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1. Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp.

   https://doi.org/10.1128/mbio.01323-25  

   Rajesh-Khanna, Dhanabala-Subhiksha; Piña_Páez, Carolina G; He, Susu; Dolan, Elora G; Mirpuri, Kiran S; Stajich, Jason E; Hogan, Deborah A (November 2025, mBio)

   Goldman, Gustavo H (Ed.)

   ABSTRACT Infections caused by the emerging pathogenic yeastClavispora (Candida) lusitaniaecan be difficult to manage due to multi-drug resistance. Resistance to the frontline antifungal fluconazole (FLZ) inCandidaspp. is commonly acquired through gain-of-function (GOF) mutations in the gene encoding the transcription factor Mrr1. These activated Mrr1 variants enhance FLZ efflux via upregulation of the multi-drug transporter geneMDR1. Recently, it was reported that, unlike in the well-studiedCandida albicansspecies,C. lusitaniaeandCandida parapsilosiswith activated Mrr1 also have high expression ofCDR1, which encodes another multi-drug transporter with overlapping but distinct transported substrate profiles and Cdr1-dependent FLZ resistance. To better understand the mechanisms of Mrr1 regulation ofMDR1andCDR1, and other co-regulated genes, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analysis of Mrr1 binding sites. Mrr1 bound the promoter regions ofMDR1andCDR1, as well asFLU1, which encodes another transporter capable of FLZ efflux. Mdr1 and Cdr1 independently contributed to the decreased susceptibility of theMRR1^GOFstrains against diverse clinical azoles and other antifungals, including 5-flucytosine. A consensus motif, CGGAGWTAR, enriched in Mrr1-boundC. lusitaniaeDNA was also conserved upstream ofMDR1andCDR1across species, includingC. albicans. CUT&RUN and RNA-seq data were used to define the Mrr1 regulon, which includes genes involved in transport, stress response, and metabolism. Activated and inducible Mrr1 bound similar regions in the promoters of Mrr1 regulon genes. Our studies provide new evolutionary insights into the coordinated regulation of multi-drug transporters and potential mechanism(s) that aid secondary resistance acquisition in emergingCandida. IMPORTANCEUnderstanding antifungal resistance in emergingCandidapathogens is essential to managing treatment failures and guiding the development of new therapeutic strategies. Like otherCandidaspecies, the environmental opportunistic fungal pathogenClavispora(Candida)lusitaniaecan acquire resistance to the antifungal fluconazole by overexpression of the multi-drug efflux pump Mdr1 through gain-of-function (GOF) mutations in the gene encoding the transcription factor Mrr1. Here, we show thatC. lusitaniaeMrr1 also directly regulatesCDR1, another major multi-drug transporter gene, along withMDR1. In strains with activated Mrr1, upregulation ofMDR1andCDR1protects against diverse antifungals, potentially aiding the rise of other resistance mutations. Mrr1 also regulates several stress response and metabolism genes, thereby providing new perspectives into the physiology of drug-resistant strains. The identification of an Mrr1 binding motif that is conserved across strains and species will advance future efforts to understand multi-drug resistance acrossCandidaspecies. 

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2. The Agrobacterium fabrum efflux pump PecM is produced in response to the plant exudate 4-hydroxybenzaldehyde to avoid disruption of central metabolism

   https://doi.org/10.1128/jb.00150-25  

   Ghosh, Arpita; Grove, Anne (July 2025, Journal of Bacteriology)

   Becker, Anke (Ed.)

   ABSTRACT Agrobacterium fabrum is a phytopathogen that causes crown gall disease. In the rhizosphere, it encounters plant exudates, some of which are toxic, such as 4-hydroxybenzaldehyde (4HBA). Others, including 4-hydroxybenzoate (4HB), participate in the induction of virulence genes.A. fabrum encodes the transcription factor PecS, which has been reported to enhance bacterial fitness in the rhizosphere. The gene encoding PecS is divergent from pecM, which encodes an efflux pump. PecS represses both pecS and pecM, as evidenced by increased expression in the presence of the PecS ligand urate and by elevated pecM expression in a pecS disruption strain. We report here that the expression ofpecM is induced selectively by 4HBA. Expression of genes encoding enzymes involved in the degradation of 4HB is induced by both 4HBA and 4HB, as expected; however, overexpression ofpecM attenuates the induction by 4HBA, suggesting that 4HBA is a substrate for PecM. Consistent with this inference, untargeted metabolomics shows that 4HBA accumulates intracellularly whenpecM is disrupted. Analysis of PecS by thermal stability assay and DNase I footprinting suggests that 4HBA is not a ligand for PecS. Taken together, our data suggest that 4HBA is a substrate for PecM.IMPORTANCEPlant roots secrete a number of compounds that may be toxic to bacteria residing in the surrounding soil. One such bacterium is Agrobacterium fabrum, which infects plants and induces tumor formation. We show here that an A. fabrum strain in which the efflux pump PecM has been disrupted accumulates 4-hydroxybenzaldehyde, and that this plant root exudate induces the expression of pecM. Our data suggest that PecM and PecS, a transcription factor that regulates pecM expression, both function to promote A. fabrum fitness in the rhizosphere. As a competitive advantage in the rhizosphere is a prerequisite for subsequent plant infection, our data contribute to a more complete understanding of the A. fabrum infection process. 

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3. Biophysical modeling reveals the transcriptional regulatory mechanism of Spo0A, the master regulator in starving Bacillus subtilis

   https://doi.org/10.1128/msystems.00072-25  

   Zhang, Yujia; Palma, Cristina_S D; Chen, Zhuo; Zarazúa-Osorio, Brenda; Fujita, Masaya; Igoshin, Oleg A; Eichenberger, Patrick (May 2025, mSystems)

   Svensson, Sarah L (Ed.)

   ABSTRACT In starvingBacillus subtilisbacteria,the initiation of two survival programs—biofilm formation and sporulation—is controlled by the same phosphorylated master regulator, Spo0A~P. Its gene,spo0A,is transcribed from two promoters, Pvand Ps,that are, respectively, regulated by RNA polymerase (RNAP) holoenzymes bearing σ^Aand σ^H. Notably, transcription is directly autoregulated by Spo0A~P binding sites known as 0A1, 0A2, and 0A3 box, located in between the two promoters. It remains unclear whether, at the onset of starvation, these boxes activate or repressspo0Aexpression, and whether the Spo0A~P transcriptional feedback plays a role in the increase inspo0Aexpression. Based on the experimental data of the promoter activities under systematic perturbation of the promoter architecture, we developed a biophysical model of transcriptional regulation ofspo0Aby Spo0A~P binding to each of the 0A boxes. The model predicts that Spo0A~P binding to its boxes does not affect the RNAP recruitment to the promoters but instead affects the transcriptional initiation rate. Moreover, the effects of Spo0A~P binding to 0A boxes are mainly repressive and saturated early at the onset of starvation. Therefore, the increase inspo0Aexpression is mainly driven by the increase in RNAP holoenzyme levels. Additionally, we reveal that Spo0A~P affinity to 0A boxes is strongest at 0A3 and weakest at 0A2 and that there are attractive forces between the occupied 0A boxes. Our findings, in addition to clarifying how the sporulation master regulator is controlled, offer a framework to predict regulatory outcomes of complex gene-regulatory mechanisms. IMPORTANCECell differentiation is often critical for survival. In bacteria, differentiation decisions are controlled by transcriptional master regulators under transcriptional feedback control. Therefore, understanding how master regulators are transcriptionally regulated is required to understand differentiation. However, in many cases, the underlying regulation is complex, with multiple transcription factor binding sites and multiple promoters, making it challenging to dissect the exact mechanisms. Here, we address this problem for theBacillus subtilismaster regulator Spo0A. Using a biophysical model, we quantitatively characterize the effect of individual transcription factor binding sites on eachspo0Apromoter. Furthermore, the model allows us to identify the specific transcription step that is affected by transcription factor binding. Such a model is promising for the quantitative study of a wide range of master regulators involved in transcriptional feedback. 

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4. Genome content reorganization in the non-model ciliate Chilodonella uncinata : insights into nuclear architecture, DNA content, and chromosome fragmentation during macronuclear development

   https://doi.org/10.1128/msphere.00075-25  

   Ahsan, Ragib; Maurer-Alcalá, Xyrus X; Katz, Laura A (June 2025, mSphere)

   Ciliates are a model lineage for studies of genome architecture given their unusual genome structures. All ciliates have both somatic macronuclei (MAC) and germline micronuclei (MIC), both of which develop from a zygotic nucleus following sex (i.e., conjugation). Nuclear developmental stages are not well documented among non-model ciliates, includingChilodonella uncinata(class Phyllopharyngea), the focus of our work. Here, we characterize nuclear architecture and genome dynamics inC. uncinataby combining 4′,6-diamidino-2-phenylindole (DAPI) staining and fluorescencein situhybridization (FISH) techniques with confocal microscopy. We developed a telomere probe for staining, which alongside DAPI allows for the identification of fragmented somatic chromosomes among the total DNA in the nuclei. We quantify both total DNA and telomere-bound signals from more than 250 nuclei sampled from 116 individual cells, and analyze changes in DNA content and nuclear architecture acrossChilodonella’s nuclear life cycle. Specifically, we find that MAC developmental stages in the ciliateC. uncinataare different from those reported from other ciliate species. These data provide insights into nuclear dynamics during development and enrich our understanding of genome evolution in non-model ciliates. IMPORTANCECiliates are a clade of diverse single-celled eukaryotic microorganisms that contain at least one somatic macronucleus (MAC) and germline micronucleus (MIC) within each cell/organism. Ciliates rely on complex genome rearrangements to generate somatic genomes from a zygotic nucleus. However, the development of somatic nuclei has only been documented for a few model ciliate genera, includingParamecium,Tetrahymena, andOxytricha. Here, we study the MAC developmental process in the non-model ciliate,C. uncinata. We analyze both total DNA and the generation of gene-sized somatic chromosomes using a laser scanning confocal microscope to describeC. uncinata’s nuclear life cycle. We show that DNA content changes dramatically during their life cycle and in a manner that differs from previous studies on model ciliates. Our study expands knowledge of genome dynamics in ciliates and among eukaryotes more broadly. 

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5. The virulence regulator bvgS controls nutrient-induced filamentation in Bordetella avium

   https://doi.org/10.1128/jb.00030-25  

   Perslow, Niklas G; Meadows-Graves, Serena J; Luallen, Robert J (September 2025, Journal of Bacteriology)

   Kendall, Melissa M (Ed.)

   ABSTRACT Bacteria can change morphology in response to stressors and changes in their environment, including infection of a host. We previously identified the bacterial species,Bordetella atropi, which uses nutrient-induced filamentation as a novel mechanism for cell-to-cell spreading in the intestinal epithelial cells of a nematode host. To further investigate the conservation of nutrient-induced filamentation in Bordetellae, we utilized the turkey-infecting speciesBordetella avium,which filamentsin vitrowhen switched from a standard growth media to an enriched media. We conducted a selection-based filamentation screen withB. aviumand isolated two independent non-filamentous mutants that failed to filament in highly enriched media. These mutants contained different alleles inbvgS,the sensor in the two-component master virulence regulator (BvgAS) conserved across theBordetellagenus. To investigate the role ofbvgSin nutrient-induced filamentation, we conducted transcriptomics and found that our allele ofbvgSresulted in loss of responsiveness to highly enriched media, especially in genes related to nutrient uptake and metabolism. The most dysregulated gene in thebvgSmutant encoded for succinyl-CoA:acetate CoA-transferase, and we were able to regulate filamentation with exogenous metabolites up and downstream of this enzyme. These data suggest thatbvgSregulates nutrient-induced filamentation by controlling metabolic capacity. Overall, we found that the virulence regulatorbvgScan control nutrient-induced filamentation inB. avium,suggesting there may be conservation in Bordetellae for utilizing this morphological change as a virulence phenotype.IMPORTANCEBordetella aviumis the causative agent of bordetellosis, an infectious disease affecting the respiratory system of birds, significantly increasing morbidity in poultry, ultimately leading to economic losses. It is long known that the pathogenesis ofB. aviumis governed by the two-component master virulence regulator, BvgAS. However, this regulon has never before been associated with nutrient-induced filamentation. In this study, we identify BvgS to be regulating nutrient-induced filamentation. We also report the first transcriptomics analysis of filamentousB. avium, showing the enzyme succinyl-CoA:acetate CoA-transferase may be involved in a metabolic shift in enriched nutrient conditions leading to filamentation. Our results suggest that virulence inB. aviumis a dynamic relationship, affected by nutrient availability, rather than a simple binary decision. 

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