Biology subjects

Brady, S. F.

Publications and source records attributed to Brady, S. F..

6 recordsLinked to original sources

bioRxiv2026

A 5-hydroxymethylcytosine DNA glycosylase provides defense against T-even bacteriophages

The most abundant prokaryotic mechanisms of defense against phage predation involve the recognition and destruction of the infecting DNA. One method of counter-defense is the incorporation of modified nucleobases into the phage genome to avoid interaction with enzymes that target the viral DNA. T-even coliphages replace cytosine with 5-hydroxymethylcytosine (5hmC) that in some cases are further decorated with glucosyl groups. To explore the diversity of immunity genes that recognize 5hmC, we infected a library of metagenomic DNA inserts from uncultured, non-sequenced soil bacteria with a mutant T4 phage that harbored only non-glucosylated 5hmC on its genome. Bacteria that resisted infection carried a DNA glycosylase, Brig3, that specifically excises 5hmC nucleobases to generate abasic sites in the phage genome and prevent viral proliferation. The crystal structure of Brig3 bound to its substrate revealed a catalytic mechanism in which the 5hmC nucleobase is flipped out of the DNA into the active site and replaced by an asparagine residue that inserts into the double helix to contact the complementary guanosine. Brig3 is encoded within an operon that also encodes BapA, a hydrolase that removes glucosyl groups from glucosyl-5hmC present in the genome of otherwise Brig3-resistant T-even phages carrying this hypermodified base. Our results uncover a defense strategy in which the combined action of BapA and Brig3 widens the immune response to restrict the infection of T-even phages with genomes that are either partially or completely glucosylated.

microbiology↗

bioRxiv2025

A CARF-HAD phosphatase effector provides immunity during the type III-A CRISPR-Cas response

CRISPR-Cas systems provide adaptive immunity against phage infection in prokaryotes using an RNA-guided complex that recognizes complementary foreign nucleic acids. Different types of CRISPR-Cas systems have been identified that differ in their mechanism of defense. Upon infection, Type III CRISPR-Cas systems employ the Cas10 complex to find phage transcripts and synthesize cyclic oligo-adenylate (cOA) messengers. These ligands bind and activate CARF immune effectors that cause cell toxicity to prevent the completion of the viral lytic cycle. Here we investigated two proteins containing an N-terminal haloacid dehalogenase (HAD) phosphatase domain followed by four predicted transmembrane helices and a C-terminal CARF domain, which we named Chp. We show that, in vivo, Chp localizes to the bacterial membrane and that its activation induces a growth arrest, leads to a depletion of ATP and IMP and prevents phage propagation during the type III CRISPR-Cas response. In vitro, the CARF domain of Chp binds cyclic tetra-adenylates and the HAD phosphatase domain dephosphorylates dATP, ATP and IMP. Our findings extend the range of molecular mechanisms employed by CARF effectors to defend prokaryotes against phage infection.

microbiology↗

bioRxiv2024

Oxydifficidin, a potent Neisseria gonorrhoeae antibiotic due to DedA assisted uptake and ribosomal protein RplL sensitivity

Gonorrhea, which is caused by Neisseria gonorrhoeae, is the second most reported sexually transmitted infection worldwide. The increasing appearance of isolates that are resistant to approved therapeutics raises the concern that gonorrhea may become untreatable. Here, we serendipitously identified oxydifficidin as a potent N. gonorrhoeae antibiotic through the observation of a Bacillus amyloliquefaciens contaminant in a lawn of N. gonorrhoeae. Oxydifficidin is active against both wild- type and multidrug-resistant N. gonorrhoeae. Its potent activity results from a combination of DedA- assisted uptake into the cytoplasm and the presence of an oxydifficidin-sensitive ribosomal protein L7/L12 (RplL). Our data indicates that oxydifficidin binds to the ribosome at a site that is distinct from other antibiotics and that L7/L12 is uniquely associated with its mode of action. This study opens a potential new avenue for addressing antibiotic resistant gonorrhea and underscores the possibility of identifying overlooked natural products from cultured bacteria, particularly those with activity against previously understudied pathogens.

microbiology↗

bioRxiv2023

Bacterial cGAS senses a viral RNA to initiate immunity

CBASS immunity protects prokaryotes from viral (phage) attack through the production of cyclic dinucleotides which activate effector proteins that trigger the death of the infected host. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a highly structured 400-nt RNA, termed CBASS-activating bacteriophage RNA (cabRNA), that binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP. Phages that escape CBASS immunity harbor mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. Since the mammalian cyclase OAS1 also binds viral dsRNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defense pathways.

microbiology↗

bioRxiv2020

Elucidating the diversity and potential function of nonribosomal peptide and polyketide biosynthetic gene clusters in the root microbiome

Polyketides (PKs) and nonribosomal peptides (NRPs) are two microbial secondary metabolite (SM) families known for their variety of functions, including antimicrobials, siderophores and others. Despite their involvement in bacteria-bacteria and bacteria-plant interactions, root-associated SMs are largely unexplored due to the limited cultivability of bacteria. Here, we analyzed the diversity and expression of SM-encoding biosynthetic gene clusters (BGCs) in root microbiomes by culture-independent amplicon sequencing, shotgun metagenomics and metatranscriptomics. Roots (tomato and lettuce) harbored distinct compositions of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) relative to the adjacent bulk soil, and specific BGC markers were both enriched and highly expressed in the root microbiomes. While several of the highly abundant and expressed sequences were remotely associated with known BGCs, the low similarity to characterized genes suggests their potential novelty. Low similarity genes were screened against a large set of soil-derived cosmid libraries, from which five whole BGCs of unknown function were retrieved. Three clusters were taxonomically affiliated with Actinobacteria, while the remaining were not associated with known bacteria. One Streptomyces-derived BGC was predicted to encode for a polyene with potential antifungal activity, while the others were too novel to predict chemical structure. Screening against a suite of metagenomic datasets revealed a higher abundance of retrieved clusters in roots and soil samples. In contrast, they were almost completely absent in aquatic and gut environments, supporting the notion that they might play an important role in root ecosystems. Overall, our results indicate that root microbiomes harbor a specific assemblage of undiscovered SMs. ImportanceWe identified distinct secondary metabolite (polyketide and nonribosomal peptide) encoding genes that are enriched (relative to adjacent bulk soil) and expressed in root ecosystems, yet almost completely absent in human gut and aquatic environments. Several of the genes were distantly related to genes encoding for antimicrobials and siderophores, and their high sequence variability relative to known sequences suggests that they may encode for novel metabolites and may have unique ecological functions. This study demonstrates that plant roots harbor a diverse array of unique secondary metabolite encoding genes that are highly enriched and expressed in the root ecosystem. The secondary metabolites encoded by these genes might assist the bacteria that produce them in colonization and persistence in the root environment. To explore this hypothesis, future investigations should assess their potential role in inter-bacterial and bacterial-plant interactions.

microbiology↗

bioRxiv2019

Mapping interactions of microbial metabolites and human receptors

Despite evidence linking the human microbiome to health and disease, the mechanistic details of how the microbiota affects human physiology remain largely unknown. Metabolites encoded by bacteria are expected to play an integral role in the microbiotas effect on its human host. Assigning function to these metabolites is therefore critical to determining the molecular underpinnings of the host-microbe relationship and ultimately developing microbiota inspired therapies. Here we use large-scale functional screening of small molecules produced by individual members of a simplified human microbiota to identify bacterial metabolites that agonize G-protein coupled receptors (GPCR). This analysis revealed a complex network of metabolite host receptor interactions and guided our identification of multiple microbiota derived agonists of GPCRs associated with diverse biological functions within the nervous and immune systems, among others. Collectively, the metabolite-receptor pairs we uncovered indicate that diverse aspects of human health are potentially modulated by structurally simple metabolites arising from primary bacterial metabolism.\n\nStatement of SignificanceBacteria residing within the human body have been shown to influence human health. It is likely that physiological responses to the human microbiota are mediated by the collection of small molecules encoded within these bacteria. In this study we use direct functional screening of small molecules produced by individual members of a simplified human microbiota to identify new G protein coupled receptor-metabolite interactions that seek to explain the molecular underpinnings of the microbiotas influence on its human host.

microbiology↗