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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. 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. The biocontrol potential of endophyte Bacillus velezensis to reduce post-harvest tomato infection caused by Rhizopus microsporus

   https://doi.org/10.1128/spectrum.01064-25  

   Kock, Alicia; Napo, Mmanoko; Viviers, Dionné; Akinmoladun, Oluwakemi V; Alayande, Kazeem A; Yusuf, Abdullahi; Uehling, Jessie; Pawlowska, Teresa E; Adeleke, Rasheed A (October 2025, Microbiology Spectrum)

   Hockett, Kevin Loren (Ed.)

   ABSTRACT Rhizopus microsporusis a necrotrophic post-harvest pathogen that causes significant economic losses in the agricultural sector. To explore alternatives to conventional management strategies for the mitigation of post-harvest infections, we investigated the potential of two previously identified endophyticBacillus velezensisstrains as biological control agents. Throughin vitroandin vivoexperiments, we examined the mechanisms of biocontrol displayed by twoB. velezensisstrains (KV10 and KV15) against threeR. microsporusstrains (W2-50, W2-51, and W2-58).In vitroassays assessed co-cultivability and the inhibitory effects ofB. velezensisagainstR. microsporus. The results demonstrated strain-specific antifungal activity with a reduction in fungal growth across treatments. Further analysis revealed that volatile organic compounds produced byB. velezensiscontributed to its antifungal properties. To evaluate the biocontrol efficacyin vivo, tomato fruits were inoculated withR. microsporusand subsequently treated withB. velezensis. The results support the strain-specific reduction in tomato spoilage, yielding various spoilage rates observed across treatments. Our findings highlight the potential ofB. velezensisas a promising biocontrol agent for the management ofR. microsporuspost-harvest infections in tomatoes. Further research is warranted to optimize the applicationof B. velezensisas a sustainable and environmentally friendly approach for controlling post-harvest diseases in tomatoes.IMPORTANCEOur study shows the significance of improving sustainable agriculture by offering an alternative to the use of chemical fungicides in post-harvest applications. Opportunistic fungal pathogens likeRhizopus microsporuscan have detrimental effects on post-harvest commodities like tomatoes. Post-harvest fungal infections are mainly controlled by chemical fungicides that pose health risks to humans and the environment. Utilizing biocontrol agents provides an environmentally safe alternative. Understanding the mechanisms of biocontrol employed by beneficial bacteria likeBacillus velezensison fungal pathogens gives insight into safer, more environmentally friendly alternatives to protect food crops. Our results suggest that targeted microbial solutions can mitigate post-harvest losses. 

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4. Cyanoexosortase B is essential for motility, biofilm formation, and scytonemin production in a filamentous cyanobacterium

   https://doi.org/10.1128/msphere.01006-24  

   Parrett, Gabriel A; Haft, Daniel H; Ruiz, Maida; Garcia-Pichel, Ferran; Ebmeier, Christopher C; Risser, Douglas D (June 2025, mSphere)

   Ellermeier, Craig D (Ed.)

   ABSTRACT Exosortases are involved in trafficking proteins containing PEP-CTERM domains to the exterior of gram-negative bacterial cells. The role of these proteins in cyanobacteria, where such homologs are common, has not been defined. The filamentous cyanobacteriumNostoc punctiformecontains a single putative exosortase, designated cyanoexosortase B (CrtB), implicated by previous work both in motility and in the production of the UV-absorbing pigment, scytonemin. To determine the role ofcrtBinN. punctiforme, acrtB-deletion strain (ΔcrtB) was generated. ΔcrtBpresented the loss of motility, biofilm formation, and scytonemin production. In the case of motility, the ΔcrtBmutant exhibited a specific defect in the ability of hormogonia (specialized motile filaments) to adhere to hormogonium polysaccharide (HPS), and several PEP-CTERM proteins expressed in motile hormogonia were differentially abundant in the exoproteome of the wild-type compared with the ΔcrtBstrain. These results are consistent with the hypothetical role of CrtB in the processing and export of PEP-CTERM proteins that play a critical role in stabilizing the interaction between the filament surface and HPS to facilitate motility and biofilm formation. In the case of scytonemin—the late biosynthetic steps of which occur in the periplasm and whose operon contains several putative PEP-CTERM proteins—ΔcrtBfailed to produce it. Given the abundance of putative PEP-CTERM proteins encoded in theN. punctiformegenome and the fact that this study only associates a fraction of them with biological functions, it seems likely that CrtB may play an important role in other biological processes in cyanobacteria.IMPORTANCEIn gram-negative bacteria, exosortases facilitate the trafficking of proteins to the exterior of the cell where they have been implicated in stabilizing the association of extracellular polymeric substances (EPS) with the cell surface to facilitate biofilm formation and flocculation, but the role of exosortases in cyanobacteria has not been explored. Here, we characterize the role of cyanoexosortase B (CrtB) in the filamentous cyanobacteriumNostoc punctiforme, demonstrating thatcrtBis essential for motility, biofilm formation, and the production of the sunscreen pigment scytonemin. These findings have important implications for understanding motility and biofilm formation in filamentous cyanobacteria as well as efforts toward the heterologous production of scytonemin in non-native hosts. 

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5. Tersicoccus phoenicis (Actinobacteria), a spacecraft clean room isolate, exhibits dormancy

   https://doi.org/10.1128/spectrum.01692-25  

   Tirumalai, Madhan; Ali, Sahar; Fox, George E; Widger, William (August 2025, Microbiology Spectrum)

   Tischler, Dirk (Ed.)

   ABSTRACT Space missions or spacecraft equipment destined for sensitive environments, such as Mars, Europa, or Enceladus, are required to be designed to avoid forward contamination. Spacecraft are assembled in clean rooms (SACs) employing treatments to eliminate microbial contamination. However, some organisms can survive the cleaning procedures. Characterization of these populations, through both culture-based and sequencing methods, reveals that the majority consists of spore-forming bacteria. However, a smaller group of non-spore-forming organisms, primarily classified within the orderMicrococcalesof the phylumActinobacteria(Actinomycetota), exists in some SACs. Despite their repeated occurrence and isolation, actinobacterial strains associated with SACs have not been studied for their dormancy potential. Here, we show for the first time that a non-spore-forming SAC isolate,Tersicoccus phoenicis(Micrococcales), enters dormancy under nutrient starvation. Dormancy inMicrococcus luteusinvolves a universal stress protein and a resuscitation-promoting factor (Rpf). Genes for these proteins are widely found in actinobacteria, includingT. phoenicis. We show that dormantT. phoenicis(Micrococcales) can be revived through the addition of the Rpf to the media. Dormancy, as observed in the SAC actinobacterial isolateT. phoenicis, could well be a common trait adopted by other actinobacterial strains under the stressful conditions of spacecraft clean rooms or the ISS (International Space Station). This has implications for the persistence, identification, and recovery of such microbes from cleanroom facilities.IMPORTANCENASA’s long-range goal of a human mission to the Mars surface raises issues relating to planetary protection. The concerns of forward contamination require that spacecraft assembly clean rooms (SACs) be maintained to inhibit microbial survival. However, despite these efforts, distinct microbial communities persist. Here, we show that a SAC isolate,T. phoenicis, exhibits dormancy, a state in which the cells are viable but not cultivable. Dormancy may help non-spore-forming organisms survive in the clean room environments utilized for space missions. This has implications for improving cleaning procedures. 

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