Metabolic resource overlap impacts competition among phyllosphere bacteria.

The phyllosphere is densely colonised by microbial communities, despite sparse and heterogeneously distributed resources. The limitation of resources is expected to drive bacterial competition resulting in exclusion or coexistence based on fitness differences and resource overlap between individual colonisers. We studied the impact of resource competition by determining the effects of different bacterial colonisers on the growth of the model epiphyte Pantoea eucalypti 299R (Pe299R). Resource overlap was predicted based on genome-scale metabolic modelling. By combining results of metabolic modelling and pairwise competitions in the Arabidopsis thaliana phyllosphere and in vitro, we found that ten resources sufficed to explain fitness of Pe299R. An effect of both resource overlap and phylogenetic relationships was found on competition outcomes in vitro as well as in the phyllosphere. However, effects of resource competition were much weaker in the phyllosphere when compared to in vitro experiments. When investigating growth dynamics and reproductive success at the single-cell resolution, resource overlap and phylogenetic relationships are only weakly correlated with epiphytic Pe299R reproductive success, indicating that the leaf's spatial heterogeneity mitigates resource competition. Although the correlation is weak, the presence of competitors led to the development of Pe299R subpopulations that experienced different life histories and cell divisions. In some in planta competitions, Pe299R benefitted from the presence of epiphytes despite high resource overlap to the competitor strain suggesting other factors having stronger effects than resource competition. This study provides fundamental insights into how bacterial communities are shaped in heterogeneous environments and a framework to predict competition outcomes.

Replicating Arabidopsis Model Leaf Surfaces for Phyllosphere Microbiology.

Artificial surfaces are commonly used in place of leaves in phyllosphere microbiology to study microbial behaviour on plant leaf surfaces. These surfaces enable a reductionist approach to be undertaken, to enable individual environmental factors influencing microorganisms to be studied. Commonly used artificial surfaces include nutrient agar, isolated leaf cuticles, and reconstituted leaf waxes. Recently, replica surfaces mimicking the complex topography of leaf surfaces for phyllosphere microbiology studies are appearing in literature. Replica leaf surfaces have been produced in agar, epoxy, polystyrene, and polydimethylsiloxane (PDMS). However, none of these protocols are suitable for replicating fragile leaves such as of the model plant Arabidopsis thaliana. This is of importance, as A. thaliana is a model system for molecular plant genetics, molecular plant biology, and microbial ecology. To overcome this limitation, we introduce a versatile replication protocol for replicating fragile leaf surfaces into PDMS. Here we demonstrate the capacity of our replication process using optical microscopy, atomic force microscopy (AFM), and contact angle measurements to compare living and PDMS replica A. thaliana leaf surfaces. To highlight the use of our replica leaf surfaces for phyllosphere microbiology, we visualise bacteria on the replica leaf surfaces in comparison to living leaf surfaces.

Comparison of replica leaf surface materials for phyllosphere microbiology.

Artificial surfaces are routinely used instead of leaves to enable a reductionist approach in phyllosphere microbiology, the study of microorganisms residing on plant leaf surfaces. Commonly used artificial surfaces include, flat surfaces, such as metal and nutrient agar, and microstructured surfaces, such as isolate leaf cuticles or reconstituted leaf waxes. However, interest in replica leaf surfaces as an artificial surface is growing, as replica surfaces provide an improved representation of the complex topography of leaf surfaces. To date, leaf surfaces have predominantly been replicated for their superhydrophobic properties. In contrast, in this paper we investigated the potential of agarose, the elastomer polydimethylsiloxane (PDMS), and gelatin as replica leaf surface materials for phyllosphere microbiology studies. Using a test pattern of pillars, we investigated the ability to replicate microstructures into the materials, as well as the degradation characteristics of the materials in environmental conditions. Pillars produced in PDMS were measured to be within 10% of the mold master and remained stable throughout the degradation experiments. In agarose and gelatin the pillars deviated by more than 10% and degraded considerably within 48 hours in environmental conditions. Furthermore, we investigated the surface energy of the materials, an important property of a leaf surface, which influences resource availability and microorganism attachment. We found that the surface energy and bacterial viability on PDMS was comparable to isolated Citrus × aurantium and Populus × canescens leaf cuticles. Hence indicating that PDMS is the most suitable material for replica leaf surfaces. In summary, our experiments highlight the importance of considering the inherent material properties when selecting a replica leaf surface for phyllosphere microbiology studies. As demonstrated, a PDMS replica leaf offers a control surface that can be used for investigating microbe-microbe and microbe-plant interactions in the phyllosphere, which will enable mitigation strategies against pathogens to be developed.

Chromatic Bacteria - A Broad Host-Range Plasmid and Chromosomal Insertion Toolbox for Fluorescent Protein Expression in Bacteria.

Differential fluorescent labeling of bacteria has become instrumental for many aspects of microbiological research, such as the study of biofilm formation, bacterial individuality, evolution, and bacterial behavior in complex environments. We designed a variety of plasmids, each bearing one of eight unique, constitutively expressed fluorescent protein genes in conjunction with one of four different antibiotic resistance combinations. The fluorophores mTagBFP2, mTurquoise2, sGFP2, mClover3, sYFP2, mOrange2, mScarlet-I, and mCardinal, encoding for blue, cyan, green, green-yellow, yellow, orange, red, and far-red fluorescent proteins, respectively, were combined with selectable markers conferring tetracycline, gentamicin, kanamycin, and/or chloramphenicol resistance. These constructs were cloned into three different plasmid backbones: a broad host-range plasmid, a Tn5 transposon delivery plasmid, and a Tn7 transposon delivery plasmid. The utility of the plasmids and transposons was tested in bacteria from the phyla Actinobacteria, Proteobacteria, and Bacteroidetes. We were able to tag representatives from the phylum Proteobacteria at least via our Tn5 transposon delivery system. The present study enables labeling bacteria with a set of plasmids available to the community. One potential application of fluorescently-tagged bacterial species is the study of bacteria-bacteria, bacteria-host, and bacteria-environment interactions.

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.