Health And Performance Promotion in Youth (HAPPY) hybrid effectiveness-implementation cluster randomised trial: comparison of two strategies to implement an injury prevention exercise programme in Danish youth handball.

OBJECTIVE: To investigate if a combination of an online and onsite implementation strategy was superior to an online-only strategy in enhancing the use of an injury prevention exercise programme (IPEP) and in reducing the risk of shoulder, knee and ankle injuries in youth community handball players (age 11-17) over a handball season. METHODS: In this 30-week hybrid effectiveness-implementation cluster randomised type 3 study, 20 youth handball clubs were randomly assigned 1:1 to either a combined online and onsite implementation strategy (coach workshop using the health action process approach behaviour change model and health service provider (HSP) support) or an online-only strategy (control group). The primary implementation outcome was coach-reported adherence, measured as the average IPEP exercise usage by the team over 30 weeks. The primary effectiveness outcome was player-reported handball playing time to any new handball-related shoulder, knee and ankle injuries, reported weekly using the Oslo Sports Trauma Research Centre Questionnaire on Health Problems. RESULTS: We enrolled 63 coaches (27% women) and 945 players (mean age 14.5 years, 55% girls). Intention-to-treat analyses showed no statistically significant difference between implementation strategies in adherence (between-group difference 1.4, 95% CI -0.5 to 3.4) or in cumulative injury risk (between-group difference 5.5% points, 95% CI -2.2 to 13.1). CONCLUSION: Our findings demonstrate that in youth community handball, a combined online and onsite implementation strategy, including a coach workshop and HSP support, was not superior to an online-only strategy regarding adherence to an IPEP or in reducing shoulder, knee and ankle injury risk. TRIAL REGISTRATION NUMBER: NCT05294237.

A deterministic, c-di-GMP-dependent program ensures the generation of phenotypically similar, symmetric daughter cells during cytokinesis.

Phenotypic heterogeneity in bacteria can result from stochastic processes or deterministic programs. The deterministic programs often involve the versatile second messenger c-di-GMP, and give rise to daughter cells with different c-di-GMP levels by deploying c-di-GMP metabolizing enzymes asymmetrically during cell division. By contrast, less is known about how phenotypic heterogeneity is kept to a minimum. Here, we identify a deterministic c-di-GMP-dependent program that is hardwired into the cell cycle of Myxococcus xanthus to minimize phenotypic heterogeneity and guarantee the formation of phenotypically similar daughter cells during division. Cells lacking the diguanylate cyclase DmxA have an aberrant motility behaviour. DmxA is recruited to the cell division site and its activity is switched on during cytokinesis, resulting in a transient increase in the c-di-GMP concentration. During cytokinesis, this c-di-GMP burst ensures the symmetric incorporation and allocation of structural motility proteins and motility regulators at the new cell poles of the two daughters, thereby generating phenotypically similar daughters with correct motility behaviours. Thus, our findings suggest a general c-di-GMP-dependent mechanism for minimizing phenotypic heterogeneity, and demonstrate that bacteria can ensure the formation of dissimilar or similar daughter cells by deploying c-di-GMP metabolizing enzymes to distinct subcellular locations.

Self-Perceived Interpersonal Problems Among Long-Term Unemployed Individuals, and Vocational Rehabilitation Programs (In)ability to Change Them.

OBJECTIVE: Self-perceived interpersonal problems can challenge one's access to the work market, making it harder to attain and keep a job while adding to the distress of being outside of the labor market. METHODS: In this study, we compared the self-perceived interpersonal problems among long-term unemployed individuals taking part in vocational rehabilitation programs (VRPs) (N = 220) with those of the general population. In addition, we examined whether their self-perceived interpersonal problems changed while taking part in the VRPs. RESULTS: We found that participants report significantly higher levels of self-perceived interpersonal problems as measured by the Inventory of Interpersonal Problems (IIP), especially with regard to feeling cold/distanced, socially inhibited, vindictive/self-centered, and non-assertive. The participants did not report a significant decrease in self-perceived interpersonal problems after being part of VRPs for one year. CONCLUSION: These results are relevant as they may inform interventions targeted this population aimed at increasing employability and/or individual well-being. Importantly, the findings may be viewed as a reflection of both social and individual processes. Long-term unemployed individuals' tendency to feel insufficiently engaged may reflect difficulty with keeping up with a job market in constant change.

Tight-packing of large pilin subunits provides distinct structural and mechanical properties for the Myxococcus xanthus type IVa pilus.

Type IVa pili (T4aP) are ubiquitous cell surface filaments important for surface motility, adhesion to surfaces, DNA uptake, biofilm formation, and virulence. T4aP are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While major pilins of structurally characterized T4aP have lengths of <165 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a conserved N-terminal domain and a variable C-terminal domain, and the additional residues of PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4aP (T4aPMx) at a resolution of 3.0 Å using cryo-EM. The T4aPMx follows the structural blueprint of other T4aP with the pilus core comprised of the interacting N-terminal α1-helices, while the globular domains decorate the T4aP surface. The atomic model of PilA built into this map shows that the large C-terminal domain has more extensive intersubunit contacts than major pilins in other T4aP. As expected from these greater contacts, the bending and axial stiffness of the T4aPMx is significantly higher than that of other T4aP and supports T4aP-dependent motility on surfaces of different stiffnesses. Notably, T4aPMx variants with interrupted intersubunit interfaces had decreased bending stiffness, pilus length, and strongly reduced motility. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4aP that expands the environmental conditions in which the T4aP system functions.

Preliminary Feasibility and Acceptability Examination of Using a Novel Ergonomic Chinrest with a Low Shoulder Rest by a Viola Player: A Case Report.

BACKGROUND: High-string players, such as violinists and violists, are prone to neck problems. One factor can be their body posture, with often a rotated and flexed neck position with the jaw placed on a flat chinrest. The Kréddle chinrest (EC) was specifically designed to promote a more neutral neck position and prevent musculoskeletal problems among high-string players. This case report aims to evaluate the preliminary feasibility and acceptability of using the EC, with a low shoulder rest, in a pain-free professional high-string player. A secondary aim was to register newly developed pain and fatigue. METHODS: A 32-year-old professional viola player was instructed to use the EC for most of her playing time during a 6-week trial period. Outcome measures such as compliance (% of use out of total playing time over the last 7 days), performance, comfort and acceptability were assessed at baseline and at the end of each trial week. The Rapid Upper Limb Assessment (RULA) tool was used to evaluate the body posture at baseline when using the EC versus her own chinrest and shoulder rest. RESULTS: The initial trial period was interrupted after 2 days due to pain. A second 6-week period was completed. While compliance was high and there was positive feedback on how the EC affected her performance and comfort while holding the instrument, the case report identified a challenge with the complex instruction material, which made it difficult to install and adjust the product. The viola player was positive towards using EC in the end despite pain and fatigue during the trial period. CONCLUSION: This case report examined a professional viola player's 6-week experience with an EC. While positive outcomes were observed in posture, performance, and product acceptance, issues with complex instructions were noted. This study marks the first exploration of individual challenges with EC use.

A miniTurbo-based proximity labeling protocol to identify conditional protein interactomes in vivo in Myxococcus xanthus.

Protein-protein interactions are foundational for many cellular processes. Such interactions are especially challenging to identify if they are transient or depend on environmental conditions. This protocol details steps to identify stable and transient protein interactomes in the bacterium Myxococcus xanthus using biotin ligase miniTurbo-based proximity labeling. We include instructions for optimizing the expression of control proteins, in vivo biotin labeling of bacteria grown on a surface or in suspension culture, enrichment of biotinylated proteins, and sample processing for proteomic analysis. For complete details on the use and execution of this protocol, please refer to Branon et al. (2018).1.

The mechanism for polar localization of the type IVa pilus machine in Myxococcus xanthus.

IMPORTANCE: Type IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in motility, surface adhesion, biofilm formation, and virulence. Different bacteria have adapted different piliation patterns. To address how these patterns are established, we focused on the bipolar localization of the T4aP machine in the model organism Myxococcus xanthus by studying the localization of the PilQ secretin, the first component of this machine that assembles at the poles. Based on experiments using a combination of fluorescence microscopy, biochemistry, and computational structural analysis, we propose that PilQ, and specifically its AMIN domains, binds septal and polar peptidoglycan, thereby enabling polar Tgl localization, which then stimulates PilQ multimerization in the outer membrane. We also propose that the presence and absence of AMIN domains in T4aP secretins contribute to the different piliation patterns across bacteria.

Large pilin subunits provide distinct structural and mechanical properties for the Myxococcus xanthus type IV pilus.

Type IV pili (T4P) are ubiquitous bacterial cell surface filaments important for surface motility, adhesion to biotic and abiotic surfaces, DNA uptake, biofilm formation, and virulence. T4P are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While the major pilins of structurally characterized T4P have lengths of up to 161 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a highly conserved N-terminal domain and a highly variable C-terminal domain, and the additional residues in the M. xanthus PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4P (T4P Mx ) at a resolution of 3.0 Å using cryo-electron microscopy (cryo-EM). The T4P Mx follows the structural blueprint observed in other T4P with the pilus core comprised of the extensively interacting N-terminal α1-helices while the globular domains decorate the T4P surface. The atomic model of PilA built into this map shows that the large C-terminal domain has much more extensive intersubunit contacts than major pilins in other T4P. As expected from these greater contacts, the bending and axial stiffness of the T4P Mx is significantly higher than that of other T4P and supports T4P-dependent motility on surfaces of different stiffnesses. Notably, T4P Mx variants with interrupted intersubunit interfaces had decreased bending stiffness and strongly reduced motility on all surfaces. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4P that expands the environmental conditions in which the T4P system functions.

Molecular basis and design principles of switchable front-rear polarity and directional migration in Myxococcus xanthus.

During cell migration, front-rear polarity is spatiotemporally regulated; however, the underlying design of regulatory interactions varies. In rod-shaped Myxococcus xanthus cells, a spatial toggle switch dynamically regulates front-rear polarity. The polarity module establishes front-rear polarity by guaranteeing front pole-localization of the small GTPase MglA. Conversely, the Frz chemosensory system, by acting on the polarity module, causes polarity inversions. MglA localization depends on the RomR/RomX GEF and MglB/RomY GAP complexes that localize asymmetrically to the poles by unknown mechanisms. Here, we show that RomR and the MglB and MglC roadblock domain proteins generate a positive feedback by forming a RomR/MglC/MglB complex, thereby establishing the rear pole with high GAP activity that is non-permissive to MglA. MglA at the front engages in negative feedback that breaks the RomR/MglC/MglB positive feedback allosterically, thus ensuring low GAP activity at this pole. These findings unravel the design principles of a system for switchable front-rear polarity.

Biomolecular condensate drives polymerization and bundling of the bacterial tubulin FtsZ to regulate cell division.

Cell division is spatiotemporally precisely regulated, but the underlying mechanisms are incompletely understood. In the social bacterium Myxococcus xanthus, the PomX/PomY/PomZ proteins form a single megadalton-sized complex that directly positions and stimulates cytokinetic ring formation by the tubulin homolog FtsZ. Here, we study the structure and mechanism of this complex in vitro and in vivo. We demonstrate that PomY forms liquid-like biomolecular condensates by phase separation, while PomX self-assembles into filaments generating a single large cellular structure. The PomX structure enriches PomY, thereby guaranteeing the formation of precisely one PomY condensate per cell through surface-assisted condensation. In vitro, PomY condensates selectively enrich FtsZ and nucleate GTP-dependent FtsZ polymerization and bundle FtsZ filaments, suggesting a cell division site positioning mechanism in which the single PomY condensate enriches FtsZ to guide FtsZ-ring formation and division. This mechanism shares features with microtubule nucleation by biomolecular condensates in eukaryotes, supporting this mechanism's ancient origin.

During heat stress in Myxococcus xanthus, the CdbS PilZ domain protein, in concert with two PilZ-DnaK chaperones, perturbs chromosome organization and accelerates cell death.

C-di-GMP is a bacterial second messenger that regulates diverse processes in response to environmental or cellular cues. The nucleoid-associated protein (NAP) CdbA in Myxococcus xanthus binds c-di-GMP and DNA in a mutually exclusive manner in vitro. CdbA is essential for viability, and CdbA depletion causes defects in chromosome organization, leading to a block in cell division and, ultimately, cell death. Most NAPs are not essential; therefore, to explore the paradoxical cdbA essentiality, we isolated suppressor mutations that restored cell viability without CdbA. Most mutations mapped to cdbS, which encodes a stand-alone c-di-GMP binding PilZ domain protein, and caused loss-of-function of cdbS. Cells lacking CdbA and CdbS or only CdbS were fully viable and had no defects in chromosome organization. CdbA depletion caused post-transcriptional upregulation of CdbS accumulation, and this CdbS over-accumulation was sufficient to disrupt chromosome organization and cause cell death. CdbA depletion also caused increased accumulation of CsdK1 and CsdK2, two unusual PilZ-DnaK chaperones. During CdbA depletion, CsdK1 and CsdK2, in turn, enabled the increased accumulation and toxicity of CdbS, likely by stabilizing CdbS. Moreover, we demonstrate that heat stress, possibly involving an increased cellular c-di-GMP concentration, induced the CdbA/CsdK1/CsdK2/CdbS system, causing a CsdK1- and CsdK2-dependent increase in CdbS accumulation. Thereby this system accelerates heat stress-induced chromosome mis-organization and cell death. Collectively, this work describes a unique system that contributes to regulated cell death in M. xanthus and suggests a link between c-di-GMP signaling and regulated cell death in bacteria.

A bipartite, low-affinity roadblock domain-containing GAP complex regulates bacterial front-rear polarity.

The Ras-like GTPase MglA is a key regulator of front-rear polarity in the rod-shaped Myxococcus xanthus cells. MglA-GTP localizes to the leading cell pole and stimulates assembly of the two machineries for type IV pili-dependent motility and gliding motility. MglA-GTP localization is spatially constrained by its cognate GEF, the RomR/RomX complex, and GAP, the MglB Roadblock-domain protein. Paradoxically, RomR/RomX and MglB localize similarly with low and high concentrations at the leading and lagging poles, respectively. Yet, GEF activity dominates at the leading and GAP activity at the lagging pole by unknown mechanisms. Here, we identify RomY and show that it stimulates MglB GAP activity. The MglB/RomY interaction is low affinity, restricting formation of the bipartite MglB/RomY GAP complex almost exclusively to the lagging pole with the high MglB concentration. Our data support a model wherein RomY, by forming a low-affinity complex with MglB, ensures that the high MglB/RomY GAP activity is confined to the lagging pole where it dominates and outcompetes the GEF activity of the RomR/RomX complex. Thereby, MglA-GTP localization is constrained to the leading pole establishing front-rear polarity.

Evidence for a Widespread Third System for Bacterial Polysaccharide Export across the Outer Membrane Comprising a Composite OPX/ß-Barrel Translocon.

In Gram-negative bacteria, secreted polysaccharides have multiple critical functions. In Wzx/Wzy- and ABC transporter-dependent pathways, an outer membrane (OM) polysaccharide export (OPX) type translocon exports the polysaccharide across the OM. The paradigm OPX protein Wza of Escherichia coli is an octamer in which the eight C-terminal domains form an α-helical OM pore and the eight copies of the three N-terminal domains (D1 to D3) form a periplasmic cavity. In synthase-dependent pathways, the OM translocon is a 16- to 18-stranded ß-barrel protein. In Myxococcus xanthus, the secreted polysaccharide EPS (exopolysaccharide) is synthesized in a Wzx/Wzy-dependent pathway. Here, using experiments, phylogenomics, and computational structural biology, we identify and characterize EpsX as an OM 18-stranded ß-barrel protein important for EPS synthesis and identify AlgE, a ß-barrel translocon of a synthase-dependent pathway, as its closest structural homolog. We also find that EpsY, the OPX protein of the EPS pathway, consists only of the periplasmic D1 and D2 domains and completely lacks the domain for spanning the OM (herein termed a D1D2OPX protein). In vivo, EpsX and EpsY mutually stabilize each other and interact in in vivo pulldown experiments supporting their direct interaction. Based on these observations, we propose that EpsY and EpsX make up and represent a third type of translocon for polysaccharide export across the OM. Specifically, in this composite translocon, EpsX functions as the OM-spanning ß-barrel translocon together with the periplasmic D1D2OPX protein EpsY. Based on computational genomics, similar composite systems are widespread in Gram-negative bacteria. IMPORTANCE Bacteria secrete a wide variety of polysaccharides that have critical functions in, e.g., fitness, surface colonization, and biofilm formation and in beneficial and pathogenic human-, animal-, and plant-microbe interactions. In Gram-negative bacteria, export of these chemically diverse polysaccharides across the outer membrane depends on two known translocons, i.e., an outer membrane OPX protein in Wzx/Wzy- and ABC transporter-dependent pathways and an outer membrane 16- to 18-stranded ß-barrel protein in synthase-dependent pathways. Here, using a combination of experiments in Myxococcus xanthus, phylogenomics, and computational structural biology, we provide evidence supporting that a third type of translocon can export polysaccharides across the outer membrane. Specifically, in this translocon, an outer membrane-spanning ß-barrel protein functions together with an entirely periplasmic OPX protein that completely lacks the domain for spanning the OM. Computational genomics support that similar composite systems are widespread in Gram-negative bacteria.

En route to flourishing - a longitudinal mixed methods study of long-term unemployed citizens in an interdisciplinary rehabilitation program.

BACKGROUND: Interdisciplinary rehabilitation programmes (IRP) are used in municipality settings to assist unemployed citizens with complex health and/or life issues. Individually tailored IRP activities help people develop their personal working life skills and increase their chances of re-entering the work force. The aims of this paper were to describe citizens' wellbeing in terms of health aspects, explore the impact of stressful life events on wellbeing and obtain understanding of how IRP activities affect the participants' development towards future employment. METHODS: A mixed methods exploratory approach has been used. For data collection a quantitative longitudinal survey (baseline and 1-year follow-up) and qualitative interviews were conducted. Descriptive statistics were used for the analysis of survey data, while the data material from interviews was analysed using directed content analysis. Results were discussed with the theory of flourishing as a framework to develop understanding. RESULTS: At baseline, 146 respondents (71% females) filled in the survey and seven participants were interviewed. The analysis of survey data and interviews revealed five themes: (1) Stressful life events, (2) Positive emotions - how IRP-activities positively impacted wellbeing and physical capacity, (3) Appreciation of engagement, (4) Relationships, and (5) Meaning and optimal functioning. Results showed that IRP participants from the outset experienced high general pain intensity as well as distress, anxiety and depression. Life events relating both to physical health and work life were significant for their wellbeing. IRP activities supported participants' positive development towards future employment in ways that were specific to each individual. CONCLUSIONS: From this study it can be derived that participants' development took place around self-acceptance, acceptance by others, physical capacity, psychological resources and capacity to balance engagement to cultivate the best version of themselves. In future programmes, it may be emphasized that participants' interest may be an important driver for wellbeing and future employment. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02641704, date of registration December 29, 2015.

Spatiotemporal regulation of switching front-rear cell polarity.

Bacterial cells are spatiotemporally highly organised with proteins localising dynamically to distinct subcellular regions. Motility in the rod-shaped Myxococcus xanthus cells represents an example of signal-induced spatiotemporal regulation of cell polarity. M. xanthus cells move across surfaces with defined front-rear polarity; occasionally, they invert polarity and, in parallel, reverse direction of movement. The polarity module establishes front-rear polarity between reversals and consists of the Ras-like GTPase MglA and its cognate GEF and GAP, that all localise asymmetrically to the cell poles. The Frz chemosensory system constitutes the polarity inversion module and interfaces with the proteins of the polarity module, thereby triggering their polar repositioning. As a result, the polarity proteins, over time, toggle between the cell poles causing cells to oscillate irregularly. Here, we review recent progress in how front-rear polarity is established by the polarity module and inverted by the Frz system and highlight open questions for future studies.

Parked on the verge: vocational rehabilitation of long-term unemployed citizens - a mixed methods study.

BACKGROUND: Vocational rehabilitation programs (VRP) developing and improving work ability are used in Denmark to assist long-term unemployed citizens with complex problems. The aims of this study were to (1) describe VRP-participants in relation to general health, well-being, work ability and self-efficacy at baseline and one-year follow-up, (2) obtain an understanding of VRP-participants' personal development towards improving work ability, and (3) explore VRP-participants' hopes and thoughts about their future. METHODS: In a mixed methods approach, data from a longitudinal survey and semi-structured interviews were collected. In the quantitative longitudinal survey, all participants completed paper questionnaires at baseline and one-year follow-up. For the qualitative semi-structured interviews, VRP participants were recruited with a maximum variation sampling strategy through VRP coordinators and personal contact. Data were analysed by descriptive statistics and systematic text condensation. Following analysis, data were merged and presented in combination according to identified themes. RESULTS: At baseline and one-year follow-up 146 (response rate 34%) and 74 participants (response rate 52%) respectively, responded to the questionnaire. Seven participants were interviewed. The analysis revealed four themes: 1) Individual explanations of life situation and health; 2) Finding the path; 3) Work as giving meaning to life; 4) Hope for the future. Despite self-reported scores indicating poor general health, lack of well-being, low work ability and low self-efficacy, VRP-activities seemed to have assisted participants in finding meaning in life. VRP-components that may be drivers of successful recovery processes were identified. CONCLUSIONS: VRP-participants experienced life situations that include multifactorial burdens, and low levels of general health, well-being, work ability, and self-efficacy at baseline and 1 year later. From the outset, most did not have a clear goal of employment, but over time, new goals were set as realistic opportunities for re-developing their work ability were explored. Successful core components of VRP were individually tailored programs and support, development of new relationships, and accommodated flexible internships and jobs. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02641704 , date of registration December 29, 2015.

A noncanonical cytochrome c stimulates calcium binding by PilY1 for type IVa pili formation.

Type IVa pili (T4aP) are versatile bacterial cell surface structures that undergo extension/adhesion/retraction cycles powered by the cell envelope-spanning T4aP machine. In this machine, a complex composed of four minor pilins and PilY1 primes T4aP extension and is also present at the pilus tip mediating adhesion. Similar to many several other bacteria, Myxococcus xanthus contains multiple minor pilins/PilY1 sets that are incompletely understood. Here, we report that minor pilins and PilY1 (PilY1.1) of cluster_1 form priming and tip complexes contingent on calcium and a noncanonical cytochrome c (TfcP) with an unusual His/Cys heme ligation. We provide evidence that TfcP is unlikely to participate in electron transport and instead stimulates calcium binding by PilY1.1 at low-calcium concentrations, thereby stabilizing PilY1.1 and enabling T4aP function in a broader range of calcium concentrations. These results not only identify a previously undescribed function of cytochromes c but also illustrate how incorporation of an accessory factor expands the environmental range under which the T4aP system functions.

A national Danish proof of concept on feasibility and safety of home -based intensive chemotherapy in patients with acute myeloid leukemia.

Technological advances have made it possible to offer home-based chemotherapy to patients without health care professionals being present. Prior studies on effects of home-based treatment lack inclusion of patients with hematologic malignancies. We present data from a multicenter single-arm feasibility and safety study of home-based intensive chemotherapy in patients with newly diagnosed acute myeloid leukemia and their quality of life and psychological wellbeing. This national study included patients from six sites in Denmark who received intensive chemotherapy on programmed CADD Solis infusion pumps through a central venous catheter and were also managed as outpatients during treatment-induced pancytopenia. Data are presented from 104 patients, receiving 272 treatments with 1.096 (mean 4.57, SD 3.0) home infusion days out of 1.644 treatment days (67 %). Sixty-two of 168 (36.9 %) reinduction and consolidation treatment cycles ensuing pancytopenia phases were solely handled in the outpatient clinic. Patients reported high satisfaction with home-based treatment, which had a positive influence on their ability to be involved in their treatment and be socially and physically active. No unexpected events occurred during the intervention. Overall, patients improved in all quality of life outcomes over time. Home-based intensive chemotherapy treatment was feasible and safe in this population. ClinicalTrials.gov identifier: NCT04904211.

Transcriptomic analysis of the Myxococcus xanthus FruA regulon, and comparative developmental transcriptomic analysis of two fruiting body forming species, Myxococcus xanthus and Myxococcus stipitatus.

BACKGROUND: The Myxococcales are well known for their predatory and developmental social processes, and for the molecular complexity of regulation of these processes. Many species within this order have unusually large genomes compared to other bacteria, and their genomes have many genes that are unique to one specific sequenced species or strain. Here, we describe RNAseq based transcriptome analysis of the FruA regulon of Myxococcus xanthus and a comparative RNAseq analysis of two Myxococcus species, M. xanthus and Myxococcus stipitatus, as they respond to starvation and begin forming fruiting bodies. RESULTS: We show that both species have large numbers of genes that are developmentally regulated, with over half the genome showing statistically significant changes in expression during development in each species. We also included a non-fruiting mutant of M. xanthus that is missing the transcriptional regulator FruA to identify the direct and indirect FruA regulon and to identify transcriptional changes that are specific to fruiting and not just the starvation response. We then identified Interpro gene ontologies and COG annotations that are significantly up- or down-regulated during development in each species. Our analyses support previous data for M. xanthus showing developmental upregulation of signal transduction genes, and downregulation of genes related to cell-cycle, translation, metabolism, and in some cases, DNA replication. Gene expression in M. stipitatus follows similar trends. Although not all specific genes show similar regulation patterns in both species, many critical developmental genes in M. xanthus have conserved expression patterns in M. stipitatus, and some groups of otherwise unstudied orthologous genes share expression patterns. CONCLUSIONS: By identifying the FruA regulon and identifying genes that are similarly and uniquely regulated in two different species, this work provides a more complete picture of transcription during Myxococcus development. We also provide an R script to allow other scientists to mine our data for genes whose expression patterns match a user-selected gene of interest.