Commensal microbiota regulate aldosterone.

The gut microbiome regulates many important host physiological processes associated with cardiovascular health and disease; however, the impact of the gut microbiome on aldosterone is unclear. Investigating whether gut microbiota regulate aldosterone can offer novel insights into how the microbiome affects blood pressure. In this study, we aimed to determine whether gut microbiota regulate host aldosterone. We used enzyme-linked immunosorbent assays (ELISAs) to assess plasma aldosterone and plasma renin activity (PRA) in female and male mice in which gut microbiota are intact, suppressed, or absent. In addition, we examined urinary aldosterone. Our findings demonstrated that when the gut microbiota is suppressed following antibiotic treatment, there is an increase in plasma and urinary aldosterone in both female and male mice. In contrast, an increase in PRA is seen only in males. We also found that when gut microbiota are absent (germ-free mice), plasma aldosterone is significantly increased compared with conventional animals (in both females and males), but PRA is not. Understanding how gut microbiota influence aldosterone levels could provide valuable insights into the development and treatment of hypertension and/or primary aldosteronism. This knowledge may open new avenues for therapeutic interventions, such as probiotics or dietary modifications to help regulate blood pressure via microbiota-based changes to aldosterone.NEW & NOTEWORTHY We explore the role of the gut microbiome in regulating aldosterone, a hormone closely linked to blood pressure and cardiovascular disease. Despite the recognized importance of the gut microbiome in host physiology, the relationship with circulating aldosterone remains largely unexplored. We demonstrate that suppression of gut microbiota leads to increased levels of plasma and urinary aldosterone. These findings underscore the potential of the gut microbiota to influence aldosterone regulation, suggesting new possibilities for treating hypertension.

Defining the concept of mental dysregulation in patients requiring ambulance and/or emergency department care: protocol for a Delphi consensus study.

INTRODUCTION: From the patient and staff perspective, care delivery for patients experiencing a mental health problem in ambulance and emergency department (ED) settings is challenging. There is no uniform and internationally accepted concept to reflect people with a mental health problem who require emergency care, be it for, or as a result of, a mental health or physical health problem. On initial presentation to the emergency service provider (ambulance or ED), the cause of their healthcare condition/s (mental health and/or physical health) is often initially unknown. Due to this (1) the prevalence and range of underlying causes (mental and/or physical) of the patients presenting condition is unknown; (2) misattribution of physical symptoms to a mental health problem can occur and (3) diagnosis and treatment of the initial somatic complaint and cause(s) of the mental/physical health problem may be hindered.This study will name and define a new concept: 'mental dysregulation' in the context of ambulance and ED settings. METHODS AND ANALYSIS: A Delphi study, informed by a rapid literature review, will be undertaken. For the literature review, a steering group (ie, persons with lived experience, ED and mental health clinicians, academics) will systematically search the literature to provide a working definition of the concept: mental dysregulation. Based on this review, statements will be generated regarding (1) the definition of the concept; (2) possible causes of mental dysregulation and (3) observable behaviours associated with mental dysregulation. These statements will be rated in three Delphi rounds to achieve consensus by an international expert panel (comprising persons with lived experience, clinicians and academics). ETHICS AND DISSEMINATION: This study has been approved by the Medical Ethical Committee of the University of Applied Sciences Utrecht (reference number: 258-000-2023_Geurt van der Glind). Results will be disseminated via peer-reviewed journal publication(s), scientific conference(s) and to key stakeholders.

Emergency nurse and physician perceptions of barriers and facilitators to optimal nutrition in the emergency department: A national cross-sectional survey.

INTRODUCTION: Optimal nutritional support is becoming increasingly important in Emergency Departments (EDs) as over half of patients presenting to ED are reported to be malnourished or at risk of malnutrition. Few studies have examined the barriers and facilitators to nutritional support in ED. AIM: To identify barriers and facilitators to providing optimal nutritional support in the ED from nurse and physician perspectives. METHODS: A cross-sectional 31-item electronic survey was developed, validated, and distributed nationally in August 2021 in China. RESULTS: A total of 1766 eligible respondents completed the survey, including 846 ED nurses and 920 ED physicians from 155 hospitals. Barriers to optimal nutrition were moderate (2.72/5 ± 0.88); the most common barrier was lack of multidisciplinary team-work support. Facilitators to support optimal nutrition were moderately high (3.58/5 ± 1.08); the most common facilitator was technical/professional support and organizational management. Respondents who received recent nutrition training and those with higher levels of nutrition knowledge (self-rated) perceived fewer barriers overall to optimal nutrition in ED (P < 0.01). CONCLUSION: Context specific barriers and facilitators both hinder and support optimal nutrition in ED. Further research is required to develop tailored interventions to address specific barriers to optimal nutrition and enhance facilitators in the ED context.

Patient and public involvement in emergency care research: a scoping review of the literature.

BACKGROUND: Establishing the benefits of patient and public involvement (PPI) in emergency care research is important to improve the quality and relevance of research. Little is known about the extent of PPI in emergency care research, its methodological and reporting quality. This scoping review aimed to establish the extent of PPI in emergency care research, identify PPI strategies and processes and assess the quality of reporting on PPI in emergency care research. METHODS: Keyword searches of five databases (OVID MEDLINE, Elsevier EMBASE, EBSCO CINAHL, PsychInfo, Cochrane Central Register of Controlled trials); hand searches of 12 specialist journals and citation searches of the included journal articles were performed. A patient representative contributed to research design and co-authored this review. RESULTS: A total of 28 studies reporting PPI from the USA, Canada, UK, Australia and Ghana were included. The quality of reporting was variable, with only seven studies satisfying all Guidance for Reporting Involvement of Patients and the Public short form reporting criteria. None of the included studies adequately described all the key aspects of reporting the impact of PPI. CONCLUSION: Relatively few emergency care studies comprehensively describe PPI. Opportunity exists to improve the consistency and quality of reporting of PPI in emergency care research. Further research is required to better understand the specific challenges for implementing PPI in emergency care research, and to determine whether emergency care researchers have adequate resources, education and funding to undertake and report involvement.

Researcher Perceptions of Involving Consumers in Health Research in Australia: A Qualitative Study.

There is growing recognition internationally of the importance of involving consumers, patients, and the public in research. This is being driven by political mandates for policies, funding, and governance that demand genuine and meaningful engagement with consumers. There are many potential benefits to involving consumers in research, including an increased relevance to patient needs, improved quality and outcomes, and enhanced public confidence in research. However, the current literature highlights that efforts to incorporate their contributions are often tokenistic and there is a limited understanding of the psychological factors that can impact researcher attitudes, intentions, and behaviours when working with consumers in research. To address this gap, this study conducted 25 semi-structured interviews with health researchers in Australia using the qualitative case study method. The study aim was to explore the underlying influences on researcher behaviour when involving consumers in health research. The results identified several factors that influence researchers' behaviour, including better quality research, emotional connection and the humanisation of research, and a shift in research culture and expectations as major drivers. However, beliefs that consumers would hinder research and must be protected from risks, paternalism, and a lack of researcher skills and resources were identified as major barriers. This article presents a theory of planned behaviour for consumer involvement in the health research model. The model offers a valuable tool for policymakers and practitioners to understand the factors that influence researcher behaviours. It can also serve as a framework for future research in this area.

Establishing critical care nursing research priorities for three Australian regional public hospitals: A mixed method priority setting study.

OBJECTIVE: To determine key priorities for critical care nursing research in three Australian regional public hospitals, representing the shared priorities of healthcare professionals and patient representatives. METHODS: A three phase priority setting study, including consensus methods (nominal group), survey, qualitative interviews and focus groups were conducted between May 2021 and March 2022. Healthcare professionals and patient representatives from critical care units in regional public hospitals in Australia participated. A patient representative contributed to research design and co-authored this paper. RESULTS: In phase one, 29 research topics were generated. In phase two, during a nominal group ranking process, the top 5 priority areas for each site were identified. In the final phase, three themes from focus groups and interviews included patient flow through intensive care, patient care through intensive care journey and intensive care patient recovery. CONCLUSION: Identifying context specific research priorities through a priority setting exercise provides insight into the topics that are important to healthcare professionals and to patients in critical care. The top research priorities for nursing research in critical care in regional Australian hospitals include patient flow, patient recovery, and evidence based patient care through the intensive care journey, such as delirium management, pain and sedation, and mobilisation. These shared priorities will be used to guide future nursing research in critical care over the next 3-5 years. IMPLICATIONS FOR CLINICAL PRACTICE: The method we used in identifying the research priorities can be used by other researchers and clinicians; close collaboration among researchers and clinicians will be beneficial for practice improvement; and how we can be reassured that our practice is evidence based is worthy of attention.

Early Life Stress in Mice Leads to Impaired Colonic Corticosterone Production and Prolonged Inflammation Following Induction of Colitis.

BACKGROUND: Early life stress (ELS) is an environmental trigger believed to promote increased risk of IBD. Our goal was to identify mechanisms whereby ELS in mice affects susceptibility to and/or severity of gut inflammation. METHODS: We utilized 2 published animal models of ELS. In the first model, newborn mice were separated from the dam daily for 4 to 8 hours starting on postnatal day 2 and then weaned early on postnatal day 17. Control mice were left undisturbed with the dams until weaning on postnatal day 21. In the second model, dams were fed dexamethasone or vehicle ad libitum in drinking water on postpartum days 1 to 14. Plasma and colonic corticosterone were measured in juvenile and adult mice. Colitis was induced in 4-week-old mice via intraperitoneal injection of interleukin (IL)-10 receptor blocking antibody every 5 days for 15 days. Five or 15 days later, colitis scores and transcripts for Tnf, glucocorticoid receptors, and steroidogenic enzymes were measured. RESULTS: Mice exposed to ELS displayed reduced plasma and colonic corticosterone. Control animals showed improvements in indices of inflammation following cessation of interleukin-10 receptor blockade, whereas ELS-exposed animals maintained high levels of Tnf and histological signs of colitis. In colitic animals, prior exposure to ELS was associated with significantly lower expression of genes associated with corticosterone synthesis and responsiveness. Finally, TNF stimulation of colonic crypt cells from ELS mice led to increased inhibition of corticosterone synthesis. CONCLUSIONS: Our study identifies impaired local glucocorticoid production and responsiveness as a potential mechanism whereby ELS predisposes to chronic colitis in susceptible hosts.

Using 2 distinct animal models, this study shows that in mice, early life stress leads to reduced colonic corticosterone and that induction of colitis after stress removal results in reduced transcription of glucocorticoid synthesis genes, increased Tnf, and enhanced chronicity of intestinal inflammation.

Patient-mediated interventions in hospital: A systematic review.

AIMS: To describe the characteristics of hospital-based, patient-mediated interventions and their impact on patient, clinician and organization outcomes. DESIGN: Systematic review. DATA SOURCES: Health literature databases (MEDLINE, CINAHL and EMBASE) were searched in August 2021. Backward and forward citation searching was conducted. REVIEW METHODS: Studies investigating patient-mediated interventions, targeted at adult hospitalized patients were eligible. Data were extracted related to study and intervention characteristics. Narrative synthesis was used to understand intervention impact on patient, clinician and organization outcomes (as per a framework). Methodological quality was assessed using the Mixed Methods Assessment Tool. RESULTS: Thirty-three studies, reporting 18 interventions, were included. Twelve interventions prompted patients to report health information about their own health/needs/concerns and six interventions encouraged patients to provide feedback about clinical practice. Across all interventions, there was evidence that patients used patient-mediated interventions and that they may improve patient communication. Healthcare professional outcomes were mixed for actual/intended use, acceptability and usefulness of interventions; yet there was some evidence of healthcare professional behaviour change. Interventions that encouraged patients to report health information about their own health/needs/concerns appeared more successful than other types of interventions. CONCLUSIONS: There is some evidence that hospital-based patient-mediated interventions may influence patient communication and healthcare professional behaviour. Patient-mediated interventions that encourage patients to report patient data before a clinical encounter may be more impactful than interventions that encourage patient feedback during or post-encounter. IMPACT: To date, most patient-mediated intervention research has been conducted in primary care settings; we uncovered the types of patient-mediated interventions that have been trialled in hospitals. We found that patient communication and healthcare professional behaviour may be influenced by these patient-mediated interventions. Future researchers could explore the suitability and effectiveness of a wider range of hospital-based patient-mediated interventions. NO PATIENT OR PUBLIC CONTRIBUTION: There was no funding to remunerate a patient/member of the public for this review.

ICOS ligand and IL-10 synergize to promote host-microbiota mutualism.

Genome-wide association studies have identified ICOSLG, which encodes the inducible costimulator ligand (ICOSLG or ICOSL) as a susceptibility locus for inflammatory bowel disease. ICOSL has been implicated in the enhancement of pattern recognition receptor signaling in dendritic cells, induction of IL-10 production by CD4 T cells, and the generation of high-affinity antibodies to specific antigens-all of which can potentially explain its involvement in gastrointestinal inflammation. Here, we show that murine ICOSL deficiency results in significant enrichment of IL-10-producing CD4 T cells particularly in the proximal large intestine. Transient depletion of IL-10-producing cells from adult ICOSL-deficient mice induced severe colonic inflammation that was prevented when mice were first treated with metronidazole. ICOSL-deficient mice displayed reduced IgA and IgG antibodies in the colon mucus and impaired serum antibody recognition of microbial antigens, including flagellins derived from mucus-associated bacteria of the Lachnospiraceae family. Confirming the synergy between ICOSL and IL-10, ICOSL deficiency coupled with CD4-specific deletion of the Il10 gene resulted in juvenile onset colitis that was impeded when pups were fostered by ICOSL-sufficient dams. In this setting, we found that both maternally acquired and host-derived antibodies contribute to the life anti-commensal antibody repertoire that mediates this protection in early life. Collectively, our findings reveal a partnership between ICOSL-dependent anti-commensal antibodies and IL-10 in adaptive immune regulation of the microbiota in the large intestine. Furthermore, we identify ICOSL deficiency as an effective platform for exploring the functions of anti-commensal antibodies in host-microbiota mutualism.

A scoping review of the role of health literacy in chronic kidney disease self-management.

BACKGROUND: Chronic kidney disease is a serious health condition and is increasing globally. Effective self-management could slow disease progression and improve health outcomes, although the contribution of health literacy and knowledge for self-management is not well known. AIM: To investigate the recent evidence of health literacy and the relationship between health literacy, knowledge and self-management of chronic kidney disease. METHODS: Arksey and O'Malley's framework informed this scoping review. Eligible studies involving adults with any grade of chronic kidney disease, measuring all dimensions of health literacy (i.e., functional, communicative, and critical), disease-specific knowledge and self-management, published in English between January 2005 and March 2020, were included. RESULTS: The scoping review found 12 eligible studies, with 11 assessing all dimensions of health literacy. No study examined health literacy, knowledge and self-management. When individuals had greater health literacy, this was associated with greater knowledge about the disease. Communicative health literacy was a significant predictor of medication, diet and fluid adherence, and overall self-management behaviours. CONCLUSION: This scoping review shows that disease-specific knowledge is important for health literacy and that health literacy is essential for effective self-management of chronic kidney disease. The implications of these relationships can inform strategies for the development of evidence-based patient education to support increased self-management. There is also a need for further research to explore these associations.

Patient involvement in surgical wound care research: A scoping review.

Active involvement of patients in planning, conducting, and disseminating research has been adopted by many organisations internationally, but the extent to which this occurs in surgical wound care is not evident. This scoping review aimed to identify how patients have been involved in surgical wound care research and the quality of its reporting. Full-text studies focused on preoperative and postoperative surgical wound care in the acute care setting, published in English between 2004 and 2019, were included in the review. Screening, data charting, and quality assessment were conducted by two reviewers independently, adjudicated by a third, and then reviewed by five others. Thematic analysis synthesised the findings. Of the eight included studies, seven explained the methods for patient involvement and five described aims related to patient involvement and commented on patient involvement in the discussion. None met all of the quality assessment criteria. Three themes emerged: involvement in modifying and refining research processes, connecting and balancing expert and patient views, and sharing personal insights. Recommendations to improve patient involvement in surgical wounds research include the following: using framework and tools to inform future research; training researcher and patients in their respective research roles; and ongoing monitoring of patient involvement.

Intradermal grass pollen immunotherapy increases TH2 and IgE responses and worsens respiratory allergic symptoms.

BACKGROUND: Repeated low-dose grass pollen intradermal allergen injection suppresses allergen-induced cutaneous late-phase responses comparably with conventional subcutaneous and sublingual immunotherapy. OBJECTIVE: We sought to evaluate the efficacy and safety of grass pollen intradermal immunotherapy in the treatment of allergic rhinitis. METHODS: We randomly assigned 93 adults with grass pollen-induced allergic rhinitis to receive 7 preseasonal intradermal allergen injections (containing 7 ng of Phl p 5 major allergen) or a histamine control. The primary end point was daily combined symptom-medication scores during the 2013 pollen season (area under the curve). Analysis was by intention to treat. Skin biopsy specimens were collected after intradermal allergen challenges, and late-phase responses were measured 4 and 7, 10, or 13 months after treatment. RESULTS: There was no significant difference in the primary end point between treatment arms (active, n = 46; control, n = 47; median difference, 14; 95% CI, -172.5 to 215.1; P = .80). Among secondary end points, nasal symptoms were worse in the intradermal treatment group, as measured based on daily (median difference, 35; 95% CI, 4.0-67.5; P = .03) and visual analog scale (median difference, 53; 95% CI, -11.6 to 125.2; P = .05) scores. In a per-protocol analysis intradermal immunotherapy was further associated with worse asthma symptoms and fewer symptom-free days. Intradermal immunotherapy increased serum Phleum pratense-specific IgE levels (P = .001) compared with those in the control arm. T cells cultured from biopsy specimens of subjects undergoing intradermal immunotherapy had higher expression of the TH2 surface marker CRTH2 (P = .04) and lower expression of the TH1 marker CXCR3 (P = .01), respectively. Late-phase responses remained inhibited 7 months after treatment (P = .03). CONCLUSION: Intradermal allergen immunotherapy suppressed skin late-phase responses but was not clinically effective and resulted in worsening of respiratory allergic symptoms.

Protocol for a double-blind randomised controlled trial of low dose intradermal grass pollen immunotherapy versus a histamine control on symptoms and medication use in adults with seasonal allergic rhinitis (PollenLITE).

BACKGROUND: Subcutaneous immunotherapy with high dose grass pollen (typically microgram quantities) was first described over 100 years ago. This treatment suppresses allergen-induced cutaneous late responses, with lesser effects on early responses. We previously reported that repeated 2-weekly intradermal injections of grass pollen - containing approximately 7 ng of major allergen Phl p 5 - led to a progressive suppression of the allergen-induced cutaneous response, and that by the sixth injection, this was inhibited by over 90%. The purpose of this trial is to investigate the clinical efficacy of intradermal desensitisation with low doses (i.e. nanogram quantities) of grass pollen allergen for seasonal allergic rhinitis. METHODS/DESIGN: The Pollen Low dose Intradermal therapy Evaluation (PollenLITE) is a single centre double-blind randomised parallel group controlled trial of the efficacy and safety of intradermal grass pollen injections plus standard treatment, versus histamine injections plus standard treatment, in adults with moderate-severe grass pollen-induced allergic rhinitis ('summer hay fever'). A minimum of ninety adults with a history of moderate-severe persistent allergic rhinitis during the UK grass pollen season will be randomised into two equal groups to receive 7 or 8 intradermal injections of grass pollen extract (containing approximately 7 ng of major allergen Phl p 5) or histamine, before the grass pollen season. In the summer, participants will score their symptoms, medication requirements, visual analogue scores, and complete EuroQOL (EQ-5D-5 L) and mini Rhinoconjunctivitis Quality of Life Questionnaires. Global assessments will also be recorded at the end of the pollen season. Blood samples will be collected from all participants for mechanistic immune assays. Skin punch biopsies will also be collected in 40 participants selected at random from intradermal injection sites after the grass pollen season for mechanistic assays. Finally, to investigate if the desensitising effect of intradermal immunotherapy on cutaneous responses is long-lasting, all participants will be randomised to receive a follow up intradermal injection after 3, 6 or 12 months with measurement of early and late response sizes. DISCUSSION: Randomisation began in February 2013 and the final participant will complete the trial protocol in August 2014. TRIAL REGISTRATION: ISRCTN 78413121EudraCT number 2012-002193-31.

Virulence of Leucobacter chromiireducens subsp. solipictus to Caenorhabditis elegans: characterization of a novel host-pathogen interaction.

We describe the pathogenic interaction between a newly described gram-positive bacterium, Leucobacter chromiireducens subsp. solipictus strain TAN 31504, and the nematode Caenorhabditis elegans. TAN 31504 pathogenesis on C. elegans is exerted primarily through infection of the adult nematode uterus. TAN 31504 enters the uterus through the external vulval opening, and the ensuing uterine infection is strongly correlated with a significant reduction in host life span. Young worms can feed and develop on TAN 31504, but not preferably over the standard food source. C. elegans worms reared on TAN 31504 as the sole food source develop into thin adults with little intestinal fat stores, produce few progeny, and subsequently cannot persist on the pathogenic food source. Within 12 h of exposure, adult worms challenged with TAN 31504 alter the expression of a number of C. elegans innate immunity-related genes, including nlp-29, which encodes a neuropeptide-like protein. C. elegans worms exposed briefly to TAN 31504 develop lethal uterine infections analogous to worms exposed continuously to pathogen, suggesting that mere contact with the pathogen is sufficient for the host to become infected. TAN 31504 produces a robust biofilm, and this behavior is speculated to play a role in the virulence exerted on the nematode host. The interaction between TAN 31504 and C. elegans provides a convenient opportunity to study bacterial virulence on nematode tissues other than the intestine and may allow for the discovery of host innate immunity elicited specifically in response to vulva-uterus infection.

Leucobacter chromiireducens subsp. solipictus subsp. nov., a pigmented bacterium isolated from the nematode Caenorhabditis elegans, and emended description of L. chromiireducens.

A yellow-pigmented, Gram-positive, aerobic, non-motile, non-spore-forming, irregular rod-shaped bacterium (strain TAN 31504(T)) was isolated from the bacteriophagous nematode Caenorhabditis elegans. Based on 16S rRNA gene sequence similarity, DNA G+C content of 69.5 mol%, 2,4-diaminobutyric acid in the cell-wall peptidoglycan, major menaquinone MK-11, abundance of anteiso- and iso-fatty acids, polar lipids diphosphatidylglycerol and phosphatidylglycerol and a number of shared biochemical characteristics, strain TAN 31504(T) was placed in the genus Leucobacter. DNA-DNA hybridization comparisons demonstrated a 91 % DNA-DNA relatedness between strain TAN 31504(T) and Leucobacter chromiireducens LMG 22506(T) indicating that these two strains belong to the same species, when the recommended threshold value of 70 % DNA-DNA relatedness for the definition of a bacterial species by the ad hoc committee on reconciliation of approaches to bacterial systematics is considered. Based on distinct differences in morphology, physiology, chemotaxonomic markers and various biochemical characteristics, it is proposed to split the species L. chromiireducens into two novel subspecies, Leucobacter chromiireducens subsp. chromiireducens subsp. nov. (type strain L-1(T)=CIP 108389(T)=LMG 22506(T)) and Leucobacter chromiireducens subsp. solipictus subsp. nov. (type strain TAN 31504(T)=DSM 18340(T)=ATCC BAA-1336(T)).

The trans-acting flagellar regulatory proteins, FliX and FlbD, play a central role in linking flagellar biogenesis and cytokinesis in Caulobacter crescentus.

The FliX/FlbD-dependent temporal transcription of late flagellar genes in Caulobacter crescentus requires the assembly of an early, class II-encoded flagellar structure. Class II flagellar-mutant strains exhibit a delay in the completion of cell division, with the accumulation of filamentous cells in culture. It is shown here that this cell-division defect is attributable to an arrest in the final stages of cell separation. Normal cell morphology could be restored in class II mutants by gain-of-function alleles of FliX or FlbD, suggesting that the timely completion of cell division requires these trans-acting factors. In synchronized cultures, inhibition of cell division by depleting FtsZ resulted in normal initial expression of the late, FlbD-dependent fliK gene; however, the cell cycle-regulated cessation of transcription was delayed, indicating that cell division may be required to negatively regulate FlbD activity. Interestingly, prolonged depletion of FtsZ resulted in an eventual loss of FlbD activity that could be bypassed by a constitutive mutant of FlbD, but not of FliX, suggesting the possible existence of a second cell cycle-dependent pathway for FlbD activation.

Role of integration host factor in the transcriptional activation of flagellar gene expression in Caulobacter crescentus.

In the Caulobacter crescentus predivisional cell, class III and IV flagellar genes, encoding the extracytoplasmic components of the flagellum, are transcribed in the nascent swarmer compartment. This asymmetric expression pattern is attributable to the compartmentalized activity of the sigma54-dependent transcriptional activator FlbD. Additionally, these temporally transcribed flagellar promoters possess a consensus sequence for the DNA-binding protein integration host factor (IHF), located between the upstream FlbD binding site and the promoter sequences. Here, we deleted the C. crescentus gene encoding the beta-subunit of the IHF, ihfB (himD), and examined the effect on flagellar gene expression. The DeltaihfB strain exhibited a mild defect in cell morphology and impaired motility. Using flagellar promoter reporter fusions, we observed that expression levels of a subset of class III flagellar promoters were decreased by the loss of IHF. However, one of these promoters, fliK-lacZ, exhibited a wild-type cell cycle-regulated pattern of expression in the absence of IHF. Thus, IHF is required for maximal transcription of several late flagellar genes. The DeltaihfB strain was found to express significantly reduced amounts of the class IV flagellin, FljL, as a consequence of reduced transcriptional activity. Our results indicate that the motility defect exhibited by the DeltaihfB strain is most likely attributable to its failure to accumulate the class IV-encoded 27-kDa flagellin subunit, FljL.

Regulation of FlbD activity by flagellum assembly is accomplished through direct interaction with the trans-acting factor, FliX.

The temporal and spatial transcription of late flagellar genes in Caulobacter crescentus is regulated by the sigma54 transcriptional activator, FlbD. One requirement for FlbD activity is the assembly of a structure encoded by early, class II flagellar genes. In this report, we show that the trans-acting factor FliX predominantly functions as a negative regulator of FlbD activity in the absence of the class II-encoded flagellar structure. In contrast, a mutant FliX that bypasses the transcriptional requirement for early flagellar assembly is incapable of repressing FlbD in a class II flagellar mutant. Expression of this mutant allele, fliX1, does not alter the temporal pattern of FlbD-dependent transcription. Remarkably, this mutation confers the correct cell cycle timing of hook operon transcription in a strain that cannot assemble the flagellum, indicating that the progression of flagellar assembly is a minor influence on temporal gene expression. Using a two-hybrid assay, we present evidence that FliX regulates FlbD through a direct interaction, a novel mechanism for this class of sigma54 transcriptional activator. Furthermore, increasing the cellular levels of FliX results in an increase in the concentration of FlbD, and a corresponding increase in FlbD-activated transcription, suggesting that FliX and FlbD form a stable complex in Caulobacter. FliX and FlbD homologues are present in several polar-flagellated bacteria, indicating that these proteins constitute an evolutionarily conserved regulatory pair in organisms where flagellar biogenesis is likely to be under control of the cell division cycle.