Biology subjects

Fire, A. Z.

Publications and source records attributed to Fire, A. Z..

7 recordsLinked to original sources

bioRxiv2026

Requirement for Type IX secretion machinery in a nematode model of Chryseobacterial pathogenesis

Bacterium-nematode interactions in soil environments hold agricultural, economic, and evolutionary significance, with the rapid killing of nematodes by nematophagous bacteria representing an extreme and potentially valuable example of such interactions. Here, we investigate rapid killing of the model nematode Caenorhabditis elegans by the nematophagous bacterial species Chryseobacterium nematophagum through development and application of bacterial genetic tools for Chryseobacterium. We show that Chryseobacterium nematophagum can be genetically manipulated through both forward genetics and homology-mediated genetic interventions, with the latter driven by the use of a pyrimidine auxotrophy system to circumvent the multidrug resistance of these bacteria. Combining these approaches, we identified the Type IX Secretion System (T9SS) machinery as a central virulence determinant. Independent mutations in multiple T9SS components each substantially attenuated nematode killing with complementation restoring virulence. This system provides a tractable experimental platform for investigating the molecular basis of nematophagous bacterial pathogenesis and of the T9SS-dependent virulence mechanisms.

microbiology↗

bioRxiv2024

CGC1, a new reference genome for Caenorhabditis elegans

The original 100.3 Mb reference genome for Caenorhabditis elegans, generated from the wild-type laboratory strain N2, has been crucial for analysis of C. elegans since 1998 and has been considered complete since 2005. Unexpectedly, this long-standing reference was shown to be incomplete in 2019 by a genome assembly from the N2-derived strain VC2010. Moreover, genetically divergent versions of N2 have arisen over decades of research and hindered reproducibility of C. elegans genetics and genomics. Here we provide a 106.4 Mb gap-free, telomere-to-telomere genome assembly of C. elegans, generated from CGC1, an isogenic derivative of the N2 strain. We used improved long-read sequencing and manual assembly of 43 recalcitrant genomic regions to overcome deficiencies of prior N2 and VC2010 assemblies, and to assemble tandem repeat loci including a 772-kb sequence for the 45S rRNA genes. While many differences from earlier assemblies came from repeat regions, unique additions to the genome were also found. Of 19,972 protein-coding genes in the N2 assembly, 19,790 (99.1%) encode products that are unchanged in the CGC1 assembly. The CGC1 assembly also may encode 183 new protein-coding and 163 new ncRNA genes. CGC1 thus provides both a completely defined reference genome and corresponding isogenic wild-type strain for C. elegans, allowing unique opportunities for model and systems biology.

genomics↗

bioRxiv2024

A genetic and microscopy toolkit for live imaging of whole-body regeneration in Macrostomum lignano

Live imaging of regenerative processes can reveal how animals restore their bodies after injury through a cascade of dynamic cellular events. Here, we present a comprehensive toolkit for live imaging of whole-body regeneration in the flatworm Macrostomum lignano, including a high throughput cloning pipeline, targeted cellular ablation, and advanced microscopy solutions. Using tissue-specific reporter expression, we examine how various structures regenerate. Enabled by a custom, low-cost luminescence/fluorescence microscope, we overcome intense stress-induced autofluorescence to demonstrate the first application of genetic cellular ablation in flatworms to reveal the limited regenerative capacity of neurons and their essential role during wound-healing. Finally, we build a novel open-source tracking microscope to continuously image moving animals throughout the week-long process of regeneration, quantifying kinetics of wound healing, nerve cord repair, body regeneration, growth, and behavioral recovery. Our findings suggest that nerve cord reconnection operates independently from primary body axis re-establishment and other downstream regenerative processes.

bioengineering↗

bioRxiv2024

Viroid-like colonists of human microbiomes

Here, we describe the "Obelisks," a previously unrecognised class of viroid-like elements that we first identified in human gut metatranscriptomic data. "Obelisks" share several properties: (i) apparently circular RNA [~]1kb genome assemblies, (ii) predicted rod-like secondary structures encompassing the entire genome, and (iii) open reading frames coding for a novel protein superfamily, which we call the "Oblins". We find that Obelisks form their own distinct phylogenetic group with no detectable sequence or structural similarity to known biological agents. Further, Obelisks are prevalent in tested human microbiome metatranscriptomes with representatives detected in [~]7% of analysed stool metatranscriptomes (29/440) and in [~]50% of analysed oral metatranscriptomes (17/32). Obelisk compositions appear to differ between the anatomic sites and are capable of persisting in individuals, with continued presence over >300 days observed in one case. Large scale searches identified 29,959 Obelisks (clustered at 90% nucleotide identity), with examples from all seven continents and in diverse ecological niches. From this search, a subset of Obelisks are identified to code for Obelisk-specific variants of the hammerhead type-III self-cleaving ribozyme. Lastly, we identified one case of a bacterial species (Streptococcus sanguinis) in which a subset of defined laboratory strains harboured a specific Obelisk RNA population. As such, Obelisks comprise a class of diverse RNAs that have colonised, and gone unnoticed in, human, and global microbiomes.

microbiology↗

bioRxiv2023

Ancient origins of complex neuronal genes

How nervous systems evolved is a central question in biology. An increasing diversity of synaptic proteins is thought to play a central role in the formation of specific synapses leading to nervous system complexity. The largest animal genes, often spanning millions of base pairs, are known to be enriched for expression in neurons at synapses and are frequently mutated or misregulated in neurological disorders and diseases. While many of these genes have been studied independently in the context of nervous system evolution and disease, general principles underlying their parallel evolution remain unknown. To investigate this, we directly compared orthologous gene sizes across eukaryotes. By comparing relative gene sizes within organisms, we identified a distinct class of large genes with origins predating the diversification of animals and in many cases the emergence of dedicated neuronal cell types. We traced this class of ancient large genes through evolution and found orthologs of the large synaptic genes driving the immense complexity of metazoan nervous systems, including in humans and cephalopods. Moreover, we found that while these genes are evolving under strong purifying selection as demonstrated by low dN/dS scores, they have simultaneously grown larger and gained the most isoforms in animals. This work provides a new lens through which to view this distinctive class of large and multi-isoform genes and demonstrates how intrinsic genomic properties, such as gene length, can provide flexibility in molecular evolution and allow groups of genes and their host organisms to evolve toward complexity.

genomics↗

bioRxiv2022

Restriction Endonuclease-based Modification-Dependent Enrichment (REMoDE) of DNA for Metagenomic Sequencing

Metagenomic sequencing is a swift and powerful tool to ascertain the presence of an organism of interest in a sample. However, sequencing coverage of the organism of interest can be insufficient due to an inundation of reads from irrelevant organisms in the sample. Here, we report a nuclease-based approach to rapidly enrich for DNA from certain organisms, including enterobacteria, based on their differential endogenous modification patterns. We exploit the ability of taxon-specific methylated motifs to resist the action of cognate methylation-sensitive restriction endonucleases that thereby digest unwanted, unmethylated DNA. Subsequently, we use a distributive exonuclease or electrophoretic separation to deplete or exclude the digested fragments, thus, enriching for undigested DNA from the organism of interest. As a proof-of-concept, we apply this method to enrich for the enterobacteria Escherichia coli and Salmonella enterica by 11- to 142-fold from mock metagenomic samples and validate this approach as a versatile means to enrich for genomes of interest in metagenomic samples. ImportancePathogens that contaminate the food supply or spread through other means can cause outbreaks that bring devastating repercussions to the health of a populace. Investigations to trace the source of these outbreaks are initiated rapidly but can be drawn out due to the labored methods of pathogen isolation. Metagenomic sequencing can alleviate this hurdle but is often insufficiently sensitive. The approach and implementations detailed here provide a rapid means to enrich for many pathogens involved in foodborne outbreaks, thereby improving the utility of metagenomic sequencing as a tool in outbreak investigations. Additionally, this approach provides a means to broadly enrich for otherwise minute levels of modified DNA which may escape unnoticed in metagenomic samples.

microbiology↗

bioRxiv2020

Identification of a pangolin niche for a 2019-nCoV-like coronavirus through an extensive meta-metagenomic search

In numerous instances, tracking the biological significance of a nucleic acid sequence can be augmented through the identification of environmental niches in which the sequence of interest is present. Many metagenomic datasets are now available, with deep sequencing of samples from diverse biological niches. While any individual metagenomic dataset can be readily queried using web-based tools, meta-searches through all such datasets are less accessible. In this brief communication, we demonstrate such a meta-meta-genomic approach, examining close matches to the Wuhan coronavirus 2019-nCoV in all high-throughput sequencing datasets in the NCBI Sequence Read Archive accessible with the keyword "virome". In addition to the homology to bat coronaviruses observed in descriptions of the 2019-nCoV sequence (F. Wu et al. 2020, Nature, doi.org/10.1038/s41586-020-2008-3; P. Zhou et al. 2020, Nature, doi.org/10.1038/s41586-020-2012-7), we note a strong homology to numerous sequence reads in a metavirome dataset generated from the lungs of deceased Pangolins reported by Liu et al. (Viruses 11:11, 2019, http://doi.org/10.3390/v11110979). Our observations are relevant to discussions of the derivation of 2019-nCoV and illustrate the utility and limitations of meta-metagenomic search tools in effective and rapid characterization of potentially significant nucleic acid sequences. ImportanceMeta-metagenomic searches allow for high-speed, low-cost identification of potentially significant biological niches for sequences of interest.

bioinformatics↗