Biology subjects

King, G. M.

Publications and source records attributed to King, G. M..

6 recordsLinked to original sources

bioRxiv2025

Automated Registration and Clustering for Enhanced Localization Atomic Force Microscopy of Flexible Membrane Proteins

Atomic Force Microscopy (AFM) can create images of biomolecules under near-native conditions but suffers from limited lateral resolution due to the finite AFM tip size and recording frequency. The recently developed Localization Atomic Force Microscopy or LAFM (Heath et al., Nature 594, 385 (2021)) enhances lateral resolution by reconstructing peak positions in AFM image stacks, but it is less effective for flexible proteins with multiple conformations. Here we introduce an unsupervised deep learning algorithm that simultaneously registers and clusters images by protein conformation, thus making LAFM applicable to more flexible proteins. Using simulated AFM images from molecular dynamics simulations of the SecYEG translocon as a model membrane protein system, we demonstrate improved resolution for individual protein conformations. This work represents a step towards a more general LAFM algorithm that can handle biological macromolecules with multiple distinct conformational states such as SecYEG. Author summaryAtomic Force Microscopy (AFM) enables high-resolution imaging of biomolecules under near-native conditions but faces lateral resolution limits due to the finite AFM tip size and recording frequency. The recently developed Localization Atomic Force Microscopy (LAFM) method addresses this by reconstructing peak positions from AFM image stacks, achieving almost atomic resolution for rigid proteins like bacteriorhodopsin (Heath et al., Nature 594, 385 (2021)). However, flexible membrane proteins with dynamic conformations, such as the SecYEG translocon, which exhibits large and highly mobile cytoplasmic loops, lead to non-physical smearing in standard LAFM reconstructions. Here, we present a computational framework combining unsupervised deep clustering and LAFM to enhance the lateral resolution of AFM images of flexible membrane proteins. Our neural network algorithm (i) groups AFM images into conformationally homogeneous clusters and (ii) registers images within each cluster. Applying LAFM separately to these clusters minimizes smearing artifacts, yielding high-resolution reconstructions for distinct conformations. We validate this approach using synthetic AFM images generated from all-atom molecular dynamics simulations of SecYEG in a solvated POPE lipid bilayer. This advancement extends LAFMs utility to encompass conformationally diverse membrane proteins.

biophysics↗

bioRxiv2024

A genome-scale screen identifies sulfated glycosaminoglycans as pivotal in epithelial cell damage by Candida albicans

Candidalysin is a cytolytic peptide produced by the opportunistic fungal pathogen Candida albicans. This peptide is a key virulence factor in mouse models of mucosal and hematogenously disseminated candidiasis. Despite intense interest in the role of candidalysin in C. albicans pathogenicity, its host cell targets have remained elusive. To fill this knowledge gap, we performed a genome-wide loss-of-function CRISPR screen in a human oral epithelial cell line to identify specific host factors required for susceptibility to candidalysin-induced cellular damage. Among the top hits were XYLT2, B3GALT6 and B3GAT3, genes that function in glycosaminoglycan (GAG) biosynthesis. Deletion of these genes led to the absence of GAGs such as heparan sulfate on the epithelial cell surface and increased resistance to damage induced by both candidalysin and live C. albicans. Biophysical analyses including surface plasmon resonance and atomic force and electron microscopy indicated that candidalysin physically binds to sulfated GAGs, facilitating its oligomerization or enrichment on the host cell surface. The addition of exogenous sulfated GAGs or the GAG analogue dextran sulfate protected cells against candidalysin-induced damage. Dextran sulfate, but not non-sulfated dextran, also inhibited epithelial cell endocytosis of C. albicans and fungal-induced epithelial cell cytokine and chemokine production. In a murine model of vulvovaginal candidiasis, topical dextran sulfate administration reduced host tissue damage and decreased intravaginal IL-1{beta} and neutrophil levels. Collectively, these data indicate that GAGs are epithelial cell targets of candidalysin and can be used therapeutically to protect cells from candidalysin-induced damage.

microbiology↗

bioRxiv2023

Carboxydovores from the Pseudomonadota colonise volcanic soils during succession

Carbon monoxide (CO) degrading microorganisms are present in volcanic deposits throughout succession, with vegetation and soil influencing the communities present. The carboxydovores are a subset of CO degraders that use CO only as an energy source, raising the question of how the physiological and metabolic features of the carboxydovores can make these bacteria more competitive in harsh volcanic ecosystems. An enrichment strategy was modified, which enabled the isolation of two carboxydovore representatives from genera that were abundant in the native soils, Cupriavidus sp. CV2T (92.3% ANI vs. Cupriavidus basilensis DSM 11853) and a putative strain of Paraburkholderia terrae (Pb. terrae COX) (96.42% ANI vs. Pb. terrae KU-64T). These isolates oxidise CO across a very broad range of concentrations, and genome sequence analysis indicated that they use form-I carbon monoxide dehydrogenase (CODH) to do so. Cupriavidus sp. CV2T and Pb. terrae COX each oxidised CO specifically at stationary phase, but the conditions for induction of CODH expression were distinct. Cupriavidus sp. CV2T expressed CODH only in the presence of CO, while Pb. terrae COX expressed CODH regardless of the presence of CO. Based on metabolic and phylogenetic analyses, Cupriavidus sp. CV2T is recommended as a novel species within the genus Cupriavidus. Therefore, we propose the name Cupriavidus ulmosensis sp. nov. for the type strain CV2T (= NCIMB 15506T, = CECT 30956T). This study provides valuable insights into the physiology and metabolism of carboxydovores, which colonise volcanic ecosystems during succession. ImportanceVolcanic ecosystems harbour many bacteria that contribute to the environmentally important process of carbon monoxide (CO) oxidation. We demonstrate a modified method for isolating bacteria, which consume CO at very low concentrations as a supplementary energy source (carboxydovory), leading to the isolation of two novel strains (Cupriavidus sp. CV2T and Paraburkholderia terrae COX) from volcanic strata that formed in 1917 and 2015, respectively. The conditions under which CO consumption occurs were investigated; each strain consumed CO during stationary phase, but Pb. terrae COX consumed CO regardless of the prior growth conditions while Cupriavidus sp. CV2 was more controlled. Cupriavidus sp. CV2 is a type strain of a new species, Cupriavidus ulmosensis str. CV2, which demonstrates relatively high tolerance for CO. These strains provide the basis for further study of the physiology, metabolism, and genetics of CO oxidation by carboxydovores, and will help us to understand how bacteria colonise harsh volcanic ecosystems.

microbiology↗

bioRxiv2020

Reconstructing genomes of carbon monoxide oxidisers in volcanic deposits including members of the class Ktedonobacteria

Microorganisms can potentially colonize volcanic rocks using the chemical energy in reduced gases such as methane, hydrogen (H2) and carbon monoxide (CO). In this study, we analysed soil metagenomes from Chilean volcanic soils, representing three different successional stages with ages of 380, 269 and 63 years, respectively. A total of 19 metagenome-assembled genomes (MAGs) were retrieved from all stages with a higher number observed in the youngest soil (1640: 2 MAGs, 1751: 1 MAG, 1957: 16 MAGs). Genomic similarity indices showed that several MAGs had amino-acid identity (AAI) values >50% to the phyla Actinobacteria, Acidobacteria, Gemmatimonadetes, Proteobacteria and Chloroflexi. Three MAGs from the youngest site (1957) belonged to the class Ktedonobacteria (Chloroflexi). Complete cellular functions of all the MAGs were characterised, including carbon fixation, terpenoid backbone biosynthesis, formate oxidation and CO oxidation. All 19 environmental genomes contained at least one gene encoding a putative carbon monoxide dehydrogenase (CODH). Three MAGs had form I coxL operon (encoding the large subunit CO-dehydrogenase). One of these MAGs (MAG-1957-2.1, Ktedonobacterales) was highly abundant in the youngest soil. MAG-1957-2.1 also contained genes encoding a [NiFe]-hydrogenase and hyp genes encoding accessory enzymes and proteins. Little is known about the Ktedonobacterales through cultivated isolates, but some species can utilize H2 and CO for growth. Our results strongly suggest that the remote volcanic sites in Chile represent a natural habitat for Ktedonobacteria and they may use reduced gases for growth.

microbiology↗

bioRxiv2019

Ecological and genomic attributes of novel bacterial taxa that thrive in subsurface soil horizons

While most bacterial and archaeal taxa living in surface soils remain undescribed, this problem is exacerbated in deeper soils owing to the unique oligotrophic conditions found in the subsurface. Additionally, previous studies of soil microbiomes have focused almost exclusively on surface soils, even though the microbes living in deeper soils also play critical roles in a wide range of biogeochemical processes. We examined soils collected from 20 distinct profiles across the U.S. to characterize the bacterial and archaeal communities that live in subsurface soils and to determine whether there are consistent changes in soil microbial communities with depth across a wide range of soil and environmental conditions. We found that bacterial and archaeal diversity generally decreased with depth, as did the degree of similarity of microbial communities to those found in surface horizons. We observed five phyla that consistently increased in relative abundance with depth across our soil profiles: Chloroflexi, Nitrospirae, Euryarchaeota, and candidate phyla GAL15 and Dormibacteraeota (formerly AD3). Leveraging the unusually high abundance of Dormibacteraeota at depth, we assembled genomes representative of this candidate phylum and identified traits that are likely to be beneficial in low nutrient environments, including the synthesis and storage of carbohydrates, the potential to use carbon monoxide (CO) as a supplemental energy source, and the ability to form spores. Together these attributes likely allow members of the candidate phylum Dormibacteraeota to flourish in deeper soils and provide insight into the survival and growth strategies employed by the microbes that thrive in oligotrophic soil environments.\n\nImportanceSoil profiles are rarely homogeneous. Resource availability and microbial abundances typically decrease with soil depth, but microbes found in deeper horizons are still important components of terrestrial ecosystems. By studying 20 soil profiles across the U.S., we documented consistent changes in soil bacterial and archaeal communities with depth. Deeper soils harbored distinct communities compared to the more commonly studied surface horizons. Most notably, we found that the candidate phylum Dormibacteraeota (formerly AD3) was often dominant in subsurface soils, and we used genomes from uncultivated members of this group to identify why these taxa are able to thrive in such resource-limited environments. Simply digging deeper into soil can reveal a surprising amount of novel microbes with unique adaptations to oligotrophic subsurface conditions.

microbiology↗

bioRxiv2019

Carbon monoxide dehydrogenases enhance bacterial survival by respiring atmospheric CO

Carbon monoxide (CO) is a ubiquitous atmospheric trace gas produced by natural and anthropogenic sources. Some aerobic bacteria can oxidize atmospheric CO and, collectively, they account for the net loss of ~250 teragrams of CO from the atmosphere each year. However, the physiological role, genetic basis, and ecological distribution of this process remain incompletely resolved. In this work, we addressed these knowledge gaps through culture-based and culture-independent work. We confirmed through shotgun proteomic and transcriptional analysis that the genetically tractable aerobic soil actinobacterium Mycobacterium smegmatis upregulates expression of a carbon monoxide dehydrogenase by 50-fold when exhausted for organic carbon substrates. Whole-cell biochemical assays in wild-type and mutant backgrounds confirmed that this organism aerobically respires CO, including at sub-atmospheric concentrations, using the enzyme. Contrary to current paradigms on CO oxidation, the enzyme did not support chemolithoautotrophic growth and was dispensable for CO detoxification. However, it significantly enhanced long-term survival, suggesting that atmospheric CO serves a supplemental energy source during organic carbon starvation. Phylogenetic analysis indicated that atmospheric CO oxidation is widespread and an ancestral trait of CO dehydrogenases. Homologous enzymes are encoded by 685 sequenced species of bacteria and archaea, including from seven dominant soil phyla, and we confirmed genes encoding this enzyme are abundant and expressed in terrestrial and marine environments. On this basis, we propose a new survival-centric model for the evolution of CO oxidation and conclude that, like atmospheric H2, atmospheric CO is a major energy source supporting persistence of aerobic heterotrophic bacteria in deprived or changeable environments.

microbiology↗