Structure and Function of Stator Units of the Bacterial Flagellar Motor.

Many bacteria use the flagellum for locomotion and chemotaxis. Its bidirectional rotation is driven by a membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport, and how these changes power rotation of the flagellum remain unknown. Here, we present ~3 Å-resolution cryoelectron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a detailed mechanistic model for motor function and switching of rotational direction.

Evaluation of a Virtual Reality Platform to Train Stress Management Skills for a Defense Workforce: Multisite, Mixed Methods Feasibility Study.

BACKGROUND: Psychological stress-related injuries within first-responder organizations have created a need for the implementation of effective stress management training. Most stress management training solutions have limitations associated with scaled adoption within the workforce. For instance, those that are effective in civilian populations often do not align with the human performance culture embedded within first-responder organizations. Programs involving expert-led instructions that are high in quality are often expensive. OBJECTIVE: This study sought to evaluate a tailored stress management training platform within the existing training schedule of the Australian Defense Force (ADF). The platform, known as Performance Edge (PE), is a novel virtual reality (VR) and biofeedback-enabled stress management skills training platform. Focusing on practical training of well-established skills and strategies, the platform was designed to take advantage of VR technology to generate an immersive and private training environment. This study aimed to assess the feasibility of delivering the VR platform within the existing group-based training context and intended training population. In this setting, the study further aimed to collect data on critical predictors of user acceptance and technology adoption in education, including perceived usability, usefulness, and engagement, while also assessing training impacts. METHODS: This study used a mixed methods, multisite approach to collect observational, self-reported, and biometric data from both training staff and trainers within a real-world "on-base" training context in the ADF. Validated scales include the Presence Questionnaire and User Engagement Scale for perceived usefulness, usability, and engagement, as well as the State Mindfulness Scale and Relaxation Inventory, to gain insights into immediate training impacts for specific training modules. Additional surveys were specifically developed to assess implementation feedback, intention to use skills, and perceived training impact and value. RESULTS: PE training was delivered to 189 ADF trainees over 372 training sessions. The platform was easy to use at an individual level and was feasible to deliver in a classroom setting. Trainee feedback consistently showed high levels of engagement and a sense of presence with the training content and environment. PE is overall perceived as an effective and useful training tool. Self-report and objective indices confirmed knowledge improvement, increased skill confidence, and increased competency after training. Specific training elements resulted in increased state mindfulness, increased physical relaxation, and reduced breathing rate. The ability to practice cognitive strategies in a diverse, private, and immersive training environment while in a group setting was highlighted as particularly valuable. CONCLUSIONS: This study found the VR-based platform (PE) to be a feasible stress management training solution for group-based training delivery in a defense population. Furthermore, the intended end users, both trainers and trainees, perceive the platform to be usable, useful, engaging, and effective for training, suggesting end-user acceptance and potential for technology adoption.

Correction: Evaluation of a Virtual Reality Platform to Train Stress Management Skills for a Defense Workforce: Multisite, Mixed Methods Feasibility Study.

[This corrects the article DOI: 10.2196/46368.].

Comparing approaches for selection, development, and deployment of extended reality (XR) teaching applications: A case study at The University of Newcastle Australia.

The use of extended reality (XR) technology in education offers many advantages for transferring knowledge and practical skills training at the higher education level. As a result, many Universities over the past 5 + years have undertaken pilot programs to both develop XR content and assess how to best implement it within existing teaching and learning systems. Unfortunately, very few of these efforts have included structured evaluation or documentation. As such, limited published evidence exists to inform processes and approaches that may assist or hinder broad scale implementation. This leads many Universities to unnecessarily commit significant time and resources to testing identical or similar approaches, resulting in repeated identification of the same or similar challenges. In response to this situation, The University of Newcastle, Australia decided to systematically document the approach for selection, development and implementation of four new virtual-reality (VR) teaching applications. The current paper contains a detailed intrinsic case study, outlining the process and critical elements that shaped the selection of suitable teaching content, software development, hardware solutions and implementation. Details are provided on how decisions were made, what components were considered helpful, challenges identified, and important lessons outlined. These findings will be useful to organisations and individuals as they look to develop pathways and processes to integrate XR technology, particularly within their existing training and educational frameworks. Supplementary Information: The online version contains supplementary material available at 10.1007/s10639-022-11364-2.

RflM mediates target specificity of the RcsCDB phosphorelay system for transcriptional repression of flagellar synthesis in Salmonella enterica.

The bacterial flagellum enables directed movement of Salmonella enterica towards favorable conditions in liquid environments. Regulation of flagellar synthesis is tightly controlled by various environmental signals at transcriptional and post-transcriptional levels. The flagellar master regulator FlhD4 C2 resides on top of the flagellar transcriptional hierarchy and is under autogenous control by FlhD4 C2 -dependent activation of the repressor rflM. The inhibitory activity of RflM depends on the presence of RcsB, the response regulator of the RcsCDB phosphorelay system. In this study, we elucidated the molecular mechanism of RflM-dependent repression of flhDC. We show that RcsB and RflM form a heterodimer that coordinately represses flhDC transcription independent of RcsB phosphorylation. RcsB-RflM complex binds to a RcsB box downstream the P1 transcriptional start site of the flhDC promoter with increased affinity compared to RcsB in the absence of RflM. We propose that RflM stabilizes binding of unphosphorylated RcsB to the flhDC promoter in absence of environmental cues. Thus, RflM is a novel auxiliary regulatory protein that mediates target specificity of RcsB for flhDC repression. The cooperative action of the RcsB-RflM repressor complex allows Salmonella to fine-tune initiation of flagellar gene expression and adds another level to the complex regulation of flagellar synthesis.

The Salmonella Spi1 virulence regulatory protein HilD directly activates transcription of the flagellar master operon flhDC.

Infection of intestinal epithelial cells is dependent on the Salmonella enterica serovar Typhimurium pathogenicity island 1 (Spi1)-encoded type III injectisome system and flagellar motility. Thus, the expression of virulence and flagellar genes is subject to tight regulatory control mechanisms in order to ensure the correct spatiotemporal production of the respective gene products. In this work, we reveal a new level of cross-regulation between the Spi1 and flagellar regulatory systems. Transposon mutagenesis identified a class of mutants that prevented flhDC autorepression by overexpressing HilD. HilD, HilC, RtsA, and HilA comprise a positive regulatory circuit for the expression of the Spi1 genes. Here, we report a novel transcriptional cross talk between the Spi1 and flagellar regulons where HilD transcriptionally activates flhDC gene expression by binding to nucleotides -68 to -24 upstream from the P5 transcriptional start site. We additionally show that, in contrast to the results of a previous report, HilA does not affect flagellar gene expression. Finally, we discuss a model of the cross-regulation network between Spi1 and the flagellar system and propose a regulatory mechanism via the Spi1 master regulator HilD that would prime flagellar genes for rapid reactivation during host infection.

Klebsiella oxytoca inhibits Salmonella infection through multiple microbiota-context-dependent mechanisms.

The Klebsiella oxytoca species complex is part of the human microbiome, especially during infancy and childhood. K. oxytoca species complex strains can produce enterotoxins, namely, tilimycin and tilivalline, while also contributing to colonization resistance (CR). The relationship between these seemingly contradictory roles is not well understood. Here, by coupling ex vivo assays with CRISPR-mutagenesis and various mouse models, we show that K. oxytoca provides CR against Salmonella Typhimurium. In vitro, the antimicrobial activity against various Salmonella strains depended on tilimycin production and was induced by various simple carbohydrates. In vivo, CR against Salmonella depended on toxin production in germ-free mice, while it was largely toxin-independent in mice with residual microbiota. This was linked to the relative levels of toxin-inducing carbohydrates in vivo. Finally, dulcitol utilization was essential for toxin-independent CR in gnotobiotic mice. Together, this demonstrates that nutrient availability is key to both toxin-dependent and substrate-driven competition between K. oxytoca and Salmonella.

Methylation of Salmonella Typhimurium flagella promotes bacterial adhesion and host cell invasion.

The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.

Characterization of Novel Factors Involved in Swimming and Swarming Motility in Salmonella enterica Serovar Typhimurium.

Salmonella enterica utilizes flagellar motility to swim through liquid environments and on surfaces. The biosynthesis of the flagellum is regulated on various levels, including transcriptional and posttranscriptional mechanisms. Here, we investigated the motility phenotype of 24 selected single gene deletions that were previously described to display swimming and swarming motility effects. Mutations in flgE, fliH, ydiV, rfaG, yjcC, STM1267 and STM3363 showed an altered motility phenotype. Deletions of flgE and fliH displayed a non-motile phenotype in both swimming and swarming motility assays as expected. The deletions of STM1267, STM3363, ydiV, rfaG and yjcC were further analyzed in detail for flagellar and fimbrial gene expression and filament formation. A ΔydiV mutant showed increased swimming motility, but a decrease in swarming motility, which coincided with derepression of curli fimbriae. A deletion of yjcC, encoding for an EAL domain-containing protein, increased swimming motility independent on flagellar gene expression. A ΔSTM1267 mutant displayed a hypermotile phenotype on swarm agar plates and was found to have increased numbers of flagella. In contrast, a knockout of STM3363 did also display an increase in swarming motility, but did not alter flagella numbers. Finally, a deletion of the LPS biosynthesis-related protein RfaG reduced swimming and swarming motility, associated with a decrease in transcription from flagellar class II and class III promoters and a lack of flagellar filaments.