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Regulation and quality control by Lon-dependent proteolysis
Research in Microbiology 160 (2009) 645e651 www.elsevier.com/locate/resmic
Regulation and quality control by Lon-dependent proteolysis Laurence Van Melderen a,*, Abram Aertsen a,b a
Ge´ne´tique et Physiologie Bacte´rienne, Universite´ Libre de Bruxelles, Faculte´ des Sciences, IBMM-DBM, 12 Rue des Professeurs Jeneer et Brachet, B-6041 Gosselies, Belgium b Center for Food and Microbial Technology, Department of Microbial and Molecular Systems (M2S), Faculty of Bioscience Engineering, K.U.Leuven, Kasteelpark Arenberg 22, B-3001 Leuven, Belgium Received 24 July 2009; accepted 20 August 2009 Available online 20 September 2009
Abstract After their first discovery in Escherichia coli, Lon homologues were found to be widely distributed among prokaryotes to eukaryotes. The ATP-dependent Lon protease belongs to the AAAþ (ATPases associated with a variety of cellular activities) superfamily, and is involved in both general quality control by degrading abnormal proteins and in the specific control of several regulatory proteins. As such, this enzyme has a pivotal role in quality control and cellular physiology. This review focuses on mechanisms of degradation both from the protease and substrate points of view, and discusses the role of Lon in global regulation, stress response and virulence. Ó 2009 Elsevier Masson SAS. All rights reserved. Keywords: Lon protease; Gene expression regulation; Stress response
1. General introduction Like many proteases, Lon belongs to the heat shock regulon of bacteria, which generally depends on chaperones and proteases to refold or degrade aberrant proteins, respectively. ATP-dependent proteases, like Lon, are also involved in the targeted and conditional degradation of regulatory proteins, thereby adding a level of posttranslational control to the tight regulation of these proteins. Although only a minority of proteins are subjected to this type of regulation in Escherichia coli, this process is essential for fine-tuning the proper and timely amount of key proteins in response to the environment. In this review, we will focus on recent advances in Lon structure and substrate recognition mechanisms, as well as its involvement in global regulatory pathways and virulence.
* Corresponding author. Tel.: þ32 2 650 97 78; fax: þ32 650 97 70. E-mail address: [email protected] (L. Van Melderen). 0923-2508/$ - see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2009.08.021
2. Structure, biochemistry and mechanism of degradation by the Lon protease 2.1. From the protease standpoint Lon is an ATP-dependent protease belonging to the AAAþ (ATPases associated with a variety of cellular activities) superfamily of enzymes. Members of this family share a highly conserved ATPase module that conveys the free energy released by ATP hydrolysis into molecular remodelling events [58,60]. This allows these proteins to perform a wide range of different cellular functions, ranging from protein unfolding and microtubule severing over membrane fusion to DNA replication and recombination. Because of this versatility, AAAþ proteins are well represented in eukaryotes, prokaryotes, archaea and viruses [58]. Obviously, the exact cellular activity of AAAþ proteins is determined by the functional partner to which the ATPase domain is linked. In the case of Lon, the central ATPase (AAAþ) domain is covalently fused to a proteolytic domain at the C-terminus. It should be noted, however, that for most ATP-dependent proteases, the ATPase and protease domains
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are not linked in a single polypeptide, but associate noncovalently. The proteolytic site of Lon is composed of a SerLys dyad, sharing no relatedness to the classical catalytic Ser-His-Asp triad of serine proteases [6,53]. In addition to these catalytic domains, Lon proteins of bacterial and eukaryotic origin (LonA subfamily) possess a large N-terminal domain which displays protein binding ability and is believed to be involved in substrate recognition and enzymatic functionality [46]. On the other hand, archaeal proteases (LonB subfamily) harbor a transmembrane domain inserted into the ATPase region that anchors the protein to the cytoplasmic side of the membrane [52]. While the Lon protease was first discovered almost 30 years ago in E. coli [62], attempts to crystallize the entire enzyme have failed, and structural insights largely stem from electron microscopy and crystallization of individual fragments and subdomains [6,46,49]. Although purification methods and cofactors seem to drastically affect the oligomeric state of the enzyme, it is currently suggested that E. coli Lon oligomerizes into a hexameric ring [49], which is consistent with the assembly of most AAAþ members. This conformation creates a ring-shaped structure with a central cavity termed the proteolytic chamber, with the proteolytic active sites being located inside this cavity. Numerous studies both on Lon and other ATP-dependent proteases have contributed to the elaboration of a general model for the mechanism of substrate degradation (for review, [36]). The Lon N-terminal domain might be involved in substrate recognition [35]. Unfolding of the substrate is mediated by the central ATPase domain which allows translocation of the polypeptide in a thread-like manner towards the proteolytic chamber. Peptide bond cleavage preferentially occurs close to hydrophobic residues, although multiple and discontinuous interactions with substrate residues in the vicinity of the scissile site have also been observed [50]. A Lon mutant for the proteolytic site is still able to unfold and sequester substrates, showing that ATP-dependent unfolding is coupled with movement of the substrate into the proteolytic chamber [69]. Thus, unfolding and translocation are powered by ATP hydrolysis and result in processive degradation of the substrate [36]. 2.2. From the substrate standpoint: degradation rules Since Lon is responsible for w50% of the turnover of abnormal proteins in E. coli, it was proposed early on that Lon recognizes patches of hydrophobic amino acids that are normally buried in properly folded substrates (for review, [44]). Studies of several Lon substrates that are naturally partially unfolded, such as l N and CcdA antitoxin, confirmed this hypothesis [43,71]. Unfolding in the case of the Yersinia RovA transcriptional regulator has been recently demonstrated as being responsible for Lon degradation. RovA is a temperaturesensing protein that loses its DNA binding property at 37 C, thereby becoming susceptible to Lon [27]. Temperature dependence for Lon degradation is also observed for the HTS enzyme (homoserine trans-succinylase), the first enzyme in
the methionine biosynthesis pathway [5]. However, there are exceptions to this rule, since all unfolded proteins are not necessarily good substrates [26]. Subsequent studies on UmuD, HemA and SoxS identified an N-terminal degradation tag rich in non-polar amino acids as being essential for Lon degradation [20,56,73], while the C-terminal end of SulA appeared important for Lon recognition [30]. Addition of the 21 amino acid ‘degradation’ tag of SoxS to the otherwise stable GFP protein targeted it for Lon-dependent proteolysis, further demonstrating that this tag is sufficient for Lon recognition [56]. Similarly, addition of an unfolded b-galactosidase variant that was proven to be a good Lon substrate to GFP and to the N-terminal domain of the l CI repressor targeted these natively folded proteins to degradation [26]. Interestingly, addition of this artificial degradation tag to the N- or C-terminal part or its insertion into an unfolded but Lon-resistant protein targeted all these variants to degradation. The nature of these degradation tags seems to vary. In the case of the unfolded b-galactosidase fragment as well as the HemA and UmuD tag, non-polar (preferentially aromatic) amino acids seem to be the key determinants of Lon-dependent degradation [20,26,73]. A variant to this rule is observed with the SoxS tag, which contains a subregion of polar amino acids that is important for Lon degradation [56]. E. coli Lon also degrades SsrA-tagged proteins, although less efficiently than ClpXP and ClpAP [10,37]. It is, however, not clear whether degradation requires partially unfolded SsrA-tagged proteins [10,37]. In Mycoplasma, which is lacking the ClpAP, ClpXP and ClpYQ proteases, Lon degrades SsrA-tagged proteins, although it should be noted that the Mycoplasma SsrA tag is rich in aromatic amino acids. As a result of this apparent coevolution of Mycoplasma SsrA tag and Lon, Mycoplasma lon has become unable to complement an E. coli lon mutation [25]. Another key determinant in Lon-dependent proteolysis is the interaction between the substrates and their ligands. In most of the cases reported so far, ligands protect the substrates against Lon. For instance, the CcdA antitoxin is stabilized upon interaction with the CcdB toxin both in vivo and in vitro [68,71]. Another example concerns the E. coli SoxS transcriptional factor that regulates the superoxide regulon: addition of Sox- box DNA or RNA polymerase partially protects SoxS from Lon degradation in an in vitro assay [55]. The case of the quorum sensing transcriptional regulator TraR of Agrobacterium tumefaciens also highlights the importance of ligand interaction: TraR attains its native conformation and is resistant to Lon-dependent proteolysis upon interaction with its cognate homoserine lactone [77,78]. 3. Biological functions of Lon Despite the fact that Lon activity is involved in the regulation of numerous pathways, it is not an essential enzyme under ‘normal’ growth conditions in many bacterial species. E. coli K-12 lon mutants are viable in se, although they are sensitive to DNA damaging agents and accumulate abnormal proteins (for review, [23]). Lon mutant colonies also present
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a typical mucoid phenotype. This is a consequence of capsular polysaccharide overproduction due to the stabilization of the transcriptional activator RcsA (for a recent review, [40]). Lon also controls toxineantitoxin systems which are notably involved in plasmid maintenance (for a recent review, [70]). Lon is also involved in cell cycle and differentiation regulation in various species; e.g. Lon regulates cell cycle progression in Caulobacter crescentus as well as sporulation in Myxococcus xanthus and Bacillus subtilis (lonD and lonA respectively, as both species contain 2 lon genes) (for a recent review, [67]). Note than one of the lon genes of M. xanthus is essential for viability (lonV) [65]. The B. subtilus LonA protease was recently shown to co-localize with the nucleoid during normal growth [57]. Interestingly, E. coli Lon has been shown to have DNA binding activity and that DNA stimulates its ATPase activity in vitro [8,9]. However, it remains to be determined whether this localization is general for bacterial Lon proteases and whether it reflects a functional association or could affect substrate specificity. Thus, Lon controls a plethora of regulatory networks. Several recently reported examples are detailed below. 3.1. Global regulation and group behavior 3.1.1. Bacterial communication Quorum sensing (QS) is a mechanism by which bacteria sense the size of their population and synchronize group behavior accordingly (for review, [74]). QS relies on intercellular communication using signal molecules and signal transduction systems to regulate gene expression. The nature of the signal molecules and of the transduction systems varies among species. In Gram-negative bacteria, the most common signal molecules are homoserine lactones (AHLs) that are synthesized by LuxI-type enzymes. At high cell densities, the AHL concentration surpasses a certain threshold, AHLs bind to LuxR-type transcriptional regulators and induce a positive feedback. QS regulates a large number of genes, e.g. more than 300 genes belong to the Pseudomonas aeruginosa QS global regulatory network (over 6% of the genome) (for review, [54]). Recent studies have involved Lon-dependent proteolysis in QS regulation of pseudomonads [4,64]. lon mutants produce more AHL than their corresponding wildtype cells, showing that Lon negatively regulates QS. In P. aeruginosa, the major Lon target appears to be one of the LuxI-type AHL synthases (2 QS systems are present in this species) [64], while the LuxR-type of transcriptional regulator appears to be controlled by Lon in Pseudomonas putida [4]. It remains to be determined, however, whether the Lon effect is direct or not. 3.1.2. Motility and biofilm formation Bacteria often form sessile multicellular communities associated on a surface that are called biofilms (for reviews, [15,48]). This lifestyle protects them against harsh environmental conditions such as predators and antibiotics. While being sedentary is advantageous, it sometimes is necessary to move and colonize new niches. Bacteria have evolved
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different types of molecular machines to enable movement on different surface types. They notably swim using flagellar structures. Flagella synthesis in E. coli and related species relies on the master activator FlhDC that positively regulates expression of the class 2 genes of the flagellar regulon. Expression of the FliA factor (Sigma28) regulates the expression of class 3 genes, and is under the control of FlhDC. Interestingly, both regulators are controlled by ATP-dependent proteolysis. FlhDC from Salmonella typhimurium and Proteus mirabilis are unstable and degraded by ClpXP and Lon respectively [11,66], suggesting that E. coli FlhDC also has this property. E. coli FliA appears to be conditionally unstable [3]. It is protected from degradation by Lon by interacting with its anti-sigma factor FlgM. FlgM is secreted upon assembly of the hook basal body, together with the flagellin subunits, leaving FliA free to interact with the RNAP core and activate the expression of class 3 genes. ATP-dependent proteolysis thus controls flagella synthesis by shutting down expression of class 2 and 3 genes. Lon-dependent proteolysis is involved in the control of other types of movement such as swarming and twitching motility. Swarming is a group behavior that requires a threshold quorum to be initiated and relies on flagellar structures. Lon is involved in the control of swarming differentiation in various bacterial species through FlhDC proteolysis [12,42,59,61]. Twitching motility enables bacteria to crawl on a surface and requires type IV pili. It is involved in early steps of biofilm formation. In P. aeruginosa, in addition to being defective in swarming, Lon mutants were shown to be impaired in twitching motility (although type IV pili are still expressed) and in biofilm formation [42], although the underlying molecular mechanisms are still unknown. 3.2. Stress response 3.2.1. The SOS system The bacterial SOS response is an essential DNA damage response that can be triggered by a variety of factors. The SOS regulon is mainly controlled by RecA and LexA, and encodes functions to repair the incurred DNA damage and restore the replication fork (for reviews, [2,16]). However, the SOS response also controls the lateral spread of mobile genetic elements, mutagenesis and cell division, and some of these phenomena involve the action of the Lon protease. Probably the best-known example of Lon control in the context of the SOS response comprises the degradation of SulA [47]. The sulA gene is under direct control of the LexA repressor, and is concomitantly induced upon DNA damage. By binding to the FtsZ cell division protein, SulA acts as a physical inhibitor of septum formation and, as such, delays cell division for the time needed to repair DNA damages. At the posttranslational level, SulA is rapidly degraded by Lon in order to resume cell division when the damages are repaired and sulA expression is shut down. Because of the severe impact of SulA on cell division, its proteolysis is crucial and other ATP-dependent proteases, such as ClpYQ (HslUV), take over SulA degradation under conditions where Lon activity is
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absent [76]. Nevertheless, Lon remains the major degrader of SulA, and lon mutants suffer filamentation and cell death upon SOS induction [1,24]. A second aspect of the SOS response in which Lon plays an essential function is SOS-induced mutagenesis, which results from the translesion bypass of error-prone polymerases. In case of severe DNA damage, passage of the replicative DNA polymerase (Pol) III can become blocked by DNA lesions. In this case, UmuC and UmuD proteins, both members of the SOS regulon, will assemble into a novel DNA polymerase (Pol V) capable of traversing the lesion and allowing normal DNA replication to resume [22]. However, this translesion bypass intrinsically requires decreased fidelity and therefore inevitably results in the incorporation of errors. Obviously, this error-prone DNA replication should be tightly controlled at different levels in order to safeguard the genetic blueprint. At the transcriptional level, the umuDC operon is only expressed in the last stage of the SOS response, in order to resolve those lesions that could not be mended with the error-free DNA repair. In addition, at the posttranslational level, cellular levels of both UmuD and UmuC are kept very low under normal conditions as they are rapidly degraded by the Lon protease. Only when RecA is activated by DNA damage, the autoproteolytic removal of the 24 N-terminal amino acids of UmuD is triggered, and the resulting UmuD0 becomes relatively resistant to Lon degradation. In turn, UmuC becomes stabilized by association with UmuD02 , which ultimately leads to the assembly of the mutagenically active Pol V complex (UmuD02 C) [21]. A more recently discovered case of Lon-mediated control of the activity of an SOS induced protein deals with the SymE protein. SymE (YjiW) is a recently discovered toxin whose expression was shown to be repressed by LexA [17,32]. It constitutes an endoribonuclease that cleaves mRNA and is believed to play a role in recycling RNAs that might have been damaged by the agents that cause the SOS response [32]. Interestingly, aside from its transcriptional control by LexA, stability and translation of the symE mRNA are controlled by a cis-encoded antisense RNA (SymR), which acts as an antitoxin. In addition to these two layers of regulation, the SymE protein itself is a Lon target, and its degradation further prevents the potential deleterious accumulation of this toxin [32]. Interestingly, while this posttranslational control of SymE typically resembles that of other potentially harmful SOS functions (such as SulA and UmuDC described above), it is in sharp contrast with the usual control of toxineantitoxin systems where, in fact, the toxin tends to be more stable than the antitoxin (for a recent review, [70]). Finally, Lon was also shown to be involved in the paradoxical survival of E. coli to high quinolone concentrations [41]. Quinolone antibiotics target the bacterial DNA gyrase and thereby result in the generation of double-stranded DNA breaks. Generation of these lesions leads to SOS induction. Although exposure to quinolones is lethal, a counterintuitive yet distinct increase in bacterial survival has been historically noted at high quinolone concentrations [14]. Surprisingly, this paradoxical survival is abolished in the absence of the Lon
protease. Furthermore, this phenomenon was demonstrated to be independent of effects on SulA and to be mediated specifically by the protease rather than the ATPase function of Lon [41]. However, how exactly Lon is molecularly involved in this enigmatic cellular behavior remains to be elucidated. 3.2.2. Acid tolerance Acid resistance is the ability to sustain very low pH conditions. Due to its lifestyle in the mammalian digestive tract, E. coli has a remarkable ability to adapt to pH stress. This capacity enables E. coli to survive gastric acidity and volatile fatty acids produced by fermentation in the intestine. Glutamate-dependent acid resistance is one of the mechanisms used by E. coli and its relatives (for review, [18]). In this pathway, GadE is the key transcriptional regulator. It positively controls acid resistance gene expression such as that of gadA and gadB, which encode glutamate decarboxylase isoenzymes, and gadC, which encodes a putative glutamate g-aminobutyric anti-porter [39]. Amino acid decarboxylase systems are thought to confer acid resistance by consuming intracellular protons. Under acid stress, glutamate is taken up by the cell using the GadC anti-porter and decarboxylation of glutamate by GadA and GadB produces g-aminobutyric acid that will expel through GadC. This results in alkalinization of the cytoplasm. Interestingly, these genes (except of gadBC ) and others involved in acid resistance are located in a cluster of sS-dependent genes called the ‘fitness island for acid adaptation’ [29]. Expression of these genes, including gadE, is strongly induced in stationary phase in a sS-dependent manner, providing a molecular explanation for the acid resistance phenotype displayed by stationary phase cells [75]. Thus, regulation of the acid resistance response is quite complex: the master regulator RpoS activates the expression of the secondary regulator GadE which in turn activates acid resistance genes expression. Recently, the group of Hengge showed that the key acid resistance genes (notably gadE, gadA and gadBC ) are upregulated in a lon mutant, raising the possibility that Lon could control the amount of the GadE secondary regulator [28]. Indeed, the authors showed that Lon has no effect on RpoS activity and degrades GadE constitutively. Lon-dependent GadE degradation is involved in acid resistance response shutoff, since GadE positively activates its own expression. In addition, the authors also identified other RpoS-controlled genes the expression of which is altered in a lon mutant, indicating that Lon-dependent degradation of secondary regulators might be widespread within the RpoS regulon. 3.2.3. Nutritional stress Under normal growth conditions, the majority of E. coli proteins are extremely stable (for a review, [44]). Around 75% of total proteins are estimated to present a half-life greater than 25 h. However, the overall degradation rate increases severalfold under starvation conditions, such as amino acid, nitrogen or carbon deprivation. Increased degradation in response to amino acid or nitrogen starvation is linked to the stringent response and the production of ppGpp. Recently, Kornberg
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and co-workers showed that during an amino acid downshift, the pool of free ribosomal proteins is degraded by Lon in a polyphosphate-dependent manner [33]. ppGpp is needed for accumulation of polyphosphate, which in turn participates in the formation of a complex between Lon and specific ribosomal proteins. Degradation will release free amino acids that are, in turn, used for synthesis of biosynthetic enzymes. Proteolysis and activation of biosynthetic gene expression allow the cell to adapt to nutrient-poor conditions. Few other examples are reported in the literature, such as Lon- and ClpAP-dependent degradation of L-glutamate dehydrogenase in carbon or nitrogen-starved cells which also appears to be ppGpp-dependent [45]. Proteolysis also controls heme biosynthesis in Salmonella typhimurium [72]. Under a normal heme level, HemA (first enzyme in the heme biosynthesis pathway) is degraded in a Lon- and ClpAPdependent fashion, while it is stable in heme-limiting conditions, making the cell ready for new heme synthesis. 3.3. Virulence and pathogenicity lon mutations can confer a reduced pathogenicity/virulence phenotype. This is the case for several Gram-negative species such as Salmonella enterica [63], Brucella abortus [51], Campylobacter jeujeni [12], A. tumefaciens [61] and Pseudomonas syringae [34]. Gram-negative pathogens often use type three secretion systems (TTSSs) to inject effectors (virulence factors) into eukaryotic cells. TTSSs are intricate molecular machines shaped as a needle and spanning the cell envelope (for review, [13]). Lon regulates TTSS expression in various bacterial species by modulating the amount of key transcriptional regulators that control TTSS gene expression (for recent reviews, [7,67]). Lon notably upregulates TTSS in Yersinia pestis by degrading the RovA negative regulator at 37 C, as mentioned above [31] as well as P. syringae TTSS by a yet unknown mechanism [34]. Furthermore, Lon controls effector secretion through TTSS by degrading the excess of effectors that were not secreted [19,38]. 4. Concluding remarks Recent advances regarding Lon structure as well as substrate characteristics have led to a better understanding of how this protease specifically selects proteins to degrade among a pool of non-substrate proteins. Lon recognizes sequences rich in hydrophobic/aromatic residues whether they are located in an N- or C-terminal degradation tag or exposed in naturally unfolded substrates and in abnormal proteins. Adaptors might be required for some substrate recognition, like Poly-P in the case of ribosomal subunits. Whether other types of adaptators such as small proteins are involved in some cases remains to be shown. Structure-function studies might still be needed to decipher the precise molecular functioning of this family of proteases. Regarding the biological roles of Lon, although multiple physiological processes have been found to be under Lon control, it is likely that not yet determined pathways in various
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bacteria are regulated at one or several steps by Lon-dependent proteolysis. Recent microarray analysis led to the discovery of the involvement of Lon in acid tolerance and flagellum synthesis [3,28]. Such global approaches will further reveal the true versatility of the Lon protease and its central role in different aspects of cellular physiology. Acknowledgements AA acknowledges a postdoctoral fellowship and a mobility grant of the Research Foundation e Flanders (FWO). References [1] Aertsen, A., Michiels, C.W. (2005) SulA-dependent hypersensitivity to high pressure and hyperfilamentation after high-pressure treatment of Escherichia coli lon mutants. Res. Microbiol. 156, 233e237. [2] Aertsen, A., Michiels, C.W. (2006) Upstream of the SOS response: figure out the trigger. Trends Microbiol. 14, 421e423. [3] Barembruch, C., Hengge, R. (2007) Cellular levels and activity of the flagellar sigma factor FliA of Escherichia coli are controlled by FlgMmodulated proteolysis. Mol. Microbiol. 65, 76e89. [4] Bertani, I., Rampioni, G., Leoni, L., Venturi, V. (2007) The Pseudomonas putida Lon protease is involved in N-acyl homoserine lactone quorum sensing regulation. BMC Microbiol. 7, 71. [5] Biran, D., Gur, E., Gollan, L., Ron, E.Z. (2000) Control of methionine biosynthesis in Escherichia coli by proteolysis. Mol. Microbiol. 37, 1436e1443. [6] Botos, I., Melnikov, E.E., Cherry, S., Khalatova, A.G., Rasulova, F.S., Tropea, J.E., Maurizi, M.R., Rotanova, T.V., Gustchina, A., Wlodawer, A. (2004) Crystal structure of the AAAþ alpha domain of E. coli Lon protease at 1.9A resolution. J. Struct. Biol. 146, 113e122. [7] Butler, S.M., Festa, R.A., Pearce, M.J., Darwin, K.H. (2006) Self-compartmentalized bacterial proteases and pathogenesis. Mol. Microbiol. 60, 553e562. [8] Charette, M.F., Henderson, G.W., Doane, L.L., Markovitz, A. (1984) DNA-stimulated ATPase activity on the lon (CapR) protein. J. Bacteriol. 158, 195e201. [9] Charette, M.F., Henderson, G.W., Markovitz, A. (1981) ATP hydrolysisdependent protease activity of the lon (capR) protein of Escherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A 78, 4728e4732. [10] Choy, J.S., Aung, L.L., Karzai, A.W. (2007) Lon protease degrades transfer-messenger RNA-tagged proteins. J. Bacteriol. 189, 6564e6571. [11] Claret, L., Hughes, C. (2000) Rapid turnover of FlhD and FlhC, the flagellar regulon transcriptional activator proteins, during Proteus swarming. J. Bacteriol. 182, 833e836. [12] Cohn, M.T., Ingmer, H., Mulholland, F., Jorgensen, K., Wells, J.M., Brondsted, L. (2007) Contribution of conserved ATP-dependent proteases of Campylobacter jejuni to stress tolerance and virulence. Appl. Environ. Microbiol. 73, 7803e7813. [13] Cornelis, G.R. (2006) The type III secretion injectisome. Nat. Rev. Microbiol. 4, 811e825. [14] Crumplin, G.C., Smith, J.T. (1975) Nalidixic acid: an antibacterial paradox. Antimicrob. Agents Chemother. 8, 251e261. [15] Davey, M.E., O’Toole, G.A. (2000) Microbial biofilms: from ecology to molecular genetics. Microbiol. Mol. Biol. Rev. 64, 847e867. [16] Erill, I., Campoy, S., Barbe, J. (2007) Aeons of distress: an evolutionary perspective on the bacterial SOS response. FEMS Microbiol. Rev. 31, 637e656. [17] Fernandez De Henestrosa, A.R., Ogi, T., Aoyagi, S., Chafin, D., Hayes, J.J., Ohmori, H., Woodgate, R. (2000) Identification of additional genes belonging to the LexA regulon in Escherichia coli. Mol. Microbiol. 35, 1560e1572. [18] Foster, J.W. (2004) Escherichia coli acid resistance: tales of an amateur acidophile. Nat. Rev. Microbiol. 2, 898e907.
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[40] Majdalani, N., Gottesman, S. (2007) Genetic dissection of signaling through the Rcs phosphorelay. Meth. Enzymol. 423, 349e362. [41] Malik, M., Capecci, J., Drlica, K. (2009) Lon protease is essential for paradoxical survival of Escherichia coli exposed to high concentrations of quinolone. Antimicrob. Agents Chemother. 53, 3103e3105. [42] Marr, A.K., Overhage, J., Bains, M., Hancock, R.E. (2007) The Lon protease of Pseudomonas aeruginosa is induced by aminoglycosides and is involved in biofilm formation and motility. Microbiology 153, 474e482. [43] Maurizi, M.R. (1987) Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli. J. Biol. Chem. 262, 2696e2703. [44] Maurizi, M.R. (1992) Proteases and protein degradation in Escherichia coli. Experientia 48, 178e201. [45] Maurizi, M.R., Rasulova, F. (2002) Degradation of L-glutamate dehydrogenase from Escherichia coli: allosteric regulation of enzyme stability. Arch. Biochem. Biophys. 397, 206e216. [46] Melnikov, E.E., Andrianova, A.G., Morozkin, A.D., Stepnov, A.A., Makhovskaya, O.V., Botos, I., Gustchina, A., Wlodawer, A., Rotanova, T.V. (2008) Limited proteolysis of E. coli ATP-dependent protease Lon e a unified view of the subunit architecture and characterization of isolated enzyme fragments. Acta Biochim. Pol. 55, 281e296. [47] Mizusawa, S., Gottesman, S. (1983) Protein degradation in Escherichia coli: the lon gene controls the stability of sulA protein. Proc. Natl. Acad. Sci. U.S.A. 80, 358e362. [48] Moons, P., Michiels, C.W., Aertsen, A. (2009) Bacterial interactions in biofilms. Crit. Rev. Microbiol. 35, 157e168. [49] Park, S.C., Jia, B., Yang, J.K., Van, D.L., Shao, Y.G., Han, S.W., Jeon, Y.J., Chung, C.H., Cheong, G.W. (2006) Oligomeric structure of the ATPdependent protease La (Lon) of Escherichia coli. Mol. Cells 21, 129e134. [50] Patterson-Ward, J., Tedesco, J., Hudak, J., Fishovitz, J., Becker, J., Frase, H., McNamara, K., Lee, I. (2009) Utilization of synthetic peptides to evaluate the importance of substrate interaction at the proteolytic site of Escherichia coli Lon protease. Biochim. Biophys. Acta. [51] Robertson, G.T., Kovach, M.E., Allen, C.A., Ficht, T.A., Roop 2nd, R.M. (2000) The Brucella abortus Lon functions as a generalized stress response protease and is required for wild-type virulence in BALB/c mice. Mol. Microbiol. 35, 577e588. [52] Rotanova, T.V., Botos, I., Melnikov, E.E., Rasulova, F., Gustchina, A., Maurizi, M.R., Wlodawer, A. (2006) Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains. Protein Sci. 15, 1815e1828. [53] Rotanova, T.V., Melnikov, E.E., Khalatova, A.G., Makhovskaya, O.V., Botos, I., Wlodawer, A., Gustchina, A. (2004) Classification of ATPdependent proteases Lon and comparison of the active sites of their proteolytic domains. Eur. J. Biochem. 271, 4865e4871. [54] Schuster, M., Greenberg, E.P. (2006) A network of networks: quorumsensing gene regulation in Pseudomonas aeruginosa. Int. J. Med. Microbiol. 296, 73e81. [55] Shah, I.M., Wolf Jr., R.E. (2006) Inhibition of Lon-dependent degradation of the Escherichia coli transcription activator SoxS by interaction with ‘soxbox’ DNA or RNA polymerase. Mol. Microbiol. 60, 199e208. [56] Shah, I.M., Wolf Jr., R.E. (2006) Sequence requirements for Londependent degradation of the Escherichia coli transcription activator SoxS: identification of the SoxS residues critical to proteolysis and specific inhibition of in vitro degradation by a peptide comprised of the N-terminal 21 amino acid residues. J. Mol. Biol. [57] Simmons, L.A., Grossman, A.D., Walker, G.C. (2008) Clp and Lon proteases occupy distinct subcellular positions in Bacillus subtilis. J. Bacteriol. 190, 6758e6768. [58] Snider, J., Thibault, G., Houry, W.A. (2008) The AAAþ superfamily of functionally diverse proteins. Genome Biol. 9, 216. [59] Stewart, B.J., Enos-Berlage, J.L., McCarter, L.L. (1997) The lonS gene regulates swarmer cell differentiation of Vibrio parahaemolyticus. J. Bacteriol. 179, 107e114. [60] Striebel, F., Kress, W., Weber-Ban, E. (2009) Controlled destruction: AAAþ ATPases in protein degradation from bacteria to eukaryotes. Curr. Opin. Struct. Biol. 19, 209e217.
L. Van Melderen, A. Aertsen / Research in Microbiology 160 (2009) 645e651 [61] Su, S., Stephens, B.B., Alexandre, G., Farrand, S.K. (2006) Lon protease of the alpha-proteobacterium Agrobacterium tumefaciens is required for normal growth, cellular morphology and full virulence. Microbiology 152, 1197e1207. [62] Swamy, K.H., Goldberg, A.L. (1981) E. coli contains eight soluble proteolytic activities, one being ATP dependent. Nature 292, 652e654. [63] Takaya, A., Suzuki, M., Matsui, H., Tomoyasu, T., Sashinami, H., Nakane, A., Yamamoto, T. (2003) Lon, a stress-induced ATP-dependent protease, is critically important for systemic Salmonella enterica serovar typhimurium infection of mice. Infect. Immun. 71, 690e696. [64] Takaya, A., Tabuchi, F., Tsuchiya, H., Isogai, E., Yamamoto, T. (2008) Negative regulation of quorum-sensing systems in Pseudomonas aeruginosa by ATP-dependent Lon protease. J. Bacteriol. 190, 4181e4188. [65] Tojo, N., Inouye, S., Komano, T. (1993) Cloning and nucleotide sequence of the Myxococcus xanthus lon gene: indispensability of lon for vegetative growth. J. Bacteriol. 175, 2271e2277. [66] Tomoyasu, T., Takaya, A., Isogai, E., Yamamoto, T. (2003) Turnover of FlhD and FlhC, master regulator proteins for Salmonella flagellum biogenesis, by the ATP-dependent ClpXP protease. Mol. Microbiol. 48, 443e452. [67] Tsilibaris, V., Maenhaut-Michel, G., Van Melderen, L. (2006) Biological roles of the Lon ATP-dependent protease. Res. Microbiol. 157, 701e713. [68] Van Melderen, L., Bernard, P., Couturier, M. (1994) Lon-dependent proteolysis of CcdA is the key control for activation of CcdB in plasmidfree segregant bacteria. Mol. Microbiol. 11, 1151e1157. [69] Van Melderen, L., Gottesman, S. (1999) Substrate sequestration by a proteolytically inactive Lon mutant. Proc. Natl. Acad. Sci. U.S.A. 96, 6064e6071. [70] Van Melderen, L., Saavedra De Bast, M. (2009) Bacterial toxineantitoxin systems: more than selfish entities? PLoS Genet. 5, e1000437.
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[71] Van Melderen, L., Thi, M.H., Lecchi, P., Gottesman, S., Couturier, M., Maurizi, M.R. (1996) ATP-dependent degradation of CcdA by Lon protease. Effects of secondary structure and heterologous subunit interactions. J. Biol. Chem. 271, 27730e27738. [72] Wang, L., Elliott, M., Elliott, T. (1999) Conditional stability of the HemA protein (glutamyl-tRNA reductase) regulates heme biosynthesis in Salmonella typhimurium. J. Bacteriol. 181, 1211e1219. [73] Wang, L., Wilson, S., Elliott, T. (1999) A mutant HemA protein with positive charge close to the N terminus is stabilized against hemeregulated proteolysis in Salmonella typhimurium. J. Bacteriol. 181, 6033e6041. [74] Waters, C.M., Bassler, B.L. (2005) Quorum sensing: cell-to-cell communication in bacteria. Annu. Rev. Cell Dev. Biol. 21, 319e346. [75] Weber, H., Polen, T., Heuveling, J., Wendisch, V.F., Hengge, R. (2005) Genome-wide analysis of the general stress response network in Escherichia coli: sigmaS-dependent genes, promoters, and sigma factor selectivity. J. Bacteriol. 187, 1591e1603. [76] Wu, W.F., Zhou, Y., Gottesman, S. (1999) Redundant in vivo proteolytic activities of Escherichia coli Lon and the ClpYQ (HslUV) protease. J. Bacteriol. 181, 3681e3687. [77] Zhu, J., Winans, S.C. (1999) Autoinducer binding by the quorum-sensing regulator TraR increases affinity for target promoters in vitro and decreases TraR turnover rates in whole cells. Proc. Natl. Acad. Sci. U.S.A. 96, 4832e4837. [78] Zhu, J., Winans, S.C. (2001) The quorum-sensing transcriptional regulator TraR requires its cognate signaling ligand for protein folding, protease resistance, and dimerization. Proc. Natl. Acad. Sci. U.S.A. 98, 1507e1512.
Regulation and quality control by Lon-dependent proteolysis Laurence Van Melderen a,*, Abram Aertsen a,b a
Ge´ne´tique et Physiologie Bacte´rienne, Universite´ Libre de Bruxelles, Faculte´ des Sciences, IBMM-DBM, 12 Rue des Professeurs Jeneer et Brachet, B-6041 Gosselies, Belgium b Center for Food and Microbial Technology, Department of Microbial and Molecular Systems (M2S), Faculty of Bioscience Engineering, K.U.Leuven, Kasteelpark Arenberg 22, B-3001 Leuven, Belgium Received 24 July 2009; accepted 20 August 2009 Available online 20 September 2009
Abstract After their first discovery in Escherichia coli, Lon homologues were found to be widely distributed among prokaryotes to eukaryotes. The ATP-dependent Lon protease belongs to the AAAþ (ATPases associated with a variety of cellular activities) superfamily, and is involved in both general quality control by degrading abnormal proteins and in the specific control of several regulatory proteins. As such, this enzyme has a pivotal role in quality control and cellular physiology. This review focuses on mechanisms of degradation both from the protease and substrate points of view, and discusses the role of Lon in global regulation, stress response and virulence. Ó 2009 Elsevier Masson SAS. All rights reserved. Keywords: Lon protease; Gene expression regulation; Stress response
1. General introduction Like many proteases, Lon belongs to the heat shock regulon of bacteria, which generally depends on chaperones and proteases to refold or degrade aberrant proteins, respectively. ATP-dependent proteases, like Lon, are also involved in the targeted and conditional degradation of regulatory proteins, thereby adding a level of posttranslational control to the tight regulation of these proteins. Although only a minority of proteins are subjected to this type of regulation in Escherichia coli, this process is essential for fine-tuning the proper and timely amount of key proteins in response to the environment. In this review, we will focus on recent advances in Lon structure and substrate recognition mechanisms, as well as its involvement in global regulatory pathways and virulence.
* Corresponding author. Tel.: þ32 2 650 97 78; fax: þ32 650 97 70. E-mail address: [email protected] (L. Van Melderen). 0923-2508/$ - see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2009.08.021
2. Structure, biochemistry and mechanism of degradation by the Lon protease 2.1. From the protease standpoint Lon is an ATP-dependent protease belonging to the AAAþ (ATPases associated with a variety of cellular activities) superfamily of enzymes. Members of this family share a highly conserved ATPase module that conveys the free energy released by ATP hydrolysis into molecular remodelling events [58,60]. This allows these proteins to perform a wide range of different cellular functions, ranging from protein unfolding and microtubule severing over membrane fusion to DNA replication and recombination. Because of this versatility, AAAþ proteins are well represented in eukaryotes, prokaryotes, archaea and viruses [58]. Obviously, the exact cellular activity of AAAþ proteins is determined by the functional partner to which the ATPase domain is linked. In the case of Lon, the central ATPase (AAAþ) domain is covalently fused to a proteolytic domain at the C-terminus. It should be noted, however, that for most ATP-dependent proteases, the ATPase and protease domains
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are not linked in a single polypeptide, but associate noncovalently. The proteolytic site of Lon is composed of a SerLys dyad, sharing no relatedness to the classical catalytic Ser-His-Asp triad of serine proteases [6,53]. In addition to these catalytic domains, Lon proteins of bacterial and eukaryotic origin (LonA subfamily) possess a large N-terminal domain which displays protein binding ability and is believed to be involved in substrate recognition and enzymatic functionality [46]. On the other hand, archaeal proteases (LonB subfamily) harbor a transmembrane domain inserted into the ATPase region that anchors the protein to the cytoplasmic side of the membrane [52]. While the Lon protease was first discovered almost 30 years ago in E. coli [62], attempts to crystallize the entire enzyme have failed, and structural insights largely stem from electron microscopy and crystallization of individual fragments and subdomains [6,46,49]. Although purification methods and cofactors seem to drastically affect the oligomeric state of the enzyme, it is currently suggested that E. coli Lon oligomerizes into a hexameric ring [49], which is consistent with the assembly of most AAAþ members. This conformation creates a ring-shaped structure with a central cavity termed the proteolytic chamber, with the proteolytic active sites being located inside this cavity. Numerous studies both on Lon and other ATP-dependent proteases have contributed to the elaboration of a general model for the mechanism of substrate degradation (for review, [36]). The Lon N-terminal domain might be involved in substrate recognition [35]. Unfolding of the substrate is mediated by the central ATPase domain which allows translocation of the polypeptide in a thread-like manner towards the proteolytic chamber. Peptide bond cleavage preferentially occurs close to hydrophobic residues, although multiple and discontinuous interactions with substrate residues in the vicinity of the scissile site have also been observed [50]. A Lon mutant for the proteolytic site is still able to unfold and sequester substrates, showing that ATP-dependent unfolding is coupled with movement of the substrate into the proteolytic chamber [69]. Thus, unfolding and translocation are powered by ATP hydrolysis and result in processive degradation of the substrate [36]. 2.2. From the substrate standpoint: degradation rules Since Lon is responsible for w50% of the turnover of abnormal proteins in E. coli, it was proposed early on that Lon recognizes patches of hydrophobic amino acids that are normally buried in properly folded substrates (for review, [44]). Studies of several Lon substrates that are naturally partially unfolded, such as l N and CcdA antitoxin, confirmed this hypothesis [43,71]. Unfolding in the case of the Yersinia RovA transcriptional regulator has been recently demonstrated as being responsible for Lon degradation. RovA is a temperaturesensing protein that loses its DNA binding property at 37 C, thereby becoming susceptible to Lon [27]. Temperature dependence for Lon degradation is also observed for the HTS enzyme (homoserine trans-succinylase), the first enzyme in
the methionine biosynthesis pathway [5]. However, there are exceptions to this rule, since all unfolded proteins are not necessarily good substrates [26]. Subsequent studies on UmuD, HemA and SoxS identified an N-terminal degradation tag rich in non-polar amino acids as being essential for Lon degradation [20,56,73], while the C-terminal end of SulA appeared important for Lon recognition [30]. Addition of the 21 amino acid ‘degradation’ tag of SoxS to the otherwise stable GFP protein targeted it for Lon-dependent proteolysis, further demonstrating that this tag is sufficient for Lon recognition [56]. Similarly, addition of an unfolded b-galactosidase variant that was proven to be a good Lon substrate to GFP and to the N-terminal domain of the l CI repressor targeted these natively folded proteins to degradation [26]. Interestingly, addition of this artificial degradation tag to the N- or C-terminal part or its insertion into an unfolded but Lon-resistant protein targeted all these variants to degradation. The nature of these degradation tags seems to vary. In the case of the unfolded b-galactosidase fragment as well as the HemA and UmuD tag, non-polar (preferentially aromatic) amino acids seem to be the key determinants of Lon-dependent degradation [20,26,73]. A variant to this rule is observed with the SoxS tag, which contains a subregion of polar amino acids that is important for Lon degradation [56]. E. coli Lon also degrades SsrA-tagged proteins, although less efficiently than ClpXP and ClpAP [10,37]. It is, however, not clear whether degradation requires partially unfolded SsrA-tagged proteins [10,37]. In Mycoplasma, which is lacking the ClpAP, ClpXP and ClpYQ proteases, Lon degrades SsrA-tagged proteins, although it should be noted that the Mycoplasma SsrA tag is rich in aromatic amino acids. As a result of this apparent coevolution of Mycoplasma SsrA tag and Lon, Mycoplasma lon has become unable to complement an E. coli lon mutation [25]. Another key determinant in Lon-dependent proteolysis is the interaction between the substrates and their ligands. In most of the cases reported so far, ligands protect the substrates against Lon. For instance, the CcdA antitoxin is stabilized upon interaction with the CcdB toxin both in vivo and in vitro [68,71]. Another example concerns the E. coli SoxS transcriptional factor that regulates the superoxide regulon: addition of Sox- box DNA or RNA polymerase partially protects SoxS from Lon degradation in an in vitro assay [55]. The case of the quorum sensing transcriptional regulator TraR of Agrobacterium tumefaciens also highlights the importance of ligand interaction: TraR attains its native conformation and is resistant to Lon-dependent proteolysis upon interaction with its cognate homoserine lactone [77,78]. 3. Biological functions of Lon Despite the fact that Lon activity is involved in the regulation of numerous pathways, it is not an essential enzyme under ‘normal’ growth conditions in many bacterial species. E. coli K-12 lon mutants are viable in se, although they are sensitive to DNA damaging agents and accumulate abnormal proteins (for review, [23]). Lon mutant colonies also present
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a typical mucoid phenotype. This is a consequence of capsular polysaccharide overproduction due to the stabilization of the transcriptional activator RcsA (for a recent review, [40]). Lon also controls toxineantitoxin systems which are notably involved in plasmid maintenance (for a recent review, [70]). Lon is also involved in cell cycle and differentiation regulation in various species; e.g. Lon regulates cell cycle progression in Caulobacter crescentus as well as sporulation in Myxococcus xanthus and Bacillus subtilis (lonD and lonA respectively, as both species contain 2 lon genes) (for a recent review, [67]). Note than one of the lon genes of M. xanthus is essential for viability (lonV) [65]. The B. subtilus LonA protease was recently shown to co-localize with the nucleoid during normal growth [57]. Interestingly, E. coli Lon has been shown to have DNA binding activity and that DNA stimulates its ATPase activity in vitro [8,9]. However, it remains to be determined whether this localization is general for bacterial Lon proteases and whether it reflects a functional association or could affect substrate specificity. Thus, Lon controls a plethora of regulatory networks. Several recently reported examples are detailed below. 3.1. Global regulation and group behavior 3.1.1. Bacterial communication Quorum sensing (QS) is a mechanism by which bacteria sense the size of their population and synchronize group behavior accordingly (for review, [74]). QS relies on intercellular communication using signal molecules and signal transduction systems to regulate gene expression. The nature of the signal molecules and of the transduction systems varies among species. In Gram-negative bacteria, the most common signal molecules are homoserine lactones (AHLs) that are synthesized by LuxI-type enzymes. At high cell densities, the AHL concentration surpasses a certain threshold, AHLs bind to LuxR-type transcriptional regulators and induce a positive feedback. QS regulates a large number of genes, e.g. more than 300 genes belong to the Pseudomonas aeruginosa QS global regulatory network (over 6% of the genome) (for review, [54]). Recent studies have involved Lon-dependent proteolysis in QS regulation of pseudomonads [4,64]. lon mutants produce more AHL than their corresponding wildtype cells, showing that Lon negatively regulates QS. In P. aeruginosa, the major Lon target appears to be one of the LuxI-type AHL synthases (2 QS systems are present in this species) [64], while the LuxR-type of transcriptional regulator appears to be controlled by Lon in Pseudomonas putida [4]. It remains to be determined, however, whether the Lon effect is direct or not. 3.1.2. Motility and biofilm formation Bacteria often form sessile multicellular communities associated on a surface that are called biofilms (for reviews, [15,48]). This lifestyle protects them against harsh environmental conditions such as predators and antibiotics. While being sedentary is advantageous, it sometimes is necessary to move and colonize new niches. Bacteria have evolved
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different types of molecular machines to enable movement on different surface types. They notably swim using flagellar structures. Flagella synthesis in E. coli and related species relies on the master activator FlhDC that positively regulates expression of the class 2 genes of the flagellar regulon. Expression of the FliA factor (Sigma28) regulates the expression of class 3 genes, and is under the control of FlhDC. Interestingly, both regulators are controlled by ATP-dependent proteolysis. FlhDC from Salmonella typhimurium and Proteus mirabilis are unstable and degraded by ClpXP and Lon respectively [11,66], suggesting that E. coli FlhDC also has this property. E. coli FliA appears to be conditionally unstable [3]. It is protected from degradation by Lon by interacting with its anti-sigma factor FlgM. FlgM is secreted upon assembly of the hook basal body, together with the flagellin subunits, leaving FliA free to interact with the RNAP core and activate the expression of class 3 genes. ATP-dependent proteolysis thus controls flagella synthesis by shutting down expression of class 2 and 3 genes. Lon-dependent proteolysis is involved in the control of other types of movement such as swarming and twitching motility. Swarming is a group behavior that requires a threshold quorum to be initiated and relies on flagellar structures. Lon is involved in the control of swarming differentiation in various bacterial species through FlhDC proteolysis [12,42,59,61]. Twitching motility enables bacteria to crawl on a surface and requires type IV pili. It is involved in early steps of biofilm formation. In P. aeruginosa, in addition to being defective in swarming, Lon mutants were shown to be impaired in twitching motility (although type IV pili are still expressed) and in biofilm formation [42], although the underlying molecular mechanisms are still unknown. 3.2. Stress response 3.2.1. The SOS system The bacterial SOS response is an essential DNA damage response that can be triggered by a variety of factors. The SOS regulon is mainly controlled by RecA and LexA, and encodes functions to repair the incurred DNA damage and restore the replication fork (for reviews, [2,16]). However, the SOS response also controls the lateral spread of mobile genetic elements, mutagenesis and cell division, and some of these phenomena involve the action of the Lon protease. Probably the best-known example of Lon control in the context of the SOS response comprises the degradation of SulA [47]. The sulA gene is under direct control of the LexA repressor, and is concomitantly induced upon DNA damage. By binding to the FtsZ cell division protein, SulA acts as a physical inhibitor of septum formation and, as such, delays cell division for the time needed to repair DNA damages. At the posttranslational level, SulA is rapidly degraded by Lon in order to resume cell division when the damages are repaired and sulA expression is shut down. Because of the severe impact of SulA on cell division, its proteolysis is crucial and other ATP-dependent proteases, such as ClpYQ (HslUV), take over SulA degradation under conditions where Lon activity is
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absent [76]. Nevertheless, Lon remains the major degrader of SulA, and lon mutants suffer filamentation and cell death upon SOS induction [1,24]. A second aspect of the SOS response in which Lon plays an essential function is SOS-induced mutagenesis, which results from the translesion bypass of error-prone polymerases. In case of severe DNA damage, passage of the replicative DNA polymerase (Pol) III can become blocked by DNA lesions. In this case, UmuC and UmuD proteins, both members of the SOS regulon, will assemble into a novel DNA polymerase (Pol V) capable of traversing the lesion and allowing normal DNA replication to resume [22]. However, this translesion bypass intrinsically requires decreased fidelity and therefore inevitably results in the incorporation of errors. Obviously, this error-prone DNA replication should be tightly controlled at different levels in order to safeguard the genetic blueprint. At the transcriptional level, the umuDC operon is only expressed in the last stage of the SOS response, in order to resolve those lesions that could not be mended with the error-free DNA repair. In addition, at the posttranslational level, cellular levels of both UmuD and UmuC are kept very low under normal conditions as they are rapidly degraded by the Lon protease. Only when RecA is activated by DNA damage, the autoproteolytic removal of the 24 N-terminal amino acids of UmuD is triggered, and the resulting UmuD0 becomes relatively resistant to Lon degradation. In turn, UmuC becomes stabilized by association with UmuD02 , which ultimately leads to the assembly of the mutagenically active Pol V complex (UmuD02 C) [21]. A more recently discovered case of Lon-mediated control of the activity of an SOS induced protein deals with the SymE protein. SymE (YjiW) is a recently discovered toxin whose expression was shown to be repressed by LexA [17,32]. It constitutes an endoribonuclease that cleaves mRNA and is believed to play a role in recycling RNAs that might have been damaged by the agents that cause the SOS response [32]. Interestingly, aside from its transcriptional control by LexA, stability and translation of the symE mRNA are controlled by a cis-encoded antisense RNA (SymR), which acts as an antitoxin. In addition to these two layers of regulation, the SymE protein itself is a Lon target, and its degradation further prevents the potential deleterious accumulation of this toxin [32]. Interestingly, while this posttranslational control of SymE typically resembles that of other potentially harmful SOS functions (such as SulA and UmuDC described above), it is in sharp contrast with the usual control of toxineantitoxin systems where, in fact, the toxin tends to be more stable than the antitoxin (for a recent review, [70]). Finally, Lon was also shown to be involved in the paradoxical survival of E. coli to high quinolone concentrations [41]. Quinolone antibiotics target the bacterial DNA gyrase and thereby result in the generation of double-stranded DNA breaks. Generation of these lesions leads to SOS induction. Although exposure to quinolones is lethal, a counterintuitive yet distinct increase in bacterial survival has been historically noted at high quinolone concentrations [14]. Surprisingly, this paradoxical survival is abolished in the absence of the Lon
protease. Furthermore, this phenomenon was demonstrated to be independent of effects on SulA and to be mediated specifically by the protease rather than the ATPase function of Lon [41]. However, how exactly Lon is molecularly involved in this enigmatic cellular behavior remains to be elucidated. 3.2.2. Acid tolerance Acid resistance is the ability to sustain very low pH conditions. Due to its lifestyle in the mammalian digestive tract, E. coli has a remarkable ability to adapt to pH stress. This capacity enables E. coli to survive gastric acidity and volatile fatty acids produced by fermentation in the intestine. Glutamate-dependent acid resistance is one of the mechanisms used by E. coli and its relatives (for review, [18]). In this pathway, GadE is the key transcriptional regulator. It positively controls acid resistance gene expression such as that of gadA and gadB, which encode glutamate decarboxylase isoenzymes, and gadC, which encodes a putative glutamate g-aminobutyric anti-porter [39]. Amino acid decarboxylase systems are thought to confer acid resistance by consuming intracellular protons. Under acid stress, glutamate is taken up by the cell using the GadC anti-porter and decarboxylation of glutamate by GadA and GadB produces g-aminobutyric acid that will expel through GadC. This results in alkalinization of the cytoplasm. Interestingly, these genes (except of gadBC ) and others involved in acid resistance are located in a cluster of sS-dependent genes called the ‘fitness island for acid adaptation’ [29]. Expression of these genes, including gadE, is strongly induced in stationary phase in a sS-dependent manner, providing a molecular explanation for the acid resistance phenotype displayed by stationary phase cells [75]. Thus, regulation of the acid resistance response is quite complex: the master regulator RpoS activates the expression of the secondary regulator GadE which in turn activates acid resistance genes expression. Recently, the group of Hengge showed that the key acid resistance genes (notably gadE, gadA and gadBC ) are upregulated in a lon mutant, raising the possibility that Lon could control the amount of the GadE secondary regulator [28]. Indeed, the authors showed that Lon has no effect on RpoS activity and degrades GadE constitutively. Lon-dependent GadE degradation is involved in acid resistance response shutoff, since GadE positively activates its own expression. In addition, the authors also identified other RpoS-controlled genes the expression of which is altered in a lon mutant, indicating that Lon-dependent degradation of secondary regulators might be widespread within the RpoS regulon. 3.2.3. Nutritional stress Under normal growth conditions, the majority of E. coli proteins are extremely stable (for a review, [44]). Around 75% of total proteins are estimated to present a half-life greater than 25 h. However, the overall degradation rate increases severalfold under starvation conditions, such as amino acid, nitrogen or carbon deprivation. Increased degradation in response to amino acid or nitrogen starvation is linked to the stringent response and the production of ppGpp. Recently, Kornberg
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and co-workers showed that during an amino acid downshift, the pool of free ribosomal proteins is degraded by Lon in a polyphosphate-dependent manner [33]. ppGpp is needed for accumulation of polyphosphate, which in turn participates in the formation of a complex between Lon and specific ribosomal proteins. Degradation will release free amino acids that are, in turn, used for synthesis of biosynthetic enzymes. Proteolysis and activation of biosynthetic gene expression allow the cell to adapt to nutrient-poor conditions. Few other examples are reported in the literature, such as Lon- and ClpAP-dependent degradation of L-glutamate dehydrogenase in carbon or nitrogen-starved cells which also appears to be ppGpp-dependent [45]. Proteolysis also controls heme biosynthesis in Salmonella typhimurium [72]. Under a normal heme level, HemA (first enzyme in the heme biosynthesis pathway) is degraded in a Lon- and ClpAPdependent fashion, while it is stable in heme-limiting conditions, making the cell ready for new heme synthesis. 3.3. Virulence and pathogenicity lon mutations can confer a reduced pathogenicity/virulence phenotype. This is the case for several Gram-negative species such as Salmonella enterica [63], Brucella abortus [51], Campylobacter jeujeni [12], A. tumefaciens [61] and Pseudomonas syringae [34]. Gram-negative pathogens often use type three secretion systems (TTSSs) to inject effectors (virulence factors) into eukaryotic cells. TTSSs are intricate molecular machines shaped as a needle and spanning the cell envelope (for review, [13]). Lon regulates TTSS expression in various bacterial species by modulating the amount of key transcriptional regulators that control TTSS gene expression (for recent reviews, [7,67]). Lon notably upregulates TTSS in Yersinia pestis by degrading the RovA negative regulator at 37 C, as mentioned above [31] as well as P. syringae TTSS by a yet unknown mechanism [34]. Furthermore, Lon controls effector secretion through TTSS by degrading the excess of effectors that were not secreted [19,38]. 4. Concluding remarks Recent advances regarding Lon structure as well as substrate characteristics have led to a better understanding of how this protease specifically selects proteins to degrade among a pool of non-substrate proteins. Lon recognizes sequences rich in hydrophobic/aromatic residues whether they are located in an N- or C-terminal degradation tag or exposed in naturally unfolded substrates and in abnormal proteins. Adaptors might be required for some substrate recognition, like Poly-P in the case of ribosomal subunits. Whether other types of adaptators such as small proteins are involved in some cases remains to be shown. Structure-function studies might still be needed to decipher the precise molecular functioning of this family of proteases. Regarding the biological roles of Lon, although multiple physiological processes have been found to be under Lon control, it is likely that not yet determined pathways in various
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bacteria are regulated at one or several steps by Lon-dependent proteolysis. Recent microarray analysis led to the discovery of the involvement of Lon in acid tolerance and flagellum synthesis [3,28]. Such global approaches will further reveal the true versatility of the Lon protease and its central role in different aspects of cellular physiology. Acknowledgements AA acknowledges a postdoctoral fellowship and a mobility grant of the Research Foundation e Flanders (FWO). References [1] Aertsen, A., Michiels, C.W. (2005) SulA-dependent hypersensitivity to high pressure and hyperfilamentation after high-pressure treatment of Escherichia coli lon mutants. Res. Microbiol. 156, 233e237. [2] Aertsen, A., Michiels, C.W. (2006) Upstream of the SOS response: figure out the trigger. Trends Microbiol. 14, 421e423. [3] Barembruch, C., Hengge, R. (2007) Cellular levels and activity of the flagellar sigma factor FliA of Escherichia coli are controlled by FlgMmodulated proteolysis. Mol. Microbiol. 65, 76e89. [4] Bertani, I., Rampioni, G., Leoni, L., Venturi, V. (2007) The Pseudomonas putida Lon protease is involved in N-acyl homoserine lactone quorum sensing regulation. BMC Microbiol. 7, 71. [5] Biran, D., Gur, E., Gollan, L., Ron, E.Z. (2000) Control of methionine biosynthesis in Escherichia coli by proteolysis. Mol. Microbiol. 37, 1436e1443. [6] Botos, I., Melnikov, E.E., Cherry, S., Khalatova, A.G., Rasulova, F.S., Tropea, J.E., Maurizi, M.R., Rotanova, T.V., Gustchina, A., Wlodawer, A. (2004) Crystal structure of the AAAþ alpha domain of E. coli Lon protease at 1.9A resolution. J. Struct. Biol. 146, 113e122. [7] Butler, S.M., Festa, R.A., Pearce, M.J., Darwin, K.H. (2006) Self-compartmentalized bacterial proteases and pathogenesis. Mol. Microbiol. 60, 553e562. [8] Charette, M.F., Henderson, G.W., Doane, L.L., Markovitz, A. (1984) DNA-stimulated ATPase activity on the lon (CapR) protein. J. Bacteriol. 158, 195e201. [9] Charette, M.F., Henderson, G.W., Markovitz, A. (1981) ATP hydrolysisdependent protease activity of the lon (capR) protein of Escherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A 78, 4728e4732. [10] Choy, J.S., Aung, L.L., Karzai, A.W. (2007) Lon protease degrades transfer-messenger RNA-tagged proteins. J. Bacteriol. 189, 6564e6571. [11] Claret, L., Hughes, C. (2000) Rapid turnover of FlhD and FlhC, the flagellar regulon transcriptional activator proteins, during Proteus swarming. J. Bacteriol. 182, 833e836. [12] Cohn, M.T., Ingmer, H., Mulholland, F., Jorgensen, K., Wells, J.M., Brondsted, L. (2007) Contribution of conserved ATP-dependent proteases of Campylobacter jejuni to stress tolerance and virulence. Appl. Environ. Microbiol. 73, 7803e7813. [13] Cornelis, G.R. (2006) The type III secretion injectisome. Nat. Rev. Microbiol. 4, 811e825. [14] Crumplin, G.C., Smith, J.T. (1975) Nalidixic acid: an antibacterial paradox. Antimicrob. Agents Chemother. 8, 251e261. [15] Davey, M.E., O’Toole, G.A. (2000) Microbial biofilms: from ecology to molecular genetics. Microbiol. Mol. Biol. Rev. 64, 847e867. [16] Erill, I., Campoy, S., Barbe, J. (2007) Aeons of distress: an evolutionary perspective on the bacterial SOS response. FEMS Microbiol. Rev. 31, 637e656. [17] Fernandez De Henestrosa, A.R., Ogi, T., Aoyagi, S., Chafin, D., Hayes, J.J., Ohmori, H., Woodgate, R. (2000) Identification of additional genes belonging to the LexA regulon in Escherichia coli. Mol. Microbiol. 35, 1560e1572. [18] Foster, J.W. (2004) Escherichia coli acid resistance: tales of an amateur acidophile. Nat. Rev. Microbiol. 2, 898e907.
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