Novel viruses of the family Partitiviridae discovered in Saccharomyces cerevisiae.

It has been 49 years since the last discovery of a new virus family in the model yeast Saccharomyces cerevisiae. A large-scale screen to determine the diversity of double-stranded RNA (dsRNA) viruses in S. cerevisiae has identified multiple novel viruses from the family Partitiviridae that have been previously shown to infect plants, fungi, protozoans, and insects. Most S. cerevisiae partitiviruses (ScPVs) are associated with strains of yeasts isolated from coffee and cacao beans. The presence of partitiviruses was confirmed by sequencing the viral dsRNAs and purifying and visualizing isometric, non-enveloped viral particles. ScPVs have a typical bipartite genome encoding an RNA-dependent RNA polymerase (RdRP) and a coat protein (CP). Phylogenetic analysis of ScPVs identified three species of ScPV, which are most closely related to viruses of the genus Cryspovirus from the mammalian pathogenic protozoan Cryptosporidium parvum. Molecular modeling of the ScPV RdRP revealed a conserved tertiary structure and catalytic site organization when compared to the RdRPs of the Picornaviridae. The ScPV CP is the smallest so far identified in the Partitiviridae and has structural homology with the CP of other partitiviruses but likely lacks a protrusion domain that is a conspicuous feature of other partitivirus particles. ScPVs were stably maintained during laboratory growth and were successfully transferred to haploid progeny after sporulation, which provides future opportunities to study partitivirus-host interactions using the powerful genetic tools available for the model organism S. cerevisiae.

Altered Iron and Microstructure in Huntington's Disease Subcortical Nuclei: Insight From 7T MRI.

BACKGROUND: Pathophysiological changes of Huntington's disease (HD) can precede symptom onset by decades. Robust imaging biomarkers are needed to monitor HD progression, especially before the clinical onset. PURPOSE: To investigate iron dysregulation and microstructure alterations in subcortical regions as HD imaging biomarkers, and to associate such alterations with motor and cognitive impairments. STUDY TYPE: Prospective. POPULATION: Fourteen individuals with premanifest HD (38.0 ± 11.0 years, 9 females; far-from-onset N = 6, near-onset N = 8), 21 manifest HD patients (49.1 ± 12.1 years, 11 females), and 33 age-matched healthy controls (43.9 ± 12.2 years, 17 females). FIELD STRENGTH/SEQUENCE: 7 T, T1 -weighted imaging, quantitative susceptibility mapping, and diffusion tensor imaging. ASSESSMENT: Volume, susceptibility, fractional anisotropy (FA), and mean diffusivity (MD) within subcortical brain structures were compared across groups, used to establish HD classification models, and correlated to clinical measures and cognitive assessments. STATISTICAL TESTS: Generalized linear model, multivariate logistic regression, receiver operating characteristics with the area under the curve (AUC), and likelihood ratio test comparing a volumetric model to one that also includes susceptibility and diffusion metrics, Wilcoxon paired signed-rank test, and Pearson's correlation. A P-value <0.05 after Benjamini-Hochberg correction was considered statistically significant. RESULTS: Significantly higher striatal susceptibility and FA were found in premanifest and manifest HD preceding atrophy, even in far-from-onset premanifest HD compared to controls (putamen susceptibility: 0.027 ± 0.022 vs. 0.018 ± 0.013 ppm; FA: 0.358 ± 0.048 vs. 0.313 ± 0.039). The model with additional susceptibility, FA, and MD features showed higher AUC compared to volume features alone when differentiating premanifest HD from HC (0.83 vs. 0.66), and manifest from premanifest HD (0.94 vs. 0.83). Higher striatal susceptibility significantly correlated with cognitive deterioration in HD (executive function: r = -0.600; socioemotional function: r = -0.486). DATA CONCLUSION: 7 T MRI revealed iron dysregulation and microstructure alterations with HD progression, which could precede volume loss, provide added value to HD differentiation, and might be associated with cognitive changes. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 2.

The Species-Specific Acquisition and Diversification of a K1-like Family of Killer Toxins in Budding Yeasts of the Saccharomycotina.

Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition.

The prevalence of killer yeasts and double-stranded RNAs in the budding yeast Saccharomyces cerevisiae.

Killer toxins are antifungal proteins produced by many species of "killer" yeasts, including the brewer's and baker's yeast Saccharomyces cerevisiae. Screening 1270 strains of S. cerevisiae for killer toxin production found that 50% are killer yeasts, with a higher prevalence of yeasts isolated from human clinical samples and winemaking processes. Since many killer toxins are encoded by satellite double-stranded RNAs (dsRNAs) associated with mycoviruses, S. cerevisiae strains were also assayed for the presence of dsRNAs. This screen identified that 51% of strains contained dsRNAs from the mycovirus families Totiviridae and Partitiviridae, as well as satellite dsRNAs. Killer toxin production was correlated with the presence of satellite dsRNAs but not mycoviruses. However, in most killer yeasts, whole genome analysis identified the killer toxin gene KHS1 as significantly associated with killer toxin production. Most killer yeasts had unique spectrums of antifungal activities compared to canonical killer toxins, and sequence analysis identified mutations that altered their antifungal activities. The prevalence of mycoviruses and killer toxins in S. cerevisiae is important because of their known impact on yeast fitness, with implications for academic research and industrial application of this yeast species.

Physiological constraints dictate toxin spatial heterogeneity in snake venom glands.

BACKGROUND: Venoms are ecological innovations that have evolved numerous times, on each occasion accompanied by the co-evolution of specialised morphological and behavioural characters for venom production and delivery. The close evolutionary interdependence between these characters is exemplified by animals that control the composition of their secreted venom. This ability depends in part on the production of different toxins in different locations of the venom gland, which was recently documented in venomous snakes. Here, we test the hypothesis that the distinct spatial distributions of toxins in snake venom glands are an adaptation that enables the secretion of venoms with distinct ecological functions. RESULTS: We show that the main defensive and predatory peptide toxins are produced in distinct regions of the venom glands of the black-necked spitting cobra (Naja nigricollis), but these distributions likely reflect developmental effects. Indeed, we detected no significant differences in venom collected via defensive 'spitting' or predatory 'biting' events from the same specimens representing multiple lineages of spitting cobra. We also found the same spatial distribution of toxins in a non-spitting cobra and show that heterogeneous toxin distribution is a feature shared with a viper with primarily predatory venom. CONCLUSIONS: Our findings suggest that heterogeneous distributions of toxins are not an adaptation to controlling venom composition in snakes. Instead, it likely reflects physiological constraints on toxin production by the venom glands, opening avenues for future research on the mechanisms of functional differentiation of populations of protein-secreting cells within adaptive contexts.

A bipartite thermodynamic-kinetic contribution by an activating mutation to RDF-independent excision by a phage serine integrase.

Streptomyces phage ϕC31 integrase (Int)-a large serine site-specific recombinase-is autonomous for phage integration (attP x attB recombination) but is dependent on the phage coded gp3, a recombination directionality factor (RDF), for prophage excision (attL x attR recombination). A previously described activating mutation, E449K, induces Int to perform attL x attR recombination in the absence of gp3, albeit with lower efficiency. E449K has no adverse effect on the competence of Int for attP x attB recombination. Int(E449K) resembles Int in gp3 mediated stimulation of attL x attR recombination and inhibition of attP x attB recombination. Using single-molecule analyses, we examined the mechanism by which E449K activates Int for gp3-independent attL x attR recombination. The contribution of E449K is both thermodynamic and kinetic. First, the mutation modulates the relative abundance of Int bound attL-attR site complexes, favoring pre-synaptic (PS) complexes over non-productively bound complexes. Roughly half of the synaptic complexes formed from Int(E449K) pre-synaptic complexes are recombination competent. By contrast, Int yields only inactive synapses. Second, E449K accelerates the dissociation of non-productively bound complexes and inactive synaptic complexes formed by Int. The extra opportunities afforded to Int(E499K) in reattempting synapse formation enhances the probability of success at fruitful synapsis.

Vaginal Isolates of Candida glabrata Are Uniquely Susceptible to Ionophoric Killer Toxins Produced by Saccharomyces cerevisiae.

Compared to other species of Candida yeasts, the growth of Candida glabrata is inhibited by many different strains of Saccharomyces killer yeasts. The ionophoric K1 and K2 killer toxins are broadly inhibitory to all clinical isolates of C. glabrata from patients with recurrent vulvovaginal candidiasis, despite high levels of resistance to clinically relevant antifungal therapeutics.

HIV-1 Accessory Protein Vpr Interacts with REAF/RPRD2 To Mitigate Its Antiviral Activity.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and, to a lesser extent, cycling T cells. Virion-packaged Vpr is released in target cells shortly after entry, suggesting it is required in the early phase of infection. Previously, we described REAF (RNA-associated early-stage antiviral factor; RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without an intact vpr gene is more highly restricted by REAF and, using delivery by virus-like particles (VLPs), that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells, and we detected, by coimmunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts quickly during the early phase of replication and induces the degradation of REAF within 30 min of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localization and interaction with cullin 4A-DBB1 (DCAF1) E3 ubiquitin ligase are required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, that impedes HIV infection in macrophages.IMPORTANCE For at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1 in vivo A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages or to explain why Vpr is carried in the virus particle. Here, we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one that inhibits virus replication during reverse transcription. REAF is degraded by Vpr within 30 min of virus entry in a manner dependent on the nuclear localization of Vpr and its interaction with the cell's protein degradation machinery.

Unexpected lack of specialisation in the flow properties of spitting cobra venom.

Venom spitting is a defence mechanism based on airborne venom delivery used by a number of different African and Asian elapid snake species ('spitting cobras'; Naja spp. and Hemachatus spp.). Adaptations underpinning venom spitting have been studied extensively at both behavioural and morphological level in cobras, but the role of the physical properties of venom itself in its effective projection remains largely unstudied. We hereby provide the first comparative study of the physical properties of venom in spitting and non-spitting cobras. We measured the viscosity, protein concentration and pH of the venom of 13 cobra species of the genus Naja from Africa and Asia, alongside the spitting elapid Hemachatus haemachatus and the non-spitting viper Bitis arietans By using published microCT scans, we calculated the pressure required to eject venom through the fangs of a spitting and a non-spitting cobra. Despite the differences in the modes of venom delivery, we found no significant differences between spitters and non-spitters in the rheological and physical properties of the studied venoms. Furthermore, all analysed venoms showed a Newtonian flow behaviour, in contrast to previous reports. Although our results imply that the evolution of venom spitting did not significantly affect venom viscosity, our models of fang pressure suggests that the pressure requirements to eject venom are lower in spitting cobras than in non-spitting cobras.

Unexpected lack of specialisation in the flow properties of spitting cobra venom.

Venom spitting is a defence mechanism based on airborne venom delivery used by a number of different African and Asian elapid snake species ('spitting cobras'; Naja spp. and Hemachatus spp.). Adaptations underpinning venom spitting have been studied extensively at both behavioural and morphological level in cobras, but the role of the physical properties of venom itself in its effective projection remains largely unstudied. We hereby provide the first comparative study of the physical properties of venom in spitting and non-spitting cobras. We measured the viscosity, protein concentration and pH of the venom of 13 cobra species of the genus Naja from Africa and Asia, alongside the spitting elapid Hemachatus haemachatus and the non-spitting viper Bitis arietans. By using published microCT scans, we calculated the pressure required to eject venom through the fangs of a spitting and a non-spitting cobra. Despite the differences in the modes of venom delivery, we found no significant differences between spitters and non-spitters in the rheological and physical properties of the studied venoms. Furthermore, all analysed venoms showed a Newtonian flow behaviour, in contrast to previous reports. Although our results imply that the evolution of venom spitting did not significantly affect venom viscosity, our models of fang pressure suggests that the pressure requirements to eject venom are lower in spitting cobras than in non-spitting cobras.

Control of yeast retrotransposons mediated through nucleoporin evolution.

Yeasts serve as hosts to several types of genetic parasites. Few studies have addressed the evolutionary trajectory of yeast genes that control the stable co-existence of these parasites with their host cell. In Saccharomyces yeasts, the retrovirus-like Ty retrotransposons must access the nucleus. We show that several genes encoding components of the yeast nuclear pore complex have experienced natural selection for substitutions that change the encoded protein sequence. By replacing these S. cerevisiae genes with orthologs from other Saccharomyces species, we discovered that natural sequence changes have affected the mobility of Ty retrotransposons. Specifically, changing the genetic sequence of NUP84 or NUP82 to match that of other Saccharomyces species alters the mobility of S. cerevisiae Ty1 and Ty3. Importantly, all tested housekeeping functions of NUP84 and NUP82 remained equivalent across species. Signatures of natural selection, resulting in altered interactions with viruses and parasitic genetic elements, are common in host defense proteins. Yet, few instances have been documented in essential housekeeping proteins. The nuclear pore complex is the gatekeeper of the nucleus. This study shows how the evolution of this large, ubiquitous eukaryotic complex can alter the replication of a molecular parasite, but concurrently maintain essential host functionalities regarding nucleocytoplasmic trafficking.

Predicting postoperative delirium severity in older adults: The role of surgical risk and executive function.

OBJECTIVES: Delirium is an important postoperative complication, yet predictive risk factors for postoperative delirium severity remain elusive. We hypothesized that the NSQIP risk calculation for serious complications (NSQIP-SC) or risk of death (NSQIP-D), and cognitive tests of executive function (Trail Making Tests A and B [TMTA and TMTB]), would be predictive of postoperative delirium severity. Further, we demonstrate how advanced statistical techniques can be used to identify candidate predictors. METHODS/DESIGN: Data from an ongoing perioperative prospective cohort study of 100 adults (65 y old or older) undergoing noncardiac surgery were analyzed. In addition to NSQIP-SC, NSQIP-D, TMTA, and TMTB, participant age, sex, American Society of Anesthesiologists (ASA) score, tobacco use, surgery type, depression, Framingham risk score, and preoperative blood pressure were collected. The Delirium Rating Scale-R-98 (DRS) measured delirium severity; the Confusion Assessment Method (CAM) identified delirium. LASSO and best subsets linear regression were employed to identify predictive risk factors. RESULTS: Ninety-seven participants with a mean age of 71.68 ± 4.55, 55% male (31/97 CAM+, 32%), and a mean peak DRS of 21.5 ± 6.40 were analyzed. LASSO and best subsets regression identified NSQIP-SC and TMTB to predict postoperative delirium severity (P < 00.001, adjusted R2 : 0.30). NSQIP-SC and TMTB were also selected as predictors for postoperative delirium incidence (AUROC 0.81, 95% CI, 0.72-0.90). CONCLUSIONS: In this cohort, we identified NSQIP risk score for serious complications and a measure of executive function, TMT-B, to predict postoperative delirium severity using advanced modeling techniques. Future studies should investigate the utility of these variables in a formal delirium severity prediction model.

XRN1 Is a Species-Specific Virus Restriction Factor in Yeasts.

In eukaryotes, the degradation of cellular mRNAs is accomplished by Xrn1 and the cytoplasmic exosome. Because viral RNAs often lack canonical caps or poly-A tails, they can also be vulnerable to degradation by these host exonucleases. Yeast lack sophisticated mechanisms of innate and adaptive immunity, but do use RNA degradation as an antiviral defense mechanism. One model is that the RNA of yeast viruses is subject to degradation simply as a side effect of the intrinsic exonuclease activity of proteins involved in RNA metabolism. Contrary to this model, we find a highly refined, species-specific relationship between Xrn1p and the "L-A" totiviruses of different Saccharomyces yeast species. We show that the gene XRN1 has evolved rapidly under positive natural selection in Saccharomyces yeast, resulting in high levels of Xrn1p protein sequence divergence from one yeast species to the next. We also show that these sequence differences translate to differential interactions with the L-A virus, where Xrn1p from S. cerevisiae is most efficient at controlling the L-A virus that chronically infects S. cerevisiae, and Xrn1p from S. kudriavzevii is most efficient at controlling the L-A-like virus that we have discovered within S. kudriavzevii. All Xrn1p orthologs are equivalent in their interaction with another virus-like parasite, the Ty1 retrotransposon. Thus, the activity of Xrn1p against totiviruses is not simply an incidental consequence of the enzymatic activity of Xrn1p, but rather Xrn1p co-evolves with totiviruses to maintain its potent antiviral activity and limit viral propagation in Saccharomyces yeasts. Consistent with this, we demonstrated that Xrn1p physically interacts with the Gag protein encoded by the L-A virus, suggesting a host-virus interaction that is more complicated than just Xrn1p-mediated nucleolytic digestion of viral RNAs.

The frenemies within: viruses, retrotransposons and plasmids that naturally infect Saccharomyces yeasts.

Viruses are a major focus of current research efforts because of their detrimental impact on humanity and their ubiquity within the environment. Bacteriophages have long been used to study host-virus interactions within microbes, but it is often forgotten that the single-celled eukaryote Saccharomyces cerevisiae and related species are infected with double-stranded RNA viruses, single-stranded RNA viruses, LTR-retrotransposons and double-stranded DNA plasmids. These intracellular nucleic acid elements have some similarities to higher eukaryotic viruses, i.e. yeast retrotransposons have an analogous lifecycle to retroviruses, the particle structure of yeast totiviruses resembles the capsid of reoviruses and segregation of yeast plasmids is analogous to segregation strategies used by viral episomes. The powerful experimental tools available to study the genetics, cell biology and evolution of S. cerevisiae are well suited to further our understanding of how cellular processes are hijacked by eukaryotic viruses, retrotransposons and plasmids. This article has been written to briefly introduce viruses, retrotransposons and plasmids that infect Saccharomyces yeasts, emphasize some important cellular proteins and machineries with which they interact, and suggest the evolutionary consequences of these interactions. Copyright © 2017 John Wiley & Sons, Ltd.

Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

The effect of species representation on the detection of positive selection in primate gene data sets.

Over evolutionary time, both host- and virus-encoded genes have been continually selected to modify their interactions with one another. This has resulted in the rapid evolution of the specific codons that govern the physical interactions between host and virus proteins. Virologists have discovered that these evolutionary signatures, acquired in nature, can provide a shortcut in the functional dissection of host-virus interactions in the laboratory. However, the use of evolution studies in this way is complicated by the fact that many nonhuman primate species are endangered, and biomaterials are often difficult to acquire. Here, we assess how the species representation in primate gene data sets affects the detection of positive natural selection. Our results demonstrate how targeted primate sequencing projects could greatly enhance research in immunology, virology, and beyond.

Temporal sequence and cell cycle cues in the assembly of host factors at the yeast 2 micron plasmid partitioning locus.

The Saccharomyces cerevisiae 2 micron plasmid exemplifies a benign but selfish genome, whose stability approaches that of the chromosomes of its host. The plasmid partitioning locus STB (stability locus) displays certain functional analogies with centromeres along with critical distinctions, a significant one being the absence of the kinetochore complex at STB. The remodels the structure of chromatin (RSC) chromatin remodeling complex, the nuclear motor Kip1, the histone H3 variant Cse4 and the cohesin complex associate with both loci. These factors appear to contribute to plasmid segregation either directly or indirectly through their roles in chromosome segregation. Assembly and disassembly of the plasmid-coded partitioning proteins Rep1 and Rep2 and host factors at STB follow a temporal hierarchy during the cell cycle. Assembly is initiated by STB association of [Rsc8-Rsc58], followed by [Rep1-Rep2-Kip1] and [Cse4-Rsc2-Sth1] recruitment, and culminates in cohesin assembly. Disassembly starts with dissociation of RSC components, is followed by cohesin disassembly and Cse4 exit during anaphase and late telophase, respectively. [Rep1-Rep2-Kip1] persists through G1 of the ensuing cell cycle. The de novo assembly of the 'partitioning complex' is cued by the innate cell cycle clock and is dependent on DNA replication. Shared functional attributes of STB and centromere (CEN) are consistent with a potential evolutionary link between them.

An apparent lack of synergy between degradative enzymes against Staphylococcus aureus biofilms.

Enzymes combat bacterial infections by degrading biomolecules to disperse Staphylococcus aureus biofilms. Commercial enzyme mixtures, like cellulase and pepsin, show concentration-dependent dispersion, but low concentrations lack synergy. Only the sequential addition of pepsin followed by Arthrobacter luteus zymolyase 20T displays synergy, effectively dispersing biofilms. Purified zymolyase 100T outperforms zymolyase 20T but lacks synergy with pepsin. This study underscores the complexity of enzymatic biofilm dispersal, highlighting the need for tailored approaches based on enzyme properties and biofilm composition.

Evolution of a Plasmid Regulatory Circuit Ameliorates Plasmid Fitness Cost.

Plasmids play a major role in rapid adaptation of bacteria by facilitating horizontal transfer of diverse genes, most notably those conferring antibiotic resistance. While most plasmids that replicate in a broad range of bacteria also persist well in diverse hosts, there are exceptions that are poorly understood. We investigated why a broad-host range plasmid, pBP136, originally found in clinical Bordetella pertussis isolates, quickly became extinct in laboratory Escherichia coli populations. Through experimental evolution we found that inactivation of a previously uncharacterized plasmid gene, upf31, drastically improved plasmid maintenance in E. coli. This gene inactivation resulted in decreased transcription of the global plasmid regulators (korA, korB, and korC) and numerous genes in their regulons. It also caused transcriptional changes in many chromosomal genes primarily related to metabolism. In silico analyses suggested that the change in plasmid transcriptome may be initiated by Upf31 interacting with the plasmid regulator KorB. Expression of upf31 in trans negatively affected persistence of pBP136Δupf31 as well as the closely related archetypal IncP-1ß plasmid R751, which is stable in E. coli and natively encodes a truncated upf31 allele. Our results demonstrate that while the upf31 allele in pBP136 might advantageously modulate gene expression in its original host, B. pertussis, it has harmful effects in E. coli. Thus, evolution of a single plasmid gene can change the range of hosts in which that plasmid persists, due to effects on the regulation of plasmid gene transcription.

yEvo: A modular eukaryotic genetics and evolution research experience for high school students.

The resources for carrying out and analyzing microbial evolution experiments have become more accessible, making it possible to expand these studies beyond the research laboratory and into the classroom. We developed five connected, standards-aligned yeast evolution laboratory modules, called "yEvo," for high school students. The modules enable students to take agency in answering open-ended research questions. In Module 1, students evolve baker's yeast to tolerate an antifungal drug, and in subsequent modules, investigate how evolved yeasts adapted to this stressful condition at both the phenotype and genotype levels. We used pre- and post-surveys from 72 students at two different schools and post-interviews with students and teachers to assess our program goals and guide module improvement over 3 years. We measured changes in student conceptions, confidence in scientific practices, and interest in STEM careers. Students who participated in yEvo showed improvements in understanding of activity-specific concepts and reported increased confidence in designing a valid biology experiment. Student experimental data replicated literature findings and has led to new insights into antifungal resistance. The modules and provided materials, alongside "proof of concept" evaluation metrics, will serve as a model for other university researchers and K - 16 classrooms interested in engaging in open-ended research questions using yeast as a model system.