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1. The Slowdown of Growth Rate Controls the Single-Cell Distribution of Biofilm Matrix Production via an SinI-SinR-SlrR Network

   https://doi.org/10.1128/msystems.00622-22  

   Chen, Zhuo; Zarazúa-Osorio, Brenda; Srivastava, Priyanka; Fujita, Masaya; Igoshin, Oleg A. (February 2023, mSystems)

   Kovács, Ákos T. (Ed.)

   ABSTRACT In Bacillus subtilis , master regulator Spo0A controls several cell-differentiation pathways. Under moderate starvation, phosphorylated Spo0A (Spo0A~P) induces biofilm formation by indirectly activating genes controlling matrix production in a subpopulation of cells via an SinI-SinR-SlrR network. Under severe starvation, Spo0A~P induces sporulation by directly and indirectly regulating sporulation gene expression. However, what determines the heterogeneity of individual cell fates is not fully understood. In particular, it is still unclear why, despite being controlled by a single master regulator, biofilm matrix production and sporulation seem mutually exclusive on a single-cell level. In this work, with mathematical modeling, we showed that the fluctuations in the growth rate and the intrinsic noise amplified by the bistability in the SinI-SinR-SlrR network could explain the single-cell distribution of matrix production. Moreover, we predicted an incoherent feed-forward loop; the decrease in the cellular growth rate first activates matrix production by increasing in Spo0A phosphorylation level but then represses it via changing the relative concentrations of SinR and SlrR. Experimental data provide evidence to support model predictions. In particular, we demonstrate how the degree to which matrix production and sporulation appear mutually exclusive is affected by genetic perturbations. IMPORTANCE The mechanisms of cell-fate decisions are fundamental to our understanding of multicellular organisms and bacterial communities. However, even for the best-studied model systems we still lack a complete picture of how phenotypic heterogeneity of genetically identical cells is controlled. Here, using B. subtilis as a model system, we employ a combination of mathematical modeling and experiments to explain the population-level dynamics and single-cell level heterogeneity of matrix gene expression. The results demonstrate how the two cell fates, biofilm matrix production and sporulation, can appear mutually exclusive without explicitly inhibiting one another. Such a mechanism could be used in a wide range of other biological systems. 

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2. Nascent flagellar basal bodies are immobilized by rod assembly in Bacillus subtilis

   https://doi.org/10.1128/mbio.00530-25  

   Dunn, Caroline M; Foust, Daniel J; Gao, Yongqiang; Biteen, Julie S; Shaw, Sidney L; Kearns, Daniel B (June 2025, mBio)

   Salama, Nina R (Ed.)

   ABSTRACT Flagella are complex, trans-envelope nanomachines that localize in species-specific patterns on the cell surface. Here, we study the localization dynamics of the earliest stage of basal body formation inBacillus subtilisusing a fluorescent fusion to the C-ring protein FliM. We find thatB. subtilisbasal bodies do not exhibit dynamic subunit exchange and are largely stationary at steady state, consistent with flagellar assembly through the peptidoglycan (PG). However, rare mobile basal bodies were observed, and the prevalence of mobile basal bodies is elevated both early in basal body assembly and when the rod is mutated. Thus, basal body mobility is a precursor to patterning, and we propose that rod polymerization probes the PG superstructure for pores of sufficient diameter to permit rod transit. Furthermore, mutation of the rod disrupts basal body patterning in a way that phenocopies mutation of the cytoplasmic flagellar patterning protein FlhF. We infer that rod synthesis and the cytoplasmic regulators coordinate flagellar assembly by interpreting a grid-like pattern of pores, pre-existent in the PG. IMPORTANCEBacteria insert flagella in a species-specific pattern on the cell body, but how patterns are achieved is poorly understood. In bacteria with a single polar flagellum, a marker protein localizes to the cell pole and nucleates the assembly of the flagellum at that site.Bacillus subtilisassembles ~25 basal bodies over the length of the cell in a grid-like pattern and lacks proteins required for their polar targeting. Here, we show thatB. subtilisbasal bodies are mobile soon after assembly and become immobilized when the flagellar rod transits the peptidoglycan (PG) wall. Moreover, defects in the flagellar rod lead to a more-random distribution of flagella and an increase in polar basal bodies. We conclude that the peritrichous patterning of flagella ofB. subtilisis different from the polar patterning of other bacteria, and we infer that theB. subtilisrod probes the PG for holes that can accommodate the machine. 

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3. Mfd deficiency decreases the abundance of complete transcripts of sporulation genes and alters sporogenesis and the structure of dormant Bacillus subtilis spores

   https://doi.org/10.3389/fmicb.2025.1680580  

   Martin, Holly Anne; Heron, Robert; Leyva-Sanchez, Hilda; Perez, Ryan King; Callaway, Amber; Ermi, Tatiana; Picard, Aude; Grifaldo, Jessica; Barnes, Jesse L; Pedraza-Reyes, Mario; et al (October 2025, Frontiers in Microbiology)

   Sporulation is a survival mechanism employed by Firmicutes, includingBacillus subtilis, when facing stressful conditions of growth (e.g., starvation). In this bacterium, the transcription repair coupling factor, Mfd, has been shown to play pivotal roles in sporulation transcription-coupled DNA repair and stress-associated mutagenesis. Recent studies have also revealed an unexpected role of Mfd in regulating gene expression duringB. subtilissporulation. This study examines the effects ofB. subtilisMfd deficiency on the expression of sporulation genes, sporulation efficiency, and spore morphology. In the absence of exogenous DNA damage, we found that Mfd deficiency does not compromise spore germination outgrowth; however, the loss of this factor promoted spore morphological defects and decreased sporulation efficiency. Also, our results confirmed an anomalous pattern of expression of sporulation genes in cells lacking Mfd. These results showed that Mfd influences bacterial physiology beyond DNA repair of actively transcribed genes. 

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4. 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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5. Bacillus subtilis Histidine Kinase KinC Activates Biofilm Formation by Controlling Heterogeneity of Single-Cell Responses

   https://doi.org/10.1128/mbio.01694-21  

   Chen, Zhuo; Srivastava, Priyanka; Zarazúa-Osorio, Brenda; Marathe, Anuradha; Fujita, Masaya; Igoshin, Oleg A. (February 2022, mBio)

   Zhulin, Igor B. (Ed.)

   ABSTRACT In Bacillus subtilis , biofilm and sporulation pathways are both controlled by a master regulator, Spo0A, which is activated by phosphorylation via a phosphorelay—a cascade of phosphotransfer reactions commencing with autophosphorylation of histidine kinases KinA, KinB, KinC, KinD, and KinE. However, it is unclear how the kinases, despite acting via the same regulator, Spo0A, differentially regulate downstream pathways, i.e., how KinA mainly activates sporulation genes and KinC mainly activates biofilm genes. In this work, we found that KinC also downregulates sporulation genes, suggesting that KinC has a negative effect on Spo0A activity. To explain this effect, with a mathematical model of the phosphorelay, we revealed that unlike KinA, which always activates Spo0A, KinC has distinct effects on Spo0A at different growth stages: during fast growth, KinC acts as a phosphate source and activates Spo0A, whereas during slow growth, KinC becomes a phosphate sink and contributes to decreasing Spo0A activity. However, under these conditions, KinC can still increase the population-mean biofilm matrix production activity. In a population, individual cells grow at different rates, and KinC would increase the Spo0A activity in the fast-growing cells but reduce the Spo0A activity in the slow-growing cells. This mechanism reduces single-cell heterogeneity of Spo0A activity, thereby increasing the fraction of cells that activate biofilm matrix production. Thus, KinC activates biofilm formation by controlling the fraction of cells activating biofilm gene expression. IMPORTANCE In many bacterial and eukaryotic systems, multiple cell fate decisions are activated by a single master regulator. Typically, the activities of the regulators are controlled posttranslationally in response to different environmental stimuli. The mechanisms underlying the ability of these regulators to control multiple outcomes are not understood in many systems. By investigating the regulation of Bacillus subtilis master regulator Spo0A, we show that sensor kinases can use a novel mechanism to control cell fate decisions. By acting as a phosphate source or sink, kinases can interact with one another and provide accurate regulation of the phosphorylation level. Moreover, this mechanism affects the cell-to-cell heterogeneity of the transcription factor activity and eventually determines the fraction of different cell types in the population. These results demonstrate the importance of intercellular heterogeneity for understanding the effects of genetic perturbations on cell fate decisions. Such effects can be applicable to a wide range of cellular systems. 

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