Tanya E. S. Dahms

Expression of the human μ opioid receptor in a stable Sf9 cell line

The cDNA that encodes the human mu opioid receptor (hMOR) has been cloned and expressed in Spodoptera frugiperda (Sf9) cells using a nonlytic vector system. The coding sequence fused to the cleavable glycoprotein signal peptide gp 64, and a C-terminal histidine tag was placed under the transcriptional control of the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus immediate-early 2 (OpIE2) promoter. Transfected cells were selected using Zeocin resistance and the receptor was constitutively expressed at approximately 12000 receptors per cell. Immunofluorescence images illustrated that more than 75% of the Sf9 cells expressed hMOR at the plasma membrane. This is the first report of the constitutive and heterologous expression of a G protein-coupled receptor in a stably transfected Sf9 cell line, under the control of the OpIE2 promoter.

Friends or foes? Emerging insights from fungal interactions with plants

Fungi interact with plants in various ways, with each interaction giving rise to different alterations in both partners. While fungal pathogens have detrimental effects on plant physiology, mutualistic fungi augment host defence responses to pathogens and/or improve plant nutrient uptake. Tropic growth towards plant roots or stomata, mediated by chemical and topographical signals, has been described for several fungi, with evidence of species-specific signals and sensing mechanisms. Fungal partners secrete bioactive molecules such as small peptide effectors, enzymes and secondary metabolites which facilitate colonization and contribute to both symbiotic and pathogenic relationships. There has been tremendous advancement in fungal molecular biology, omics sciences and microscopy in recent years, opening up new possibilities for the identification of key molecular mechanisms in plant–fungal interactions, the power of which is often borne out in their combination. Our fragmentary knowledge on the interactions between plants and fungi must be made whole to understand the potential of fungi in preventing plant diseases, improving plant productivity and understanding ecosystem stability. Here, we review innovative methods and the associated new insights into plant–fungal interactions.

Quantifying the Importance of Galactofuranose in Aspergillus nidulans Hyphal Wall Surface Organization by Atomic Force Microscopy

ABSTRACT The fungal wall mediates cell-environment interactions. Galactofuranose (Galf), the five-member ring form of galactose, has a relatively low abundance in Aspergillus walls yet is important for fungal growth and fitness. Aspergillus nidulans strains deleted for Galf biosynthesis enzymes UgeA (UDP-glucose-4-epimerase) and UgmA (UDP-galactopyranose mutase) lacked immunolocalizable Galf, had growth and sporulation defects, and had abnormal wall architecture. We used atomic force microscopy and force spectroscopy to image and quantify cell wall viscoelasticity and surface adhesion of ugeAΔ and ugmAΔ strains. We compared the results for ugeAΔ and ugmAΔ strains with the results for a wild-type strain (AAE1) and the ugeB deletion strain, which has wild-type growth and sporulation. Our results suggest that UgeA and UgmA are important for cell wall surface subunit organization and wall viscoelasticity. The ugeAΔ and ugmAΔ strains had significantly larger surface subunits and lower cell wall viscoelastic moduli than those of AAE1 or ugeBΔ hyphae. Double deletion strains (ugeAΔ ugeBΔ and ugeAΔ ugmAΔ) had more-disorganized surface subunits than single deletion strains. Changes in wall surface structure correlated with changes in its viscoelastic modulus for both fixed and living hyphae. Wild-type walls had the largest viscoelastic modulus, while the walls of the double deletion strains had the smallest. The ugmAΔ strain and particularly the ugeAΔ ugmAΔ double deletion strain were more adhesive to hydrophilic surfaces than the wild type, consistent with changes in wall viscoelasticity and surface organization. We propose that Galf is necessary for full maturation of A. nidulans walls during hyphal extension.

Atomic force microscopy of a ctpA mutant in Rhizobium leguminosarum reveals surface defects linking CtpA function to biofilm formation

Atomic force microscopy was used to investigate the surface ultrastructure, adhesive properties and biofilm formation of Rhizobium leguminosarum and a ctpA mutant strain. The surface ultrastructure of wild-type R. leguminosarum consists of tightly packed surface subunits, whereas the ctpA mutant has much larger subunits with loose lateral packing. The ctpA mutant strain is not capable of developing fully mature biofilms, consistent with its altered surface ultrastructure, greater roughness and stronger adhesion to hydrophilic surfaces. For both strains, surface roughness and adhesive forces increased as a function of calcium ion concentration, and for each, biofilms were thicker at higher calcium concentrations.

Oxidative stress and metabolic perturbations in Escherichia coli exposed to sublethal levels of 2,4-dichlorophenoxyacetic acid

The chlorophenoxy herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is used extensively worldwide despite its known toxicity and our limited understanding of how it affects non-target organisms. Escherichia coli is a suitable model organism to investigate toxicity and adaptation mechanisms in bacteria exposed to xenobiotic chemicals. We developed a methodical platform that uses atomic force microscopy, metabolomics and biochemical assays to quantify the response of E. coli exposed to sublethal levels of 2,4-D. This herbicide induced a filamentous phenotype in E. coli BL21 and a similar phenotype was observed in a selection of genotypically diverse E. coli strains (A0, A1, B1, and D) isolated from the environment. The filamentous phenotype was observed at concentrations 1000 times below field levels and was reversible upon supplementation with polyamines. Cells treated with 2,4-D had more compliant envelopes, significantly remodeled surfaces that were rougher and altered vital metabolic pathways including oxidative phosphorylation, the ABC transport system, peptidoglycan biosynthesis, amino acid, nucleotide and sugar metabolism. Most of the observed effects could be attributed to oxidative stress, consistent with increases in reactive oxygen species as a function of 2,4-D exposure. This study provides direct evidence that 2,4-D at sublethal levels induces oxidative stress and identifies the associated metabolic changes in E. coli.

Saskatchewan's (Canada) Regional Centre of Expertise on Education for Sustainable Development

Purpose – The purpose of this paper is to describe the unique collaborative process initiated at the University of Regina in Saskatchewan, Canada, to develop a Regional Centre of Expertise (RCE) on Education for Sustainable Development (ESD) through the United Nations University-Institute of Advanced Studies (UNU-IAS). Design/methodology/approach – The innovative aspects of developing RCE Saskatchewan are elaborated as a function of regional characteristics and features. The founding members used a unique combination of facilitation methods and technology to establish a flexible governance model in support of ESD themes defined by participating members. The paper provides a critical analysis of the governance structure and educational approaches supported by RCE Saskatchewan. Findings – The strength-based model, in conjunction with open meetings and online discussion, has proven to be a highly effective method for establishing an organization in which members have diverse expertise and affiliation. The process used to develop the RCE was democratic, inclusive and transparent. Each member and member group contributes existing knowledge, with the common goal of advancing research on ESD as part of a regional and global network. The RCE was established in just over one year, a testament to the efficiency of the process. The web site provides a flexible host for the RCE virtual structure, facilitating communication within and between groups, documentation, announcements/postings, and the creation of searchable research databases with local and global public access. Flexibility of the governance model and partnerships with higher education institutions ensures long-term sustainability. The use of non-traditional educational modes (non-formal and informal) has accelerated ESD awareness in the region, and their combination with formal education leads to programs that span different sectors. Originality/value – This paper outlines the process developed by the founding members of RCE Saskatchewan to mobilise people, organizations and resources during the de novo establishment of an organization dedicated to promoting ESD. The virtual home and governance model, taken together, describe a completely unique structure that ensures long-term sustainability for RCE Saskatchewan. Combining educational modes has so far proven successful in promoting ESD.

Viscoelasticity in Biological Systems: A Special Focus on Microbes

Over billions of years of evolution, living organisms have developed into complex biosystems, of which the basic unit is the cell. Cells have a complex molecular structure with a certain level of rigidity. Living cells, whether isolated or part of a larger collective, live under constant mechanical stress from their external environments. Cells have developed adaptive mechanisms to maintain homeostasis and viability, which interestingly follow the basic principles of classical mechanics.

Rhizobium leguminosarum bv. viciae 3841 Adapts to 2,4-Dichlorophenoxyacetic Acid with "Auxin-Like" Morphological Changes, Cell Envelope Remodeling and Upregulation of Central Metabolic Pathways

There is a growing need to characterize the effects of environmental stressors at the molecular level on model organisms with the ever increasing number and variety of anthropogenic chemical pollutants. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), as one of the most widely applied pesticides in the world, is one such example. This herbicide is known to have non-targeted undesirable effects on humans, animals and soil microbes, but specific molecular targets at sublethal levels are unknown. In this study, we have used Rhizobium leguminosarum bv. viciae 3841 (Rlv) as a nitrogen fixing, beneficial model soil organism to characterize the effects of 2,4-D. Using metabolomics and advanced microscopy we determined specific target pathways in the Rlv metabolic network and consequent changes to its phenotype, surface ultrastructure, and physical properties during sublethal 2,4-D exposure. Auxin and 2,4-D, its structural analogue, showed common morphological changes in vitro which were similar to bacteroids isolated from plant nodules, implying that these changes are related to bacteroid differentiation required for nitrogen fixation. Rlv showed remarkable adaptation capabilities in response to the herbicide, with changes to integral pathways of cellular metabolism and the potential to assimilate 2,4-D with consequent changes to its physical and structural properties. This study identifies biomarkers of 2,4-D in Rlv and offers valuable insights into the mode-of-action of 2,4-D in soil bacteria.

Exposure to Sub-lethal 2,4-Dichlorophenoxyacetic Acid Arrests Cell Division and Alters Cell Surface Properties in Escherichia coli

Escherichia coli is a robust, easily adaptable and culturable bacterium in vitro, and a model bacterium for studying the impact of xenobiotics in the environment. We have used correlative atomic force – laser scanning confocal microscopy (AFM-LSCM) to characterize the mechanisms of cellular response to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). One of the most extensively used herbicides world-wide, 2,4-D is known to cause hazardous effects in diverse non-target organisms. Sub-lethal concentrations of 2,4-D caused DNA damage in E. coli WM1074 during short exposure periods which increased significantly over time. In response to 2,4-D, FtsZ and FtsA relocalized within seconds, coinciding with the complete inhibition of cell septation and cell elongation. Exposure to 2,4-D also resulted in increased activation of the SOS response. Changes to cell division were accompanied by concomitant changes to surface roughness, elasticity and adhesion in a time-dependent manner. This is the first study describing the mechanistic details of 2,4-D at sub-lethal levels in bacteria. Our study suggests that 2,4-D arrests E. coli cell division within seconds after exposure by disrupting the divisome complex, facilitated by dissipation of membrane potential. Over longer exposures, 2,4-D causes filamentation as a result of an SOS response to oxidative stress induced DNA damage.

Future Directions in Advanced Mycological Microscopy

The field of mycology is poised to exploit the many recent advances in microscopic tools and instrumentation for cell biology. This chapter first outlines the latest developments in biosensors that will prove useful for targeting specific events in the context of single fungal cells, mycelia, or fungal-plant systems. Next, we focus on microscopic methods, in particular electron based, capable of generating three-dimensional (3D) data at single-molecule resolution. Combining the capabilities of any of the powerful microscopy platforms discussed in this volume (Chaps. 1–7) leads to correlative microscopy which vastly expands the range of image scale and data complexity. This sophisticated approach combines data collected separately or simultaneously from individual or hybrid microscopes, respectively, offering complementary internal and external spatial, structural, biochemical, and biophysical information on one sample. Finally, we review the recent select advances in imaging technology that we believe hold special promise to glean new insights from the inner working of fungal cells with unprecedented spatial and temporal resolutions.