assemble into a dodecamer that displays different enzymatic properties, and the hexamer-dodecamer equilibrium appears to play a role in controlling Lon activity in cells (Vieux et al. 2013) . In E.coli, Lon is responsible for ∼50% of the turnover of proteins (Kowit & Goldberg 1977). The mechanism by which Lon recognizes diverse substrates involves synergistic recognition of multiple signals in unfolded polypeptides, and significant variation is allowed in sequences that mediate this recognition (Gur & Sauer 2008).
Non-proteolytic activity of AAA+ proteins
Disassembly of protein aggregates and stable protein complexes
In addition to proteolytic functions, AAA+ unfoldases also function in disassembly reactions, and display chaperone or remodeling functions in the absence of their peptidase partners (Sauer & Baker 2011). During environmental stress, proteolytic systems not always can cope with the accumulation of unfolded proteins leading to accumulation of protein aggregates. For example, around 3% of the total cellular proteins aggregate after heat shock at 45°C) (Gragerov et al. 1992; Laskowska et al. 1996; Winkler et al. 2010), and such high levels of protein aggregation can be toxic to the bacterial cell (Maisonneuve et al. 2008). AAA + proteins can act as chaperones to rescue the proteins contained the aggregates by resolubilizing and refolding them (Goloubinoff et al. 1999; Motohashi et al. 1999; Zolkiewski 1999). These proprieties of AAA+ proteins are best illustrated by the role of ClpB AAA+ chaperone in the development of thermotolerance (the ability of microorganisms to transiently survive otherwise lethal heat shock after acclimation by sublethal heat-treatment)(Squires et al. 1991; Weibezahn et al. 2004). The ability of ClpB to resolve protein aggregates requires its cooperation with the DnaK chaperone system (Glover & Lindquist 1998). Initially, DnaK binds of aggregated proteins, thus restricting access of other chaperones and proteases. Aggregated polypeptides are then transferred to the ClpB, probably by direct contact between DnaK and ClpB that is mediated by the unique M-domain of ClpB, not present in other AAA+ family members (Haslberger et al. 2007; Haslberger et al. 2008). ClpB uses energy from APT hydrolysis to thread large aggregates through its central pore, thus breaking aggregates into smaller fragments that are subsequently refolded by DnaK. The process of assist disaggregation by the DnaK/ClpB is facilitated by small heat shock proteins (sHsps) that binds to misfolded proteins and alters the process of aggregation (Haslbeck et al. 2005) .
The unique capacity of the AAA+ protein family to act on protein aggregates is not limited to ClpB. Other AAA+ chaperones, such as ClpA, and ClpC were shown to possess a disaggregation activity in vitro (Dougan, Reid et al. 2002b; Schlothauer et al. 2003) . Several recent studies suggest that in Bacillus subtilis the ClpC/ClpP and ClpE/ClpP AAA+ proteases are recruited to stress-induced protein aggregates in vivo, and contribute to protein disaggregation (Kruger et al. 2000; Kock et al. 2004; Miethke et al. 2006; Kirstein et al. 2008). ClpL, another member of the AAA+ protein family, is present in Gram positive bacteria, and has been implicated in thermotolerance development (Suokko et al. 2008; Frees, Brondsted et al. 2013).
In addition to destabilizing protein aggregates in bacterial cells, AAA+ proteins also function as disassemblers of stable protein complexes. One example of such activity is ClpX-mediated destabilization of DNA-bound strand-transfer complex (STC) of bacterial virus Mu that inhibits bacterial DNA replication (Surette et al. 1987; Mizuuchi et al. 1995). The destabilization of the STC by ClpX is independent of ClpP, and requires ATP hydrolysis (Levchenko et al. 1995; Levchenko et al. 1997; Burton et al. 2001). The mechanism of protein destabilization involves binding and unfoding of viral MuA subunits by ClpX , that leads to the release of the MuA monomers from the STC (Burton & Baker 2003).
Enzyme activation by AAA+ proteins
Another interesting function of bacterial AAA+ proteins is best illustrated by the activity of AAA+ protein homologues CbbQ and CbbO, expressed in chemoautotrophic proteobacteria. CbbQ/CbbO form a complex, CbbQO, that acts as an activator of the bacterial photosynthetic CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) (Badger & Bek 2008). CbbQO binds to inhibited rubisco complexes via the acidic surface residue of the rubisco large subunit using the binding site located on CbbO, while CbbQ hexamer also interact with rubisco directly, possibly via the C terminus of the large subunit. Binding of CbbQO to rubisco results in a stimulation of ATPase activity, and provides the energy necessary to remodel the inhibited rubisco active site to allow release of the inhibitor (Tsai et al. 2015).
Conclusions
There has been a major progress in understanding the structure and mechanism of action of bacterial AAA+ proteins. However, further research is needed to better understand multiple aspects of AAA+ mediated processes. Numerous questions remain unanswered, such as is how conformational changes induced by substrate binding and ATP hydrolysis contribute to substrate unfolding, translocation and remodelling; what factors determine the fate of partially unfolded proteins that can be either released by the AAA+ proteases or degraded; what is a full repertoire of degrons and adaptor proteins recognized by the different bacterial AAA+. Answering these questions will move forward our understanding of AAA+ protein function.