Biology subjects

Uehling, J.

Publications and source records attributed to Uehling, J..

3 recordsLinked to original sources

bioRxiv2024

Inferring the composition of a mixed culture of natural microbial isolates by deep sequencing

Next generation sequencing has unlocked a wealth of genotype information for microbial populations, but phenotyping remains a bottleneck for exploiting this information, particularly for pathogens that are difficult to manipulate. Here, we establish a method for high-throughput phenotyping of mixed cultures, in which the pattern of naturally occurring single-nucleotide polymorphisms in each isolate is used as intrinsic barcodes which can be read out by sequencing. We demonstrate that our method can correctly deconvolute strain proportions in simulated mixed-strain pools. As an experimental test of our method, we perform whole genome sequencing of 66 natural isolates of the thermally dimorphic pathogenic fungus Coccidioides posadasii and infer the strain compositions for large mixed pools of these strains after competition at 37{degrees}C and room temperature. We validate the results of these selection experiments by recapitulating the temperature-specific enrichment results in smaller pools. Additionally, we demonstrate that strain fitness estimated by our method can be used as a quantitative trait for genome-wide association studies. We anticipate that our method will be broadly applicable to natural populations of microbes and allow high-throughput phenotyping to match the rate of genomic data acquisition. Author summaryThe diversity of the gene pool in natural populations encodes a wealth of information about its molecular biology. This is an especially valuable resource for non-model organisms, from humans to many microbial pathogens, lacking traditional genetic approaches. An effective method for reading out this population genetic information is a genome wide association study (GWAS) which searches for genotypes correlated with a phenotype of interest. With the advent of cheap genotyping, high throughput phenotyping is the primary bottleneck for GWAS, particularly for microbes that are difficult to manipulate. Here, we take advantage of the fact that the naturally occurring genetic variation within each individual strain can be used as an intrinsic barcode, which can be used to read out relative abundance of each strain as a quantitative phenotype from a mixed culture. Coccidioides posadasii, the causative agent of Valley Fever, is a fungal pathogen that must be manipulated under biosafety level 3 conditions, precluding many high-throughput phenotyping approaches. We apply our method to pooled competitions of C. posadasii strains at environmental and host temperatures. We identify robustly growing and temperature-sensitive strains, confirm these inferences in validation pooled growth experiments, and successfully demonstrate their use in GWAS.

genetics↗

bioRxiv2023

Prevalence and diversity of TAL effector-like proteins in fungal endosymbiotic Mycetohabitans spp.

Endofungal Mycetohabitans (formerly Burkholderia) spp. rely on a type III secretion system to deliver mostly unidentified effector proteins when colonizing their host fungus, Rhizopus microsporus. The one known secreted effector family from Mycetohabitans consists of homologs of transcription activator-like (TAL) effectors, which are used by plant pathogenic Xanthomonas and Ralstonia spp. to activate host genes that promote disease. These Burkholderia TAL-like (Btl) proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of two Mycetohabitans strains revealed that the single Btl in one M. endofungorum strain enhances fungal membrane stress tolerance, while others in a M. rhizoxinica strain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nine Mycetohabitans spp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal-bacterial metagenomes. We show that btl genes are present across diverse Mycetohabitans strains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested that Mycetohabitans might contain more species than previously recognized. Within our data set, Btl proteins were more conserved across Mycetohabitans rhizoxinica strains than across Mycetohabitans endofungorum, but there was also evidence of greater overall strain diversity within the latter clade. Overall, the results suggest that Btl proteins contribute to bacterial-fungal symbioses in myriad ways. Impact StatementMany Mucoromycota fungi harbor endosymbiotic bacteria, including Rhizopus spp. that are food fermenters and pathogens of plants and immunocompromised people. Rhizopus microsporus has endofungal Mycetohabitans (formerly Burkholderia) spp. that deploy proteins related to DNA-binding transcription activator-like effectors of plant pathogens, which enter plant nuclei and activate disease susceptibility genes. By sequencing isolated bacteria and mining fungal holobiont sequences, we found Btl proteins in diverse Mycetohabitans strains, varying in predicted DNA binding specificity, thus in potential host targets. Btl proteins were more conserved within M. rhizoxinica, suggesting distinctions among the two named species. The results in the context of phenotypic differences observed in other studies suggest that Btl proteins contribute to symbiosis in diverse ways, providing insight into effector evolution and arguing for functional characterization of additional Btl proteins to understand establishment and maintenance of these important fungal-bacterial interactions.

microbiology↗

bioRxiv2022

Developmental stage-dependent effects of perceived predation risk on physiology and fledging success of tree swallows (Tachycineta bicolor)

The risk of predation directly affects physiology, behavior, and fitness of wild birds. Social interactions with conspecifics may affect how individuals respond to stressors such as predators. Strong social connections could help individuals recover from a stressful experience; however, competitive interactions also have the potential to exacerbate stress. Few studies have investigated the interaction between environmental stressors and the social landscape in wild bird populations. Here, we experimentally simulated predation attempts on breeding female tree swallows (Tachicyneta bicolor). At the same time, we manipulated female breast plumage color, a key social signal. Simulated predation events on tree swallows negatively affected their nestlings condition, telomere lengths, and fledging success. However, the effects of experimental manipulations were timing-dependent: simulated predation during the early nestling period was more detrimental than "predation" during incubation. Contrary to our expectations, manipulation of the social environment did not affect the response of tree swallows to simulated predation. However, manipulating female plumage during the nestling period did affect nestling size, indicating an effect of the social environment on reproductive success. Our data demonstrate that transient stressors on breeding female birds can have carry-over effects on their nestlings, some of which may be long-lasting.

ecology↗