Biology subjects

Gallagher, L. A.

Publications and source records attributed to Gallagher, L. A..

10 recordsLinked to original sources

bioRxiv2026

Oral Actinobacteria sense and defend against parasitic epibionts

Bacteria contend with a wide range of antagonistic interactions from neighboring microbes. One widespread but poorly understood threat is posed by Patescibacteria, which proliferate by colonizing host bacteria surfaces and extracting cellular resources. Despite the ubiquity of Patescibacteria in the environment, how hosts defend against these epibionts remains unknown. Here we show that Actinomycetota fend off Patescibacteria using the Esx secretion system. We identify a secreted effector protein that mediates this defensive behavior and demonstrate that Esx induction is part of a multifaceted, epibiont-specific response that includes multiple predicted cell surface modifications. Finally, we show that this response occurs at the transcriptional level and is mediated by a previously undescribed threat sensing pathway whose activation leads to threonine phosphorylation of an FHA domain-containing output protein. Together with bioinformatic evidence linking this threat sensing pathway to Esx across Actinomycetales, our experimental findings establish a functional role for the elusive and broadly distributed Esx pathway in defense against epibionts. Furthermore, they help resolve the ecological relationship between Patescibacteria and their hosts as one of antagonism, not mutualism.

microbiology↗

bioRxiv2025

A conserved electron transport chain sensitizes Bacteroides to a pore-forming type VI secretion toxin

Data suggest that antagonism between bacteria is prevalent within the gut microbiome. Such antagonism could have profound consequences on the fitness of species; however, the susceptibility determinants to even the most pervasive antagonistic factors in this ecosystem remain incompletely understood. Here, we screened for genetic factors that impact the susceptibility of Bacteroides to type VI secretion system (T6SS)-delivered toxins. This revealed that the Bte2 family of pore-forming toxins, which are widespread in B. fragilis and other human gut-associated Bacteroidales, strictly require the H+/Na+-translocating ferredoxin:NAD+ reductase (Rnf) electron transport chain within target cells in order to intoxicate. In Bacteriodes, the precise metabolic role of the conserved Rnf pathway has not been defined. We establish that the Rnf complex is important for redox balancing within cells utilizing sugars derived from dietary fiber and is critical for fitness in vivo. Surprisingly, we find that while the intact Rnf membrane complex is required for Bte2 intoxication, Rnf-catalyzed electron transport is dispensable. We propose that the Rnf complex facilitates Bte2 membrane insertion, leading to intoxication via membrane depolarization. Our data suggest that T6SS toxins may avoid collateral damage within a complex ecosystem by recognizing discriminatory features of competitor species. SignificancePathways for interbacterial antagonism are prevalent in the gut microbiome. The breadth of targeting and specificity determinants of these systems remain largely uncharacterized. We discovered that a widespread pore-forming toxin produced by gut Bacteroidales requires the conserved Rnf protein complex in target organisms. Although this complex is dispensable during in vitro growth, we show it is required for Bacteroides fitness during colonization of the mammalian gut. Our data support a model in which transient interaction between the toxin and Rnf enables rearrangement of the protein, facilitating membrane insertion. Related toxins found in Proteobacteria lack the requirement for Rnf, suggesting that competition between Bacteroidales species in the gut may be driving specialization of their antibacterial toxins.

microbiology↗

bioRxiv2025

Pseudomonads coordinate innate defense against viruses and bacteria with a single regulatory system

Bacterial cells live under the constant existential threats imposed by other bacteria and viruses. Their mechanisms for contending with these threats are well documented; however, the regulation of these diverse defense elements remains poorly understood. Here we show that bacteria can mount a genome-wide, coordinated, and highly effective immune response against bacterial and viral threats using a single regulatory pathway. Bioinformatic analyses revealed that Pseudomonas species broadly possess a specialized form of the Gac/Rsm regulatory pathway (GRP), which our prior work in Pseudomonas aeruginosa implicated in activating interbacterial antagonism defense mechanisms in response to neighbor cell death. Proteomic studies comparing GRP-activated and -inactivated strains derived from diverse Pseudomonas species showed that the pathway regulates a large and variable suite of factors implicated in defense against both bacterial and phage threats. Focusing on P. protegens, we identify profound phenotypic consequences of these factors against multiple forms of bacterial antagonism and several phage. Together, our results reveal that bacteria, like more complex organisms, couple danger sensing to the activation of an immune system with antibacterial and antiviral arms.

microbiology↗

bioRxiv2024

A biofilm-tropic Pseudomonas aeruginosa bacteriophage uses the exopolysaccharide Psl as receptor

Bacteria in nature can exist in multicellular communities called biofilms. Biofilms also form in the course of many infections. Pseudomonas aeruginosa infections frequently involve biofilms, which contribute materially to the difficulty to treat these infections with antibiotic therapy. Many biofilm-related characteristics are controlled by the second messenger, cyclic-di-GMP, which is upregulated on surface contact. Among these factors is the exopolysaccharide Psl, which is a critically important component of the biofilm matrix. Here we describe the discovery of a P. aeruginosa bacteriophage, which we have called Clew-1, that directly binds to and uses Psl as a receptor. While this phage does not efficiently infect planktonically growing bacteria, it can disrupt P. aeruginosa biofilms and replicate in biofilm bacteria. We further demonstrate that the Clew-1 can reduce the bacterial burden in a mouse model of P. aeruginosa keratitis, which is characterized by the formation of a biofilm on the cornea. Due to its reliance on Psl for infection, Clew-1 does not actually form plaques on wild-type bacteria under standard in vitro conditions. This argues that our standard isolation procedures likely exclude bacteriophage that are adapted to using biofilm markers for infection. Importantly, the manner in which we isolated Clew-1 can be easily extended to other strains of P. aeruginosa and indeed other bacterial species, which will fuel the discovery of other biofilm-tropic bacteriophage and expand their therapeutic use.

microbiology↗

bioRxiv2023

Umbrella toxin particles produced by Streptomyces block mycelial growth of competing species

The Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species. Here we show that in addition to small molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-EM structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, leading to our naming them umbrella particles. S. coelicolor encodes three umbrella particles with distinct toxin and lectin composition, and supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, S. griseus, we find inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative mycelial growth. Our data show that Streptomyces umbrella particles mediate competition between vegetative mycelia of related species, a function distinct from small molecule antibiotics, which are produced at the onset of reproductive growth and act broadly. Sequence analyses suggest this role of umbrella particles extends beyond Streptomyces, as we find umbrella loci in nearly one-thousand species across Actinobacteria.

microbiology↗

bioRxiv2023

Genetic manipulation of candidate phyla radiation bacteria provides functional insights into microbial dark matter

The study of bacteria has yielded fundamental insights into cellular biology and physiology, biotechnological advances and many therapeutics. Yet due to a lack of suitable tools, the significant portion of bacterial diversity held within the candidate phyla radiation (CPR) remains inaccessible to such pursuits. Here we show that CPR bacteria belonging to the phylum Saccharibacteria exhibit natural competence. We exploit this property to develop methods for their genetic manipulation, including the insertion of heterologous sequences and the construction of targeted gene deletions. Imaging of fluorescent protein-labeled Saccharibacteria provides high spatiotemporal resolution of phenomena accompanying epibiotic growth and a transposon insertion sequencing genome-wide screen reveals the contribution of enigmatic Saccharibacterial genes to growth on their Actinobacteria hosts. Finally, we leverage metagenomic data to provide cutting-edge protein structure-based bioinformatic resources that support the strain Southlakia epibionticum and its corresponding host, Actinomyces israelii, as a model system for unlocking the molecular underpinnings of the epibiotic lifestyle.

microbiology↗

bioRxiv2023

Metabolic reprogramming and flux to cell envelope precursors in a pentose phosphate pathway mutant increases MRSA resistance to β-lactam antibiotics

Central metabolic pathways controls virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to {beta}-lactam antibiotics, particularly in chemically defined media with glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased {beta}-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. Further evidence of the pleiotropic effect of the pgl mutation was reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Reduced binding of wheat germ agglutinin (WGA) to pgl was indicative of lower wall teichoic acid/lipoteichoic acid levels or altered teichoic acid structures. Mutations in the vraFG or graRS loci reversed the increased OX resistance phenotype and restored WGA binding to wild-type levels. VraFG/GraRS was previously implicated in susceptibility to cationic antimicrobial peptides and vancomycin, and these data reveal a broader role for this multienzyme membrane complex in the export of cell envelope precursors or modifying subunits required for resistance to diverse antimicrobial agents. Altogether our study highlights important roles for the PPP and VraFG/GraRS in {beta}-lactam resistance, which will support efforts to identify new drug targets and reintroduce {beta}-lactams in combination with adjuvants or other antibiotics for infections caused by MRSA and other {beta}-lactam resistant pathogens. Author summaryHigh-level resistance to penicillin-type ({beta}-lactam) antibiotics significantly limits the therapeutic options for patients with MRSA infections necessitating the use of newer agents, for which reduced susceptibility has already been described. Here we report for the first time that the central metabolism pentose phosphate pathway controls MRSA resistance to penicillin-type antibiotics. We comprehensively demonstrated that mutation of the PPP gene pgl perturbed metabolism in MRSA leading to increased flux to cell envelope precursors to drive increased antibiotic resistance. Moreover, increased resistance was dependent on the VraRG/GraRS multienzyme membrane complex previously implicated in resistance to antimicrobial peptides and vancomycin. Our data thus provide new insights on MRSA mechanisms of {beta}-lactam resistance, which will support efforts to expand the treatment options for infections caused by this and other antimicrobial resistant pathogens.

microbiology↗

bioRxiv2023

Discovery of a unique pathway for glutathione utilization in Francisella

Glutathione (GSH) is an abundant metabolite that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella spp. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

microbiology↗

bioRxiv2021

Coordinately regulated interbacterial antagonism defense pathways constitute a bacterial innate immune system

Bacterial survival is fraught with antagonism, including that deriving from viruses and competing bacterial cells1-3 4. It is now appreciated that bacteria mount complex antiviral responses; however, whether a coordinated defense against bacterial threats is undertaken is not well understood. Previously we showed that Pseudomonas aeruginosa possess a danger sensing pathway that is a critical fitness determinant during competition against other bacteria5, 6. Here, we conducted genome-wide screens in P. aeruginosa that reveal three conserved and widespread interbacterial antagonism resistance clusters (arc1-3). We find that although arc1-3 are coordinately activated by the Gac/Rsm danger sensing system, they function independently and provide idiosyncratic defense capabilities, distinguishing them from general stress response pathways. Our findings demonstrate that Arc3 family proteins provide specific protection against phospholipase toxins by preventing the accumulation of lysophospholipids in a manner distinct from previously characterized membrane repair systems. These findings liken the response of P. aeruginosa to bacterial threats to that of eukaryotic innate immunity, wherein threat detection leads to the activation of specialized defense systems.

microbiology↗

bioRxiv2021

Genome-wide protein-DNA interaction site mapping using a double strand DNA-specific cytosine deaminase

DNA-protein interactions (DPIs) are central to such fundamental cellular processes as transcription and chromosome maintenance and organization. The spatiotemporal dynamics of these interactions dictate their functional consequences; therefore, there is great interest in facile methods for defining the sites of DPI within cells. Here, we present a general method for mapping DPI sites in vivo using the double stranded DNA-specific cytosine deaminase toxin DddA. Our approach, which we term DddA-sequencing (3D-seq), entails generating a translational fusion of DddA to a DNA binding protein of interest, inactivating uracil DNA glycosylase, modulating DddA activity via its natural inhibitor protein, and DNA sequencing for genome-wide DPI detection. We successfully applied this method to three Pseudomonas aeruginosa transcription factors that represent divergent protein families and bind variable numbers of chromosomal locations. 3D-seq offers several advantages over existing technologies including ease of implementation and the possibility to measure DPIs at single-cell resolution.

genetics↗