Filament formation activates protease and ring nuclease activities of CRISPR Lon-SAVED.

To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cAn) signaling to activate various auxiliary effectors, including the CRISPR-associated Lon-SAVED protease CalpL, which forms a tripartite effector system together with an anti-σ factor, CalpT, and an ECF-like σ factor, CalpS. Here, we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA4 binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E. coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA4 in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity.

Activator of one protease transforms into inhibitor of another in response to nutritional signals.

All cells use proteases to adjust protein amounts. Proteases maintain protein homeostasis by degrading nonfunctional toxic proteins and play regulatory roles by targeting particular substrates in response to specific signals. Here we address how cells tune protease specificity to nutritional signals. We report that Salmonella enterica increases the specificity of the broadly conserved proteases Lon and ClpSAP by transforming the Lon activator and substrate HspQ into an inhibitor of the N-degron recognin ClpS, the adaptor of the ClpAP protease. We establish that upon acetylation, HspQ stops being a Lon activator and substrate and that the accumulated HspQ binds to ClpS, hindering degradation of ClpSAP substrates. Growth on glucose promotes HspQ acetylation by increasing acetyl-CoA amounts, thereby linking metabolism to proteolysis. By altering protease specificities but continuing to degrade junk proteins, cells modify the abundance of particular proteins while preserving the quality of their proteomes. This rapid response mechanism linking protease specificity to nutritional signals is broadly conserved.

Exogenous hydrogen sulfide prevents SOD2 degradation to safeguard renal function in diabetic kidney disease.

Diabetic kidney disease (DKD) is a major contributor to chronic kidney disease. Hydrogen sulfide (H2S) serves as an endogenous gaseous signaling molecule capable of safeguarding renal function within the context of DKD. However, the underlying mechanisms need to be elucidated. This study was undertaken to unveil the mechanisms by which H2S counteracts against DKD. Utilizing mice and human renal tubular epithelial (HK-2) cells, we demonstrated a reduction in cystathionine-γ-lyase/H2S levels within renal tissues of db/db mice and in HK-2 cells subjected to hyperglycemic and hyperlipidemic environments. Notably, we observed that sodium hydrosulfide (NaHS) supplementation could serve as an exogenous source of H2S. Exogenous H2S exhibited the capacity to mitigate the accumulation of reactive oxygen species and attenuate the degradation of superoxide dismutase 2 (SOD2) by Lon protease homolog 1 induced by hyperglycemia and hyperlipidemia, thus affording cellular protection against mitochondrial apoptosis. Consequently, NaHS treatment led to decreased serum levels of blood urea nitrogen and serum creatinine, reflecting alleviated renal damage and thereby preserving renal function in db/db mice. Based on these findings, we propose that exogenous H2S exerts a protective role against DKD by inhibiting SOD2 degradation.

Combined effect of polyphosphate kinase and lon protease in Streptomyces coelicolor A3(2) antibiotic production.

The bacterial stringent response is a global regulatory process in which polyphosphate kinase (Ppk) and lon protease are important players. Previous studies have shown that overexpression of the lon gene and deletion of the ppk gene significantly increased actinorhodin production in Streptomyces coelicolor (SCO). In this study, a recombinant SCOΔppk-lon cell, expressing the extra lon gene in Δppk cells, was simulated using a modified in silico (computational) model, ecSco-GEM, and the negative effect of Ppk on actinorhodin production was confirmed. In addition, we identified key enzymes that play a positive role in actinorhodin production. Of these, NADH dehydrogenase/complex-I, beta-ketoacyl-[acyl-carrier-protein] synthase III, glycine cleavage system, and superoxide dismutase were identified as the most significant. By confirming these results with experiments, we have shown that GEMs can be a reliable starting point for in vitro (lab-based) studies of Streptomyces..

Divergent Roles of Escherichia Coli Encoded Lon Protease in Imparting Resistance to Uncouplers of Oxidative Phosphorylation: Roles of marA, rob, soxS and acrB.

Uncouplers of oxidative phosphorylation dissipate the proton gradient, causing lower ATP production. Bacteria encounter several non-classical uncouplers in the environment, leading to stress-induced adaptations. Here, we addressed the molecular mechanisms responsible for the effects of uncouplers in Escherichia coli. The expression and functions of genes involved in phenotypic antibiotic resistance were studied using three compounds: two strong uncouplers, i.e., Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-Dinitrophenol (DNP), and one moderate uncoupler, i.e., Sodium salicylate (NaSal). Quantitative expression studies demonstrated induction of transcripts encoding marA, soxS and acrB with NaSal and DNP, but not CCCP. Since MarA and SoxS are degraded by the Lon protease, we investigated the roles of Lon using a lon-deficient strain (Δlon). Compared to the wild-type strain, Δlon shows compromised growth upon exposure to NaSal or 2, 4-DNP. This sensitivity is dependent on marA but not rob and soxS. On the other hand, the Δlon strain shows enhanced growth in the presence of CCCP, which is dependent on acrB. Interestingly, NaSal and 2,4-DNP, but not CCCP, induce resistance to antibiotics, such as ciprofloxacin and tetracycline. This study addresses the effects of uncouplers and the roles of genes involved during bacterial growth and phenotypic antibiotic resistance. Strong uncouplers are often used to treat wastewater, and these results shed light on the possible mechanisms by which bacteria respond to uncouplers. Also, the rampant usage of some uncouplers to treat wastewater may lead to the development of antibiotic resistance.

Proteomic survey of the DNA damage response in Caulobacter crescentus.

The bacterial DNA damage response is a critical, coordinated response to endogenous and exogenous sources of DNA damage. Response dynamics are dependent on coordinated synthesis and loss of relevant proteins. While much is known about its global transcriptional control, changes in protein abundance that occur upon DNA damage are less well characterized at the system level. Here, we perform a proteome-wide survey of the DNA damage response in Caulobacter crescentus. We find that while most protein abundance changes upon DNA damage are readily explained by changes in transcription, there are exceptions. The survey also allowed us to identify the novel DNA damage response factor, YaaA, which has been overlooked by previously published, transcription-focused studies. A similar survey in a ∆lon strain was performed to explore lon's role in DNA damage survival. The ∆lon strain had a smaller dynamic range of protein abundance changes in general upon DNA damage compared to the wild-type strain. This system-wide change to the dynamics of the response may explain this strain's sensitivity to DNA damage. Our proteome survey of the DNA damage response provides additional insight into the complex regulation of stress response and nominates a novel response factor that was overlooked in prior studies. IMPORTANCE The DNA damage response helps bacteria to react to and potentially survive DNA damage. The mutagenesis induced during this stress response contributes to the development of antibiotic resistance. Understanding how bacteria coordinate their response to DNA damage could help us to combat this growing threat to human health. While the transcriptional regulation of the bacterial DNA damage response has been characterized, this study is the first to our knowledge to assess the proteomic response to DNA damage in Caulobacter.

Regulation of the general stress response sigma factor σT by Lon-mediated proteolysis.

IMPORTANCE: Regulated protein degradation is a critical process in all cell types, which contributes to the precise regulation of protein amounts in response to internal and external cues. In bacteria, protein degradation is carried out by ATP-dependent proteases. Although past work revealed detailed insights into the operation principles of these proteases, there is limited knowledge about the substrate proteins that are degraded by distinct proteases and the regulatory role of proteolysis in cellular processes. This study reveals a direct role of the conserved protease Lon in regulating σT, a transcriptional regulator of the general stress response in α-proteobacteria. Our work is significant as it underscores the importance of regulated proteolysis in modulating the levels of key regulatory proteins under changing conditions.

PrkA is an ATP-dependent protease that regulates sporulation in Bacillus subtilis.

In Bacillus subtilis, sporulation is a sequential and highly regulated process. Phosphorylation events by histidine kinases are key points in the phosphorelay that initiates sporulation, but serine/threonine protein kinases also play important auxiliary roles in this regulation. PrkA has been proposed to be a serine protein kinase expressed during the initiation of sporulation and involved in this differentiation process. Additionally, the role of PrkA in sporulation has been previously proposed to be mediated via the transition phase regulator ScoC, which in turn regulates the transcriptional factor σK and its regulon. However, the kinase activity of PrkA has not been clearly demonstrated, and neither its autophosphorylation nor phosphorylated substrates have been unambiguously established in B. subtilis. We demonstrated here that PrkA regulation of ScoC is likely indirect. Following bioinformatic homology searches, we revealed sequence similarities of PrkA with the ATPases associated with diverse cellular activities ATP-dependent Lon protease family. Here, we showed that PrkA is indeed able to hydrolyze α-casein, an exogenous substrate of Lon proteases, in an ATP-dependent manner. We also showed that this ATP-dependent protease activity is essential for PrkA function in sporulation since mutation in the Walker A motif leads to a sporulation defect. Furthermore, we found that PrkA protease activity is tightly regulated by phosphorylation events involving one of the Ser/Thr protein kinases of B. subtilis, PrkC. Taken together, our results clarify the key role of PrkA in the complex process of B. subtilis sporulation.

Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease.

The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. While the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the amino-terminal FinO domain and the variable carboxy-terminal region that are required for ProQ activity. Two carboxy-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. In contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of nonfunctional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.

Structure, Substrate Specificity and Role of Lon Protease in Bacterial Pathogenesis and Survival.

Proteases are the group of enzymes that carry out proteolysis in all forms of life and play an essential role in cell survival. By acting on specific functional proteins, proteases affect the transcriptional and post-translational pathways in a cell. Lon, FtsH, HslVU and the Clp family are among the ATP-dependent proteases responsible for intracellular proteolysis in bacteria. In bacteria, Lon protease acts as a global regulator, governs an array of important functions such as DNA replication and repair, virulence factors, stress response and biofilm formation, among others. Moreover, Lon is involved in the regulation of bacterial metabolism and toxin-antitoxin systems. Hence, understanding the contribution and mechanisms of Lon as a global regulator in bacterial pathogenesis is crucial. In this review, we discuss the structure and substrate specificity of the bacterial Lon protease, as well as its ability to regulate bacterial pathogenesis.

Lon protease downregulates phenazine-1-carboxamide biosynthesis by degrading the quorum sensing signal synthase PhzI and exhibits negative feedback regulation of Lon itself in Pseudomonas chlororaphis HT66.

Pseudomonas chlororaphis HT66 exhibits strong antagonistic activity against various phytopathogenic fungi due to its main antibiotic phenazine-1-carboxamide (PCN). PCN gene cluster consists of phzABCDEFG, phzH, phzI, and phzR operons. phzABCDEFG transcription is activated by the PhzI/R quorum sensing system. Deletion of the lon gene encoding an ATP-dependent protease resulted in significant enhancement of PCN production in strain HT66. However, the regulatory pathway and mechanism of Lon on PCN biosynthesis remain unknown. Here, lon mutation was shown to significantly improve antimicrobial activity of strain HT66. The N-acyl-homoserine lactone synthase PhzI mediates the negative regulation of PCN biosynthesis and phzABCDEFG transcription by Lon. Western blot showed that PhzI protein abundance and stability were significantly enhanced by lon deletion. The in vitro degradation assay suggested that Lon could directly degrade PhzI protein. However, Lon with an amino acid replacement (S674 -A) could not degrade PhzI protein. Lon-recognized region was located within the first 50 amino acids of PhzI. In addition, Lon formed a new autoregulatory feedback circuit to modulate its own degradation by other potential proteases. In summary, we elucidated the Lon-regulated pathway mediated by PhzI during PCN biosynthesis and the molecular mechanism underlying the degradation of PhzI by Lon in P. chlororaphis HT66.

Mitochondrial oxidative stress in the tumor microenvironment and cancer immunoescape: foe or friend?

The major concept of "oxidative stress" is an excess elevated level of reactive oxygen species (ROS) which are generated from vigorous metabolism and consumption of oxygen. The precise harmonization of oxidative stresses between mitochondria and other organelles in the cell is absolutely vital to cell survival. Under oxidative stress, ROS produced from mitochondria and are the major mediator for tumorigenesis in different aspects, such as proliferation, migration/invasion, angiogenesis, inflammation, and immunoescape to allow cancer cells to adapt to the rigorous environment. Accordingly, the dynamic balance of oxidative stresses not only orchestrate complex cell signaling events in cancer cells but also affect other components in the tumor microenvironment (TME). Immune cells, such as M2 macrophages, dendritic cells, and T cells are the major components of the immunosuppressive TME from the ROS-induced inflammation. Based on this notion, numerous strategies to mitigate oxidative stresses in tumors have been tested for cancer prevention or therapies; however, these manipulations are devised from different sources and mechanisms without established effectiveness. Herein, we integrate current progress regarding the impact of mitochondrial ROS in the TME, not only in cancer cells but also in immune cells, and discuss the combination of emerging ROS-modulating strategies with immunotherapies to achieve antitumor effects.

Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation.

The cell cycle-regulated methylation state of Caulobacter DNA mediates the temporal control of transcriptional activation of several key regulatory proteins. Temporally controlled synthesis of the CcrM DNA methyltransferase and Lon-mediated proteolysis restrict CcrM to a specific time in the cell cycle, thereby allowing the maintenance of the hemimethylated state of the chromosome during the progression of DNA replication. We determined that a chromosomal DNA-based platform stimulates CcrM degradation by Lon and that the CcrM C terminus both binds to its DNA substrate and is recognized by the Lon protease. Upon asymmetric cell division, swarmer and stalked progeny cells employ distinct mechanisms to control active CcrM. In progeny swarmer cells, CcrM is completely degraded by Lon before its differentiation into a replication-competent stalked cell later in the cell cycle. In progeny stalked cells, however, accumulated CcrM that has not been degraded before the immediate initiation of DNA replication is sequestered to the cell pole. Single-molecule imaging demonstrated physical anticorrelation between sequestered CcrM and chromosomal DNA, thus preventing DNA remethylation. The distinct control of available CcrM in progeny swarmer and stalked cells serves to protect the hemimethylated state of DNA during chromosome replication, enabling robustness of cell cycle progression.

Roles of LonP1 in Oral-Maxillofacial Developmental Defects and Tumors: A Novel Insight.

Recent studies have indicated a central role for LonP1 in mitochondrial function. Its physiological functions include proteolysis, acting as a molecular chaperone, binding mitochondrial DNA, and being involved in cellular respiration, cellular metabolism, and oxidative stress. Given its vital role in energy metabolism, LonP1 has been suggested to be associated with multi-system neoplasms and developmental disorders. In this study, we investigated the roles, possible mechanisms of action, and therapeutic roles of LonP1 in oral and maxillofacial tumor development. LonP1 was highly expressed in oral-maxillofacial cancers and regulated their development through a sig-naling network. LonP1 may therefore be a promising anticancer therapy target. Mutations in LONP1 have been found to be involved in the etiology of cerebral, ocular, dental, auricular, and skeletal syndrome (CODAS). Only patients carrying specific LONP1 mutations have certain dental abnormalities (delayed eruption and abnormal morphology). LonP1 is therefore a novel factor in the development of oral and maxillofacial tumors. Greater research should therefore be conducted on the diagnosis and therapy of LonP1-related diseases to further define LonP1-associated oral phenotypes and their underlying molecular mechanisms.

Shotgun Proteomics Revealed Preferential Degradation of Misfolded In Vivo Obligate GroE Substrates by Lon Protease in Escherichia coli.

The Escherichia coli chaperonin GroEL/ES (GroE) is one of the most extensively studied molecular chaperones. So far, ~80 proteins in E. coli are identified as GroE substrates that obligately require GroE for folding in vivo. In GroE-depleted cells, these substrates, when overexpressed, tend to form aggregates, whereas the GroE substrates expressed at low or endogenous levels are degraded, probably due to misfolded states. However, the protease(s) involved in the degradation process has not been identified. We conducted a mass-spectrometry-based proteomics approach to investigate the effects of three ATP-dependent proteases, Lon, ClpXP, and HslUV, on the E. coli proteomes under GroE-depleted conditions. A label-free quantitative proteomic method revealed that Lon protease is the dominant protease that degrades the obligate GroE substrates in the GroE-depleted cells. The deletion of DnaK/DnaJ, the other major E. coli chaperones, in the ∆lon strain did not cause major alterations in the expression or folding of the obligate GroE substrates, supporting the idea that the folding of these substrates is predominantly dependent on GroE.

Lon Protease- and Temperature-Dependent Activity of a Lysis Cassette Located in the Insecticidal Island of Yersinia enterocolitica.

The Yersinia genus comprises pathogens that can adapt to an environmental life cycle stage as well as to mammals. Yersinia enterocolitica strain W22703 exhibits both insecticidal and nematocidal activity conferred by the tripartite toxin complex (Tc) that is encoded on the 19-kb pathogenicity island Tc-PAI Ye All tc genes follow a strict temperature regulation in that they are silenced at 37°C but activated at lower temperatures. Four highly conserved phage-related genes, located within the Tc-PAI Ye , were recently demonstrated to encode a biologically functional holin-endolysin gene cassette that lyses its own host W22703 at 37°C. Conditions transcriptionally activating the cassette are not yet known. In contrast to Escherichia coli, the overproduction of holin and endolysin did not result in cell lysis of strain W22703 at 15°C. When the holin-endolysin genes were overexpressed at 15°C in four Y. enterocolitica biovars and in four other Yersinia spp., a heterogenous pattern of phenotypes was observed, ranging from lysis resistance of a biovar 1A strain to the complete growth arrest of a Y. kristensenii strain. To decipher the molecular mechanism underlying this temperature-dependent lysis, we constructed a Lon protease-negative mutant of W22703 in which the overexpression of the lysis cassette leads to cell death at 15°C. Overexpressed endolysin exhibited a high proteolytic susceptibility in strain W22703 but remained stable in the W22703 Δlon strain or in Y. pseudotuberculosis Although artificial overexpression was applied here, the data indicate that Lon protease plays a role in the control of the temperature-dependent lysis in Y. enterocolitica W22703.IMPORTANCE The investigation of the mechanisms that help pathogens survive in the environment is a prerequisite to understanding their evolution and their virulence capacities. In members of the genus Yersinia, many factors involved in virulence, metabolism, motility, or biofilm formation follow a strict temperature-dependent regulation. While the molecular mechanisms underlying the activation of determinants at body temperature have been analyzed in detail, the molecular basis of low-temperature-dependent phenotypes is largely unknown. Here, we demonstrate that a novel phage-related lysis cassette, which is part of the insecticidal and nematocidal pathogenicity island of Y. enterocolitica, does not lyse its own host following overexpression at 15°C and that the Lon protease is involved in this phenotype.

Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid ß1-42 pathologies.

Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-ß 1-42 (Aß42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin-proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies.

A structure and function relationship study to identify the impact of the R721G mutation in the human mitochondrial lon protease.

Lon is an ATP-dependent protease belonging to the "ATPase associated with diverse cellular activities" (AAA+) protein family. In humans, Lon is translated as a precursor and imported into the mitochondria matrix through deletion of the first 114 amino acid residues. In mice, embryonic knockout of lon is lethal. In humans, some dysfunctional lon mutations are tolerated but they cause a developmental disorder known as the CODAS syndrome. To gain a better understanding on the enzymology of human mitochondrial Lon, this study compares the structure-function relationship of the WT versus one of the CODAS mutants R721G to identify the mechanistic features in Lon catalysis that are affected. To this end, steady-state kinetics were used to quantify the difference in ATPase and ATP-dependent peptidase activities between WT and R721G. The Km values for the intrinsic as well as protein-stimulated ATPase were increased whereas the kcat value for ATP-dependent peptidase activity was decreased in the R721G mutant. The mutant protease also displayed substrate inhibition kinetics. In vitro studies revealed that R721G did not degrade the endogenous mitochondrial Lon substrate pyruvate dehydrogenase kinase isoform 4 (PDK4) effectively like WT hLon. Furthermore, the pyruvate dehydrogenase complex (PDH) protected PDK4 from hLon degradation. Using hydrogen deuterium exchange/mass spectrometry and negative stain electron microscopy, structural perturbations associated with the R721G mutation were identified. To validate the in vitro findings under a physiologically relevant condition, the intrinsic stability as well as proteolytic activity of WT versus R721G mutant towards PDK 4 were compared in cell lysates prepared from immortalized B lymphocytes expressing the respective protease. The lifetime of PDK4 is longer in the mutant cells, but the lifetime of Lon protein is longer in the WT cells, which corroborate the in vitro structure-functional relationship findings.

Lon Protease Removes Excess Signal Recognition Particle Protein in Escherichia coli.

Correct targeting of membrane proteins is essential for membrane integrity, cell physiology, and viability. Cotranslational targeting depends on the universally conserved signal recognition particle (SRP), which is a ribonucleoprotein complex comprised of the protein component Ffh and the 4.5S RNA in Escherichia coli About 25 years ago it was reported that Ffh is an unstable protein, but the underlying mechanism has never been explored. Here, we show that Lon is the primary protease responsible for adjusting the cellular Ffh level. When overproduced, Ffh is particularly prone to degradation during transition from exponential to stationary growth and the cellular Ffh amount is lowest in stationary phase. The Ffh protein consists of two domains, the NG domain, responsible for GTP hydrolysis and docking to the membrane receptor FtsY, and the RNA-binding M domain. We find that the NG domain alone is stable, whereas the isolated M domain is degraded. Consistent with the importance of Lon in this process, the M domain confers synthetic lethality to the lon mutant. The Ffh homolog from the model plant Arabidopsis thaliana, which forms a protein-protein complex rather than a protein-RNA complex, is stable, suggesting that the RNA-binding ability residing in the M domain of E. coli Ffh is important for proteolysis. Our results support a model in which excess Ffh not bound to 4.5S RNA is subjected to proteolysis until an appropriate Ffh concentration is reached. The differential proteolysis adjusts Ffh levels to the cellular demand and maintains cotranslational protein transport and membrane integrity.IMPORTANCE Since one-third of all bacterial proteins reside outside the cytoplasm, protein targeting to the appropriate address is an essential process. Cotranslational targeting to the membrane relies on the signal recognition particle (SRP), which is a protein-RNA complex in bacteria. We report that the protein component Ffh is a substrate of the Lon protease. Regulated proteolysis of Ffh provides a simple mechanism to adjust the concentration of the essential protein to the cellular demand. This is important because elevated or depleted SRP levels negatively impact protein targeting and bacterial fitness.

Highly Contingent Phenotypes of Lon Protease Deficiency in Escherichia coli upon Antibiotic Challenge.

Evolutionary trajectories and mutational landscapes of drug-resistant bacteria are influenced by cell-intrinsic and extrinsic factors. In this study, I demonstrated that loss of the Lon protease altered susceptibility of Escherichia coli to trimethoprim and that these effects were strongly contingent on the drug concentration and genetic background. Lon, an AAA+ ATPase, is a bacterial master regulator protease involved in cytokinesis, suppression of transposition events, and clearance of misfolded proteins. I show that Lon deficiency enhances intrinsic drug tolerance at sub-MIC levels of trimethoprim. As a result, loss of Lon, though disadvantageous under drug-free conditions, has a selective advantage at low concentrations of trimethoprim. At high drug concentrations, however, Lon deficiency is detrimental for E. coli I show that the former is explained by suppression of drug efflux by Lon, while the latter can be attributed to SulA-dependent hyperfilamentation. On the other hand, deletion of lon in a trimethoprim-resistant mutant E. coli strain (harboring the Trp30Gly dihydrofolate reductase [DHFR] allele) directly potentiates resistance by enhancing the in vivo stability of mutant DHFR. Using extensive mutational analysis at 3 hot spots of resistance, I show that many resistance-conferring mutations render DHFR prone to proteolysis. This trade-off between gaining resistance and losing in vivo stability limits the number of mutations in DHFR that can confer trimethoprim resistance. Loss of Lon expands the mutational capacity for acquisition of trimethoprim resistance. This paper identifies the multipronged action of Lon in trimethoprim resistance in E. coli and provides mechanistic insight into how genetic backgrounds and drug concentrations may alter the potential for antimicrobial resistance evolution.IMPORTANCE Understanding the evolutionary dynamics of antimicrobial resistance is vital to curb its emergence and spread. Being fundamentally similar to natural selection, the fitness of resistant mutants is a key parameter to consider in the evolutionary dynamics of antimicrobial resistance (AMR). Various intrinsic and extrinsic factors modulate the fitness of resistant bacteria. This study demonstrated that Lon, a bacterial master regulator protease, influences drug tolerance and resistance. Lon is a key regulator of several fundamental processes in bacteria, including cytokinesis. I demonstrated that Lon deficiency produces highly contingent phenotypes in E. coli challenged with trimethoprim and can expand the mutational repertoire available to E. coli to evolve resistance. This multipronged influence of Lon on drug resistance provides an illustrative instance of how master regulators shape the response of bacteria to antibiotics.