Plasmid-free Cells Research Articles

Integration Site Library for Efficient Construction of Plasmid-Free Microbial Cell Factories in Escherichia coli.

Enhanced production stability and efficiency along with a decrease in production costs are required to build efficient microbial cell factories. Target genes can be integrated into the genome to enhance genetic stability, reduce reliance on antibiotics, and alleviate the metabolic burden. However, selecting the optimal insertion site for the desired gene expression levels remains challenging. Therefore, 18 commonly usedEscherichia coliintegration sites were systematically characterized in this study. Promoters of different strengths were combined with integration sites, yielding a differential intensity range of up to 93-fold. This indicated the versatility and precision of this approach for controlling gene expression levels. Referring to the library, pathway genes were strategically integrated into theE. coligenome based on their respective expression levels. Genetically stable and highly efficient engineered strains that could biosynthesize arbutin and p-aminobenzoic acid were constructed.

Coupling antitoxins and blue/white screening with parAB/resolvase mutation as a strategy for Salmonella spp. plasmid curing.

Plasmids play an important role in bacterial physiology, adaptation, evolution, virulence, and antibiotic resistance. An in-depth study of these roles partly depends on the generation of plasmid-free cells. This study shows that vector tools that target genes required for plasmid stability in the presence of an antitoxin-expressing helper plasmid are a viable approach to cure specific plasmids. Expression of bgaB from target plasmids can greatly facilitate visual detection of plasmid cured colonies avoiding time-consuming screening procedures. This approach can be refined for the development of a universal plasmid curing system that can be used to generate plasmid-free cells in other human bacterial pathogens including Gram positives and Gram negatives.

The histone-like nucleoid-structuring protein encoded by the plasmid pMBL6842 regulates both plasmid stability and host physiology of Pseudoalteromonas rubra SCSIO 6842

Plasmids orchestrate bacterial adaptation across diverse environments and facilitate lateral gene transfer within bacterial communities. Their presence can perturb host metabolism, creating a competitive advantage for plasmid-free cells. Plasmid stability hinges on efficient replication and partition mechanisms. While plasmids commonly encode histone-like nucleoid-structuring (H-NS) family proteins, the precise influence of plasmid-encoded H-NS proteins on stability remains elusive. In this study, we examined the conjugative plasmid pMBL6842, harboring the hns gene, and observed its positive regulation of parAB transcription, critical for plasmid segregation. Deletion of hns led to rapid plasmid loss, which was remedied by hns complementation. Further investigations unveiled adverse effects of hns overexpression on the bacterial host. Transcriptome analysis revealed hns's role in regulating numerous bacterial genes, impacting both host growth and swimming motility in the presence of the hns gene. Therefore, our study unveils the multifaceted roles of H-NS in both plasmid stability and host physiology, underscoring its biological significance and paving the way for future inquiries into the involvement of H-NS in horizontal gene transfer events.

Modelling Plasmid-Mediated Horizontal Gene Transfer in Biofilms

In this study, we present a mathematical model for plasmid spread in a growing biofilm, formulated as a nonlocal system of partial differential equations in a 1-D free boundary domain. Plasmids are mobile genetic elements able to transfer to different phylotypes, posing a global health problem when they carry antibiotic resistance factors. We model gene transfer regulation influenced by nearby potential receptors to account for recipient-sensing. We also introduce a promotion function to account for trace metal effects on conjugation, based on literature data. The model qualitatively matches experimental results, showing that contaminants like toxic metals and antibiotics promote plasmid persistence by favoring plasmid carriers and stimulating conjugation. Even at higher contaminant concentrations inhibiting conjugation, plasmid spread persists by strongly inhibiting plasmid-free cells. The model also replicates higher plasmid density in biofilm’s most active regions.

Plasmid-free cheater cells commonly evolve during laboratory growth.

It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, extracellular beta-lactamases produced by resistant cells that subsequently degrade penicillin and related antibiotics allow neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show in multiple bacterial species that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface-grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss was still observed. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated.IMPORTANCEPlasmids are routinely used in microbiology as readouts of cell biology or tools to manipulate cell function. Central to these studies is the assumption that all cells in an experiment contain the plasmid. Plasmid maintenance in a host cell typically depends on a plasmid-encoded antibiotic resistance marker, which provides a selective advantage when the plasmid-containing cell is grown in the presence of antibiotic. Here, we find that growth of plasmid-containing bacteria on a surface and to a lesser extent in liquid culture in the presence of three distinct antibiotic families leads to the evolution of a significant number of plasmid-free cells, which rely on the resistance mechanisms of the plasmid-containing cells. This process generates a heterogenous population of plasmid-free and plasmid-containing bacteria, an outcome which could confound further experimentation.

Plasmids Increase the Competitive Ability of Plasmid-Bearing Cells Even When Transconjugants Are Poor Donors, as Shown by Computer Simulations.

Bacterial cells often suffer a fitness cost after conjugative plasmids' entry because these cells replicate slower than plasmid-free cells. Compensatory mutations may appear after tens of or a few hundred generations, reducing or eliminating this cost. A previous work based on a mathematical model and computer simulations has shown that plasmid-bearing cells already adapted to the plasmid may gain a fitness advantage when plasmids transfer into neighboring plasmid-free cells because these cells are still unadapted to the plasmid. These slow-growing transconjugants use fewer resources, which can benefit donor cells. However, opportunities for compensatory mutations in transconjugants increase if these cells become numerous (through replication or conjugation). Moreover, transconjugants also gain an advantage when transferring the plasmid, but the original donors may be too distant from conjugation events to gain an advantage. To understand which consequence prevails, we performed further computer simulations allowing versus banning transfer from transconjugants. The advantage to donors is higher if transconjugants do not transfer plasmids, mainly when donors are rare and when the plasmid transfer rate (from donors) is high. These results show that conjugative plasmids are efficient biological weapons even if the transconjugant cells are poor plasmid donors. After some time, conjugative plasmids gain other host-benefit genes, such as virulence and drug-resistance.

Plasmid Costs Explain Plasmid Maintenance, Irrespective of the Nature of Compensatory Mutations.

Conjugative plasmids often carry virulence and antibiotic-resistant genes. Therefore, understanding the behavior of these extra-chromosomal DNA elements gives insights into their spread. Bacteria frequently replicate slower after plasmids' entry, an observation inconsistent with the plasmids' ubiquity in nature. Several hypotheses explain the maintenance of plasmids among bacterial communities. However, the numerous combinations of bacterial species and strains, plasmids, and environments claim a robust elucidatory mechanism of plasmid maintenance. Previous works have shown that donor cells already adapted to the plasmid may use the plasmid as a 'weapon' to compete with non-adapted plasmid-free cells. Computer simulations corroborated this hypothesis with a wide range of parameters. Here we show that donor cells benefit from harboring conjugative plasmids even if compensatory mutations in transconjugant cells occur in the plasmid, not on chromosomes. The advantage's leading causes are as follows: mutations take time to appear, many plasmids remain costly, and re-transfer of mutated plasmids usually occurs in sites distant to the original donors, implying little competition between these cells. Research in previous decades cautioned against uncritical acceptance of the hypothesis that resistance cost helps to preserve antibiotics' effectiveness. This work gives a new twist to this conclusion by showing that costs help antibiotic-resistant bacteria to compete with plasmid-free cells even if compensatory mutations appear in plasmids.

Is cooperation favored by horizontal gene transfer?

It has been hypothesized that horizontal gene transfer on plasmids can facilitate the evolution of cooperation, by allowing genes to jump between bacteria, and hence increase genetic relatedness at the cooperative loci. However, we show theoretically that horizontal gene transfer only appreciably increases relatedness when plasmids are rare, where there are many plasmid-free cells available to infect (many opportunities for horizontal gene transfer). In contrast, when plasmids are common, there are few opportunities for horizontal gene transfer, meaning relatedness is not appreciably increased, and so cooperation is not favored. Plasmids, therefore, evolve to be rare and cooperative, or common and noncooperative, meaning plasmid frequency and cooperativeness are never simultaneously high. The overall level of plasmid-mediated cooperation, given by the product of plasmid frequency and cooperativeness, is therefore consistently negligible or low.

Plasmids Can Shift Bacterial Morphological Response against Antibiotic Stress.

Bacterial cell filamentation is a morphological change wherein cell division is blocked, which can improve bacterial survival under unfavorable conditions (e.g., antibiotic stress that causes DNA damage). As an extrachromosomal DNA molecule, plasmids can confer additionally advantageous traits including antibiotic resistance on the host. However, little is known about whether plasmids could shift bacterial morphological responses to antibiotic stress. Here, it is reported that plasmid-free cells, rather than plasmid-bearing cells, exhibit filamentation and asymmetrical cell division under exposure to sub-inhibitory concentrations of antibiotics (ciprofloxacin and cephalexin). The underlying mechanism is revealed by investigating DNA damage, cell division inhibitor sulA, the SOS response, toxin-antitoxin module (parDE) located on plasmids, and efflux pumps. Significantly higher expression of sulA is observed in plasmid-free cells, compared to plasmid-bearing cells. Plasmid carriage enables the hosts to suffer less DNA damage, exhibit stronger efflux pump activities, and thus have a higher antibiotic tolerance. These benefits are attributed to the parDE module that mediates stress responses from plasmid-bearing cells and mainly contributes to cell morphological changes. Collectively, the findings demonstrate that plasmids can confer additional innate defenses on the host to antibiotics, thus advancing the understanding of how plasmids affect bacterial evolution in hostile environments.

Harmful behaviour through plasmid transfer: a successful evolutionary strategy of bacteria harbouring conjugative plasmids.

Conjugative plasmids are extrachromosomal mobile genetic elements pervasive among bacteria. Plasmids' acquisition often lowers cells' growth rate, so their ubiquity has been a matter of debate. Chromosomes occasionally mutate, rendering plasmids cost-free. However, these compensatory mutations typically take hundreds of generations to appear after plasmid arrival. By then, it could be too late to compete with fast-growing plasmid-free cells successfully. Moreover, arriving plasmids would have to wait hundreds of generations for compensatory mutations to appear in the chromosome of their new host. We hypothesize that plasmid-donor cells may use the plasmid as a 'weapon' to compete with plasmid-free cells, particularly in structured environments. Cells already adapted to plasmids may increase their inclusive fitness through plasmid transfer to impose a cost to nearby plasmid-free cells and increase the replication opportunities of nearby relatives. A mathematical model suggests conditions under which the proposed hypothesis works, and computer simulations tested the long-term plasmid maintenance. Our hypothesis explains the maintenance of conjugative plasmids not coding for beneficial genes. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Vertical and Horizontal Transmission of ESBL Plasmid from Escherichia coli O104:H4.

Multidrug resistance (MDR) often results from the acquisition of mobile genetic elements (MGEs) that encode MDR gene(s), such as conjugative plasmids. The spread of MDR plasmids is founded on their ability of horizontal transference, as well as their faithful inheritance in progeny cells. Here, we investigated the genetic factors involved in the prevalence of the IncI conjugative plasmid pESBL, which was isolated from the Escherichia coli O104:H4 outbreak strain in Germany in 2011. Using transposon-insertion sequencing, we identified the pESBL partitioning locus (par). Genetic, biochemical and microscopic approaches allowed pESBL to be characterized as a new member of the Type Ib partitioning system. Inactivation of par caused mis-segregation of pESBL followed by post-segregational killing (PSK), resulting in a great fitness disadvantage but apparent plasmid stability in the population of viable cells. We constructed a variety of pESBL derivatives with different combinations of mutations in par, conjugational transfer (oriT) and pnd toxin-antitoxin (TA) genes. Only the triple mutant exhibited plasmid-free cells in viable cell populations. Time-lapse tracking of plasmid dynamics in microfluidics indicated that inactivation of pnd improved the survival of plasmid-free cells and allowed oriT-dependent re-acquisition of the plasmid. Altogether, the three factors—active partitioning, toxin-antitoxin and conjugational transfer—are all involved in the prevalence of pESBL in the E. coli population.

Plasmid Interactions Can Improve Plasmid Persistence in Bacterial Populations.

It is difficult to understand plasmid maintenance in the absence of selection and theoretical models predict the conditions for plasmid persistence to be limited. Plasmid-associated fitness costs decrease bacterial competitivity, while imperfect partition allows the emergence of plasmid-free cells during cell division. Although plasmid conjugative transfer allows mobility into plasmid-free cells, the rate of such events is generally not high enough to ensure plasmid persistence. Experimental data suggest several factors that may expand the conditions favorable for plasmid maintenance, such as compensatory mutations and accessory genes that allow positive selection. Most of the previous studies focus on bacteria that carry a single plasmid. However, there is increasing evidence that multiple plasmids inhabit the same bacterial population and that interactions between them affect their transmission and persistence. Here, we adapt previous mathematical models to include multiple plasmids and perform computer simulations to study how interactions among them affect plasmid maintenance. We tested the contribution of different plasmid interaction parameters that impact three biological features: host fitness, conjugative transfer and plasmid loss – which affect plasmid persistence. The interaction affecting conjugation was studied in the contexts of intracellular and intercellular interactions, i.e., the plasmids interact when present in the same cell or when in different cells, respectively. First, we tested the effect of each type of interaction alone and concluded that only interactions affecting fitness (epistasis) prevented plasmid extinction. Although not allowing plasmid maintenance, intracellular interactions increasing conjugative efficiencies had a more determinant impact in delaying extinction than the remaining parameters. Then, we allowed multiple interactions between plasmids and concluded that, in a few cases, a combined effect of (intracellular) interactions increasing conjugation and fitness lead to plasmid maintenance. Our results show a hierarchy among these interaction parameters. Those affecting fitness favor plasmid persistence more than those affecting conjugative transfer and lastly plasmid loss. These results suggest that interactions between different plasmids can favor their persistence in bacterial communities.

Ratiometric quorum sensing governs the trade-off between bacterial vertical and horizontal antibiotic resistance propagation.

Plasmid-mediated horizontal gene transfer of antibiotic resistance and virulence in pathogenic bacteria underlies a major public health issue. Understanding how, in the absence of antibiotic-mediated selection, plasmid-bearing cells avoid being outnumbered by plasmid-free cells is key to developing counterstrategies. Here, we quantified the induction of the plasmidial sex pheromone pathway of Enterococcus faecalis to show that the integration of the stimulatory (mate-sensing) and inhibitory (self-sensing) signaling modules from the pCF10 conjugative plasmid provides a precise measure of the recipient-to-donor ratio, agnostic to variations in population size. Such ratiometric control of conjugation favors vertical plasmid transfer under low mating likelihood and allows activation of conjugation functions only under high mating likelihood. We further show that this strategy constitutes a cost-effective investment into mating effort because overstimulation produces unproductive self-aggregation and growth rate reduction. A mathematical model suggests that ratiometric control of conjugation increases plasmid fitness and predicts a robust long-term, stable coexistence of donors and recipients. Our results demonstrate how population-level parameters can control transfer of antibiotic resistance in bacteria, opening the door for biotic control strategies.

Inter and intra species social exploitation of antimicrobial resistance alters antibiotic efficacy

The widespread use and misuse of antibiotics has led to the global spread of antimicrobial resistance. It is increasingly evident that very low concentrations of antibiotics, well below the MIC of sensitive strains, can select for antimicrobial resistance. However, it is less clear how social interactions within bacterial communities can alter sub-MIC selection dynamics, potentially confounding the outcomes of antibiotic treatments. Here we explore how antimicrobial resistances that inactivate antibiotics can be socially exploited by sensitive members of the microbial community at the inter and intra species level. We first show that a beta-lactamase encoded multi-drug resistance plasmid provides high levels of protection to plasmid-free antibiotic-sensitive cells within single-species populations in a frequency-dependent manner. Second, a similar protection can also occur between different species during polymicrobial infections. Using model Cystic Fibrosis lung communities, we demonstrate that the focal pathogen Pseudomonas aeruginosa can socially exploit antibiotic resistance in the presence of Stenotrophomonas maltophilia bacterium that can hydrolyse imipenem antibiotic. In contrast, the presence of Staphylococcus aureus, another commonly co-occurring CF pathogen, provided P. aeruginosa with no protection, but instead, made P. aeruginosa more susceptible to antibiotic due to intensified competition. These findings reveal that social exploitation of pre-occurring antimicrobial resistance, and inter-specific competition, can have a large effect on the efficacy of antibiotic treatments, highlighting the importance of microbial ecology for understanding antibiotic resistance evolution.

Plasmid expression level heterogeneity monitoring via heterologous eGFP production at the single-cell level in Cupriavidus necator.

A methodology for plasmid expression level monitoring of eGFP expression suitable for dynamic processes was assessed during fermentation. This technique was based on the expression of a fluorescent biosensor (eGFP) encoded on a recombinant plasmid coupled to single-cell analysis. Fluorescence intensity at single-cell level was measured by flow cytometry. We demonstrated that promoter evaluation based on single-cell analysis versus classic global analysis brings valuable insights. Single-cell analysis pointed out the fact that intrinsic fluorescence increased with the strength of the promoter up to a threshold. Beyond that, cell permeability increases to excrete the fluorescent protein in the medium. The metabolic load due to the increase in the eGFP production in the case of strong constitutive promoters leads to slower growth kinetics compared with plasmid-free cells. With the strain Cupriavidus necator Re2133, growth rate losses were measured from 3% with the weak constitutive promoter Plac to 56% with the strong constitutive promoter Pj5. Through this work, it seems crucial to find a compromise between the fluorescence intensity in single cells and the metabolic load; in our conditions, the best compromise found was the weak promoter Plac. The plasmid expression level monitoring method was tested in the presence of a heterogeneous population induced by plasmid-curing methods. For all the identified subpopulations, the plasmid expression level heterogeneity was significantly detected at the level of fluorescence intensity in single cells. After cell sorting, growth rate and cultivability were assessed for each subpopulation. In conclusion, this eGFP biosensor makes it possible to follow the variations in the level of plasmid expression under conditions of population heterogeneity.Key Points•Development of a plasmid expression level monitoring method at the single-cell level by flow cytometry.•Promoter evaluation by single-cell analysis: cell heterogeneity and strain robustness.•Reporter system optimization for efficient subpopulation detection in pure cultures.

Two New Plasmid Post-segregational Killing Mechanisms for the Implementation of Synthetic Gene Networks in Escherichia coli

SummaryPlasmids are the workhorse of both industrial biotechnology and synthetic biology, but ensuring they remain in bacterial cells is a challenge. Antibiotic selection cannot be used to stabilize plasmids in most real-world applications, and inserting dynamical gene networks into the genome remains challenging. Plasmids have evolved several mechanisms for stability, one of which, post-segregational killing (PSK), ensures that plasmid-free cells do not survive. Here we demonstrate the plasmid-stabilizing capabilities of the axe/txe toxin-antitoxin system and the microcin-V bacteriocin system in the probiotic bacteria Escherichia coli Nissle 1917 and show that they can outperform the commonly used hok/sok. Using plasmid stability assays, automated flow cytometry analysis, mathematical models, and Bayesian statistics we quantified plasmid stability in vitro. Furthermore, we used an in vivo mouse cancer model to demonstrate plasmid stability in a real-world therapeutic setting. These new PSK systems, plus the developed Bayesian methodology, will have wide applicability in clinical and industrial biotechnology.

Parasitic plasmid-host dynamics and host competition in flowing habitats

Competition and coexistence were examined for two bacterial species, each potentially carrying a fitness-reducing, parasitic plasmid that was vertically transmitted with possible loss through segregation. Here, the fitness reduction of hosts was due to a toxin produced by plasmid-bearing cells and inhibiting plasmid-free cells. These populations were placed in a flow reactor habitat representing an idealized mammal gut. It was numerically shown that parasitic plasmids can mediate coexistence of competing host species, in conditions where plasmid-free hosts could not coexist. Numerical construction of a coexistence example suggests that it arises only for a narrow parameter range. In particular, both rates of segregation and the growth costs of plasmid carriage must be relatively low.

Harnessing novel bacterial peptides for antimicrobial activity in the gut microbiome

The Enterococci are a resilient collection of species, found in the human intestine, river sediment and even certain cheeses. Human infection by this genus is dominated by E. faecalis and E. faecium. Vancomycin resistant enterococci (VRE) are associated with higher mortality rates over non-VRE strains. Enterococci can utilise the highly efficient pheromone responsive plasmid (PRP) system to transfer plasmid DNA between cells. Plasmid containing donor cells respond to small peptide pheromones (7–8 amino acids) and transfer plasmid DNA to pheromone-producing plasmid-free recipient cells. PRP can encode antibiotic resistance (including vancomycin) and virulence enhancing factors. Investigation into the PRP system between donor and recipient E. faecalis environmental isolates has indicated a 40 % decrease in PRP transfer in colder environments. Additionally, PRP efficiencies under other conditions, including in presence of synthetic pheromone peptides, have been calculated. Future assays will utilise pheromone imitative fluorescently labelled synthetic peptides to visualise the pheromone binding receptor (PrgZ) on the E. faecalis donor cell membrane. Later experiments will focus on varying the synthetic pheromone amino acid composition so to interfere with the PRP system machinery, with the aim of reducing PRP transfer efficiency or preventing PRP transfer completely.

Plasmid curing resulted in improved heterologous gene expression inLeuconostoc citreumEFEL2700

Leuconostoc citreum EFEL2700 isolated from kimchi was used as a host strain for genetic and metabolic engineering in our previous studies, but the cells of EFEL2700 contained a cryptic plasmid (P-cells). Thus, we created plasmid-free cells (F-cells) using the CRISPR/Cas9 system. In this study, we compared the microbial characteristics of P- and F-cells in terms of growth rate, biochemical properties, transformation efficiency, plasmid copy number and protein expression level. When the growth rate was measured in MRS medium at 30°C, no significant difference (P>0·01) was observed. Biochemical properties, tested using an API 50CHL kit, showed no differences. Transformation efficiency of F-cells, measured using pCB4270, was higher (1·3×104 CFU per μg DNA) than that of P-cells (5·0×103 CFU per μg DNA). Copy number after transformation of pCBBgl was 4-fold higher for F-cells than for P-cells. When β-glucosidase activity was assayed in the above experiment, F-cells showed 3·4-fold higher values than P-cells. In conclusion, this study demonstrates that plasmid curing in L. citreum EFEL2700 improves its characteristics as a gene expression host. SIGNIFICANCE AND IMPACT OF THE STUDY: Leuconostoc citreum EFEL2700 (P-cell) isolated from kimchi is a useful food-grade host for expressing heterologous genes. The presence of a cryptic plasmid is thought to limit efficient gene expression. In this study, we compared the microbial and genetic changes after plasmid curing in this strain. The plasmid-free strain showed improved levels of transformation efficiency, copy number and heterologous gene expression without alterations in phenotypes such as the growth rates and biochemical properties. The resulting strain of L. citreum EFEL2701 (F-cell) can be used as an efficient host for genetic engineering.

ClpAP protease is a universal factor that activates the parDE toxin-antitoxin system from a broad host range RK2 plasmid

The activity of type II toxin-antitoxin systems (TA), which are responsible for many important features of bacterial cells, is based on the differences between toxin and antitoxin stabilities. The antitoxin lability results from bacterial protease activity. Here, we investigated how particular Escherichia coli cytosolic proteases, namely, Lon, ClpAP, ClpXP, and ClpYQ, affect the stability of both the toxin and antitoxin components of the parDE system from the broad host range plasmid RK2. The results of our in vivo and in vitro experiments show that the ParD antitoxin is degraded by the ClpAP protease, and dsDNA stimulates this process. The ParE toxin is not degraded by any of these proteases and can therefore cause growth inhibition of plasmid-free cells after an unequal plasmid distribution during cell division. We also demonstrate that the ParE toxin interaction with ParD prevents antitoxin proteolysis by ClpAP; however, this interaction does not prevent the ClpAP interaction with ParD. We show that ClpAP protease homologs affect plasmid stability in other bacterial species, indicating that ClpAP is a universal activator of the parDE system and that ParD is a universal substrate for ClpAP.