Zinc-finger protein Zpr1 is a bespoke chaperone essential for eEF1A biogenesis.

The conserved regulon of heat shock factor 1 in budding yeast contains chaperones for general protein folding as well as zinc-finger protein Zpr1, whose essential role in archaea and eukaryotes remains unknown. Here, we show that Zpr1 depletion causes acute proteotoxicity driven by biosynthesis of misfolded eukaryotic translation elongation factor 1A (eEF1A). Prolonged Zpr1 depletion leads to eEF1A insufficiency, thereby inducing the integrated stress response and inhibiting protein synthesis. Strikingly, we show by using two distinct biochemical reconstitution approaches that Zpr1 enables eEF1A to achieve a conformational state resistant to protease digestion. Lastly, we use a ColabFold model of the Zpr1-eEF1A complex to reveal a folding mechanism mediated by the Zpr1's zinc-finger and alpha-helical hairpin structures. Our work uncovers the long-sought-after function of Zpr1 as a bespoke chaperone tailored to the biogenesis of one of the most abundant proteins in the cell.

Correction: Ploidy and recombination proficiency shape the evolutionary adaptation to constitutive DNA replication stress.

[This corrects the article DOI: 10.1371/journal.pgen.1009875.].

Heterozygous mutations cause genetic instability in a yeast model of cancer evolution.

Genetic instability, a heritable increase in the rate of genetic mutation, accelerates evolutionary adaptation1 and is widespread in cancer2,3. In mammals, instability can arise from damage to both copies of genes involved in DNA metabolism and cell cycle regulation4 or from inactivation of one copy of a gene whose product is present in limiting amounts (haploinsufficiency5); however, it has proved difficult to determine the relative importance of these two mechanisms. In Escherichia coli6, the application of repeated, strong selection enriches for genetic instability. Here we have used this approach to evolve genetic instability in diploid cells of the budding yeast Saccharomyces cerevisiae, and have isolated clones with increased rates of point mutation, mitotic recombination, and chromosome loss. We identified candidate, heterozygous, instability-causing mutations; engineering these mutations, as heterozygotes, into the ancestral diploid strain caused genetic instability. Mutations that inactivated one copy of haploinsufficient genes were more common than those that dominantly altered the function of the mutated gene copy. The mutated genes were enriched for genes functioning in transport, protein quality control, and DNA metabolism, and have revealed new targets for genetic instability7-11, including essential genes. Although only a minority (10 out of 57 genes with orthologues or close homologues) of the targets we identified have homologous human genes that have been implicated in cancer2, the remainder are candidates to contribute to human genetic instability. To test this hypothesis, we inactivated six examples in a near-haploid human cell line; five of these mutations increased instability. We conclude that single genetic events cause genetic instability in diploid yeast cells, and propose that similar, heterozygous mutations in mammalian homologues initiate genetic instability in cancer.

Ploidy and recombination proficiency shape the evolutionary adaptation to constitutive DNA replication stress.

In haploid budding yeast, evolutionary adaptation to constitutive DNA replication stress alters three genome maintenance modules: DNA replication, the DNA damage checkpoint, and sister chromatid cohesion. We asked how these trajectories depend on genomic features by comparing the adaptation in three strains: haploids, diploids, and recombination deficient haploids. In all three, adaptation happens within 1000 generations at rates that are correlated with the initial fitness defect of the ancestors. Mutations in individual genes are selected at different frequencies in populations with different genomic features, but the benefits these mutations confer are similar in the three strains, and combinations of these mutations reproduce the fitness gains of evolved populations. Despite the differences in the selected mutations, adaptation targets the same three functional modules in strains with different genomic features, revealing a common evolutionary response to constitutive DNA replication stress.

Many, but not all, lineage-specific genes can be explained by homology detection failure.

Genes for which homologs can be detected only in a limited group of evolutionarily related species, called "lineage-specific genes," are pervasive: Essentially every lineage has them, and they often comprise a sizable fraction of the group's total genes. Lineage-specific genes are often interpreted as "novel" genes, representing genetic novelty born anew within that lineage. Here, we develop a simple method to test an alternative null hypothesis: that lineage-specific genes do have homologs outside of the lineage that, even while evolving at a constant rate in a novelty-free manner, have merely become undetectable by search algorithms used to infer homology. We show that this null hypothesis is sufficient to explain the lack of detected homologs of a large number of lineage-specific genes in fungi and insects. However, we also find that a minority of lineage-specific genes in both clades are not well explained by this novelty-free model. The method provides a simple way of identifying which lineage-specific genes call for special explanations beyond homology detection failure, highlighting them as interesting candidates for further study.

Evolutionary repair: Changes in multiple functional modules allow meiotic cohesin to support mitosis.

The role of proteins often changes during evolution, but we do not know how cells adapt when a protein is asked to participate in a different biological function. We forced the budding yeast, Saccharomyces cerevisiae, to use the meiosis-specific kleisin, recombination 8 (Rec8), during the mitotic cell cycle, instead of its paralog, Scc1. This perturbation impairs sister chromosome linkage, advances the timing of genome replication, and reduces reproductive fitness by 45%. We evolved 15 parallel populations for 1,750 generations, substantially increasing their fitness, and analyzed the genotypes and phenotypes of the evolved cells. Only one population contained a mutation in Rec8, but many populations had mutations in the transcriptional mediator complex, cohesin-related genes, and cell cycle regulators that induce S phase. These mutations improve sister chromosome cohesion and delay genome replication in Rec8-expressing cells. We conclude that changes in known and novel partners allow cells to use an existing protein to participate in new biological functions.

Erector Spinae Plane Block With Liposomal Bupivacaine: Analgesic Adjunct in Adult Pectus Surgery.

INTRODUCTION: Pain management may be challenging in patients undergoing pectus excavatum (PE) bar removal surgery. To enhance recovery, opioid sparing strategies with regional anesthesia including ultrasound-guided erector spinae plane block (ESPB) have been implemented. The purpose of this study was to evaluate the safety and efficacy of bilateral ESPB with a liposomal bupivacaine/traditional bupivacaine mixture as part of an enhanced patient recovery pathway. MATERIALS AND METHODS: A retrospective review of adult patients who underwent PE bar removal from January 2019 to December 2020 was performed. Perioperative data were reviewed and recorded. Patients who received ESPB were compared to historical controls (non-ESPB patients). RESULTS: A total of 202 patients were included (non-ESPB: 124 patients; ESPB: 78 patients). No adverse events were attributed to ESPB. Non-ESPB patients received more intraoperative opioids (milligram morphine equivalents; 41.8 ± 17.0 mg versus 36.7 ± 17.1, P = 0.05) and were more likely to present to the emergency department within 7 d postoperatively (4.8% versus 0%, P = 0.05) when compared to ESPB patients. No significant difference in total perioperative milligram morphine equivalents, severe pain in postanesthesia care unit (PACU), time from PACU arrival to analgesic administration, PACU length of stay, or postprocedure admission rates between groups were observed. CONCLUSIONS: In patients undergoing PE bar removal surgery, bilateral ESPB with liposomal bupivacaine was performed without complications. ESPB with liposomal bupivacaine may be considered as an analgesic adjunct to enhance recovery in patients undergoing cardiothoracic procedures but further prospective randomized clinical trials comparing liposomal bupivacaine to traditional local anesthetics with and without indwelling nerve catheters are necessary.

Perioperative anaphylactic reactions to central venous and pulmonary artery catheters containing chlorhexidine, sulfadiazine, or latex: a historical cohort study.

PURPOSE: Central venous catheters (CVCs) and pulmonary artery catheters (PACs) containing chlorhexidine, silver sulfadiazine, or latex can cause perioperative anaphylaxis. We examined the incidence of and outcomes associated with anaphylaxis caused by CVCs/PACs. METHODS: In a historical cohort study, we retrospectively identified adult patients fitted with CVCs/PACs at the Mayo Clinics in Minnesota, Arizona, and Florida from 1 January 2008 to 1 March 2018. Potential and confirmed cases of perioperative anaphylactic reactions were individually reviewed and classified. RESULTS: During the study period, 39,505 procedures were performed during which CVCs/PACs were inserted. Of these, 2,937 patients with pre-existing chlorhexidine, sulfonamide (sulfa), and/or latex allergies had CVCs/PACs inserted that contained these substances. Perioperative anaphylaxis, in which CVCs/PACs were the confirmed or potential causative agent, occurred during 53 procedures. Seven patients had a preoperatively reported sulfa or latex allergy; no patients had a preoperative chlorhexidine allergy. Six of the seven patients with reported allergies to sulfa or latex had a CVC/PAC inserted that contained these substances. Twenty-four patients with anaphylaxis had postoperative allergic disease consultation; ten of these (42%) underwent skin testing. CONCLUSION: Perioperative anaphylactic reactions related to CVCs/PACs containing chlorhexidine, silver sulfadiazine, or latex were rare in this large historical cohort study. We identified 2,937 patients with pre-existing chlorhexidine, sulfa, and/or latex allergies and had CVCs/PACs inserted that contained these substances. Although few cases of perioperative anaphylaxis attributable to these substances were observed in patients with corresponding allergies, the potential for substantial complication exists. Providers should be aware of the potential for these hidden exposures.

RéSUMé: OBJECTIF: Les cathéters veineux centraux (CVC) et les cathéters artériels pulmonaires (CAP) contenant de la chlorhexidine, de la sulfadiazine argentique ou du latex peuvent provoquer une anaphylaxie périopératoire. Nous avons examiné l'incidence et les devenirs associés à l'anaphylaxie causée par les CVC/CAP. MéTHODE: Dans une étude de cohorte historique, nous avons identifié rétrospectivement des patients adultes chez lesquels un CVC/CAP avait été installé aux cliniques Mayo du Minnesota, de l'Arizona et de la Floride du 1er janvier 2008 au 1er mars 2018. Les cas potentiels et confirmés de réactions anaphylactiques périopératoires ont été examinés et classés individuellement. RéSULTATS: Au cours de la période à l'étude, 39 505 interventions ont été réalisées au cours desquelles des CVC/CAP ont été insérés. Parmi celles-ci, des CVC/CAP contenant de la chlorhexidine, des sulfamides et/ou du latex ont été insérés chez 2937 patients présentant des allergies préexistantes à ces substances. Une anaphylaxie périopératoire, dont l'agent causal confirmé ou potentiel était le CVC/CAP, s'est produite dans 53 interventions. Sept patients présentaient une allergie aux sulfamides ou au latex signalée avant l'opération; aucun patient n'a eu d'allergie préopératoire à la chlorhexidine. Un CVC/CAP contenant des sulfamides ou du latex a été inséré chez six des sept patients ayant signalé des allergies à ces substances. Vingt-quatre patients atteints d'anaphylaxie ont eu une consultation postopératoire pour une maladie allergique; dix d'entre eux (42 %) ont subi des tests cutanés. CONCLUSION: Les réactions anaphylactiques périopératoires liées aux CVC/CAP contenant de la chlorhexidine, de la sulfadiazine argentique ou du latex étaient rares dans cette vaste étude de cohorte historique. Nous avons identifié 2937 patients présentant des allergies préexistantes à la chlorhexidine, aux sulfamides et/ou au latex chez lesquels des CVC/CAP contenant ces substances ont été insérés. Bien que peu de cas d'anaphylaxie périopératoire attribuable à ces substances aient été observés chez des patients présentant des allergies correspondantes, il existe un risque de complication importante. Les fournisseurs doivent être conscients du potentiel de ces expositions cachées.

Cell-size regulation in budding yeast does not depend on linear accumulation of Whi5.

Cells must couple cell-cycle progress to their growth rate to restrict the spread of cell sizes present throughout a population. Linear, rather than exponential, accumulation of Whi5, was proposed to provide this coordination by causing a higher Whi5 concentration in cells born at a smaller size. We tested this model using the inducible GAL1 promoter to make the Whi5 concentration independent of cell size. At an expression level that equalizes the mean cell size with that of wild-type cells, the size distributions of cells with galactose-induced Whi5 expression and wild-type cells are indistinguishable. Fluorescence microscopy confirms that the endogenous and GAL1 promoters produce different relationships between Whi5 concentration and cell volume without diminishing size control in the G1 phase. We also expressed Cln3 from the GAL1 promoter, finding that the spread in cell sizes for an asynchronous population is unaffected by this perturbation. Our findings indicate that size control in budding yeast does not fundamentally originate from the linear accumulation of Whi5, contradicting a previous claim and demonstrating the need for further models of cell-cycle regulation to explain how cell size controls passage through Start.

Modeling the impact of single-cell stochasticity and size control on the population growth rate in asymmetrically dividing cells.

Microbial populations show striking diversity in cell growth morphology and lifecycle; however, our understanding of how these factors influence the growth rate of cell populations remains limited. We use theory and simulations to predict the impact of asymmetric cell division, cell size regulation and single-cell stochasticity on the population growth rate. Our model predicts that coarse-grained noise in the single-cell growth rate λ decreases the population growth rate, as previously seen for symmetrically dividing cells. However, for a given noise in λ we find that dividing asymmetrically can enhance the population growth rate for cells with strong size control (between a "sizer" and an "adder"). To reconcile this finding with the abundance of symmetrically dividing organisms in nature, we propose that additional constraints on cell growth and division must be present which are not included in our model, and we explore the effects of selected extensions thereof. Further, we find that within our model, epigenetically inherited generation times may arise due to size control in asymmetrically dividing cells, providing a possible explanation for recent experimental observations in budding yeast. Taken together, our findings provide insight into the complex effects generated by non-canonical growth morphologies.

Context Analysis of Continued Citation of Retracted Manuscripts Published in Anesthesiology Journals.

The continued citation of retracted publications from the medical literature is a well-known and persistent problem. We describe the contexts of ongoing citations to manuscripts that have been retracted from a selection of anesthesiology journals. We also examine how bibliographic databases and publisher websites document the retracted status of these manuscripts. The authors performed an analysis of retracted publications from anesthesiology journals using the Retraction Watch database. We then examined how the retraction information was displayed on bibliographic databases, search engines, and publisher websites. The primary outcome was the context of continued citation after retraction of flawed publications within the specialty of anesthesiology. Secondary outcomes included comparison of the documentation, bibliographic databases, search engines, and publisher websites used in identifying the retracted status of these publications and provision of access to the respective retraction notices. A total of 245 original publications were retracted over a 28-year period from 9 anesthesiology journals. PubMed, compared to the other databases and search engines, was the most consistent (98.8%) in documenting the retracted status of the publications examined, as well as providing a direct link to the retraction notice. From the 211 publications retracted before January 2020, there were 1307 postretraction citations accessed from Scopus. The median number of postretraction citations was 3.5 (range, 0-88, with at least 1 citation in 164 publications) in Scopus. Of the postretraction citations, 80% affirmed the validity of the retracted publications, while only 5.2% of citations acknowledged the retraction or misconduct. In 10.2% of the citations from original research studies, retracted manuscripts appeared to influence the decision to pursue or the methods used in subsequent original research studies. The frequency of citation of the 15 most cited retracted publications declined in a similar pattern during the 10 years after retraction. Citation of manuscripts retracted from anesthesiology journals remains a common occurrence. Technological innovations and application of standards for handling retracted publications, as suggested by coalitions of researchers across the spectrum of scientific investigation, may serve to reduce the persistence of this error.

Rapid toxin sequestration modifies poison frog physiology.

Poison frogs sequester chemical defenses from their diet of leaf litter arthropods for defense against predation. Little is known about the physiological adaptations that confer this unusual bioaccumulation ability. We conducted an alkaloid-feeding experiment with the Diablito poison frog (Oophaga sylvatica) to determine how quickly alkaloids are accumulated and how toxins modify frog physiology using quantitative proteomics. Diablito frogs rapidly accumulated the alkaloid decahydroquinoline within 4 days, and dietary alkaloid exposure altered protein abundance in the intestines, liver and skin. Many proteins that increased in abundance with decahydroquinoline accumulation are plasma glycoproteins, including the complement system and the toxin-binding protein saxiphilin. Other protein classes that change in abundance with decahydroquinoline accumulation are membrane proteins involved in small molecule transport and metabolism. Overall, this work shows that poison frogs can rapidly accumulate alkaloids, which alter carrier protein abundance, initiate an immune response, and alter small molecule transport and metabolism dynamics across tissues.

Physical interactions reduce the power of natural selection in growing yeast colonies.

Microbial populations often assemble in dense populations in which proliferating individuals exert mechanical forces on the nearby cells. Here, we use yeast strains whose doubling times depend differently on temperature to show that physical interactions among cells affect the competition between different genotypes in growing yeast colonies. Our experiments demonstrate that these physical interactions have two related effects: they cause the prolonged survival of slower-growing strains at the actively-growing frontier of the colony and cause faster-growing strains to increase their frequency more slowly than expected in the absence of physical interactions. These effects also promote the survival of slower-growing strains and the maintenance of genetic diversity in colonies grown in time-varying environments. A continuum model inspired by overdamped hydrodynamics reproduces the experiments and predicts that the strength of natural selection depends on the width of the actively growing layer at the colony frontier. We verify these predictions experimentally. The reduced power of natural selection observed here may favor the maintenance of drug-resistant cells in microbial populations and could explain the apparent neutrality of interclone competition within tumors.

Multicellularity makes somatic differentiation evolutionarily stable.

Many multicellular organisms produce two cell lineages: germ cells, whose descendants produce the next generation, and somatic cells, which support, protect, and disperse the germ cells. This germ-soma demarcation has evolved independently in dozens of multicellular taxa but is absent in unicellular species. A common explanation holds that in these organisms, inefficient intercellular nutrient exchange compels the fitness cost of producing nonreproductive somatic cells to outweigh any potential benefits. We propose instead that the absence of unicellular, soma-producing populations reflects their susceptibility to invasion by nondifferentiating mutants that ultimately eradicate the soma-producing lineage. We argue that multicellularity can prevent the victory of such mutants by giving germ cells preferential access to the benefits conferred by somatic cells. The absence of natural unicellular, soma-producing species previously prevented these hypotheses from being directly tested in vivo: to overcome this obstacle, we engineered strains of the budding yeast Saccharomyces cerevisiae that differ only in the presence or absence of multicellularity and somatic differentiation, permitting direct comparisons between organisms with different lifestyles. Our strains implement the essential features of irreversible conversion from germ line to soma, reproductive division of labor, and clonal multicellularity while maintaining sufficient generality to permit broad extension of our conclusions. Our somatic cells can provide fitness benefits that exceed the reproductive costs of their production, even in unicellular strains. We find that nondifferentiating mutants overtake unicellular populations but are outcompeted by multicellular, soma-producing strains, suggesting that multicellularity confers evolutionary stability to somatic differentiation.

Genetic drift and selection in many-allele range expansions.

We experimentally and numerically investigate the evolutionary dynamics of four competing strains of E. coli with differing expansion velocities in radially expanding colonies. We compare experimental measurements of the average fraction, correlation functions between strains, and the relative rates of genetic domain wall annihilations and coalescences to simulations modeling the population as a one-dimensional ring of annihilating and coalescing random walkers with deterministic biases due to selection. The simulations reveal that the evolutionary dynamics can be collapsed onto master curves governed by three essential parameters: (1) an expansion length beyond which selection dominates over genetic drift; (2) a characteristic angular correlation describing the size of genetic domains; and (3) a dimensionless constant quantifying the interplay between a colony's curvature at the frontier and its selection length scale. We measure these parameters with a new technique that precisely measures small selective differences between spatially competing strains and show that our simulations accurately predict the dynamics without additional fitting. Our results suggest that the random walk model can act as a useful predictive tool for describing the evolutionary dynamics of range expansions composed of an arbitrary number of genotypes with different fitnesses.

Tethering sister centromeres to each other suggests the spindle checkpoint detects stretch within the kinetochore.

The spindle checkpoint ensures that newly born cells receive one copy of each chromosome by preventing chromosomes from segregating until they are all correctly attached to the spindle. The checkpoint monitors tension to distinguish between correctly aligned chromosomes and those with both sisters attached to the same spindle pole. Tension arises when sister kinetochores attach to and are pulled toward opposite poles, stretching the chromatin around centromeres and elongating kinetochores. We distinguished between two hypotheses for where the checkpoint monitors tension: between the kinetochores, by detecting alterations in the distance between them, or by responding to changes in the structure of the kinetochore itself. To distinguish these models, we inhibited chromatin stretch by tethering sister chromatids together by binding a tetrameric form of the Lac repressor to arrays of the Lac operator located on either side of a centromere. Inhibiting chromatin stretch did not activate the spindle checkpoint; these cells entered anaphase at the same time as control cells that express a dimeric version of the Lac repressor, which cannot cross link chromatids, and cells whose checkpoint has been inactivated. There is no dominant checkpoint inhibition when sister kinetochores are held together: cells expressing the tetrameric Lac repressor still arrest in response to microtubule-depolymerizing drugs. Tethering chromatids together does not disrupt kinetochore function; chromosomes are successfully segregated to opposite poles of the spindle. Our results indicate that the spindle checkpoint does not monitor inter-kinetochore separation, thus supporting the hypothesis that tension is measured within the kinetochore.

Genetic drift opposes mutualism during spatial population expansion.

Mutualistic interactions benefit both partners, promoting coexistence and genetic diversity. Spatial structure can promote cooperation, but spatial expansions may also make it hard for mutualistic partners to stay together, because genetic drift at the expansion front creates regions of low genetic and species diversity. To explore the antagonism between mutualism and genetic drift, we grew cross-feeding strains of the budding yeast Saccharomyces cerevisiae on agar surfaces as a model for mutualists undergoing spatial expansions. By supplying varying amounts of the exchanged nutrients, we tuned strength and symmetry of the mutualistic interaction. Strong mutualism suppresses genetic demixing during spatial expansions and thereby maintains diversity, but weak or asymmetric mutualism is overwhelmed by genetic drift even when mutualism is still beneficial, slowing growth and reducing diversity. Theoretical modeling using experimentally measured parameters predicts the size of demixed regions and how strong mutualism must be to survive a spatial expansion.

Spatially Constrained Growth Enhances Conversional Meltdown.

Cells that mutate or commit to a specialized function (differentiate) often undergo conversions that are effectively irreversible. Slowed growth of converted cells can act as a form of selection, balancing unidirectional conversion to maintain both cell types at a steady-state ratio. However, when one-way conversion is insufficiently counterbalanced by selection, the original cell type will ultimately be lost, often with negative impacts on the population's overall fitness. The critical balance between selection and conversion needed for preservation of unconverted cells and the steady-state ratio between cell types depends on the spatial circumstances under which cells proliferate. We present experimental data on a yeast strain engineered to undergo irreversible conversion: this synthetic system permits cell-type-specific fluorescent labeling and exogenous variation of the relative growth and conversion rates. We find that populations confined to grow on a flat agar surface are more susceptible than their well-mixed counterparts to fitness loss via a conversion-induced "meltdown." We then present analytical predictions for growth in several biologically relevant geometries-well-mixed liquid media, radially expanding two-dimensional colonies, and linear fronts in two dimensions-by employing analogies to the directed-percolation transition from nonequilibrium statistical physics. These simplified theories are consistent with the experimental results.

How Obstacles Perturb Population Fronts and Alter Their Genetic Structure.

As populations spread into new territory, environmental heterogeneities can shape the population front and genetic composition. We focus here on the effects of an important building block of heterogeneous environments, isolated obstacles. With a combination of experiments, theory, and simulation, we show how isolated obstacles both create long-lived distortions of the front shape and amplify the effect of genetic drift. A system of bacteriophage T7 spreading on a spatially heterogeneous Escherichia coli lawn serves as an experimental model system to study population expansions. Using an inkjet printer, we create well-defined replicates of the lawn and quantitatively study the population expansion of phage T7. The transient perturbations of the population front found in the experiments are well described by a model in which the front moves with constant speed. Independent of the precise details of the expansion, we show that obstacles create a kink in the front that persists over large distances and is insensitive to the details of the obstacle's shape. The small deviations between experimental findings and the predictions of the constant speed model can be understood with a more general reaction-diffusion model, which reduces to the constant speed model when the obstacle size is large compared to the front width. Using this framework, we demonstrate that frontier genotypes just grazing the side of an isolated obstacle increase in abundance, a phenomenon we call 'geometry-enhanced genetic drift', complementary to the founder effect associated with spatial bottlenecks. Bacterial range expansions around nutrient-poor barriers and stochastic simulations confirm this prediction. The effect of the obstacle on the genealogy of individuals at the front is characterized by simulations and rationalized using the constant speed model. Lastly, we consider the effect of two obstacles on front shape and genetic composition of the population illuminating the effects expected from complex environments with many obstacles.

A model for the evolution of biological specificity: a cross-reacting DNA-binding protein causes plasmid incompatibility.

Few biological systems permit rigorous testing of how changes in DNA sequence give rise to adaptive phenotypes. In this study, we sought a simplified experimental system with a detailed understanding of the genotype-to-phenotype relationship that could be altered by environmental perturbations. We focused on plasmid fitness, i.e., the ability of plasmids to be stably maintained in a bacterial population, which is dictated by the plasmid's replication and segregation machinery. Although plasmid replication depends on host proteins, the type II plasmid partitioning (Par) machinery is entirely plasmid encoded and relies solely on three components: parC, a centromere-like DNA sequence, ParR, a DNA-binding protein that interacts with parC, and ParM, which forms actin-like filaments that push two plasmids away from each other at cell division. Interactions between the Par operons of two related plasmids can cause incompatibility and the reduced transmission of one or both plasmids. We have identified segregation-dependent plasmid incompatibility between the highly divergent Par operons of plasmids pB171 and pCP301. Genetic and biochemical studies revealed that the incompatibility is due to the functional promiscuity of the DNA-binding protein ParRpB171, which interacts with both parC DNA sequences to direct plasmid segregation, indicating that the lack of DNA binding specificity is detrimental to plasmid fitness in this environment. This study therefore successfully utilized plasmid segregation to dissect the molecular interactions between genotype, phenotype, and fitness.