Novel assay to measure the plasmid mobilizing potential of mixed microbial communities

This is the final version of the article. Available from the publisher via the DOI in this record.Mobilizable plasmids lack necessary genes for complete conjugation and are therefore non-self-transmissible. Instead, they rely on the conjugation system of conjugal plasmids to be horizontally transferred to new recipients. While community permissiveness, the fraction of a mixed microbial community that can receive self-transmissible conjugal plasmids, has been studied, the intrinsic ability of a community to mobilize plasmids that lack conjugation systems is unexplored. Here, we present a novel framework and experimental method to estimate the mobilization potential of mixed communities. We compare the transfer frequency of a mobilizable plasmid to that of a mobilizing and conjugal plasmid measured for a model strain and for the assayed community. With Pseudomonas putida carrying the gfp-tagged mobilizable IncQ plasmid RSF1010 as donor strain, we conducted solid surface mating experiments with either a P. putida strain carrying the mobilizing IncP-1α plasmid RP4 or a model bacterial community that was extracted from the inner walls of a domestic shower conduit. Additionally, we estimated the permissiveness of the same community for RP4 using P. putida as donor strain. The permissiveness of the model community for RP4 [at 1.16 × 10(-4) transconjugants per recipient (T/R)] was similar to that previously measured for soil microbial communities. RSF1010 was mobilized by the model community at a frequency of 1.16 × 10(-5) T/R, only one order of magnitude lower than its permissiveness to RP4. This mobilization frequency is unexpectedly high considering that (i) mobilization requires the presence of mobilizing conjugal plasmids within the permissive fraction of the recipients; (ii) in pure culture experiments with P. putida retromobilization of RSF1010 through RP4 only took place in approximately half of the donors receiving the conjugal plasmid in the first step. Further work is needed to establish how plasmid mobilization potential varies within and across microbial communities. This method has the potential to provide such insights; in addition it allows for the direct isolation of in situ mobilizing plasmids together with their endogenous hosts.We thank L. Riber and S. J. Sørensen for access to the tagged RSF1010 plasmid, L. K. Jensen for technical assistance in the laboratory and S. M. Milani for assistance in FACS sorting. This work was funded by the Villum Kann Rasmussen Foundation Center of Excellence CREAM (Center for Environmental and Agricultural Microbiology)

Skeletal Muscle HIF-1α Expression Is Dependent on Muscle Fiber Type

Oxygen homeostasis is an essential regulation system for cell energy production and survival. The oxygen-sensitive subunit α of the hypoxia inducible factor-1 (HIF-1) complex is a key protein of this system. In this work, we analyzed mouse and rat HIF-1α protein and mRNA expression in parallel to energetic metabolism variations within skeletal muscle. Two physiological situations were studied using HIF-1α–specific Western blotting and semiquantitative RT-PCR. First, we compared HIF-1α expression between the predominantly oxidative soleus muscle and three predominantly glycolytic muscles. Second, HIF-1α expression was assessed in an energy metabolism switch model that was based on muscle disuse. These two in vivo situations were compared with the in vitro HIF-1α induction by CoCl2 treatment on C2C12 mouse muscle cells. HIF-1α mRNA and protein levels were found to be constitutively higher in the more glycolytic muscles compared with the more oxidative muscles. Our results gave rise to the hypothesis that the oxygen homeostasis regulation system depends on the fiber type

Methods for improving teaching using student feedback techniques

Les professeurs peuvent apprendre des élèves comment mieux enseigner. Il y a de nombreuses façons d’avoir des retours pour évaluer la qualité de l’enseignement : l’auto-évaluation, l’évaluation par les collègues, les techniques de recherche dans les cours et les évaluations des enseignants ou des cours. Presque tous les établissements d’enseignement supérieur aux États-Unis utilisent des méthodes d’évaluation des enseignants ou des cours. Le succès de l’amélioration de l’enseignement dépend de la qualité des informations amassées et du désir de les appliquer.Teachers can learn from students how to teach better. There are many ways to get feedback to evaluate the quality of teaching: self-evaluation, peer-evaluation, classroom research techniques, and faculty/course evaluations. Virtually every post-secondary educational facility in the United States utilizes some method of faculty/course evaluation. The success of improving teaching depends on the quality of the information gathered and the desire to apply the information

Long-term manure exposure increases soil bacterial community potential for plasmid uptake.

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this record.Microbial communities derived from soils subject to different agronomic treatments were challenged with three broad host range plasmids, RP4, pIPO2tet and pRO101, via solid surface filter matings to assess their permissiveness. Approximately 1 in 10 000 soil bacterial cells could receive and maintain the plasmids. The community permissiveness increased up to 100% in communities derived from manured soil. While the plasmid transfer frequency was significantly influenced by both the type of plasmid and the agronomic treatment, the diversity of the transconjugal pools was purely plasmid dependent and was dominated by β- and γ-Proteobacteria

Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

This is the author accepted manuscript. The final version is available from Springer Nature via the DOI in this recordConjugal plasmids can provide microbes with full complements of new genes and constitute potent vehicles for horizontal gene transfer. Conjugal plasmid transfer is deemed responsible for the rapid spread of antibiotic resistance among microbes. While broad host range plasmids are known to transfer to diverse hosts in pure culture, the extent of their ability to transfer in the complex bacterial communities present in most habitats has not been comprehensively studied. Here, we isolated and characterized transconjugants with a degree of sensitivity not previously realized to investigate the transfer range of IncP- and IncPromA-type broad host range plasmids from three proteobacterial donors to a soil bacterial community. We identified transfer to many different recipients belonging to 11 different bacterial phyla. The prevalence of transconjugants belonging to diverse Gram-positive Firmicutes and Actinobacteria suggests that inter-Gram plasmid transfer of IncP-1 and IncPromA-type plasmids is a frequent phenomenon. While the plasmid receiving fractions of the community were both plasmid- and donor- dependent, we identified a core super-permissive fraction that could take up different plasmids from diverse donor strains. This fraction, comprising 80% of the identified transconjugants, thus has the potential to dominate IncP- and IncPromA-type plasmid transfer in soil. Our results demonstrate that these broad host range plasmids have a hitherto unrecognized potential to transfer readily to very diverse bacteria and can, therefore, directly connect large proportions of the soil bacterial gene pool. This finding reinforces the evolutionary and medical significances of these plasmids.This work was funded by the Villum Kann Rasmussen Foundation Center of Excellence CREAM (Center for Environmental and Agricultural Microbiology)

Risicoprofilering heronderzoek bijstandsuitkering = Risk profiling for social welfare re-examination

Normative advice commissionAbderrahman El Aazani, Researcher at the Ombudsman Rotterdam-RijnmondFrancien Dechesne, Associate Professor Law and Digital Technologies, Leiden UniversityMaarten van Asten, Alderman Finance, Digitalization, Sports and Events Municipality of TilburgMunish Ramlal, Ombudsman Metropole region AmsterdamOskar Gstrein, Assistant Professor Governance and Innovation, University of GroningenEffective Protection of Fundamental Rights in a pluralist worl

Nanopods: A New Bacterial Structure and Mechanism for Deployment of Outer Membrane Vesicles

Background: Bacterial outer membrane vesicles (OMV) are packets of periplasmic material that, via the proteins and other molecules they contain, project metabolic function into the environment. While OMV production is widespread in proteobacteria, they have been extensively studied only in pathogens, which inhabit fully hydrated environments. However, many (arguably most) bacterial habitats, such as soil, are only partially hydrated. In the latter, water is characteristically distributed as films on soil particles that are, on average thinner, than are typical OMV (ca. ≤10 nm water film vs. 20 to >200 nm OMV;). Methodology/Principal Findings: We have identified a new bacterial surface structure, termed a "nanopod", that is a conduit for projecting OMV significant distances (e.g., ≥6 µm) from the cell. Electron cryotomography was used to determine nanopod three-dimensional structure, which revealed chains of vesicles within an undulating, tubular element. By using immunoelectron microscopy, proteomics, heterologous expression and mutagenesis, the tubes were determined to be an assembly of a surface layer protein (NpdA), and the interior structures identified as OMV. Specific metabolic function(s) for nanopods produced by Delftia sp. Cs1-4 are not yet known. However, a connection with phenanthrene degradation is a possibility since nanopod formation was induced by growth on phenanthrene. Orthologs of NpdA were identified in three other genera of the Comamonadaceae family, and all were experimentally verified to form nanopods. Conclusions/Significance: Nanopods are new bacterial organelles, and establish a new paradigm in the mechanisms by which bacteria effect long-distance interactions with their environment. Specifically, they create a pathway through which cells can effectively deploy OMV, and the biological activity these transmit, in a diffusion-independent manner. Nanopods would thus allow environmental bacteria to expand their metabolic sphere of influence in a manner previously unknown for these organisms

Soil aggregates as massively concurrent evolutionary incubators

Soil aggregation, a key component of soil structure, has mostly been examined from the perspective of soil management and the mediation of ecosystem processes such as soil carbon storage. However, soil aggregation is also a major factor to consider in terms of the fine-scale organization of the soil microbiome. For example, the physico-chemical conditions inside of aggregates usually differ from the conditions prevalent in the bulk soil and aggregates therefore increase the spatial heterogeneity of the soil. In addition, aggregates can provide a refuge for microbes against predation since their interior is not accessible to many predators. Soil aggregates are thus clearly important for microbial community ecology in soils (for example, Vos et al., 2013; Rillig et al., 2016) and for microbially driven biogeochemistry, and soil microbial ecologists are increasingly appreciating these aspects of soil aggregation. Soil aggregates have, however, so far been neglected when it comes to evolutionary considerations (Crawford et al., 2005) and we here propose that the process of soil aggregation should be considered as an important driver of evolution in the soil microbial community

Visualizing the Needle in the Haystack: In Situ Hybridization With Fluorescent Dendrimers

In situ hybridization with 3DNA™ dendrimers is a novel tool for detecting low levels of mRNA in tissue sections and whole embryos. Fluorescently labeled dendrimers were used to identify cells that express mRNA for the skeletal muscle transcription factor MyoD in the early chick embryo. A small population of MyoD mRNA positive cells was found in the epiblast prior to the initiation of gastrulation, two days earlier than previously detected using enzymatic or radiolabeled probes for mRNA. When isolated from the epiblast and placed in culture, the MyoD mRNA positive cells were able to differentiate into skeletal muscle cells. These results demonstrate that DNA dendrimers are sensitive and precise tools for identifying low levels of mRNA in single cells and tissues