Chromatic Bacteria v.2-A Himar1 Transposon-Based Delivery Vector to Extend the Host Range of a Toolbox to Fluorescently Tag Bacteria

A recent publication described the construction and utility of a comprehensive “Chromatic Bacteria” toolbox containing a set of genetic tools that allows for fluorescently tagging a variety of Proteobacteria. In an effort to expand the range of bacteria taggable with the Chromatic Bacteria toolbox, a series of Himar1 transposon vectors was constructed to mediate insertion of fluorescent protein and antibiotic resistant genes. The Himar1 transposon was chosen as it is known to function in a wide range of bacterial species. To test the suitability of the new Himar1 Chromatic Bacteria plasmid derivatives, conjugations were attempted on recently isolated non-model organisms. Although we were unsuccessful in delivering the plasmids into Gram-positive bacterial isolates, we successfully modified previously recalcitrant isolates to the first set of the Chromatic Bacteria toolbox, such as Sphingomonas sp. Leaf357 and Acidovorax sp. Leaf84. This manuscript reports on the currently available plasmids and transposition success in different bacteria

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

The Cytokinin Complex Associated With Rhodococcus fascians: Which Compounds Are Critical for Virulence?

Virulent strains of Rhodococcus fascians cause a range of disease symptoms, many of which can be mimicked by application of cytokinin. Both virulent and avirulent strains produce a complex of cytokinins, most of which can be derived from tRNA degradation. To test the three current hypotheses regarding the involvement of cytokinins as virulence determinants, we used PCR to detect specific genes, previously associated with a linear virulence plasmid, including two methyl transferase genes (mt1 and mt2) and fas4 (dimethyl transferase), of multiple strains of R. fascians. We inoculated Pisum sativum (pea) seeds with virulent and avirulent strains of R. fascians, monitored the plants over time and compared these to mock-inoculated controls. We used RT-qPCR to monitor the expression of mt1, mt2, and fas4 in inoculated tissues and LC-MS/MS to obtain a comprehensive picture of the cytokinin complement of inoculated cotyledons, roots and shoots over time. The presence and expression of mt1 and mt2 was associated with those strains of R. fascians classed as virulent, and not those classed as avirulent. Expression of mt1, mt2, and fas4 peaked at 9 days post-inoculation (dpi) in cotyledons and at 15 dpi in shoots and roots developed from seeds inoculated with virulent strain 602. Pea plants inoculated with virulent and avirulent strains of R. fascians both contained cytokinins likely to have been derived from tRNA turnover including the 2-methylthio cytokinins and cis-zeatin-derivatives. Along with the isopentenyladenine-type cytokinins, the levels of these compounds did not correlate with virulence. Only the novel 1- and 2-methylated isopentenyladenine cytokinins were uniquely associated with infection by the virulent strains and are, therefore, the likely causative factors of the disease symptoms

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

Driving factors of bacterial interactions and spatial patterns in the phyllosphere.

The phyllosphere includes all the aboveground parts of a plant that represent microbial habitats. Bacteria are among the microorganisms that dominate this environment, and the size of the global phyllosphere-associated bacterial population can alter nutrient cycling and other ecosystem functions. However, the factors driving the assembly of these communities are not well understood. As resources are limited and unevenly distributed in the phyllosphere, exploitative competition between bacteria is expected to be promoted. The overall aim of this thesis was to investigate factors that determine interactions and spatial arrangements between phyllosphere-associated bacteria, with an emphasis on resource competition. In order to visualise bacterial populations in the phyllosphere and to study species interactions in situ, a wide range of plasmids were developed –the “Chromatic bacteria toolbox”. These plasmids allowed the stable genetic modification of a range of phyllosphere-associated bacterial strains. The fluorescently-tagged bacterial strains were used to determine similarities in resource usage between them in vitro. These profiles were then compared to results obtained in an in silico approach based on genome-scale metabolic models to evaluate the predictability of resource usage in bacteria using genomic information, resulting in an accuracy of 70% and a 40% false negative rate. In silico predictions showed that metabolic diversity strongly correlated with phylogenetic relationships. Additionally, a resource overlap index based on the similarity of predicted resource requirements between species pairs was calculated. The relationship between resource overlap and phylogenetic relationship was investigated in pairwise interactions between representative members of the alphaproteobacterial families Methylobacteriaceae and Sphingomonadaceae. Spatial analyses strongly indicated that pairs of species co-aggregated regardless of phylogenetic or metabolic similarities. The underlying interactions were further investigated in vitro and in the phyllosphere. Results showed that coexistence was facilitated by positive effects for at least one competing species both in vitro and in situ. However, neither phylogeny nor resource overlap had an effect on the observed patterns for these bacterial groups in the phyllosphere. To investigate the effect of species interactions at a wider taxonomic range and at single-cell resolution, members of the Proteobacteria and Actinobacteria were competed against the model phyllosphere bacterium Pantoea eucalypti 299R. Here, the outcome of in vitro competition could be explained by the phylogenetic relationship and resource overlap between competitors. In contrast, in the phyllosphere, resource overlap but not phylogenetic relationship showed a negative relationship with the fitness of the focal species. In conclusion, the hypothesis “Spatial distribution and competitive interactions in the phyllosphere depend on the resource overlap between bacterial species” was only partially supported. Although resource overlap impacted on bacterial fitness in the phyllosphere, the strength of competition and potentially stabilising mechanisms also facilitated spatial aggregation and coexistence in the phyllosphere

Chromatic Bacteria v.2-A Himar1 Transposon-Based Delivery Vector to Extend the Host Range of a Toolbox to Fluorescently Tag Bacteria

A recent publication described the construction and utility of a comprehensive “Chromatic Bacteria” toolbox containing a set of genetic tools that allows for fluorescently tagging a variety of Proteobacteria. In an effort to expand the range of bacteria taggable with the Chromatic Bacteria toolbox, a series of Himar1 transposon vectors was constructed to mediate insertion of fluorescent protein and antibiotic resistant genes. The Himar1 transposon was chosen as it is known to function in a wide range of bacterial species. To test the suitability of the new Himar1 Chromatic Bacteria plasmid derivatives, conjugations were attempted on recently isolated non-model organisms. Although we were unsuccessful in delivering the plasmids into Gram-positive bacterial isolates, we successfully modified previously recalcitrant isolates to the first set of the Chromatic Bacteria toolbox, such as Sphingomonas sp. Leaf357 and Acidovorax sp. Leaf84. This manuscript reports on the currently available plasmids and transposition success in different bacteria

Fluorescent Protein Expression as a Proxy for Bacterial Fitness in a High-Throughput Assay

Bacterial growth is classically assessed by measuring the increases in optical density of pure cultures in shaken liquid media. Measuring growth using optical density has severe limitations when studying multistrain interactions, as it is not possible to measure the growth of individual strains within mixed cultures. Here, we demonstrated that constitutively expressed fluorescent proteins can be used to track the growth of individual strains in different liquid media. Fluorescence measurements were highly correlated with optical density measurements and cell counts. This allowed us to assess bacterial growth not only in pure cultures but also in mixed bacterial cultures and determine the impact of a competitor on a focal strain, thereby assessing relative fitness. Furthermore, we were able to track the growth of two different strains simultaneously by using fluorescent proteins with differential excitation and emission wavelengths. Bacterial densities measured by fluorescence yielded more consistent data between technical replicates than optical density measurements. Our setup employs fluorescence microplate readers that allow high throughput and replication. IMPORTANCE We expand on an important limitation of the concept of measuring bacterial growth, which is classically limited to one strain at a time. By adopting our approach, it is possible to measure the growth of several bacterial strains simultaneously with high temporal resolution and in a high-throughput manner. This is important to investigate bacterial interactions, such as competition and facilitation

RUN1 and REN1 Pyramiding in Grapevine (Vitis vinifera cv. Crimson Seedless) Displays an Improved Defense Response Leading to Enhanced Resistance to Powdery Mildew (Erysiphe necator)

Fungal pathogens are the cause of the most common diseases in grapevine and among them powdery mildew represents a major focus for disease management. Different strategies for introgression of resistance in grapevine are currently undertaken in breeding programs. For example, introgression of several resistance genes (R) from different sources for making it more durable and also strengthening the plant defense response. Taking this into account, we cross-pollinated P09-105/34, a grapevine plant carrying both RUN1 and REN1 pyramided loci of resistance to Erysiphe necator inherited from a pseudo-backcrossing scheme with Muscadinia rotundifolia and Vitis vinifera ‘Dzhandzhal Kara,’ respectively, with the susceptible commercial table grape cv. ‘Crimson Seedless.’ We developed RUN1REN1 resistant genotypes through conventional breeding and identified them by marker assisted selection. The characterization of defense response showed a highly effective defense mechanism against powdery mildew in these plants. Our results reveal that RUN1REN1 grapevine plants display a robust defense response against E. necator, leading to unsuccessful fungal establishment with low penetration rate and poor hypha development. This resistance mechanism includes reactive oxygen species production, callose accumulation, programmed cell death induction and mainly VvSTS36 and VvPEN1 gene activation. RUN1REN1 plants have a great potential as new table grape cultivars with durable complete resistance to E. necator, and are valuable germplasm to be included in grape breeding programs to continue pyramiding with other sources of resistance to grapevine diseases

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