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1. Pan-genome-scale metabolic modeling of Bacillus subtilis reveals functionally distinct groups

   https://doi.org/10.1128/msystems.00923-24  

   Neal, Maxwell; Brakewood, William; Betenbaugh, Michael; Zengler, Karsten (November 2024, mSystems)

   Faust, Karoline (Ed.)

   ABSTRACT Bacillus subtilisis an important industrial and environmental microorganism known to occupy many niches and produce many compounds of interest. Although it is one of the best-studied organisms, much of this focus including the reconstruction of genome-scale metabolic models has been placed on a few key laboratory strains. Here, we substantially expand these prior models to pan-genome-scale, representing 481 genomes ofB. subtiliswith 2,315 orthologous gene clusters, 1,874 metabolites, and 2,239 reactions. Furthermore, we incorporate data from carbon utilization experiments for eight strains to refine and validate its metabolic predictions. This comprehensive pan-genome model enables the assessment of strain-to-strain differences related to nutrient utilization, fermentation outputs, robustness, and other metabolic aspects. Using the model and phenotypic predictions, we divideB. subtilisstrains into five groups with distinct patterns of behavior that correlate across these features. The pan-genome model offers deep insights intoB. subtilis’metabolism as it varies across environments and provides an understanding as to how different strains have adapted to dynamic habitats. IMPORTANCEAs the volume of genomic data and computational power have increased, so has the number of genome-scale metabolic models. These models encapsulate the totality of metabolic functions for a given organism.Bacillus subtilisstrain 168 is one of the first bacteria for which a metabolic network was reconstructed. Since then, several updated reconstructions have been generated for this model microorganism. Here, we expand the metabolic model for a single strain into a pan-genome-scale model, which consists of individual models for 481B. subtilisstrains. By evaluating differences between these strains, we identified five distinct groups of strains, allowing for the rapid classification of any particular strain. Furthermore, this classification into five groups aids the rapid identification of suitable strains for any application. 

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2. Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

   https://doi.org/10.1038/s41467-021-25691-4  

   Stork, Devon A.; Squyres, Georgia R.; Kuru, Erkin; Gromek, Katarzyna A.; Rittichier, Jonathan; Jog, Aditya; Burton, Briana M.; Church, George M.; Garner, Ethan C.; Kunjapur, Aditya M. (September 2021, Nature Communications)

   Abstract Bacillus subtilisis a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins inB. subtilis, here we report broad and efficient genetic code expansion inB. subtilisby incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences betweenE. coliandB. subtilisstop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system inB. subtiliswill help uncover an abundance of biological insights and aid genetic code expansion in other organisms. 

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3. Negative Interplay between Biofilm Formation and Competence in the Environmental Strains of Bacillus subtilis

   https://doi.org/10.1128/mSystems.00539-20  

   She, Qianxuan; Hunter, Evan; Qin, Yuxuan; Nicolau, Samantha; Zalis, Eliza A.; Wang, Hongkai; Chen, Yun; Chai, Yunrong (October 2020, mSystems)

   Methe, Barbara (Ed.)

   ABSTRACT Environmental strains of the soil bacterium Bacillus subtilis have valuable applications in agriculture, industry, and biotechnology; however, environmental strains are genetically less accessible. This reduced accessibility is in sharp contrast to laboratory strains, which are well known for their natural competence, and a limitation in their applications. In this study, we observed that robust biofilm formation by environmental strains of B. subtilis greatly reduced the frequency of competent cells in the biofilm. By using model strain 3610, we revealed a cross-pathway regulation that allows biofilm matrix producers and competence-developing cells to undergo mutually exclusive cell differentiation. We further demonstrated that the competence activator ComK represses the key biofilm regulatory gene sinI by directly binding to the sinI promoter, thus blocking competent cells from simultaneously becoming matrix producers. In parallel, the biofilm activator SlrR represses competence through three distinct mechanisms involving both genetic regulation and cell morphological changes. Finally, we discuss the potential implications of limiting competence in a bacterial biofilm. IMPORTANCE The soil bacterium Bacillus subtilis can form robust biofilms, which are important for its survival in the environment. B. subtilis also exhibits natural competence. By investigating competence development in B. subtilis in situ during biofilm formation, we reveal that robust biofilm formation often greatly reduces the frequency of competent cells within the biofilm. We then characterize a cross-pathway regulation that allows cells in these two developmental events to undergo mutually exclusive cell differentiation during biofilm formation. Finally, we discuss potential biological implications of limiting competence in a bacterial biofilm. 

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4. Bacillus subtilis Cell Differentiation, Biofilm Formation and Environmental Prevalence

   https://doi.org/10.3390/microorganisms10061108  

   Qin, Yuxuan; Angelini, Leticia Lima; Chai, Yunrong (June 2022, Microorganisms)

   Bacillus subtilis is a soil-dwelling, spore-forming Gram-positive bacterium capable of cell differentiation. For decades, B. subtilis has been used as a model organism to study development of specialized cell types. In this minireview, we discuss cell differentiation in B. subtilis, covering both past research and recent progresses, and the role of cell differentiation in biofilm formation and prevalence of this bacterium in the environment. We review B. subtilis as a classic model for studies of endospore formation, and highlight more recent investigations on cell fate determination and generation of multiple cell types during biofilm formation. We present mechanistic details of how cell fate determination and mutually exclusive cell differentiation are regulated during biofilm formation. 

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5. Metabolic Remodeling during Biofilm Development of Bacillus subtilis

   https://doi.org/10.1128/mBio.00623-19  

   Pisithkul, Tippapha; Schroeder, Jeremy W.; Trujillo, Edna A.; Yeesin, Ponlkrit; Stevenson, David M.; Chaiamarit, Tai; Coon, Joshua J.; Wang, Jue D.; Amador-Noguez, Daniel; Ghigo, Jean-Marc; et al (June 2019, mBio)

   ABSTRACT Biofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilis has been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilis biofilm development that will serve as a useful road map for future studies on biofilm physiology. IMPORTANCE Bacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such as Bacillus subtilis , very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism. 

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