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1. Experimental evolution of Bacillus subtilis: Experimental evolution of Bacillus subtilis

   https://doi.org/10.1111/1462-2920.13831  

   Zeigler, Daniel R.; Nicholson, Wayne L. (September 2017, Environmental Microbiology)
2. Genome Editing Methods for Bacillus subtilis

   https://doi.org/10.1007/978-1-0716-2233-9_11  

   Wozniak, Katherine J; Simmons, Lyle A (July 2022, Methods in molecular biology)

   Reisch, Christopher R (Ed.)

   Bacillus subtilis is a widely studied Gram-positive bacterium that serves as an important model for understanding processes critical for several areas of biology including biotechnology and human health. B. subtilis has several advantages as a model organism: it is easily grown under laboratory conditions, it has a rapid doubling time, it is relatively inexpensive to maintain, and it is nonpathogenic. Over the last 50 years, advancements in genetic engineering have continued to make B. subtilis a genetic workhorse in scientific discovery. In this chapter, we describe methods for traditional gene disruptions, use of gene deletion libraries from the Bacillus Genetic Stock Center, allelic exchange, CRISPRi, and CRISPR/Cas9. Additionally, we provide general materials and equipment needed, strengths and limitations, time considerations, and troubleshooting notes to perform each method. Use of the methods outlined in this chapter will allow researchers to create gene insertions, deletions, substitutions, and RNA interference strains through a variety of methods custom to each application. 

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3. Deciphering metabolic differentiation during Bacillus subtilis sporulation

   https://doi.org/10.1038/s41467-024-55586-z  

   Tibocha-Bonilla, Juan_D; Lyda, Jelani; Riley, Eammon; Pogliano, Kit; Zengler, Karsten (January 2025, Nature Communications)

   Abstract The bacteriumBacillus subtilisundergoes asymmetric cell division during sporulation, producing a mother cell and a smaller forespore connected by the SpoIIQ-SpoIIIA (or Q-A) channel. The two cells differentiate metabolically, and the forespore becomes dependent on the mother cell for essential building blocks. Here, we investigate the metabolic interactions between mother cell and forespore using genome-scale metabolic and expression models as well as experiments. Our results indicate that nucleotides are synthesized in the mother cell and transported in the form of nucleoside di- or tri-phosphates to the forespore via the Q-A channel. However, if the Q-A channel is inactivated later in sporulation, then glycolytic enzymes can form an ATP and NADH shuttle, providing the forespore with energy and reducing power. Our integrated in silico and in vivo approach sheds light into the intricate metabolic interactions underlying cell differentiation inB. subtilis, and provides a foundation for future studies of metabolic differentiation. 

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4. Diadenosine-tetraphosphate regulates biosynthesis of GTP in Bacillus subtilis

   https://doi.org/10.1038/s41564-022-01193-x  

   Giammarinaro Pietro I.; Young, Megan; Steinchen, Wieland; Mais, Christopher N.; Hochberg, Georg; Yang, Jin; Stevenson, David; Amador-Noguez, Daniel; Paulus, Anja; Wang, Jue (July 2022, Nature microbiology)

   Emma Overmaat (Ed.)

   Diadenosine tetraphosphate (Ap4A) is a putative second messenger molecule that is conserved from bacteria to man. Nevertheless, its physiological role, and the underlying molecular mechanisms, are poorly characterized. We investigated the molecular mechanism by which Ap4A regulates inosine-5’-monophosphate dehydrogenase (IMPDH, a key branching point enzyme for the biosynthesis of adenosine or guanosine nucleotides) in Bacillus subtilis. We solved the crystal structure of BsIMPDH bound to Ap4A at a resolution of 2.45 Å to show that Ap4A binds to the interface between two IMPDH subunits, acting as the glue that switches active IMPDH tetramers into less active octamers. Guided by these insights, we engineered mutant strains of B. subtilis that bypass Ap4A-dependent IMPDH regulation without perturbing intracellular Ap4A pools themselves. We used metabolomics suggesting that these mutants have a dysregulated purine, and in particular GTP, metabolome and phenotypic analysis showing increased sensitivity of B. subtilis IMPDH mutant strains to heat compared with wild-type. Our study identifies a central role for IMPDH in remodelling metabolism and heat resistance, and provides evidence that Ap4A can function as an alarmone. 

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5. 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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