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Background: Cultural sensitivity (CS) training is vital to pediatric emergency medicine (PEM) curricula. This study aimed to explore CS in Yale PEM fellows and emergency medicine (EM) residents at Indus Hospital and Health Network (IHHN) in Pakistan through distance simulation activities., Methods: This mixed-methods analysis of an educational intervention was conducted at Yale University in collaboration with IHHN. We approached seven U.S. PEM fellows and 22 Pakistani EM residents. We performed a baseline CS assessment using the Clinical Cultural Competency Questionnaire (CCCQ). Afterward, the U.S. PEM fellows facilitated the Pakistani EM residents through six distance simulation sessions. Qualitative data were collected through online focus groups. The CCCQ was analyzed using descriptive statistics, and content analysis was used to analyze the data from the focus groups., Results: Seven U.S. PEM fellows and 18 of 22 Pakistani EM residents responded to the CCCQ at the beginning of the module. The mean (±SD) CCCQ domain scores for the U.S. PEM fellows versus the Pakistani EM residents were 2.56 (±0.37) versus 2.87 (±0.72) for knowledge, 3.02 (±0.41) versus 3.33 (±0.71) for skill, 2.86 (±0.32) versus 3.17 (±0.73) for encounter/situation, and 3.80 (±0.30) versus 3.47 (±0.47) for attitude (each out of 5 points). Our qualitative data analysis showed that intercultural interactions were valuable. There is a common language of medicine among the U.S. PEM fellows and Pakistani EM residents. The data also highlighted a power distance between the facilitators and learners, as the United States was seen as the standard of "how to practice PEM." The challenges identified were time differences, cultural practices such as prayer times, the internet, and technology. The use of local language during debriefing was perceived to enhance engagement., Conclusion: The distance simulation involving U.S. PEM fellows and Pakistani EM residents was an effective approach in assessing various aspects of intercultural education, such as language barriers, technical challenges, and religious considerations., Competing Interests: The authors have no conflicts of interest to disclose., (© 2023 Society for Academic Emergency Medicine.)

Background: To compare validity evidence for dichotomous and trichotomous versions of a neonatal intubation (NI) procedural skills checklist., Methods: NI skills checklists were developed utilizing an existing framework. Experts were trained on scoring using dichotomous and trichotomous checklists, and rated recordings of 23 providers performing simulated NI. Videolaryngoscope recordings of glottic exposure were evaluated using Cormack-Lehane (CL) and Percent of Glottic Opening scales. Internal consistency and reliability of both checklists were analyzed, and correlations between checklist scores, airway visualization, entrustable professional activities (EPA), and global skills assessment (GSA) were calculated., Results: During rater training, raters gave significantly higher scores on better provider performance in standardized videos (both p < 0.001). When utilized to evaluate study participants' simulated NI attempts, both dichotomous and trichotomous checklist scores demonstrated very good internal consistency (Cronbach's alpha 0.868 and 0.840, respectively). Inter-rater reliability was higher for dichotomous than trichotomous checklists [Fleiss kappa of 0.642 and 0.576, respectively (p < 0.001)]. Sum checklist scores were significantly different among providers in different disciplines (p < 0.001, dichotomous and trichotomous). Sum dichotomous checklist scores correlated more strongly than trichotomous scores with GSA and CL grades. Sum dichotomous and trichotomous checklist scores correlated similarly well with EPA., Conclusions: Neither dichotomous or trichotomous checklist was superior in discriminating provider NI skill when compared to GSA, EPA, or airway visualization assessment. Sum scores from dichotomous checklists may provide sufficient information to assess procedural competence, but trichotomous checklists may permit more granular feedback to learners and educators. The checklist selected may vary with assessment needs., (© 2022. The Author(s).)

Background: The majority of children in the United States seek emergency care at community-based general emergency departments (GEDs); however, the quality of GED pediatric emergency care varies widely. This may be explained by a number of factors, including residency training environments and postgraduate knowledge decay. Emergency medicine (EM) residents train in academic pediatric EDs, but didactic and clinical experience vary widely between programs, and little is known about the pediatric skills of these EM residents. This study aimed to assess the performance of senior EM residents in treating simulated pediatric patients at the end of their training., Methods: This was a prospective, cross-sectional, simulation-based cohort study assessing the simulated performance of senior EM resident physicians from two Massachusetts programs leading medical teams caring for three critically ill patients. Sessions were video recorded and scored separately by three reviewers using a previously published simulation assessment tool. Self-efficacy surveys were completed prior to each session. The primary outcome was a median total performance score (TPS), calculated by the mean of individualized domain scores (IDS) for each case. Each IDS was calculated as a percentage of items performed on a checklist-based instrument., Results: A total of 18 EM resident physicians participated (PGY-3 = 8, PGY-4 = 10). Median TPS for the cohort was 61% (IQR = 56%-70%). Median IDSs by case were as follows: sepsis 67% (IQR = 50%-67%), seizure 67% (IQR = 50%-83%), and cardiac arrest 67% (IQR = 43%-70%). The overall cohort self-efficacy for pediatric EM (PEM) was 64% (IQR = 60%-70%)., Conclusions: This study has begun the process of benchmarking clinical performance of graduating EM resident physicians. Overall, the EM resident cohort in this study performed similar to prior GED teams. Self-efficacy related to PEM correlated well with performance, with the exception of knowledge relative to intravenous fluid and vasopressor administration in pediatric septic shock. A significant area of discrepancy and missed checklist items were those related to cardiopulmonary resuscitation and basic life support maneuvers., (© 2020 by the Society for Academic Emergency Medicine.)

Introduction: Disaster triage training for emergency medical service (EMS) providers is unstandardised. We hypothesised that disaster triage training with the paediatric disaster triage (PDT) video game '60 s to Survival' would be a cost-effective alternative to live simulation-based PDT training., Methods: We synthesised data for a cost-effectiveness analysis from two previous studies. The video game data were from the intervention arm of a randomised controlled trial that compared triage accuracy in a live simulation scenario of exposed vs unexposed groups to the video game. The live simulation and feedback data were from a prospective cohort study evaluating live simulation and feedback for improving disaster triage skills. Postintervention scores of triage accuracy were measured for participants via live simulations and compared between both groups. Cost-effectiveness between the live simulation and video game groups was assessed using (1) A net benefit regression model at various willingness-to-pay (WTP) values. (2) A cost-effectiveness acceptability curve (CEAC)., Results: The total cost for the live simulation and feedback training programme was $81 313.50 and the cost for the video game was $67 822. Incremental net benefit values at various WTP values revealed positive incremental net benefit values, indicating that the video game is more cost-effective compared with live simulation and feedback. Moreover, the CEAC revealed a high probability (>0.6) at various WTP values that the video game is more cost-effective., Conclusions: A video game-based simulation disaster triage training programme was more cost-effective than a live simulation and feedback-based programme. Video game-based training could be a simple, scalable and sustainable solution to training EMS providers., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Emergency department (ED) visits for behavioural conditions are rising, with 1.7 million associated episodes of patient agitation occurring annually in acute care settings. When de-escalation techniques fail during agitation management, patients are subject to use of physical restraints and sedatives, which are associated with up to 37% risk of hypotension, apnoea and physical injuries. At the same time, ED staff report workplace violence due to physical assaults during agitation events. We recently developed a theoretical framework to characterise ED agitation, which identified teamwork as a critical component to reduce harm. Currently, no structured team response protocol for ED agitation addressing both patient and staff safety exists., Methods and Analysis: Our proposed study aims to develop and implement the agitation code team (ACT) response intervention, which will consist of a standardised, structured process with defined health worker roles/responsibilities, work processes and clinical protocols. First, we will develop the ACT response intervention in a two-step design loop; conceptual design will engage users in the creation of the prototype, and iterative refinement will occur through in situ simulated agitated patient encounters in the ED to assess and improve the design. Next, we will pilot the intervention in the clinical environment and use a controlled interrupted time series design to evaluate its effect on our primary outcome of patient restraint use. The intervention will be considered efficacious if we effectively lower the rate of restraint use over a 6-month period., Ethics and Dissemination: Ethical approval by the Yale University Human Investigation Committee was obtained in 2019 (HIC #2000025113). Results will be disseminated through peer-reviewed publications and presentations at scientific meetings for each phase of the study. If this pilot is successful, we plan to formally integrate the ACT response intervention into clinical workflows at all EDs within our entire health system., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are associated with a number of skin diseases. Here, we present the design of an engineered commensal organism, Staphylococcus epidermidis , for use as a live biotherapeutic product (LBP) candidate for skin diseases. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. We therefore constructed an auxotrophic strain of S. epidermidis that requires exogenously supplied d-alanine. The S. epidermidis NRRL B-4268 Δ alr1 Δ alr2 Δ dat strain (SE ΔΔΔ ) contains deletions of three biosynthetic genes: two alanine racemase genes, alr1 and alr2 (SE1674 and SE1079), and the d-alanine aminotransferase gene, dat (SE1423). These three deletions restricted growth in d-alanine-deficient medium, pooled human blood, and skin. In the presence of d-alanine, SE ΔΔΔ colonized and increased expression of human β-defensin 2 in cultured human skin models in vitro. SE ΔΔΔ showed a low propensity to revert to d-alanine prototrophy and did not form biofilms on plastic in vitro. These studies support the potential safety and utility of SE ΔΔΔ as a live biotherapeutic strain whose growth can be controlled by d-alanine. IMPORTANCE The skin microbiome is rich in opportunities for novel therapeutics for skin diseases, and synthetic biology offers the advantage of providing novel functionality or therapeutic benefit to live biotherapeutic products. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. This study presents the design and in vitro evidence of a skin commensal whose growth can be controlled through d-alanine. The basis of this strain will support future clinical studies of this strain in humans., (Copyright © 2020 Dodds et al.)

Introduction: Simulation-based training is essential for high-quality medical care, but it requires access to equipment and expertise. Technology can facilitate connecting educators to training in simulation. We aimed to explore the use of remote simulation faculty development in Latvia using telesimulation and telementoring with an experienced debriefer located in the USA., Methods: This was a prospective, simulation-based longitudinal study. Over the course of 16 months, a remote simulation instructor (RI) from the USA and a local instructor (LI) in Latvia cofacilitated with teleconferencing. Responsibility gradually transitioned from the RI to the LI. At the end of each session, students completed the Debriefing Assessment for Simulation in Healthcare (DASH) student version form (DASH-SV) and a general feedback form, and the LI completed the instructor version of the DASH form (DASH-IV). Outcome measures were the changes in DASH scores over time., Results: A total of eight simulation sessions were cofacilitated of 16 months. As the role of the LI increased over time, the debrief quality measured with the DASH-IV did not change significantly (from 89 to 87), although the DASH-SV score decreased from a total median score of 89 (IQR 86-98) to 80 (IQR 78-85) (p = 0.005)., Conclusion: In this study, telementoring with telesimulations resulted in high-quality debriefing. The quality-perceived by the students-was higher with the involvement of the remote instructor and declined during the transition to the LI. This concept requires further investigation and could potentially build local simulation expertise promoting sustainability of high-quality simulation., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The delivery and initial resuscitation of a newborn infant are required but rarely practised skills in emergency medicine. Deliveries in the emergency department are high-risk events and deviations from best practices are associated with poor outcomes., Introduction: Telemedicine can provide emergency medicine providers real-time access to a Neonatal Resuscitation Program (NRP)-trained paediatric specialist. We hypothesised that adherence to NRP guidelines would be higher for participants with access to a remotely located NRP-trained paediatric specialist via telemedicine compared with participants without access., Materials and Methods: Prospective single-centre randomised trial. Emergency Medicine residents were randomised into a telemedicine or standard care group. The participants resuscitated a simulated, apnoeic and bradycardic neonate. In the telemedicine group a remote paediatric specialist participated in the resuscitation. Simulations were video recorded and assessed for adherence to guidelines using four critical actions. The secondary outcome of task load was measured through participants' completion of the NASA Task Load Index (NASA-TLX) and reviewers completed a detailed NRP checklist., Results: Twelve participants were included. The use of telemedicine was associated with significantly improved adherence to three of the four critical actions reflecting NRP guidelines as well as a significant improvement in the overall score (p<0.001). On the NASA-TLX, no significant difference was seen in overall subjective workload assessment, but of the subscore components, frustration was statistically significantly greater in the control group (p<0.001)., Conclusions: In this study, telemedicine improved adherence to NRP guidelines. Future work is needed to replicate these findings in the clinical environment., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Pediatric airway management is a challenging process at community emergency departments (CEDs) due to lower pediatric volume, a lack of pediatric expertise among staff, and a lack of pediatric-specific equipment and resources. This has contributed to increased mortality in pediatric patients presenting to CEDs in comparison to pediatric academic medical centers (AMCs). We hypothesized that a collaborative program between CEDs and the state AMC would improve the quality of pediatric airway management provided by CEDs in simulated settings and the CEDs' pediatric emergency readiness scores., Methods: This prospective, pre- and post-intervention study utilized in situ simulation and was conducted in 10 CEDs in the state of Indiana. A team from the pediatric AMC led a multi-faceted improvement program, which included post-simulation debriefing, addressing pediatric airway management issues, targeted assessment reports, access to pediatric resources, and ongoing communication with the AMC. The primary outcome of the study was improvement of simulated pediatric airway management in the CEDs. The secondary outcome was improvement of the CEDs' pediatric emergency readiness scores score., Results: A total of 35 multidisciplinary teams participated in pre-intervention sessions, and 40 teams participated in post-intervention sessions. Overall adherence to a critical action checklist improved from 52% at the pre-intervention visits to 71% post-intervention ( P = .003). There were significant improvements in the use of appropriate endotracheal tube (ETT) size (from 67% to 100%, P = .02), cuffed ETT (from 8% to 71%, P < .001), appropriate blade size (from 58% to 100%, P = .03), and availability of suction catheter (from 10% to 42%, P = .049). The CEDs' total pediatric emergency readiness scores score improved from 58.8 ± 15.6 pre-intervention to 75.8 ± 9.3 post-intervention ( P = .01)., Conclusions: A collaborative improvement program between a pediatric AMC and CEDs improved the CEDs' simulated pediatric emergency airway management. This model can be utilized to improve management of other pediatric critical conditions in these CEDs., (Copyright © 2019 by Daedalus Enterprises.)

Background and Objectives: Pediatric out-of-hospital cardiac arrest survival outcomes are dismal (<10%). Care that is provided in adherence to established guidelines has been associated with improved survival. Lower mortality rates have been reported in higher-volume hospitals, teaching hospitals, and trauma centers. The primary objective of this article was to explore the relationship of hospital characteristics, such as annual pediatric patient volume, to adherence to pediatric cardiac arrest guidelines during an in situ simulation. Secondary objectives included comparing adherence to other team, provider, and system factors., Methods: This prospective, multicenter, observational study evaluated interprofessional teams in their native emergency department (ED) resuscitation bays caring for a simulated 5-year-old child presenting in cardiac arrest. The primary outcome, adherence to the American Heart Association pediatric guidelines, was assessed using a 14-item tool including three component domains: basic life support (BLS), pulseless electrical activity (PEA), and ventricular fibrillation (VF). Provider, team, and hospital-level data were collected as independent data. EDs were evaluated in four pediatric volume groups (low < 1,800/year; medium 1,800-4,999; medium-high 5,000-9,999; high > 10,000). Cardiac arrest adherence and domains were evaluated by pediatric patient volume and other team and hospital-level characteristics, and path analyses were performed to evaluate the contribution of patient volume, systems readiness, and teamwork on BLS, PEA, and VF adherence., Results: A total of 101 teams from a spectrum of 50 EDs participated including nine low pediatric volume (<1,800/year), 36 medium volume (1,800-4,999/year), 24 medium-high (5,000-9,999/year), and 32 high volume (≥10000/year). The median total adherence score was 57.1 (interquartile range = 50.0-78.6). This was not significantly different across the four volume groups. The highest level of adherence for BLS and PEA domains was noted in the medium-high-volume sites, while no difference was noted for the VF domain. The lowest level of BLS adherence was noted in the lowest-volume EDs. Improved adherence was not directly associated with higher pediatric readiness survey (PRS) score provider experience, simulation teamwork performance, or more providers with Pediatric Advanced Life Support (PALS) training. EDs in teaching hospitals with a trauma center designation that served only children demonstrated higher adherence compared to nonteaching hospitals (64.3 vs 57.1), nontrauma centers (64.3 vs. 57.1), and mixed pediatric and adult departments (67.9 vs. 57.1), respectively. The overall effect sizes for total cardiac adherence score are ED type γ = 0.47 and pediatric volume (low and medium vs. medium-high and high) γ = 0.41. A series of path analyses models was conducted that indicated that overall pediatric ED volume predicted significantly better guideline adherence, but the effect of volume on performance was only mediated by the PRS for the VF domain., Conclusions: This study demonstrated variable adherence to pediatric cardiac arrest guidelines across a spectrum of EDs. Overall adherence was not associated with ED pediatric volume. Medium-high-volume EDs demonstrated the highest levels of adherence for BLS and PEA. Lower-volume EDs were noted to have lower adherence to BLS guidelines. Improved adherence was not directly associated with higher PRS score provider experience, simulation teamwork performance, or more providers with PALS training. This study demonstrates that current approaches optimizing the care of children in cardiac arrest in the ED (provider training, teamwork training, environmental preparation) are insufficient., (© 2018 by the Society for Academic Emergency Medicine.)

Background: More than 30 million children are cared for across 5,000 U.S. emergency departments (EDs) each year. Most of these EDs are not facilities designed and operated solely for children. A Web-based survey provided a national and state-by-state assessment of pediatric readiness and noted a national average score was 69 on a 100-point scale. This survey noted wide variations in ED readiness with scores ranging from 61 in low-pediatric-volume EDs to 90 in the high-pediatric-volume EDs. Additionally, the mean score at the state level ranged from 57 (Wyoming) to 83 (Florida) and for individual EDs ranged from 22 to 100. The majority of prior efforts made to improve pediatric readiness have involved providing Web-based resources and online toolkits. This article reports on the first year of a program that aimed to improve pediatric readiness across community hospitals in our state through in situ simulation-based assessment facilitated by our academic medical center. The primary aim was to improve the pediatric readiness scores in the 10 participating hospitals. The secondary aim was to explore the correlation of simulation-based performance of hospital teams with pediatric readiness scores., Methods: This interventional study measured the Pediatric Readiness Survey (PRS) prior to and after implementation of an improvement program. This program consisted of three components: 1) in situ simulations, 2) report-outs, and 3) access to online pediatric readiness resources and content experts. The simulations were conducted in situ (in the ED resuscitation bay) by multiprofessional teams of doctors, nurses, respiratory therapists, and technicians. Simulations and debriefings were facilitated by an expert team from a pediatric academic medical center. Three scenarios were conducted for all teams and include: a 6-month-old with respiratory failure, an 8-year-old with diabetic ketoacidosis (DKA), and a 6-month-old with supraventricular tachycardia (SVT). A performance score was calculated for each scenario. The improvement of PRS was compared before and after the simulation program. The correlation of the simulation performance of each hospital and the PRS was calculated., Results: Forty-one multiprofessional teams from 10 EDs in Indiana participated in the study, five were of medium pediatric volume and five were medium- to high-volume EDs. The PRS significantly improved from the first to the second on-site verification assessment (58.4 ± 4.8 to 74.7 ± 2.9, p = 0.009). Total adherence scores to scenario guidelines were 54.7, 56.4, and 62.4% in the respiratory failure, DKA, and SVT scenarios, respectively. We found no correlation between simulation performance and PRS scores. Medium ED pediatric volume significantly predicted higher PRS scores compared to medium-high pediatric ED volume (β = 8.7; confidence interval = 0.72-16.8, p = 0.034)., Conclusions: Our collaborative improvement program that involved simulation was associated with improvement in pediatric readiness scores in 10 EDs participating statewide. Future work will focus on further expanding of the network and establishing a national model for pediatric readiness improvement., (© 2017 by the Society for Academic Emergency Medicine.)

Objectives: Paramedics and emergency medical technicians (EMTs) perform triage at disaster sites. There is a need for disaster triage training. Live simulation training is costly and difficult to deliver. Screen-based simulations may overcome these training barriers. We hypothesized that a screen-based simulation, 60 Seconds to Survival (60S), would be associated with in-game improvements in triage accuracy., Methods: This was a prospective cohort study of a screen-based simulation intervention, 60S. Participants included emergency medical services (EMS) personnel from 21 EMS agencies across 12 states. Participants performed assessments (e.g., check for pulse) and actions (e.g., reposition the airway) for 12 patients in each scenario and assigned color-coded triage levels (red, yellow, green, or black) to each patient. Participants received on-screen feedback about triage performance immediately after each scenario. A scoring system was designed to encourage accurate and timely triage decisions. Participants who played 60S included practicing EMTs, paramedics, and nurses as well as students studying to assume these roles. Participants played the game at least three times over 13 weeks., Results: In total, 2,234 participants began game play and 739 completed the study and were included in the analysis. Overall, the median number of plays of the game was just above the threshold inclusion criteria (three or more plays) with a median of four plays during the study period (interquartile range [IQR] = 3-7). There was a significant difference in triage accuracy from the first play of the game to the last play of the game. Median baseline triage accuracy in the game was 89.7% (IQR = 82.1%-94.9%), which then increased to a median of 100% at the last game play (IQR = 87.5%-100.0%; p < 0.001). There was some variability in median triage accuracy on fourth through 11th game plays, ranging from 95% to 100%, and on the 12th to 16th plays, the median accuracy was sustained at 100%. There was a significant decrease in the rate of undertriage: from 10.3% (IQR = 5.1%-18.0%) to 0 (IQR = 0%-12.5%; p < 0.001)., Conclusion: 60 Seconds to Survival is associated with improved in-game triage accuracy. Further study of the correlation between in-game triage accuracy and improvements in live simulation or real-world triage decisions is warranted.

Background: Determining when to entrust trainees to perform procedures is fundamental to patient safety and competency development., Objective: To determine whether simulation-based readiness assessments of first year residents immediately prior to their first supervised infant lumbar punctures (LPs) are associated with success., Methods: This prospective cohort study enrolled paediatric and other first year residents who perform LPs at 35 academic hospitals from 2012 to 2014. Within a standardised LP curriculum, a validated 4-point readiness assessment of first year residents was required immediately prior to their first supervised LP. A score ≥3 was required for residents to perform the LP. The proportion of successful LPs (<1000 red blood cells on first attempt) was determined. Process measures included success on any attempt, number of attempts, analgesia usage and use of the early stylet removal technique., Results: We analysed 726 LPs reported from 1722 residents (42%). Of the 432 who underwent readiness assessments, 174 (40%, 95% CI 36% to 45%) successfully performed their first LP. Those who were not assessed succeeded in 103/294 (35%, 95% CI 30% to 41%) LPs. Assessed participants reported more frequent direct attending supervision of the LP (diff 16%; 95% CI 8% to 22%), greater use of topical analgesia (diff 6%; 95% CI 1% to 12%) and greater use of the early stylet removal technique (diff 11%; 95% CI 4% to 19%) but no difference in number of attempts or overall procedural success., Conclusions: Simulation-based readiness assessments performed in a point-of-care fashion were associated with several desirable behaviours but were not associated with greater clinical success with LP., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

All cells must sense and adapt to changing nutrient availability. However, detailed molecular mechanisms coordinating such regulatory pathways remain poorly understood. In Bacillus subtilis, nitrogen homeostasis is controlled by a unique circuitry composed of the regulator TnrA, which is deactivated by feedback-inhibited glutamine synthetase (GS) during nitrogen excess and stabilized by GlnK upon nitrogen depletion, and the repressor GlnR. Here we describe a complete molecular dissection of this network. TnrA and GlnR, the global nitrogen homeostatic transcription regulators, are revealed as founders of a new structural family of dimeric DNA-binding proteins with C-terminal, flexible, effector-binding sensors that modulate their dimerization. Remarkably, the TnrA sensor domains insert into GS intersubunit catalytic pores, destabilizing the TnrA dimer and causing an unprecedented GS dodecamer-to-tetradecamer conversion, which concomitantly deactivates GS. In contrast, each subunit of the GlnK trimer "templates" active TnrA dimers. Unlike TnrA, GlnR sensors mediate an autoinhibitory dimer-destabilizing interaction alleviated by GS, which acts as a GlnR chaperone. Thus, these studies unveil heretofore unseen mechanisms by which inducible sensor domains drive metabolic reprograming in the model Gram-positive bacterium B. subtilis., (© 2015 Schumacher et al.; Published by Cold Spring Harbor Laboratory Press.)

Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-β. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-α-specific residue, Arg(62), from an adjacent subunit. Notably, Arg(62) must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens.

The spatial and temporal control of Filamenting temperature sensitive mutant Z (FtsZ) Z-ring formation is crucial for proper cell division in bacteria. In Escherichia coli, the synthetic lethal with a defective Min system (SlmA) protein helps mediate nucleoid occlusion, which prevents chromosome fragmentation by binding FtsZ and inhibiting Z-ring formation over the nucleoid. However, to perform its function, SlmA must be bound to the nucleoid. To deduce the basis for this chromosomal requirement, we performed biochemical, cellular, and structural studies. Strikingly, structures show that SlmA dramatically distorts DNA, allowing it to bind as an orientated dimer-of-dimers. Biochemical data indicate that SlmA dimer-of-dimers can spread along the DNA. Combined structural and biochemical data suggest that this DNA-activated SlmA oligomerization would prevent FtsZ protofilament propagation and bundling. Bioinformatic analyses localize SlmA DNA sites near membrane-tethered chromosomal regions, and cellular studies show that SlmA inhibits FtsZ reservoirs from forming membrane-tethered Z rings. Thus, our combined data indicate that SlmA DNA helps block Z-ring formation over chromosomal DNA by forming higher-order protein-nucleic acid complexes that disable FtsZ filaments from coalescing into proper structures needed for Z-ring creation.

The E. coli chromosome is condensed into insulated regions termed macrodomains (MDs), which are essential for genomic packaging. How chromosomal MDs are specifically organized and compacted is unknown. Here, we report studies revealing the molecular basis for Terminus-containing (Ter) chromosome condensation by the Ter-specific factor MatP. MatP contains a tripartite fold with a four-helix bundle DNA-binding motif, ribbon-helix-helix and C-terminal coiled-coil. Strikingly, MatP-matS structures show that the MatP coiled-coils form bridged tetramers that flexibly link distant matS sites. Atomic force microscopy and electron microscopy studies demonstrate that MatP alone loops DNA. Mutation of key coiled-coil residues destroys looping and causes a loss of Ter condensation in vivo. Thus, these data reveal the molecular basis for a protein-mediated DNA-bridging mechanism that mediates condensation of a large chromosomal domain in enterobacteria., (Copyright © 2012 Elsevier Inc. All rights reserved.)