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ClpA is a widely conserved protease in bacteria that plays a key role in virulence. To investigate its specific mechanism of action in the pathogenicity of Paracidovorax citrulli (formerly Acidovorax citrulli), we constructed a ClpA deletion mutant, 1ClpA. The 1ClpA mutant of P. citrulli displayed reduced virulence on melon seedlings, and reduced motility, swarming ability, and antioxidant capacity. On the other hand, the ClpA deletion of P. citrulli mutant reduced the resistance to elevated temperature and enhanced biofilm formation ability. Using qRT-PCR, we observed that ClpA negatively regulates the expression of the virulence-related genes virB, pilR, pilA, and fliM, while positively regulating hrpG, hrcQ, and trbC. Bacterial double hybrid and Glutathione-Stransferase pulldown (GST-pulldown) results showed that ClpA interacts directly with RepA, and negatively regulates the expression of RepA. After deletion of the RepA gene, the pathogenicity of P. citrulli was lost, biofilm formation ability was enhanced, and the expression of hrpG, pilR, and trbC was positively regulated. These results indicate that ClpA plays a key role in the regulation of several virulence traits of P. citrulli, paving the way for future studies to better elucidate the virulence mechanisms of this bacterial plant pathogen. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is an effector delivery system used by Gram-negative bacteria to kill other bacteria or eukaryotic hosts to gain fitness. The plant pathogen Agrobacterium tumefaciens utilizes its T6SS to kill other bacteria, such as Escherichia coli. We observed that the A. tumefaciens T6SS-dependent killing outcome differs when using different T6SS-lacking, K-12 E. coli strains as a recipient cell. Thus, we hypothesized that the A. tumefaciens T6SS killing outcome not only relies on the T6SS activity of the attacker cells but also depends on the recipient cells. Here, we developed a high-throughput interbacterial competition platform to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. Among the 3,909 strains in the E. coli Keio library screened, 16 mutants with less susceptibility to A. tumefaciens C58 T6SS-dependent killing were identified, and four of them were validated by complementation test. Among the four, the clpP encoding ClpP protease, which is universal and highly conserved in both prokaryotes and eukaryotic organelles, was selected for further characterizations. We demonstrated that ClpP is responsible for enhancing susceptibility to the T6SS killing. Because ClpP protease depends on other adapter proteins such as ClpA and ClpX for substrate recognition, further mutant studies followed by complementation tests were carried out to reveal that ClpP-associated AAA+ ATPase ClpA, but not ClpX, is involved in enhancing susceptibility to A. tumefaciens T6SS killing. Moreover, functional and biochemical studies of various ClpP amino acid substitution variants provided evidence that ClpA-ClpP interaction is critical in enhancing susceptibility to the T6SS killing. This study highlights the importance of recipient factors in determining the outcome of the T6SS killing and shows the universal ClpP protease as a novel recipient factor hijacked by the T6SS of A. tumefaciens.

The type VI secretion system (T6SS) is an effector delivery system used by Gram-negative bacteria to kill other bacteria or eukaryotic hosts to gain fitness. The plant pathogen Agrobacterium tumefaciens utilizes its T6SS to kill other bacteria, such as Escherichia coli. We observed that the A. tumefaciens T6SS-dependent killing outcome differs when using different T6SS-lacking, K-12 E. coli strains as a recipient cell. Thus, we hypothesized that the A. tumefaciens T6SS killing outcome not only relies on the T6SS activity of the attacker cells but also depends on the recipient cells. Here, we developed a high-throughput interbacterial competition platform to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. Among the 3,909 strains in the E. coli Keio library screened, 16 mutants with less susceptibility to A. tumefaciens C58 T6SS-dependent killing were identified, and four of them were validated by complementation test. Among the four, the clpP encoding ClpP protease, which is universal and highly conserved in both prokaryotes and eukaryotic organelles, was selected for further characterizations. We demonstrated that ClpP is responsible for enhancing susceptibility to the T6SS killing. Because ClpP protease depends on other adapter proteins such as ClpA and ClpX for substrate recognition, further mutant studies followed by complementation tests were carried out to reveal that ClpP-associated AAA+ ATPase ClpA, but not ClpX, is involved in enhancing susceptibility to A. tumefaciens T6SS killing. Moreover, functional and biochemical studies of various ClpP amino acid substitution variants provided evidence that ClpA-ClpP interaction is critical in enhancing susceptibility to the T6SS killing. This study highlights the importance of recipient factors in determining the outcome of the T6SS killing and shows the universal ClpP protease as a novel recipient factor hijacked by the T6SS of A. tumefaciens.

ClpA is a widely conserved protease in bacteria that plays a key role in virulence. To investigate its specific mechanism of action in the pathogenicity of Paracidovorax citrulli (formerly Acidovorax citrulli) , we constructed a ClpA deletion mutant, Δ ClpA . The Δ ClpA mutant of P. citrulli displayed reduced virulence on melon seedlings, and reduced motility, swarming ability, and antioxidant capacity. On the other hand, the ClpA deletion of P. citrulli mutant reduced the resistance to elevated temperature and enhanced biofilm formation ability. Using qRT-PCR, we observed that ClpA negatively regulates the expression of the virulence-related genes virB , pilR , pilA , and fliM , while positively regulating hrpG , hrcQ , and trbC . Bacterial double hybrid and Glutathione-S-transferase pulldown (GST-pulldown) results showed that ClpA interacts directly with RepA, and negatively regulates the expression of RepA . After deletion of the RepA gene, the pathogenicity of P. citrulli was lost, biofilm formation ability was enhanced, and the expression of hrpG , pilR , and trbC was positively regulated. These results indicate that ClpA plays a key role in the regulation of several virulence traits of P. citrulli , paving the way for future studies to better elucidate the virulence mechanisms of this bacterial plant pathogen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ziye, Yuqiang, Shitong, Ling, Chenchao, Jun, Weirong, Yanli and Baishi.)

The type VI secretion system (T6SS) is an effector delivery system used by Gram-negative bacteria to kill other bacteria or eukaryotic hosts to gain fitness. The plant pathogen Agrobacterium tumefaciens utilizes its T6SS to kill other bacteria, such as Escherichia coli. We observed that the A. tumefaciens T6SS-dependent killing outcome differs when using different T6SS-lacking, K-12 E. coli strains as a recipient cell. Thus, we hypothesized that the A. tumefaciens T6SS killing outcome not only relies on the T6SS activity of the attacker cells but also depends on the recipient cells. Here, we developed a high-throughput interbacterial competition platform to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. Among the 3,909 strains in the E. coli Keio library screened, 16 mutants with less susceptibility to A. tumefaciens C58 T6SS-dependent killing were identified, and four of them were validated by complementation test. Among the four, the clpP encoding ClpP protease, which is universal and highly conserved in both prokaryotes and eukaryotic organelles, was selected for further characterizations. We demonstrated that ClpP is responsible for enhancing susceptibility to the T6SS killing. Because ClpP protease depends on other adapter proteins such as ClpA and ClpX for substrate recognition, further mutant studies followed by complementation tests were carried out to reveal that ClpP-associated AAA+ ATPase ClpA, but not ClpX, is involved in enhancing susceptibility to A. tumefaciens T6SS killing. Moreover, functional and biochemical studies of various ClpP amino acid substitution variants provided evidence that ClpA–ClpP interaction is critical in enhancing susceptibility to the T6SS killing. This study highlights the importance of recipient factors in determining the outcome of the T6SS killing and shows the universal ClpP protease as a novel recipient factor hijacked by the T6SS of A. tumefaciens.

In peer influence marketing (PIM), friendship can significantly influence customers' purchase decisions. While so far, existing researches ignore friendship construction among dissimilar influencers and target users in social networks, which help greatly to expand the friend count of influencers. Therefore, this study proposed a model to construct friendship among dissimilar users by smartly recommend friends to them. Specifically, for the sake of overcoming the influence of the scale-free topology on prediction accuracy, consensus link prediction algorithm (CLPA) is proposed to predict possible friendship between users in social networks by adaptively constructing the composite similarity index (SI) for specific user pairs. Based on the prediction results of CLPA, hill climbing algorithm (HCA) is developed to construct friendship among dissimilar users by increasing their similarity in social relationship. To share more consistency with other SIs in calculating user pair similarity, CLPA smartly combines the multiple SIs into one composite SI by using their total mean rough classification uniformity (TMRCU) and taboo search algorithm (TSA), where TMRCU is used to measure the consistency of various SIs in classifying the similar users and TSA is adopted to optimize the weights of SIs with different TMRCU. Furthermore, in CLPA, cluster consistency of user pairs is developed to identify the similar user pairs in the light of the high clustering consensus between the composite SI and other SIs. Finally, the experimental results in real social networks show that the proposed method is promising for precise PIM. [ABSTRACT FROM AUTHOR]

Regulated proteolysis is required in all organisms for the removal of misfolded or degradation-tagged protein substrates in cellular quality control pathways. The molecular machines that catalyze this process are known as ATP-dependent proteases with examples that include ClpAP and ClpCP. Clp/Hsp100 subunits form ring-structures that couple the energy of ATP binding and hydrolysis to protein unfolding and subsequent translocation of denatured protein into the compartmentalized ClpP protease for degradation. Copies of the clpA , clpC, clpE, clpK, and clpL genes are present in all characterized bacteria and their gene products are highly conserved in structure and function. However, the evolutionary relationship between these proteins remains unclear. Here we report a comprehensive phylogenetic analysis that suggests divergent evolution yielded ClpA from an ancestral ClpC protein and that ClpE/ClpL represent intermediates between ClpA/ClpC. This analysis also identifies a group of proteobacterial ClpC proteins that are likely not functional in regulated proteolysis. Our results strongly suggest that bacterial ClpC proteins should not be assumed to all function identically due to the structural differences identified here. [ABSTRACT FROM AUTHOR]

Cellular proteostasis involves not only the expression of proteins in response to environmental needs, but also the timely repair or removal of damaged or unneeded proteins. AAA+ motor proteins are critically involved in these pathways. Here, we review the structure and function of AAA+ proteins ClpA, ClpB, and Hsp104. ClpB and Hsp104 rescue damaged proteins from toxic aggregates and do not partner with any protease. ClpA functions as the regulatory component of the ATP dependent protease complex ClpAP, and also remodels inactive RepA dimers into active monomers in the absence of the protease. Because ClpA functions both with and without a proteolytic component, it is an ideal system for developing strategies that address one of the major challenges in the study of protein remodeling machines: how do we observe a reaction in which the substrate protein does not undergo covalent modification? Here, we review experimental designs developed for the examination of polypeptide translocation catalyzed by the AAA+ motors in the absence of proteolytic degradation. We propose that transient state kinetic methods are essential for the examination of elementary kinetic mechanisms of these motor proteins. Furthermore, rigorous kinetic analysis must also account for the thermodynamic properties of these complicated systems that reside in a dynamic equilibrium of oligomeric states, including the biologically active hexamer.

Summary Persisters are phenotypic variants present within isogenic bacterial populations that exhibit extreme tolerance toward antibiotic stress. We previously elucidated a mechanistic pathway by which Escherichia coli persisters to ofloxacin form in response to a carbon source transition. Here, we examine how persisters to ampicillin form from the same metabolic stress and identify the shared and unique elements of the persister formation pathways. We discovered that diauxie-dependent formation of ampicillin persisters required RelA and that loss of clpA , ssrA , or smpB eliminated persister formation through relaxation of the stringent response. Further, we found that tolerance to ampicillin was achieved through broad inhibition of peptidoglycan biosynthesis, as evidenced by the formation of persisters to antibiotics that target enzymes in different areas of that biosynthetic pathway. Interestingly, ppGpp was required for formation of both ampicillin and ofloxacin persisters, and we demonstrated that higher synthesis of the alarmone was needed to increase persisters to ampicillin compared to ofloxacin. Further, we found trans-translation and DksA to be common mediators of both pathways, whereas ClpA was unique for ampicillin persisters and nucleoid-associated proteins were unique for ofloxacin persisters. These results highlight the need to consider an antibiotic’s mode of action when analyzing persister formation, demonstrate that individual stresses can produce persister heterogeneity, and emphasize the importance of identifying each respective pathway to identify common mediators that possess the most therapeutic potential to combat persisters. [ABSTRACT FROM AUTHOR]

Rate and level of DnaKJE-dependent refolding of the thermoinactivated Aliivibrio fischeri luciferase are considerably lower in Escherichia coli ibpA and ibpB, clpA mutants than in wild type cells. The rate and level of refolding are lower in E. coli ibpB:: kan than in ibpA:: kan cells. The decline of refolding level in E. coli clpA:: kan makes progress only with the increase of thermoinactivation time of luciferase. Plasmids with genes ibpAB do not compensate clpA mutation. It is supposed that small chaperones IpbAB and chaperone ClpA operate independently in a process of DnaKJE-dependent refolding of proteins at the different stages. [ABSTRACT FROM AUTHOR]

Abstract: ATP-dependent protein degradation in bacteria is carried out by barrel-shaped proteases architecturally related to the proteasome. In Escherichia coli, ClpP interacts with two alternative ATPases, ClpA or ClpX, to form active protease complexes. ClpAP and ClpXP show different but overlapping substrate specificities. ClpXP is considered the primary recipient of ssrA-tagged substrates while ClpAP in complex with ClpS processes N-end rule substrates. Notably, in its free form, but not in complex with ClpS, ClpAP also degrades ssrA-tagged substrates and its own chaperone component, ClpA. To reveal the mechanism of ClpAP-mediated ClpA degradation, termed autodegradation, and its possible role in regulating ClpAP levels, we dissected ClpA to show that the flexible C-terminus of the second AAA module serves as the degradation signal. We demonstrate that ClpA becomes largely resistant to autodegradation in the absence of its C-terminus and, conversely, transfer of the last 11 residues of ClpA to the C-terminus of green fluorescent protein (GFP) renders GFP a substrate of ClpAP. This autodegradation tag bears similarity to the ssrA-tag in its degradation behavior, displaying similar catalytic turnover rates when coupled to GFP but a twofold lower apparent affinity constant compared to ssrA-tagged GFP. We show that, in analogy to the prevention of ssrA-mediated recognition, the adaptor ClpS inhibits autodegradation by a specificity switch as opposed to direct masking of the degradation signal. Our results demonstrate that in the presence of ssrA-tagged substrates, ClpA autodegradation will be competitively reduced. This simple mechanism allows for dynamic reallocation of free ClpAP versus ClpAPS in response to the presence of ssrA-tagged substrates. [Copyright &y& Elsevier]

The rate and level of DnaK-dependent refolding of heat-inactivated Vibrio fischeri luciferase in the clp A mutant ( clp A:: kan) were considerably lower then in wild-type cells. The decline in refolding level progressed with increasing heat inactivation time. A mutation of clp P had no influence on the kinetics and level of luciferase refolding. Approximately equal amounts of the DnaKJE chaperone were synthesized upon heat shock induction in E. coli clp A + and E. coli clpA:: kan cells. It was assumed that, like homologous chaperone ClpB, ClpA is involved in disaggregation of denatured proteins, increasing the refolding efficiency. This in vivo phenomenon occurred only upon a prolonged incubation of cells at a higher temperature, which led to the formation of large protein aggregates that were poorly refoldable by the DnaKJE system. [ABSTRACT FROM AUTHOR]

Protein degradation in the cytosol of Escherichia coli is carried out by a variety of different proteolytic machines, including ClpAP. The ClpA component is a hexameric AAA+ (ATPase associated with various cellular activities) chaperone that utilizes the energy of ATP to control substrate recognition and unfolding. The precise role of the N-domains of ClpA in this process, however, remains elusive. Here, we have analysed the role of five highly conserved basic residues in the N-domain of ClpA by monitoring the binding, unfolding and degradation of several different substrates, including short unstructured peptides, tagged and untagged proteins. Interestingly, mutation of three of these basic residues within the N-domain of ClpA (H94, R86 and R100) did not alter substrate degradation. In contrast mutation of two conserved arginine residues (R90 and R131), flanking a putative peptide-binding groove within the N-domain of ClpA, specifically compromised the ability of ClpA to unfold and degrade selected substrates but did not prevent substrate recognition, ClpS-mediated substrate delivery or ClpP binding. In contrast, a highly conserved tyrosine residue lining the central pore of the ClpA hexamer was essential for the degradation of all substrate types analysed, including both folded and unstructured proteins. Taken together, these data suggest that ClpA utilizes two structural elements, one in the N-domain and the other in the pore of the hexamer, both of which are required for efficient unfolding of some protein substrates. [ABSTRACT FROM AUTHOR]

ClpB and Hsp104, members of the AAA+ superfamily of proteins, protect cells from the devastating effects of protein inactivation and aggregation that arise after extreme heat stress. They exist as a hexameric ring and contain two nucleotide-binding sites per monomer. CIpB and Hsp104 are able to dissolve protein aggregates in conjunction with the DnaK/Hsp70 chaperone system, although the roles of the individual chaperones in disaggregation are not well understood. In the absence of the DnaK/Hsp70 system, CIpB and Hsp104 alone are able to perform protein remodeling when their ATPase activity is asymmetrically slowed either by providing a mixture of ATP and ATPγS, a nonphysiological and slowly hydrolyzed ATP analog, or by inactivating one of the two nucleotide-binding domains by mutation. To gain insight into the roles of ClpB and the DnaK system in protein remodeling, we tested whether there was a further stimulation by the DnaK chaperone system under conditions that elicited remodeling activity by CIpB alone. Our results demonstrate that ClpB and the DnaK system act synergistically to remodel proteins and dissolve aggregates. The results further show that ATP is required and that both nucleotide- binding sites of CIpB must be able to hydrolyze ATP to permit functional collaboration between CIpB and the DnaK system. [ABSTRACT FROM AUTHOR]

A study was made of the refolding of bacterial luciferases of Vibrio fischeri, V. harveyi, Photobacterium phosphoreum, and Photorhabdus luminescens. By reaction rate, luciferases were divided into two groups. The reaction rate constants of fast luciferases of V. fischeri and Ph. phosphoreum were about tenfold higher than those of slow luciferases of Ph. luminescens and V. harveyi. The order of increasing luciferase thermostability was Ph. phosphoreum, V. fischeri, V. harveyi, and Ph. luminescens. The refolding of thermoinactivated luciferases completely depended on the active DnaK–DnaJ–GrpE chaperone system. Thermolabile fast luciferases of V. fischeri and Ph. phosphoreum showed highly efficient rapid refolding. Slower and less efficient refolding was characteristic of thermostable slow luciferases of V. harveyi and Ph. luminescens. Chaperones of the Clp family were tested for effect on the efficiency of DnaK-dependent refolding of bacterial luciferases in Escherichia coli cells. The rate and extent of refolding were considerably lower in the clpB mutant than in wild-type cells. In E. coli cells with mutant clpA, clpP, of clpX showed a substantially lower luciferase refolding after heat shock. [ABSTRACT FROM AUTHOR]

Escherichia coli ClpA, an Hsp100/Clp chaperone and an integral component of the ATP-dependent ClpAP protease, participates in the dissolution and degradation of regulatory proteins and protein aggregates. ClpA consists of three functional domains: an N-terminal domain and two ATPase domains, D1 and D2. The N-domain is attached to D1 by a mobile linker and is made up of two tightly bound, identically folded α-helical bundles related by a pseudo 2-fold symmetry. Between the halves of the pseudo-dimer is a large flexible acidic loop that becomes better ordered upon binding of the small adaptor protein, ClpS. We have identified a number of structural features in the N-domain, including a Zn++ binding motif, several interfaces for binding to ClpS, and a prominent hydrophobic surface area that binds peptides in different configurations. These structural motifs may contribute to binding of protein or peptide substrates with weak affinity and broad specificity. Kinetic studies comparing wild-type ClpA to a mutant ClpA with its N-domain deleted show that the N-domains contribute to the binding of a non-specific protein substrate but not of a folded substrate with the specific SsrA recognition tag. A functional model is proposed in which the N-domains in ClpA function as tentacles to weakly hold on to proteins thereby enhancing local substrate concentration. [Copyright &y& Elsevier]

ClpS is a small protein, usually encoded immediately upstream of ClpA in the genomes of proteobacteria. Recent results show that it is a molecular adaptor for substrate recognition by ClpA in Escherichia coli. We analyzed ClpS by bioinformatic methods and found that ClpS homologs are also found in organisms that lack ClpA, such as actinobacteria, cyanobacteria, and plant chloroplasts. Furthermore, ClpS is homologous to a domain in the eukaryotic E3 ubiquitin ligase, N-recognin. This domain has previously been described as responsible for the recognition of type 2 N-end rule substrates. Despite very low levels of sequence similarity to proteins of known structure, there appears to be substantial structural similarity between ClpS and the C-terminal domain of ribosomal protein L7/12 (1CTF). [Copyright &y& Elsevier]

The role of chaperones Hsp70 (DnaK–DnaJ–GrpE) and Hsp100 (ClpA–ClpB–ClpX) in refolding of thermoinactivated luciferase from the marine bacterium Photobacterium fischeri and the terrestrial bacterium Photorhabdus luminescens has been studied. These luciferases are homologous, but differ greatly in the rate of thermal inactivation and the rate constant for the luminescence reaction. It was shown that refolding of thermoinactivated luciferases is completely determined by the DnaK–DnaJ–GrpE system. However these luciferases markedly differ in the rate and degree of refolding. The degree of refolding of thermolabile “quick” Ph. fischeri luciferase reaches 80% of the initial level over several minutes, whereas renaturation of thermostable “slow” Ph. luminescens luciferase proceeds substantially slower (the degree of renaturation reaches only ∼7-8% of the initial level over tens of minutes). The measurement of the rate of thermal inactivation of luciferases in vivo in the cells of Escherichia coli wild strain and strains containing mutations in genes clpA, clpB, clpX showed that Ph. luminescens luciferase revealed reduced thermostability in mutant strain E. coli clpA–. It was shown that this effect was not connected with DnaK-dependent refolding. In the case of thermolabile Ph. fischeri luciferase, mutation in gene clpA has no effect on the shape of the curve of thermal inactivation. These data suggest that denatured Ph. luminescens luciferase has enhanced affinity with respect to chaperone ClpA in comparison with DnaK, whereas thermolabile Ph. fischeri luciferase is characterized by enhanced affinity with respect to chaperone DnaK. Denatured luciferase bound to ClpA does not aggregate and following refolding proceeds probably spontaneously and very quickly (over 1-2 min). It is evident that the process under discussion requires ATP, since the addition of uncoupler of oxidative phosphorylation carbonyl cyanide 3-chlorophenylhydra-zone results in a sharp decrease in thermal stability of luciferase to the level typical of the enzyme in vitro. The enhanced thermosensitivity of luciferases was observed also in E. coli containing mutations in gene clpB. However, this effect, which takes place for Ph. fischeri luciferase as well as for Ph. LuminescensM luciferase, is determined by DnaK-dependent refolding and probably connected with the ability of chaperone ClpB to provide disaggregation of the proteins, resulting in their interaction with chaperones of the Hsp70 family (DnaK–DnaJ–GrpE). [ABSTRACT FROM AUTHOR]

Numerous ATPases associated with diverse cellular activities (AAA+) proteins form hexameric, ring-shaped complexes that function via ATPase-coupled translocation of substrates across the central channel. Cryo-electron microscopy of AAA+ proteins processing substrate has revealed non-symmetric, staircase-like hexameric structures that indicate a sequential clockwise/2-residue step translocation model for these motors. However, for many of the AAA+ proteins that share similar structural features, their translocation properties have not yet been experimentally determined. In the cases where translocation mechanisms have been determined, a two-residue translocation step-size has not been resolved. In this review, we explore Hsp104, ClpB, ClpA and ClpX as examples to review the experimental methods that have been used to examine, in solution, the translocation mechanisms employed by AAA+ motor proteins. We then ask whether AAA+ motors sharing similar structural features can have different translocation mechanisms. Finally, we discuss whether a single AAA+ motor can adopt multiple translocation mechanisms that are responsive to different challenges imposed by the substrate or the environment. We suggest that AAA+ motors adopt more than one translocation mechanism and are tuned to switch to the most energetically efficient mechanism when constraints are applied., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The type VI secretion system (T6SS) is an effector delivery system used by Gram-negative bacteria to kill other bacteria or eukaryotic hosts to gain fitness. The plant pathogen Agrobacterium tumefaciens utilizes its T6SS to kill other bacteria, such as Escherichia coli . We observed that the A. tumefaciens T6SS-dependent killing outcome differs when using different T6SS-lacking, K-12 E. coli strains as a recipient cell. Thus, we hypothesized that the A. tumefaciens T6SS killing outcome not only relies on the T6SS activity of the attacker cells but also depends on the recipient cells. Here, we developed a high-throughput interbacterial competition platform to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. Among the 3,909 strains in the E. coli Keio library screened, 16 mutants with less susceptibility to A. tumefaciens C58 T6SS-dependent killing were identified, and four of them were validated by complementation test. Among the four, the clpP encoding ClpP protease, which is universal and highly conserved in both prokaryotes and eukaryotic organelles, was selected for further characterizations. We demonstrated that ClpP is responsible for enhancing susceptibility to the T6SS killing. Because ClpP protease depends on other adapter proteins such as ClpA and ClpX for substrate recognition, further mutant studies followed by complementation tests were carried out to reveal that ClpP-associated AAA + ATPase ClpA, but not ClpX, is involved in enhancing susceptibility to A. tumefaciens T6SS killing. Moreover, functional and biochemical studies of various ClpP amino acid substitution variants provided evidence that ClpA-ClpP interaction is critical in enhancing susceptibility to the T6SS killing. This study highlights the importance of recipient factors in determining the outcome of the T6SS killing and shows the universal ClpP protease as a novel recipient factor hijacked by the T6SS of A. tumefaciens ., (Copyright © 2020 Lin, Yu, Sriramoju, Hsu, Liu and Lai.)

Cellular proteostasis involves not only the expression of proteins in response to environmental needs, but also the timely repair or removal of damaged or unneeded proteins. AAA+ motor proteins are critically involved in these pathways. Here, we review the structure and function of AAA+ proteins ClpA, ClpB, and Hsp104. ClpB and Hsp104 rescue damaged proteins from toxic aggregates and do not partner with any protease. ClpA functions as the regulatory component of the ATP dependent protease complex ClpAP, and also remodels inactive RepA dimers into active monomers in the absence of the protease. Because ClpA functions both with and without a proteolytic component, it is an ideal system for developing strategies that address one of the major challenges in the study of protein remodeling machines: how do we observe a reaction in which the substrate protein does not undergo covalent modification? Here, we review experimental designs developed for the examination of polypeptide translocation catalyzed by the AAA+ motors in the absence of proteolytic degradation. We propose that transient state kinetic methods are essential for the examination of elementary kinetic mechanisms of these motor proteins. Furthermore, rigorous kinetic analysis must also account for the thermodynamic properties of these complicated systems that reside in a dynamic equilibrium of oligomeric states, including the biologically active hexamer.