Encephalitozoon cuniculi and Vittaforma corneae (Phylum Microsporidia) inhibit staurosporine-induced apoptosis in human THP-1 macrophages in vitro.

Obligately intracellular microsporidia regulate their host cell life cycles, including apoptosis, but this has not been evaluated in phagocytic host cells such as macrophages that can facilitate infection but also can be activated to kill microsporidia. We examined two biologically dissimilar human-infecting microsporidia species, Encephalitozoon cuniculi and Vittaforma corneae, for their effects on staurosporine-induced apoptosis in the human macrophage-differentiated cell line, THP1. Apoptosis was measured after exposure of THP-1 cells to live and dead mature organisms via direct fluorometric measurement of Caspase 3, colorimetric and fluorometric TUNEL assays, and mRNA gene expression profiles using Apoptosis RT2 Profiler PCR Array. Both species of microsporidia modulated the intrinsic apoptosis pathway. In particular, live E. cuniculi spores inhibited staurosporine-induced apoptosis as well as suppressed pro-apoptosis genes and upregulated anti-apoptosis genes more broadly than V. corneae. Exposure to dead spores induced an opposite effect. Vittaforma corneae, however, also induced inflammasome activation via Caspases 1 and 4. Of the 84 apoptosis-related genes assayed, 42 (i.e. 23 pro-apoptosis, nine anti-apoptosis, and 10 regulatory) genes were more affected including those encoding members of the Bcl2 family, caspases and their regulators, and members of the tumour necrosis factor (TNF)/TNF receptor R superfamily.

Fast Technology Analysis Enables Identification of Species and Genotypes of Latent Microsporidia Infections in Healthy Native Cameroonians.

Several enteric microsporidia species have been detected in humans and other vertebrates and their identifications at the genotype level are currently being elucidated. As advanced methods, reagents, and disposal kits for detecting and identifying pathogens become commercially available, it is important to test them in settings other than in laboratories with "state-of-the-art" equipment and well-trained staff members. In the present study, we sought to detect microsporidia DNA preserved and extracted from FTA (fast technology analysis) cards spotted with human fecal suspensions obtained from Cameroonian volunteers living in the capital city of Yaoundé to preclude the need for employing spore-concentrating protocols. Further, we tested whether amplicon nucleotide sequencing approaches could be used on small aliquots taken from the cards to elucidate the diversity of microsporidia species and strains infecting native residents. Of 196 samples analyzed, 12 (6.1%) were positive for microsporidia DNA; Enterocytozoon bieneusi (Type IV and KIN-1), Encephalitozoon cuniculi, and Encephalitozoon intestinalis were identified. These data demonstrate the utility of the FTA cards in identifying genotypes of microsporidia DNA in human fecal samples that may be applied to field testing for prevalence studies.

Spelling Facilitates Reading: A Tutorial on the Spell-to-Read Approach.

PURPOSE: According to the American Speech-Language-Hearing Association (ASHA), prevention, identification, assessment, and intervention of children who are learning to read and write are within the scope of practice for school-based speech-language pathologists (SLPs). Furthermore, for SLPs who work in the school setting, it is not uncommon to have struggling readers and poor spellers on their caseloads. Importantly, for students who have difficulty in spelling, their spelling errors are among the early indicators of dyslexia and can provide a means for identifying readers who may benefit from early intervention. SLPs can both assess spelling skills and implement evidence-based spelling and literacy diagnosis and instruction. Spelling instruction in kindergarten through the high school grades that is heavily grounded in metalinguistic activities can provide access to mental representations of word spellings, pronunciations, and meanings; links between whole-word and phonics approaches to reading instruction; and a foundation for reading fluency and comprehension. CONCLUSIONS: Learning to spell is essential for learning to read. Accordingly, this tutorial aims to elucidate how to (a) assess and identify phases of spelling development and (b) teach spelling to facilitate reading through a listening-first approach.

Child word learning in song and speech.

Listening to sung words rather than spoken words can facilitate word learning and memory in adults and school-aged children. To explore the development of this effect in young children, this study examined word learning (assessed as forming word-object associations) in 1- to 2-year olds and 3- to 4-year olds, and word long-term memory (LTM) in 4- to 5-year olds several days after the initial learning. In an intermodal preferential looking paradigm, children were taught a pair of words utilising adult-directed speech (ADS) and a pair of sung words. Word learning performance was better with sung words than with ADS words in 1- to 2-year olds (Experiments 1a and 1b), 3- to 4-year olds (Experiment 1a), and 4- to 5-year olds (Experiment 2b), revealing a benefit of song in word learning in all age ranges recruited. We also examined whether children successfully learned the words by comparing their performance against chance. The 1- to 2-year olds only learned sung words, but the 3- to 4-year olds learned both sung and ADS words, suggesting that the reliance on music features in word learning observed at ages 1-2 decreased with age. Furthermore, song facilitated the word mapping-recognition processes. Results on children's LTM performance showed that the 4- to 5-year olds' LTM performance did not differ between sung and ADS words. However, the 4- to 5-year olds reliably recalled sung words but not spoken words. The reliable LTM of sung words arose from hearing sung words during the initial learning rather than at test. Finally, the benefit of song on word learning and the reliable LTM of sung words observed at ages 3-5 cannot be explained as an attentional effect.

Transcriptome analysis of the parasite Encephalitozoon cuniculi: an in-depth examination of pre-mRNA splicing in a reduced eukaryote.

BACKGROUND: The microsporidian Encephalitozoon cuniculi possesses one of the most reduced and compacted eukaryotic genomes. Reduction in this intracellular parasite has affected major cellular machinery, including the loss of over fifty core spliceosomal components compared to S. cerevisiae. To identify expression changes throughout the parasite's life cycle and also to assess splicing in the context of this reduced system, we examined the transcriptome of E. cuniculi using Illumina RNA-seq. RESULTS: We observed that nearly all genes are expressed at three post-infection time-points examined. A large fraction of genes are differentially expressed between the first and second (37.7%) and first and third (43.8%) time-points, while only four genes are differentially expressed between the latter two. Levels of intron splicing are very low, with 81% of junctions spliced at levels below 50%. This is dramatically lower than splicing levels found in two other fungal species examined. We also describe the first case of alternative splicing in a microsporidian, an unexpected complexity given the reduction in spliceosomal components. CONCLUSIONS: Low levels of splicing observed are likely the result of an inefficient spliceosome; however, at least in one case, splicing appears to be playing a functional role. Although several RNA decay genes are encoded in E. cuniculi, the lack of a few key players could be reducing decay levels and therefore increasing the proportion of unspliced transcripts. Significant proportions of genes are differentially expressed in the first forty-eight hours but not after, indicative of genetic changes that precede the intracellular to infective stage transition.

Rethinking Literacy Intervention: Addressing a Practice Gap With Best Practices From Multisensory Structured Language Approaches.

PURPOSE: Dyslexia is increasingly being defined, assessed, diagnosed, and treated in the educational system. The purpose of this clinical focus article is to elucidate ways in which speech-language pathologists (SLPs) can rethink how to implement literacy interventions to incorporate best practices from multisensory structured language (MSL) approaches and how they can be influential participants in the conversations of how to define and implement services for students who have written language disorders, including dyslexia, in the school setting. METHOD: This clinical focus article provides an operational definition of dyslexia, discusses the various roles and responsibilities of SLPs with respect to dyslexia, and describes the well-established evidence-based practices of MSL approaches as a means of rethinking literacy intervention. RESULTS: Using a case study scenario based on an individual diagnosed with dyslexia, this clinical focus article presents similarities and differences between traditional speech-language pathology intervention approaches and MSL approaches to literacy intervention. CONCLUSIONS: MSL strategies may be considered in literacy intervention as a means to optimize the academic gains of children with dyslexia in a school setting. Furthermore, SLPs should be considered integral participants in discussions of policies and practices related to the diagnosis and treatment of literacy disorders, including dyslexia. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.23228483.

Dataset of user interactions across four large pilots on the use of augmented reality in learning experiences.

Augmented Reality in education can support students in a wide range of cognitive tasks-fostering understanding, remembering, applying, analysing, evaluating, and creating learning-relevant information more easily. It can help keep up engagement, and it can render learning more fun. Within the framework of a multi-year investigation encompassing primary and secondary schools across Europe, the ARETE project developed several Augmented Reality applications, providing tools for user interaction and data collection in the education sector. The project developed innovative AR learning technology and methodology, validating these in four comprehensive pilot studies, in total involving more than 2,900 students and teachers. Each pilot made use of a different Augmented Reality application covering specific subjects (English literacy skills, Mathematics and Geography, Positive Behaviour, plus, additionally, an Augmented Reality authoring tool applied in a wide range of subjects). In this paper, we introduce the datasets collected during the pilots, describe how the data enabled the validation of the technology, and how the approach chosen could enhance existing augmented reality applications in data exploration and modelling.

Immune correlates of aging in outdoor-housed captive rhesus macaques (Macaca mulatta).

BACKGROUND: Questions remain about whether inflammation is a cause, consequence, or coincidence of aging. The purpose of this study was to define baseline immunological characteristics from blood to develop a model in rhesus macaques that could be used to address the relationship between inflammation and aging. Hematology, flow cytometry, clinical chemistry, and multiplex cytokine/chemokine analyses were performed on a group of 101 outdoor-housed captive rhesus macaques ranging from 2 to 24 years of age, approximately equivalent to 8 to 77 years of age in humans. RESULTS: These results extend earlier reports correlating changes in lymphocyte subpopulations and cytokines/chemokines with increasing age. There were significant declines in numbers of white blood cells (WBC) overall, as well as lymphocytes, monocytes, and polymorphonuclear cells with increasing age. Among lymphocytes, there were no significant declines in NK cells and T cells, whereas B cell numbers exhibited significant declines with age. Within the T cell populations, there were significant declines in numbers of CD4+ naïve T cells and CD8+ naïve T cells. Conversely, numbers of CD4+CD8+ effector memory and CD8+effector memory T cells increased with age. New multiplex analyses revealed that concentrations of a panel of ten circulating cytokines/chemokines, IFNγ, IL1b, IL6, IL12, IL15, TNFα, MCP1, MIP1α, IL1ra, and IL4, each significantly correlated with age and also exhibited concordant pairwise correlations with every other factor within this group. To also control for outlier values, mean rank values of each of these cytokine concentrations in relation to age of each animal and these also correlated with age. CONCLUSIONS: A panel of ten cytokines/chemokines were identified that correlated with aging and also with each other. This will permit selection of animals exhibiting relatively higher and lower inflammation status as a model to test mechanisms of inflammation status in aging with susceptibility to infections and vaccine efficacy.

"I was born full deaf." Written language outcomes after 1 year of strategic and interactive writing instruction.

Nonstandard grammatical forms are often present in the writing of deaf students that are rarely, if ever, seen in the writing of hearing students. With the implementation of Strategic and Interactive Writing Instruction (SIWI) in previous studies, students have demonstrated significant gains in high-level writing skills (e.g., text structure) but have also made gains with English grammar skills. This 1-year study expands on prior research by longitudinally examining the written language growth (i.e., writing length, sentence complexity, sentence awareness, and function words) of 29 deaf middle-school students. A repeated-measures analysis of variance with a between-subjects variable for literacy achievement level was used to examine gains over time and the intervention's efficacy when used with students of various literacy levels. Students, whether high or low achieving, demonstrated statistically significant gains with writing length, sentence complexity, and sentence awareness. Subordinate clauses were found to be an area of difficulty, and follow up strategies are suggested. An analysis of function word data, specifically prepositions and articles, revealed different patterns of written language growth by language group (e.g., American Sign Language users, oral students, users of English-based sign).

Training School Professionals in a Global Pandemic: Insights From Elementary Education and Speech-Language Pathology.

PURPOSE: The purpose of this research was to explore the preparation of practitioners from two disciplines-speech-language pathology and elementary education-who often work together in a school setting to identify ways to best support future professionals in their educational practicum settings. The primary research questions guiding this investigation were as follows: Did the student teaching and supervision experiences of preservice K-6 teachers (PSTs) and their mentors and speech-language pathology graduate student clinicians and their supervisors differ during the COVID-19 pandemic, and if so, in what ways? METHOD: A total of 54 participants from one university participated in this study from four groups: 15 graduate student clinicians in a speech-language pathology program, 14 speech-language pathology supervisors, 14 PSTs, and 11 teacher mentors. Survey questions were designed to capture the perceptions of students and supervisors in the fields of speech-language pathology and elementary education who were in the school setting during the fall 2020 semester. Quantitative and qualitative questions were included to obtain information related to the following areas: planning, environment, supervision preferences, team experiences, professional development, and telepractice. RESULTS: Speech-language pathologists and teacher mentors reported being able to successfully mentor and supervise students in the school setting despite significant challenges presented by the global pandemic. Findings also identified several significant differences in the experiences of PSTs and speech-language pathology graduate student clinicians who were in the schools for field experiences. CONCLUSIONS: Overall, speech-language pathology graduate student clinicians and their supervisors as well as PSTs and their teacher mentors reported adequately navigating their field experiences during the COVID-19 pandemic. Implications for how these findings can inform professional preparation programs to optimize future educational and therapy outcomes for students are discussed.

Emerging microsporidian infections in Russian HIV-infected patients.

Microsporidia were identified in stool specimens by histochemistry and PCR of 30 (18.9%) of 159 HIV-infected patients presenting to the S. P. Botkin Memorial Clinical Hospital of Infectious Diseases, St. Petersburg, Russia. The higher prevalence of Encephalitozoon intestinalis, in 21 (12.8%) patients, than of Enterocytozoon bieneusi, in 2 patients (1.2%), was unexpected. Encephalitozoon cuniculi was detected in three patients: one with strain I and two with strain II. Encephalitozoon hellem was detected in one patient, and two patients were identified as being infected by Microsporidium species. One patient was infected with both E. intestinalis and E. cuniculi. In two patients, the microsporidian species were not identifiable. No statistically significant differences in gender, age, and stage of AIDS were observed between the microsporidian-positive and -negative HIV-infected patients. HIV-infected patients diagnosed with microsporidian infection, however, were significantly more likely to exhibit ≤ 100 CD4(+) T cells/µl blood (20/30 patients [67%]; odds ratio [OR], 3.150; 95% confidence interval [CI(95)], 1.280 to 7.750; P = 0.0116) and weight loss of >10% of the baseline (19/30 patients [63%]; odds ratio, 2.995; CI(95), 1.100 to 8.158; P = 0.0352) than HIV-infected patients not diagnosed with microsporidian infection. In summary, this is the first report describing the diagnosis of microsporidian infection of HIV-infected patients in Russia and the first detection of E. cuniculi strain II in a human.

An immune evasion mechanism for spirochetal persistence in Lyme borreliosis.

Borrelia burgdorferi, the Lyme disease spirochete, persistently infects mammalian hosts despite the development of strong humoral responses directed against the pathogen. Here we describe a novel mechanism of immune evasion by B. burgdorferi. In immunocompetent mice, spirochetes that did not express ospC (the outer-surface protein C gene) were selected within 17 d after inoculation, concomitantly with the emergence of anti-OspC antibody. Spirochetes with no detectable OspC transcript that were isolated from immunocompetent mice reexpressed ospC after they were either cultured in vitro or transplanted to naive immunocompetent mice, but not in OspC-immunized mice. B. burgdorferi persistently expressed ospC in severe combined immune-deficient (SCID) mice. Passive immunization of B. burgdorferi-infected SCID mice with an anti-OspC monoclonal antibody selectively eliminated ospC-expressing spirochetes but did not clear the infection. OspC-expressing spirochetes reappeared in SCID mice after the anti-OspC antibody was eliminated. We submit that selection of surface-antigen nonexpressers is an immune evasion mechanism that contributes to spirochetal persistence.

The BAM complex subunit BamE (SmpA) is required for membrane integrity, stalk growth and normal levels of outer membrane {beta}-barrel proteins in Caulobacter crescentus.

The outer membrane of Gram-negative bacteria is an essential compartment containing a specific complement of lipids and proteins that constitute a protective, selective permeability barrier. Outer membrane beta-barrel proteins are assembled into the membrane by the essential hetero-oligomeric BAM complex, which contains the lipoprotein BamE. We have identified a homologue of BamE, encoded by CC1365, which is located in the outer membrane of the stalked alpha-proteobacterium Caulobacter crescentus. BamE associates with proteins whose homologues in other bacteria are known to participate in outer membrane protein assembly: BamA (CC1915), BamB (CC1653) and BamD (CC1984). Caulobacter cells lacking BamE grow slowly in rich medium and are hypersensitive to anionic detergents, some antibiotics and heat exposure, which suggest that the membrane integrity of the mutant is compromised. Membranes of the DeltabamE mutant have normal amounts of the outer membrane protein RsaF, a TolC homologue, but are deficient in CpaC*, an aggregated form of the outer membrane secretin for type IV pili. Delta bamE membranes also contain greatly reduced amounts of three TonB-dependent receptors that are abundant in wild-type cells. Cells lacking BamE have short stalks and are delayed in stalk outgrowth during the cell cycle. Based on these findings, we propose that Caulobacter BamE participates in the assembly of outer membrane beta-barrel proteins, including one or more substrates required for the initiation of stalk biogenesis.

Mechanism of origin activation by monomers of R6K-encoded pi protein.

One recurring theme in plasmid duplication is the recognition of the origin of replication (ori) by specific Rep proteins that bind to DNA sequences called iterons. For plasmid R6K, this process involves a complex interplay between monomers and dimers of the Rep protein, pi, with seven tandem iterons of gamma ori. Remarkably, both pi monomers and pi dimers can bind to iterons, a new paradigm in replication control. Dimers, the predominant form in the cell, inhibit replication, while monomers facilitate open complex formation and activate the ori. Here, we investigate a mechanism by which pi monomers out-compete pi dimers for iteron binding, and in so doing activate the ori. With an in vivo plasmid incompatibility assay, we find that pi monomers bind cooperatively to two adjacent iterons. Cooperative binding is eliminated by insertion of a half-helical turn between two iterons but is diminished only slightly by insertion of a full helical turn between two iterons. These studies show also that pi bound to a consensus site promotes occupancy of an adjacent mutated site, another hallmark of cooperative interactions. pi monomer/iteron interactions were quantified using a monomer-biased pi variant in vitro with the same collection of two-iteron constructs. The cooperativity coefficients mirror the plasmid incompatibility results for each construct tested. pi dimer/iteron interactions were quantified with a dimer-biased mutant in vitro and it was found that pi dimers bind with negligible cooperativity to two tandem iterons.

SucA-dependent uptake of sucrose across the outer membrane of Caulobacter crescentus.

Caulobacter crescentus is an aquatic Gram-negative bacterium that lives in nutrient-poor environments. Like several other aquatic and phytopathogenic bacteria, Caulobacter cells have a relatively large number of genes predicted to encode TonB-dependent receptors (TBDRs). TBDRs transport nutrients across the outer membrane using energy from the proton motive force. We identified one TBDR gene, sucA, which is situated within a cluster of genes predicted to encode a lacI-family transcription factor (sucR), amylosucrase (sucB), fructokinase (sucC), and an inner membrane transporter (sucD). Given its genomic neighborhood, we proposed that sucA encodes a transporter for sucrose. Using RT-qPCR, we determined that expression of sucABCD is strongly induced by sucrose in the media and repressed by the transcription factor, SucR. Furthermore, cells with a deletion of sucA have a reduced uptake of sucrose. Although cells with a non-polar deletion of sucA can grow with sucrose as the sole carbon source, cells with a polar deletion that eliminates expression of sucABCD cannot grow with sucrose as the sole carbon source. These results show that the suc locus is essential for sucrose utilization while SucA functions as one method of sucrose uptake in Caulobacter crescentus. This work sheds light on a new carbohydrate utilization locus in Caulobacter crescentus.

Phonological working memory in developmental stuttering: Potential insights from the neurobiology of language and cognition.

The current review examines how neurobiological models of language and cognition could shed light on the role of phonological working memory (PWM) in developmental stuttering (DS). Toward that aim, we review Baddeley's influential multicomponent model of PWM and evidence for load-dependent differences between children and adults who stutter and typically fluent speakers in nonword repetition and dual-task paradigms. We suggest that, while nonword repetition and dual-task findings implicate processes related to PWM, it is unclear from behavioral studies alone what mechanisms are involved. To address how PWM could be related to speech output in DS, a third section reviews neurobiological models of language proposing that PWM is an emergent property of cyclic sensory and motor buffers in the dorsal stream critical for speech production. We propose that anomalous sensorimotor timing could potentially interrupt both fluent speech in DS and the emergent properties of PWM. To further address the role of attention and executive function in PWM and DS, we also review neurobiological models proposing that prefrontal cortex (PFC) and basal ganglia (BG) function to facilitate working memory under distracting conditions and neuroimaging evidence implicating the PFC and BG in stuttering. Finally, we argue that cognitive-behavioral differences in nonword repetition and dual-tasks are consistent with the involvement of neurocognitive networks related to executive function and sensorimotor integration in PWM. We suggest progress in understanding the relationship between stuttering and PWM may be accomplished using high-temporal resolution electromagnetic experimental approaches.

Ecological risk assessment for aquatic invertebrate communities exposed to imidacloprid as a result of labeled agricultural and nonagricultural uses in the United States.

A probabilistic ecological risk assessment (ERA) was conducted to determine the potential effects of acute and chronic exposure of aquatic invertebrate communities to imidacloprid arising from labeled agricultural and nonagricultural uses in the United States. Aquatic exposure estimates were derived using a higher-tier refined modeling approach that accounts for realistic variability in environmental and agronomic factors. Toxicity was assessed using refined acute and chronic community-level effect metrics for aquatic invertebrates (i.e., species or taxon sensitivity distributions) developed using the best available data. Acute and chronic probabilistic risk estimates were derived by integrating the exposure distributions for different use patterns with the applicable species or taxon sensitivity distributions to generate risk curves, which plot cumulative probability of exceedance versus the magnitude of effect. Overall, the results of this assessment indicated that the aquatic invertebrate community is unlikely to be adversely affected by acute or chronic exposure to imidacloprid resulting from currently registered uses of imidacloprid in the United States. Environ Toxicol Chem 2017;36:1375-1388. © 2016 SETAC.

Sugar concentration in nectar: a quantitative metric of crop attractiveness for refined pollinator risk assessments.

Those involved with pollinator risk assessment know that agricultural crops vary in attractiveness to bees. Intuitively, this means that exposure to agricultural pesticides is likely greatest for attractive plants and lowest for unattractive plants. While crop attractiveness in the risk assessment process has been qualitatively remarked on by some authorities, absent is direction on how to refine the process with quantitative metrics of attractiveness. At a high level, attractiveness of crops to bees appears to depend on several key variables, including but not limited to: floral, olfactory, visual and tactile cues; seasonal availability; physical and behavioral characteristics of the bee; plant and nectar rewards. Notwithstanding the complexities and interactions among these variables, sugar content in nectar stands out as a suitable quantitative metric by which to refine pollinator risk assessments for attractiveness. Provided herein is a proposed way to use sugar nectar concentration to adjust the exposure parameter (with what is called a crop attractiveness factor) in the calculation of risk quotients in order to derive crop-specific tier I assessments. This Perspective is meant to invite discussion on incorporating such changes in the risk assessment process. © 2016 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Antimicrosporidial activity of (fluoro)quinolones in vitro and in vivo.

Microsporidia are a cause of emerging and opportunistic infections in humans and animals. Although two drugs are currently being used to treat microsporidiosis, concerns exist that albendazole is only selective for inhibiting some species of microsporidia that infect mammals, and fumagillin appears to have been found to be toxic. During a limited sequence survey of the Vittaforma corneae genome, a partial gene encoding for the ParC topoisomerase IV subunit was identified. The purpose of this set of studies was to determine if fluoroquinolones, which target topoisomerase IV, exert activity against Encephalitozoon intestinalis and V. corneae in vitro, and whether these compounds could prolong survival of V. corneae-infected athymic mice. Fifteen fluoroquinolones were tested. Of these, norfloxacin and ofloxacin inhibited E. intestinalis replication by more than 70% compared with non-treated control cultures, while gatifloxacin, lomefloxacin, moxifloxacin, and nalidixic acid (sodium salt) inhibited both E. intestinalis and V. corneae by at least 60% at concentrations not toxic to the host cells. These drugs were tested in vivo also, where gatifloxacin, lomefloxacin, norfloxacin, and ofloxacin prolonged survival of V. corneae-infected athymic mice (P < 0.05), whereas moxifloxacin and nalidixic acid failed to prolong survival. Therefore, these results support continued studies for evaluating the efficacy of the fluoroquinolones for treating microsporidiosis and for characterizing the target(s) of these fluoroquinolones in the microsporidia.

Bacterial expression system with tightly regulated gene expression and plasmid copy number.

A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and gamma origin (ori) plasmid copy number. Regulation of gamma ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, pi, whose gene was integrated into the chromosome of the host strain under control of the P(BAD) promoter. gamma ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium. This strain avoids the problem of all-or-nothing induction of P(BAD) because it is deficient in both arabinose uptake and degradation genes. Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome. Although this strain could be compatible with any gamma ori plasmid, we describe the utility of a gamma ori expression vector that allows especially tight regulation of gene expression. With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products. We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein. This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any gamma ori vector at precise copy levels in a cell.