Biology subjects

Schlechter, R. O.

Publications and source records attributed to Schlechter, R. O..

10 recordsLinked to original sources

bioRxiv2025

Comparative genomics of epiphytic and endophytic bacterial culture collections from Arabidopsis thaliana in Otautahi (Christchurch), Aotearoa New Zealand

Bacterial culture collections are essential resources for exploring the diversity of microorganisms and their interactions with each other and their hosts. Here, we report on the sequencing of the first 129 bacterial isolates, representing 34 genera, from a culture collection of more than 600 bacterial strains originally isolated from leaves of a naturalised Arabidopsis thaliana population from [O]tautahi (Christchurch), Aotearoa New Zealand. Epiphytic (leaf surface), and endophytic (apoplastic) bacteria were isolated separately from the same leaves, providing complementary insights into both compartments. The recovered isolates encompass the dominant taxa typically associated with the Arabidopsis phyllosphere, including Pseudomonas, Sphingomonas, Methylobacterium, and Flavobacterium. Their full genome assemblies (BUSCO average completeness > 99%, checkM average completeness > 97% and average contamination < 1%) were analysed and compared to assess genomic features across epiphytic and endophytic lineages. While the epiphytic and endophytic strain collections did not show large genomic differences, certain functional categories differ, such as terpene biosynthesis and biofilm formation being enriched in epiphytic strains, while arginine biosynthesis and carbohydrate degradation were associated with endophytic strains. These data provide a genomic foundation for future experimental work on leaf-associated microbial ecology and plant-microbe interactions. To our knowledge, this is the first Arabidopsis leaf culture collection established from a Southern Hemisphere source.

microbiology↗

bioRxiv2025

The alkane 1-monooxigenase gene alkB of Pseudomonas sp. FF2 is upregulated during colonisation of Arabidopsis thaliana leaves

Bacteria on leaf surfaces encounter highly variable access to nutrients and water. This oligotrophic environment is partly due to the presence of cuticular waxes that render the leaf surface hydrophobic, reducing the leaching of nutrients and water loss from inside the leaf. Bacteria have evolved adaptations to survive under these conditions. While alkB, an alkane hydroxylase gene, is widely prevalent in leaf-associated bacteria, its role and activity is unclear. Here, we developed a bioreporter in Pseudomonas sp. FF2 (PFF2) to investigate alkB promoter activity in diesel and on Arabidopsis thaliana leaves. In general, alkB promoter activity is highly heterogeneous, with a subpopulation exhibiting strong activation, suggesting bet-hedging in alkane metabolism. In planta, the promoter remained active over the course of seven days, indicating constant access of alkanes over time. Single-cell fluorescence intensity was heterogeneous, reflecting differences in microhabitats on the leaf surface or bet-hedging. While our results support a role of alkB in bacterial adaptation to the phyllosphere, direct evidence of cuticular wax degradation is missing. Future studies should trace the incorporation of plant-derived aliphatic compounds to elucidate the potential use of alkanes and other aliphatic compounds as resources for bacteria in the leaf environment.

microbiology↗

bioRxiv2025

Correlation of in vitro biofilm formation capacity with persistence of antibiotic-resistant Escherichia coli on gnotobiotic lamb's lettuce

Bacterial contamination of fresh produce is a growing concern for food safety, as apart from human pathogens, antibiotic-resistant bacteria can persist on fresh leafy produce. A prominent persistence trait in bacteria is biofilm formation, as it provides increased tolerance to stressful conditions. We screened a comprehensive collection of 174 antibiotic-susceptible and -resistant Escherichia coli originating from fresh leafy produce and its production environment. We tested the ability of these strains to produce biofilms, ranging from none or weak to extreme biofilm-forming bacteria. Next, we tested the ability of selected antibiotic-resistant isolates to colonize gnotobiotic lambs lettuce (Valerianella locusta) plants. We hypothesized that a higher in vitro biofilm formation capacity correlates with increased colonization of gnotobiotic plant leaves. Despite a marked difference in the ability to form in vitro biofilms for a number of E. coli strains, in vitro biofilm formation was not associated with increased survival on gnotobiotic V. locusta leaf surfaces. However, all tested strains persisted for at least 21 days, highlighting potential food safety risks through unwanted ingestion of resistant bacteria. Population densities of biofilm-forming E. coli exhibited a complex pattern, with subpopulations more successful in colonizing gnotobiotic V. locusta leaves. These findings emphasise the complex behaviour of antibiotic-resistant bacteria on leaf surfaces and their implications to human safety. ImportanceEach raw food contains a collection of microorganisms, including bacteria. This is of special importance for fresh produce such as leafy salads or herbs, as these foods are usually consumed raw or after minimal processing, whereby higher loads of living bacteria are ingested than with a food that is heated before consumption. A common bacterial lifestyle involves living in large groups embedded in secreted protective substances. Such bacterial assemblies, so-called biofilms, confer high persistence and resistance of bacteria to external harsh conditions. In our research we investigated whether stronger in vitro biofilm formation of antibiotic-resistant Escherichia coli correlates with a better survival on lambs lettuce leaves. Although no clear correlation was observed between biofilm formation capacity and population density on the salad, all tested isolates could survive for at least three weeks with no significant decline over time, highlighting a potential food safety risk independently of in vitro biofilm formation.

microbiology↗

bioRxiv2023

Bacterial community complexity in the phyllosphere penalises specialists over generalists

The leaf surface, i.e. the phylloplane, is an oligotrophic and heterogeneous environment due to its topography and uneven distribution of resources. Despite being a limiting environment, leaves host bacteria that are abundant and establish spatially-structured communities. However, factors that drive spatial distribution patterns are not well understood. Since leaf-associated bacteria can have beneficial effects to their host, understanding the rules of the community assembly can lead to novel strategies for crop protection. To investigate changes in population density and spatial distribution of bacteria in synthetic communities, we examined the behaviour of two prevalent bacterial groups in the Arabidopsis thaliana leaf microbiota: Methylobacterium spp. (specialists) and Sphingomonas spp. (generalists). We designed synthetic communities composed of two (S2) or three strains (S3) in a full factorial design and tested whether density and spatial structure of communities in S3 can be explained by pairwise comparisons in S2. Our results showed that specialists are more susceptible to changes in population densities and spatial distribution patterns than generalists, with lower densities and aggregation patterns when a specialist is in S3 than in S2. Additionally, pairwise comparisons were not sufficient to explain the observed patterns in S3, suggesting that higher-order interactions play a role in the resulting structure of complex communities at the micrometre scale.

microbiology↗

bioRxiv2023

Hitching a ride in the phyllosphere: Surfactant production of Pseudomonas spp. causes co-swarming of Pantoea eucalypti 299R

Here we demonstrate the beneficial effect of surfactant-producing Pseudomonads on the phyllosphere model strain Pantoea eucalypti 299R. To do so, we conducted a series of experiments in environments of increasing complexity. P. eucalypti 299R and Pseudomonas sp. FF1 (Pff1) or P. eucalypti 299R and the surfactant-deficient mutant P. sp. FF1::{Delta}viscB (Pff1{Delta}viscB) were co-inoculated in broth, on swarming agar plates, and on plants. In broth, there were no differences in the growth dynamics of P. eucalypti 299R when growing in the presence of Pff1 or Pff1{Delta}viscB. By contrast, on swarming agar plates, P. eucalypti was able to co-swarm with Pff1. Co-swarming led to a significant increase in the area colonized and, consequently, a boost in total biomass when compared to P. eucalypti growing with Pff1{Delta}viscB or in monoculture. Finally in planta, there were no significant effects on the population density of P. eucalypti 299R during co-colonization of Arabidopsis thaliana. However, utilizing the single cell bioreporter for reproductive success (CUSPER), we found a temporally distinct beneficial effect of Pff1 on co-inoculated P. eucalypti 299R subpopulations that did not occur in presence of Pff1{Delta}viscB. This led us to formulate a model for the positive effect of surfactant production during leaf colonization. To generalize our results, we tested the effect of three additional surfactant-producing Pseudomonads and their respective surfactant knockout mutants on P. eucalypti 299R on swarming agar. Resulting in similar co-swarming patterns in P. eucalypti 299R and showing that this organism is able to take advantage of competitors during surface colonization. Our results indicate that surfactant-dependent co-motility might be common during leaf colonization and add yet another facet to the already manyfold roles of surfactants.

microbiology↗

bioRxiv2022

The influence of resource overlap on bacterial reproductive success in the phyllosphere

The phyllosphere is densely colonised by rich 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 leafs 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. Surprisingly, 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 provides a framework to predict competition outcomes.

microbiology↗

bioRxiv2021

Chromatic bacteria version 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 Himar1 transposon vector was constructed to mediate insertion of fluorescent protein and abiotic resistant genes. The Himar1 transposon was chosen as it is known to function in a wide range of bacteria. To test the use of the Himar1 derivative of the Chromatic Bacteria plasmid series for fluorescently tagging bacteria, recently isolated non-model organisms were conjugated with the Himar1 transposon plasmids. We were unsuccessful in delivering the plasmids into Gram positive bacterial isolates but were able to successfully integrate the transposon in isolates that we were previously unable to modify such as Sphingomonas sp. Leaf357 and Acidovorax sp. Leaf84. This manuscript reports on the currently available plasmids and transposition success in different bacteria. The manuscript will be updated regularly until a full plasmid series is available and a large range of recipient strains was tested.

microbiology↗

bioRxiv2020

Constitutively expressed fluorescent proteins allow to track bacterial growth and to determine relative fitness of bacteria in mixed cultures

Bacterial growth is classically assessed by measuring the increase 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 competitors 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 fluorescent microplate readers that allow for high throughput and replication. ImportanceWe expand on an important limitation of the concept of measuring bacterial growth which is classically limited to one strain at a time. By adopting this approach, it is possible to measure growth of several bacterial strains simultaneously in high temporal resolution and in a high throughput manner. This is important to investigate bacterial interactions such as competition and facilitation.

microbiology↗

bioRxiv2020

Litterbox - A gnotobiotic zeolite-clay system to investigate Arabidopsis-microbe interactions

Plants are colonised by millions of microorganisms representing thousands of species with varying effects on plant growth and health. The microbial communities found on plants are compositionally consistent and their overall positive effect on the plant is well known. However, the effects of individual microbiota members on plant hosts and vice versa, as well as the underlying mechanisms remain largely unknown. Here, we describe Litterbox, a highly controlled system to investigate plant-microbe interactions. Plants were grown gnotobiotically on zeolite-clay, an excellent soil replacement that retains enough moisture to avoid subsequent watering. Plants grown on zeolite phenotypically resemble plants grown under environmental conditions. Further, bacterial densities on leaves in the Litterbox system resembled those in temperate environments. A PDMS sheet was used to cover the zeolite, thereby significantly lowering the bacterial load in the zeolite and rhizosphere. This reduced the likelihood of potential systemic responses in leaves induced by microbial rhizosphere colonisation. We present results of example experiments studying the transcriptional responses of leaves to defined microbiota members and the spatial distribution of bacteria on leaves. We anticipate that this versatile and affordable plant growth system will promote microbiota research and help in elucidating plant-microbe interactions and their underlying mechanisms.

plant biology↗

bioRxiv2018

Chromatic Bacteria - A broad host-range plasmid and chromosomal insertion toolbox for fluorescent protein expression in bacteria

Differential fluorescent labelling of bacteria has become instrumental for many aspects of microbiological research, such as the study of biofilm formation, bacterial individuality, evolution, and bacterial behaviour 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 here constructed plasmids are available to the community and provide a valuable tool to investigate bacteria-bacteria, bacteria-host, and bacteria-environmental interactions.

microbiology↗