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1. 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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2. 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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3. Receptor-associated kinases control the lipid provisioning program in plant–fungal symbiosis

   https://doi.org/10.1126/science.ade1124  

   Ivanov, Sergey; Harrison, Maria J (January 2024, Science)

   The mutualistic association between plants and arbuscular mycorrhizal (AM) fungi requires intracellular accommodation of the fungal symbiont and maintenance by means of lipid provisioning. Symbiosis signaling through lysin motif (LysM) receptor-like kinases and a leucine-rich repeat receptor-like kinase DOES NOT MAKE INFECTIONS 2 (DMI2) activates transcriptional programs that underlie fungal passage through the epidermis and accommodation in cortical cells. We show that twoMedicago truncatulacortical cell–specific, membrane-bound proteins of a CYCLIN-DEPENDENT KINASE-LIKE (CKL) family associate with, and are phosphorylation substrates of, DMI2 and a subset of the LysM receptor kinases.CKL1andCKL2are required for AM symbiosis and control expression of transcription factors that regulate part of the lipid provisioning program. Onset of lipid provisioning is coupled with arbuscule branching and with the REDUCED ARBUSCULAR MYCORRHIZA 1 (RAM1) regulon for complete endosymbiont accommodation. 

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4. Mechanism underlying the DNA-binding preferences of the Vibrio cholerae and vibriophage VP882 VqmA quorum-sensing receptors

   https://doi.org/10.1371/journal.pgen.1009550  

   Duddy, Olivia P.; Huang, Xiuliang; Silpe, Justin E.; Bassler, Bonnie L. (July 2021, PLOS Genetics)

   Crosson, Sean (Ed.)

   Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. In the global pathogen Vibrio cholerae , one quorum-sensing receptor and transcription factor, called VqmA (VqmA Vc ), activates expression of the vqmR gene encoding the small regulatory RNA VqmR, which represses genes involved in virulence and biofilm formation. Vibriophage VP882 encodes a VqmA homolog called VqmA Phage that activates transcription of the phage gene qtip , and Qtip launches the phage lytic program. Curiously, VqmA Phage can activate vqmR expression but VqmA Vc cannot activate expression of qtip . Here, we investigate the mechanism underlying this asymmetry. We find that promoter selectivity is driven by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. A protein sequence-guided mutagenesis approach revealed that the residue E194 of VqmA Phage and A192, the equivalent residue in VqmA Vc , in the helix-turn-helix motifs contribute to promoter-binding specificity. A genetic screen to identify VqmA Phage mutants that are incapable of binding the qtip promoter but maintain binding to the vqmR promoter delivered additional VqmA Phage residues located immediately C-terminal to the helix-turn-helix motif as required for binding the qtip promoter. Surprisingly, these residues are conserved between VqmA Phage and VqmA Vc . A second, targeted genetic screen revealed a region located in the VqmA Vc DNA-binding domain that is necessary to prevent VqmA Vc from binding the qtip promoter, thus restricting DNA binding to the vqmR promoter. We propose that the VqmA Vc helix-turn-helix motif and the C-terminal flanking residues function together to prohibit VqmA Vc from binding the qtip promoter. 

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5. An N-terminal domain specifies developmental control by the SMAX1-LIKE family of transcriptional regulators in Arabidopsis thaliana

   https://doi.org/10.1073/pnas.2412793122  

   Chang, Sun Hyun; George, Wesley J; Nelson, David C (June 2025, Proceedings of the National Academy of Sciences)

   SMAX1-LIKE (SMXL) proteins in plants are cellular signaling hubs, many of which are posttranslationally regulated by karrikins from smoke, the plant hormones strigolactones (SLs), and/or cues such as light and nutrients. SMXL proteins control diverse aspects of growth, development, and environmental adaptation in plants through transcriptional corepression and interactions with transcriptional regulator proteins. In flowering plants, the SMXL family comprises four phylogenetic clades with different roles. Functions of the aSMAX1 clade include control of germination and seedling development, while the SMXL78 clade controls shoot architecture. We investigated how SMXL roles are specified inArabidopsis thaliana.Through promoter-swapping experiments, we found thatSMXL7can partially replicateSMAX1function, butSMAX1cannot replaceSMXL7. This implies that the distinct roles of these genes are primarily due to differences in protein sequences rather than expression patterns. To determine which part of SMXL proteins specifies downstream control, we tested a series of protein chimeras and domain deletions of SMAX1 and SMXL7. We found an N-terminal region that is necessary and sufficient to specify control of germination, seedling growth, or axillary branching. We screened 158 transcription factors (TFs) for interactions with SMAX1 and SMXL7 in yeast two-hybrid assays. The N-terminal domain was necessary and/or sufficient for most of the 33 potential protein–protein interactions that were identified for SMAX1. This finding unlocks different ways to engineer plant growth control through cross-wiring SMXL regulatory “input” and developmental “output” domains from different clades and lays a foundation for understanding how functional differences evolved in the SMXL family. 

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