Biology subjects

van der Meer, J. R.

Publications and source records attributed to van der Meer, J. R..

29 records · Page 2Linked to original sources

bioRxiv2021

Reproducible Propagation of Species-Rich Soil Microbiomes Suggests Robust Underlying Deterministic Principles of Community Formation

Microbiomes are typically characterised by high species diversity but it is poorly understood how such system-level complexity can be generated and propagated. Here, we used soils as a relevant model to study microbiome development. Despite inherent stochastic variation in manipulating species-rich communities, both laboratory-mixed medium complexity (21 soil bacterial isolates in equal proportions) and high-diversity natural top-soil communities followed highly reproducible succession paths, maintaining distinct soil microbiome signatures. Development trajectories and compositional states were different for communities propagated in soils than in liquid suspension. Microbiome states were maintained over multiple renewed growth cycles but could be diverged by short-term pollutant exposure. The different but robust trajectories demonstrated that deterministic taxa-inherent characteristics underlie reproducible development and self-organized complexity of soil microbiomes within their environmental boundary conditions. Our findings also have direct implications for potential strategies to achieve controlled restoration of desertified land. TEASERSpecies-rich soil microbiomes grow and propagate reproducibly despite inherent stochastic complexity, paving the way for soil restoration.

microbiology↗

bioRxiv2020

An analog to digital converter controls bistable transfer competence of a widespread integrative and conjugative element

Conjugative transfer of the integrative and conjugative element ICEclc in Pseudomonas requires development of a transfer competence state in stationary phase, which arises only in 3-5% of individual cells. The mechanisms controlling this bistable switch between non-active and transfer competent cells have long remained enigmatic. Using a variety of genetic tools and epistasis experiments in P. putida, we uncovered an upstream cascade of three consecutive transcription factor-nodes, which controls transfer competence initiation. Initiation activates a feedback loop, which stochastic modeling and experimental data demonstrated acts as a scalable converter of unimodal input to bistable output. The feedback loop further enables prolonged production of a transcription factor that ensures downstream transfer competence formation in activated cells. Phylogenetic analyses showed that the ICEclc regulatory factors are widespread among Gamma- and Beta-proteobacteria, highlighting its evolutionary conservation and prime importance to control the behaviour of this wide family of conjugative elements.

genomics↗

bioRxiv2019

A four-step regulatory cascade controls bistable transfer competence development of the integrative and conjugative element ICEclc in Pseudomonas

Genetic bistability controls different phenotypic programs in defined subpopulations of genetically identical bacteria. Conjugative transfer of the integrative and conjugative element ICEclc in Pseudomonas requires development of a transfer competence state in stationary phase, but this state arises only in 3-5% of individual cells. The mechanisms controlling and underlying the bistable switch between non-active and transfer competence cells have long remained enigmatic. Using a variety of genetic tools combined with stochastic modeling, we characterize here the factors and overall network architecture controlling bistable ICEclc activation of transfer competence. Two new key regulators (BisR and BisDC) were uncovered, that link the hierarchical cascade of ICEclc transfer competence activation to in total four regulatory nodes. The final activator complex named BisDC drives a positive feedback on its own transcription, and directly controls the "late" ICE promoters for excision and transfer. Stochastic mathematical modeling conceptually explained the arisal and maintenance of bistability by the feedback loop, and demonstrated its importance to guarantee consistent prolonged downstream output in activated cells. A minimized gene set allowing controllable bistable output in a Pseudomonas putida in absence of the ICEclc largely confirmed model predictions. Phylogenetic analyses further showed that the two new ICEclc regulatory factors are widespread among putative ICEs found in Gamma- and Beta- proteobacteria, highlighting the conceptual importance of our findings for the behaviour of this wide family of conjugative elements. Author summaryIntegrative and conjugative elements (ICEs) are mobile genetic elements present in virtually every bacterial species, which can confer adaptive functions to their host, such as antibiotic resistance or xenometabolic pathways. Integrated ICEs maintain by replication along with the genome of their bacterial host, but in order to transfer, the ICE excises and conjugates into a new recipient cell. Single-cell studies on a unique but widely representative ICE model from Pseudomonas (ICEclc) showed that transfer only occurs from a small dedicated subpopulation of cells that arises during stationary phase conditions. This bistable subpopulation differentiation is highly significant for ICE behaviour and fitness, but how it is regulated has remained largely unknown. The present work unveiled the architecture of the ICEclc transfer competence regulation, and showed its widespread occurrence among ICEs of the same family. Stochastic mathematical modeling explained how bistability is generated and maintained, prolonging the capacity of stationary phase cells to complete all stages of ICE activation.

microbiology↗

bioRxiv2019

Acetobacteraceae in the honey bee gut comprise two distant clades with diverging metabolism and ecological niches

Various bacteria of the family Acetobacteraceae are associated with the gut environment of insects. Honey bees harbor two distinct Acetobacteraceae in their gut, Alpha2.1 and Alpha2.2. While Alpha2.1 seems to be a gut specialist, Alpha2.2 is also found in the diet (e.g. royal jelly), the hypopharyngeal glands, and the larvae of honey bees. Here, we combined amplicon and genome sequencing to better understand functional differences associated with the ecology of Alpha2.1 and Alpha2.2. We find that the two phylotypes are differentially distributed along the worker and queen bee gut. Phylogenetic analysis shows that Alpha2.2 is nested within the acetic acid bacteria and consists of two separate sub-lineages, whereas Alpha2.1 belongs to a basal lineage with an unusual GC content for Acetobacteraceae. Gene content analysis revealed major differences in the central carbon and respiratory metabolism between the two phylotypes. While Alpha2.2 encodes two periplasmic dehydrogenases to carry out oxidative fermentation, Alpha2.1 lacks this capability, but instead harbors a diverse set of cytoplasmic dehydrogenases. These differences are accompanied by the loss of the TCA cycle in Alpha2.2, but not in Alpha2.1. We speculate that Alpha2.2 has specialized for fast-resource utilization through incomplete carbohydrate oxidation, giving it an advantage in sugar-rich environments such as royal jelly. On the contrary, the broader metabolic range of Alpha2.1 may provide an advantage in the worker bee hindgut, where competition with other bacteria and flexibility in resource utilization may be relevant for persistence. Our results show that bacteria belonging to the same family may utilize vastly different strategies to colonize niches associated with the animal gut.

genomics↗

bioRxiv2019

Close-Range Interactions Favor Growth in Random-Paired Extracted Soil Bacteria

Species interactions at the cellular level are thought to govern the formation and functioning of microbial communities, but direct measurements of species interactions are difficult to perform between the hundreds of different species that constitute most microbial ecosystems. We developed a methodology to examine interactive growth of random cell pairs encapsulated inside 40-70 {micro}m diameter agarose beads. We focused on a sandy soil as a test microbial ecosystem. By using gentle washing procedures, we detached microbial cells from sand and encapsulated them either in the absence or presence of pure culture inoculants. We then tested whether inoculants had on average positive or negative effects on the growth of resident community members depending on the growth substrate. Surprisingly, all the tested inoculants (including Pseudomonas veronii 1YdBTEX2, Pseudomonas putida F1, Pseudomonas protegens CHA0 and Escherichia coli MG1655) stimulated the growth of 40-80 percent of sand-derived cells when grown pair-wise in close proximity (i.e., within the same bead). This was true essentially irrespective of the growth substrate. Beneficial inoculant-sand cell partnerships resulted in up to 100-fold increase in productivity of the sand cell partner and up to 100-fold decrease in that of the inoculant. However, the maximum productivity attained by inoculant-sand cell partners within beads did not surpass that of inoculants alone. Further surprisingly, random pairs of sand cells encapsulated within the same bead also benefited growth in comparison to individual sand cells in a mutualistic manner (i.e., productivity when grown together was greater than the sum of individual productivities), but less than productivities observed in partnerships with the tested inoculants. This suggests that partnerships between inoculants and sand cells are not so much characterized by competition for substrate as by carbon loss through metabolite provision of the inoculant to sand cells (competitive exploitation).

microbiology↗

bioRxiv2019

Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol

Bacterial periplasmic-binding proteins have been acclaimed as general biosensing platform, but their range of natural ligands is too limited for optimal development of chemical compound detection. Computational redesign of the ligand-binding pocket of periplasmic-binding proteins may yield variants with new properties, but, despite earlier claims, genuine changes of specificity to non-natural ligands have so far not been achieved. In order to better understand the reasons of such limited success, we revisited here the Escherichia coli RbsB ribose-binding protein, aiming to achieve perceptible transition from ribose to structurally related chemical ligands 1,3-cyclohexanediol and cyclohexanol. Combinations of mutations were computationally predicted for nine residues in the RbsB binding pocket, then synthesized and tested in an E. coli reporter chassis. Two million variants were screened in a microcolony-in-bead fluorescence-assisted sorting procedure, which yielded six mutants no longer responsive to ribose but with 1.2-1.5 times induction in presence of 1 mM 1,3-cyclohexanediol, one of which responded to cyclohexanol as well. Isothermal microcalorimetry confirmed 1,3-cyclohexanediol binding, although only two mutant proteins were sufficiently stable upon purification. Circular dichroism spectroscopy indicated discernable structural differences between these two mutant proteins and wild-type RbsB. This and further quantification of periplasmic-space abundance suggested most mutants to be prone to misfolding and/or with defects in translocation compared to wild-type. Our results thus affirm that computational design and library screening can yield RbsB mutants with recognition of non-natural but structurally similar ligands. The inherent arisal of protein instability or misfolding concomitant with designed altered ligand-binding pockets should be overcome by new experimental strategies or by improved future protein design algorithms.

synthetic biology↗

bioRxiv2018

Genomic changes underlying host specialization in the bee gut symbiont Lactobacillus Firm5

Bacteria that engage in longstanding associations with particular hosts are expected to evolve host-specific adaptations that limit their capacity to thrive in other environments. Consistent with this, many gut symbionts seem to have a limited host range, based on community profiling and phylogenomics. However, few studies have experimentally investigated host specialization of gut symbionts and underlying mechanisms have largely remained elusive. Here, we studied host specialization of a dominant gut symbiont of social bees, Lactobacillus Firm5. We show that Firm5 strains isolated from honey bees and bumble bees separate into deep-branching phylogenetic lineages. Despite their divergent evolution, colonization experiments show that bumble bee strains are capable of colonizing the honey bee gut. However, they were less successful than honey bee strains, and competition with honey bee strains completely abolished their colonization, whereas honey bee strains were able to coexist. This suggests that both host selection and interbacterial competition play important roles for host specialization. Using comparative genomics of 27 Firm5 isolates, we identified candidate genomic changes underlying host specialization. We found that honey bee strains harbored a larger and more diverse gene pool of carbohydrate-related functions than bumble bee strains. As dietary-derived carbohydrates are the main energy source for strains of the Firm5 phylotype, the metabolic flexibility of honey bee strains may give these bacteria a competitive advantage over bumble bee strains in colonizing the gut niche and hence contribute to host specialization.

microbiology↗

bioRxiv2018

Transient Replication in Specialized Cells Favors Conjugative Transfer of a Selfish DNA Element

Bacterial evolution is driven to a large extent by horizontal gene transfer (HGT) - the processes that distribute genetic material between species rather than by vertical descent. HGT is mostly mediated by an assortment of different selfish DNA elements, several of which have been characterized in great molecular detail. In contrast, very little is known on adaptive features optimizing horizontal fitness. By using single DNA molecule detection and time-lapse microscopy, we analyze here the fate of an integrative and conjugative element (ICE) in individual cells of the bacterium Pseudomonas putida. We uncover how the ICE excises and irregularly replicates, exclusively in a sub-set of specialized host cells. As postulated, ICE replication is dependent on its origin of transfer and its DNA relaxase. Rather than being required for ICE maintenance, however, we find that ICE replication serves more effective conjugation to recipient cells, providing selectable benefit to its horizontal transfer.

microbiology↗

bioRxiv2018

Probing chemotaxis activity in Escherichia coli using fluorescent protein fusions

Chemotaxis is based on ligand-receptor interactions that are transmitted via protein-protein interactions to the flagellar motors. Ligand-receptor interactions in chemotaxis can be deployed for the development of rapid biosensor assays, but there is no consensus as to what the best readout of such assays would have to be. Here we explore two potential fluorescent readouts of chemotactically active Escherichia coli cells. In the first, we probed interactions between the chemotaxis signaling proteins CheY and CheZ by fusing them individually with non-fluorescent parts of a split-Green Fluorescent Protein. Wild-type chemotactic cells but not mutants lacking the CheA kinase produced distinguishable fluorescence foci, two-thirds of which localize at the cell poles with the chemoreceptors and one-third at motor complexes. Cells expressing fusion proteins only were attracted to serine sources, demonstrating measurable functional interactions between CheY~P and CheZ. Fluorescent foci based on stable split-eGFP displayed small fluctuations in cells exposed to attractant or repellent, but those based on an unstable ASV-tagged eGFP showed a higher dynamic behaviour both in the foci intensity changes and the number of foci per cell. For the second readout, we expressed the pH-sensitive fluorophore pHluorin in the cyto- and periplasm of chemotactically active E. coli. Calibrations of pHluorin fluorescence as a function of pH demonstrated that cells accumulating near a chemo-attractant temporally increase cytoplasmic pH while decreasing periplasmic pH. Both readouts thus show promise as proxies for chemotaxis activity, but will have to be further optimized in order to deliver practical biosensor assays.\n\nIMPORTANCEBacterial chemotaxis may be deployed for future biosensing purposes with the advantages of its chemoreceptor ligand-specificity and its minute-scale response time. On the downside, chemotaxis is ephemeral and more difficult to quantitatively read out than, e.g., reporter gene expression. It is thus important to investigate different alternative ways to interrogate chemotactic response of cells. Here we gauge the possibilities to measure dynamic response in the Escherichia coli chemotaxis pathway resulting from phosphorylated CheY-CheZ interactions by using (unstable) split-fluorescent proteins. We further test whether pH differences between cyto- and periplasm as a result of chemotactic activity can be measured with help of pH-sensitive fluorescent proteins. Our results show that both approaches conceptually function, but will need further improvement in terms of detection and assay types to be practical for biosensing.

microbiology↗

bioRxiv2018

Heterologous Expression of Pseudomonas putida Methyl-Accepting Chemotaxis Proteins Yields Escherichia coli Chemotactic to Aromatic Compounds

Escherichia coli, commonly used in chemotaxis studies, is attracted mostly by amino acids, sugars and peptides. We envisioned modifying chemotaxis specificity of E. coli by expressing heterologous chemoreceptors from Pseudomonas putida enabling attraction either to toluene or benzoate. The mcpT gene encoding the type 40H methyl-accepting chemoreceptor for toluene from Pseudomonas putida MT53 and the pcaY gene for the type 40H receptor for benzoate and related molecules from P. putida F1 were expressed from the trg promoter on a plasmid in motile wild-type E. coli MG1655. E. coli cells expressing McpT accumulated in chemoattraction assays to sources with 60-200 M toluene; less strongly than the response to 100 M serine, but statistically significantly stronger than to sources without any added attractant. An McpT-mCherry fusion protein was detectably expressed in E. coli and yielding weak but distinguishable membrane and polar foci in 1% of cells. E. coli expressing PcaY showed weak attraction to 0.1-1 mM benzoate but 50-70% of cells localized the PcaY-mCherry fusion to their membrane. We conclude that implementing heterologous receptors in the E. coli chemotaxis network is possible and, upon improvement of the compatibility of the type 40H chemoreceptors, may bear interest for biosensing.\n\nIMPORTANCEBacterial chemotaxis might be harnessed for the development of rapid biosensors, in which chemical availability is deduced from cell accumulation to chemoattractants over time. Chemotaxis of Escherichia coli has been well-studied, but the bacterium is not attracted to chemicals of environmental concern, such as aromatic solvents. We show here that heterologous chemoreceptors for aromatic compounds from Pseudomonas putida at least partly functionally complement the E. coli chemotaxis network, yielding cells attracted to toluene or benzoate. Complementation was still inferior to native chemoattractants like serine, but our study demonstrates the potential for obtaining selective sensing for aromatic compounds in E. coli.

microbiology↗

bioRxiv2017

Adaptation to chronic malnutrition leads to reduced dependence on microbiota in Drosophila

Numerous studies have shown that animal nutrition is tightly linked to gut microbiota, especially under nutritional stress. In Drosophila, microbiota are known to promote juvenile growth, development and survival on poor diets, mainly through enhanced digestion leading to changes in hormonal signaling. Here we show that this reliance on microbiota is greatly reduced in replicated Drosophila populations that adapted to a poor larval diet in the course of over 170 generations of experimental evolution. Protein and polysaccharide digestion in these malnutrition-adapted populations became much less dependent on colonization with microbiota. This was accompanied by changes in at least some targets of dFOXO transcription factor, which is a key regulator of cell growth and survival. Our study suggests that some metazoans have retained the evolutionary potential to adapt their physiology such that association with microbiota may become optional rather than essential.

evolutionary biology↗