Biology subjects

Welch, R. D.

Publications and source records attributed to Welch, R. D..

5 recordsLinked to original sources

bioRxiv2022

Phenotypic similarity is a measure of functional redundancy within homologous gene families

Robustness to the impact of mutation can mitigate phenotypes that have the potential to inform gene function. This robustness is often encoded into the genome through gene duplication, among other mechanisms. Duplication is a source of structurally similar genes that can retain some functional overlap as they diverge, and as such contribute to functional redundancy in the face of mutation. While redundancies have been explored in groups of two or three paralogs by generating double and triple mutants, it is unclear to what extent larger homologous gene families contribute to robustness through functional redundancy. Here, we used phenotypic similarity as an indicator of functional redundancy to explore the extent to which homologous gene families contribute to redundancy in function. We hypothesize that, since functional redundancy is more likely to occur within gene families where genes are structurally similar, mutant strains within the same gene families would be more phenotypically similar. We generated 265 single-gene disruptions in four homologous gene families of Myxococcus xanthus, used time-lapse microscopy to generate time series of multicellular development, and developed an image analysis pipeline to compare phenotypic characteristics among different strains. We show that mutant strains cluster by gene family in the phenotypic feature space with principal component analysis, demonstrating that families of homologs can contain extensive functional redundancy networks.

systems biology↗

bioRxiv2021

Unraveling a Bacterial Starvation Response Through the Direct Targets of a Starvation-Induced Transcriptional Activator

Organisms frequently encounter environments with nutrient shortages and their survival depends on changes in physiology and the ability to conserve resources. In bacteria, many physiological changes associated with starvation have been identified, but the underlying genetic components and regulatory networks that direct these physiological changes are often poorly defined. Here, we aimed to better define the gene regulatory networks that mediate the starvation response in Myxococcus xanthus, a bacterium that copes with starvation by producing fruiting bodies filled with dormant and stress-resistant spores. We focused on the direct promoter/gene targets of Nla28, a transcriptional activator/enhancer binding protein (EBP) that is important for early rounds of gene expression following starvation. Using expression profiling to identify genes that are downregulated in nla28 mutant cells and bioinformatics to identify the putative promoters of these genes, 12 potential promoter targets (37 genes) of Nla28 were identified. The results of in vitro promoter binding assays, coupled with in vitro and in vivo mutational analyses, suggested that the 12 promoters are in vivo targets of Nla28 and that Nla28 dimers use tandem, imperfect repeats of an 8-bp sequence for binding. Interestingly, nine of the Nla28 target promoters are intragenic, located in the protein coding sequence of an upstream gene or in the protein coding sequence of one gene within an operon (internal promoters). Based on mutational analyses, we concluded that the 12 Nla28 target loci contain at least one gene important for production of stress-resistant spores following starvation. Most of these loci contain genes predicted to be involved in regulatory or defense-related functions. Using the consensus Nla28 binding sequence, followed by bioinformatics and expression profiling, 58 additional promoters and 102 genes were tagged as potential Nla28 targets. Among these putative Nla28 targets, functions such as regulatory, metabolic and cell envelope biogenesis were commonly assigned to genes.

microbiology↗

bioRxiv2021

Quantification of Myxococcus xanthus Aggregation and Rippling Behaviors: Deep-learning Transformation of Phase-contrast into Fluorescence Microscopy Images

Myxococcus xanthus bacteria are a model system for understanding pattern formation and collective cell behaviors. When starving, cells aggregate into fruiting bodies to form metabolically inert spores. During predation, cells self-organize into traveling cell-density waves termed ripples. Both phase-contrast and fluorescence microscopy are used to observe these patterns but each has its limitations. Phase-contrast images have higher contrast, but the resulting image intensities lose their correlation with cell density. The intensities of fluorescence microscopy images, on the other hand, are well correlated with cell density, enabling better segmentation of aggregates and better visualization of streaming patterns in between aggregates. However, fluorescence microscopy requires the engineering of cells to express fluorescent proteins and can be phototoxic to the cells. To combine the advantages of both imaging methodologies, we develop a generative adversarial network that converts phase-contrast into fluorescent images. By including an additional histogram-equalized output to the state-of-art pix2pixHD algorithm, our model generates accurate images of aggregates and streams, enabling the estimation of aggregate positions and sizes, but with small shifts of their boundaries. Further training on ripple patterns enables accurate estimation of the rippling wavelength. Our methods are thus applicable for many other phenotypic behaviors and pattern formation studies.

systems biology↗

bioRxiv2021

Profiling Myxococcus xanthus swarming phenotypes through mutation and environmental variation

Myxococcus xanthus is a bacterium that lives on surfaces as a predatory biofilm called a swarm.As a growing swarm feeds on prey and expands, it displays dynamic multicellular patterns such as traveling waves called ripples and branching protrusions called flares. The rate at which a swarm expands across a surface, and the emergence of the coexisting patterns, are all controlled through coordinated cell movement. M. xanthus cells move using two motility systems known as Adventurous (A) and Social (S). Both are involved in swarm expansion and pattern formation. In this study, we describe a set of M. xanthus swarming genotype-to-phenotype associations that include both genetic and environmental perturbations. We identified new features of the swarming phenotype; recorded and measured swarm expansion using time-lapse microscopy; and compared the impact of mutation on different surfaces. These observations and analyses have increased our ability to discriminate between swarming phenotypes and provided context that allowed us to identify some phenotypes as improbable outliers within the M. xanthus swarming phenome. ImportanceMyxococcus xanthus grows on surfaces as a predatory biofilm called a swarm. A feeding swarm expands by moving over and consuming prey bacteria. In the laboratory, a swarm is created by spotting cell suspension onto nutrient agar in lieu of prey. The cells quickly settle on the surface and the new swarm then expands radially. An assay that measures the expansion rate of a swarm of mutant cells is the first, and sometimes only, measurement used to decide whether a particular mutation impacts swarm motility. We have broadened the scope of this assay by increasing the accuracy of measurements and reintroducing prey, resulting in new identifiable and quantifiable features that can be used to improve genotype-to-phenotype associations.

microbiology↗

bioRxiv2019

Rev-erba heterozygosity produces a dose-dependent phenotypic advantage in mice

Numerous mutational studies have demonstrated that circadian clock proteins regulate behavior and metabolism. Nr1d1(Rev-erb) is a key regulator of circadian gene expression and a pleiotropic regulator of skeletal muscle homeostasis and lipid metabolism. Loss of Rev-erb expression induces muscular atrophy, high adiposity, and metabolic syndrome in mice. Here we show that, unlike knockout mice, Nr1d1 heterozygous mice are not susceptible to muscular atrophy and in fact paradoxically possess larger myofiber diameters and improved neuromuscular function, compared to wildtype mice. Heterozygous mice lacked dyslipidemia, a characteristic of Nr1d1 knockout mice and displayed increased whole-body fatty-acid oxidation during periods of inactivity (light cycle). Heterozygous mice also exhibited higher rates of glucose uptake when fasted, and had elevated basal rates of gluconeogenesis compared to wildtype and knockout littermates. Rev-erb ablation suppressed glycolysis and fatty acid-oxidation in white-adipose tissue (WAT), whereas partial Rev-erb loss, curiously stimulated these processes. Our investigations revealed that Rev-erb dose-dependently regulates glucose metabolism and fatty acid oxidation in WAT and muscle.

molecular biology↗