Identification of genes involved in the mutualistic colonization of the nematode Heterorhabditis bacteriophora by the bacterium Photorhabdus luminescens

<p>Abstract</p> <p>Background</p> <p><it>Photorhabdus </it>are Gram negative entomopathogenic bacteria that also have a mutualistic association with nematodes from the family <it>Heterorhabditis</it>. An essential part of this symbiosis is the ability of the bacterium to colonize the gut of the freeliving form of the nematode called the infective juvenile (IJ). Although the colonization process (also called transmission) has been described phenomonologically very little is known about the underlying molecular mechanisms. Therefore, in this study, we were interested in identifying genes in <it>Photorhabdus </it>that are important for IJ colonization.</p> <p>Results</p> <p>In this work we genetically tagged <it>P. luminescens </it>TT01 with <it>gfp </it>and constructed a library containing over 3200 mutants using the suicide vector, pUT-Km2. Using a combination of <it>in vitro </it>symbiosis assays and fluorescent microscopy we screened this library for mutants that were affected in their ability to colonize the IJ i.e. with decreased transmission frequencies. In total 8 mutants were identified with transmission frequencies of ≤ 30% compared to wild-type. These mutants were mapped to 6 different genetic loci; the <it>pbgPE </it>operon, <it>galE</it>, <it>galU</it>, <it>proQ</it>, <it>asmA </it>and <it>hdfR</it>. The <it>pbgPE</it>, <it>galE </it>and <it>galU </it>mutants were all predicted to be involved in LPS biosynthesis and, in support of this, we have shown that these mutants are avirulent and sensitive to the cationic antimicriobial peptide, polymyxin B. On the other hand the <it>proQ</it>, <it>asmA </it>and <it>hdfR </it>mutants were not affected in virulence and were either as resistant (<it>proQ</it>) or slightly more sensitive (<it>asmA, hdfR</it>) to polymyxin B than the wild-type (WT).</p> <p>Conclusions</p> <p>This is the first report describing the outcome of a comprehensive screen looking for transmission mutants in <it>Photorhabdus</it>. In total 6 genetic loci were identified and we present evidence that all of these loci are involved in the assembly and/or maintenance of LPS and other factors associated with the cell surface. Interestingly several, but not all, of the transmission mutants identified were also avirulent suggesting that there is a significant, but not complete, genetic overlap between pathogenicity and mutualism. Therefore, this study highlights the importance of the cell surface in mediating the symbiotic and pathogenic interactions of <it>Photorhabdus</it>.</p

Characterization of Heterorhabditis isolates by PCR amplification of segments of mtDNA and rDNA genes

Restriction digests of amplified DNA from the mitochondrial genome and the nuclear ribosomal internally transcribed spacer region have been evaluated as genetic markers for species groups in Heterorhabditis. Six RFLP profiles have been identified. These profiles supported groupings determined by cross-breeding studies and were in agreement with less definitive groupings based on other biochemical and molecular methods. Digestion patterns of both amplification products provided strong evidence for the recognition of species groups, which include Irish, NW European, tropical, and a H. bacteriophora complex. The H. bacteriophora complex could be further resolved into three genotypes represented by H. zealandica, the H. bacteriophora, Brecon (Australian) type isolate for H. bacteriophora, and a grouping composed of isolates NC1, V16, HI82, and HP88. All cultures obtained of the H. megidis isolate were identical to the NW European group. These results could be used to aid monitoring of field release of Heterorhabditis as well as allowing a rapid initial assessment of taxonomic grouping

Characterization of \u3ci\u3eHeterorhabditis\u3c/i\u3e Isolates by PCR Amplification of Segments of mtDNA and rDNA Genes

Restriction digests of amplified DNA from the mitochondrial genome and the nuclear ribosomal internally transcribed spacer region have been evaluated as genetic markers for species groups in Heterorhabditis. Six RFLP profiles have been identified. These profiles supported groupings determined by cross-breeding studies and were in agreement with less definitive groupings based on other biochemical and molecular methods. Digestion patterns of both amplification products provided strong evidence for the recognition of species groups, which include Irish, NW European, tropical, and a H. bacteriophora complex. The H. bacteriophora complex could be further resolved into three genotypes represented by H. zealandica, the H. bacteriophora, Brecon (Australian) type isolate for H. bacteriophora, and a grouping composed of isolates NC1, V16, HI82, and HP88. All cultures obtained of the H. megidis isolate were identical to the NW European group. These results could be used to aid monitoring of field release of Heterorhabditis as well as allowing a rapid initial assessment of taxonomic grouping

Dietary fibre modulates the gut microbiota

peer-reviewedDietary fibre has long been established as a nutritionally important, health-promoting food ingredient. Modern dietary practices have seen a significant reduction in fibre consumption compared with ancestral habits. This is related to the emergence of low-fibre “Western diets” associated with industrialised nations, and is linked to an increased prevalence of gut diseases such as inflammatory bowel disease, obesity, type II diabetes mellitus and metabolic syndrome. The characteristic metabolic parameters of these individuals include insulin resistance, high fasting and postprandial glucose, as well as high plasma cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL). Gut microbial signatures are also altered significantly in these cohorts, suggesting a causative link between diet, microbes and disease. Dietary fibre consumption has been hypothesised to reverse these changes through microbial fermentation and the subsequent production of short-chain fatty acids (SCFA), which improves glucose and lipid parameters in individuals who harbour diseases associated with dysfunctional metabolism. This review article examines how different types of dietary fibre can differentially alter glucose and lipid metabolism through changes in gut microbiota composition and function

Isolation and characterisation of Heterorhabditis spp. (Nematoda: Heterorhabditidae) from Hungary, Estonia and Denmark

Targeted surveys were conducted for the entomopathogenic nematode Heterorhabditis in areas of Denmark, Estonia and Hungary. Isolateswere identiŽ ed by IEF, PCR and cross-fertilitytests as belonging to three distinct taxonomic groups: H. bacteriophora, the north-west European (NWE) type of H. megidis and the Irish type of Heterorhabditis. The Irish and NWE types of Heterorhabditis were both present in Denmark (at six and four sites, respectively),while only the NWE type was recovered in Estonia. H. bacteriophora was the dominant heterorhabditididentiŽ ed in Hungary (ten sites), but the Irish typewas also detected at two sites. This is the Ž rst report of the Irish type of Heterorhabditis on continental Europe. Co-occurrence of two Heterorhabditis types at a single site was noted in Denmark (Irish and NWE) and in Hungary (Irish and H. bacteriophora). Heterorhabditiswas recovered at 38.5% of sites (n = 26) in Denmark (north coast of Sjælland), 27.3% of the coastal sites (n = 22) in Estonia, and 32.6% of sites (n = 46) in Hungary

The cholesterol-lowering effect of oats and oat beta glucan: modes of action and potential role of bile acids and the microbiome

Consumption of sufficient quantities of oat products has been shown to reduce host cholesterol and thereby modulate cardiovascular disease risk. The effects are proposed to be mediated by the gel-forming properties of oat β-glucan which modulates host bile acid and cholesterol metabolism and potentially removes intestinal cholesterol for excretion. However, the gut microbiota has emerged as a major factor regulating cholesterol metabolism in the host. Oat β-glucan has been shown to modulate the gut microbiota, particularly those bacterial species that influence host bile acid metabolism and production of short chain fatty acids, factors which are regulators of host cholesterol homeostasis. Given a significant role for the gut microbiota in cholesterol metabolism it is likely that the effects of oat β-glucan on the host are multifaceted and involve regulation of microbe-host interactions at the gut interface. Here we consider the potential for oat β-glucan to influence microbial populations in the gut with potential consequences for bile acid metabolism, reverse cholesterol transport (RCT), short-chain fatty acid (SCFA) production, bacterial metabolism of cholesterol and microbe-host signaling

The impact of the gut microbiota on drug metabolism and clinical outcome

The significance of the gut microbiota as a determinant of drug pharmacokinetics and accordingly therapeutic response is of increasing importance with the advent of modern medicines characterised by low solubility and/or permeability, or modified-release. These physicochemical properties and release kinetics prolong drug residence times within the gastrointestinal tract, wherein biotransformation by commensal microbes can occur. As the evidence base in support of this supplementary metabolic “organ” expands, novel opportunities to engineer the microbiota for clinical benefit have emerged. This review provides an overview of microbe-mediated alteration of drug pharmacokinetics, with particular emphasis on studies demonstrating proof of concept in vivo. Additionally, recent advances in modulating the microbiota to improve clinical response to therapeutics are explored

Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during mitochondrial reactive oxygen stress in macrophages

AbstractUncoupling protein-2 (UCP2) is a member of the inner mitochondrial membrane anion-carrier superfamily. Although mRNA for UCP2 is widely expressed, protein expression is detected in only a few cell types, including macrophages. UCP2 functions by an incompletely defined mechanism, to reduce reactive oxygen species production during mitochondrial electron transport. We observed that the abundance of UCP2 in macrophages increased rapidly in response to treatments (rotenone, antimycin A and diethyldithiocarbamate) that increased mitochondrial superoxide production, but not in response to superoxide produced outside the mitochondria or in response to H2O2. Increased UCP2 protein was not accompanied by increases in ucp2 gene expression or mRNA abundance, but was due to enhanced translational efficiency and possibly stabilization of UCP2 protein in the inner mitochondrial membrane. This was not dependent on mitochondrial membrane potential. These findings extend our understanding of the homeostatic function of UCP2 in regulating mitochondrial reactive oxygen production by identifying a feedback loop that senses mitochondrial reactive oxygen production and increases inner mitochondrial membrane UCP2 abundance and activity. Reactive oxygen species-induction of UCP2 may facilitate survival of macrophages and retention of function in widely variable tissue environments

Building Evidence-Based Nursing Practice Capacity in a Large Statewide Health System

Integration of evidence-based practice (EBP) into the culture of a healthcare organization is essential to provide safe patient care and promote a thriving culture for the nurses within the healthcare organization. Collaboration and utilization of both clinical and academic experts facilitate the removal of barriers to EBP. This article describes the successful partnership between a healthcare system and school of nursing in executing a 3-phased multimodal approach to an EBP training program