Browsing by Author "Fujita, Masaya"

Now showing 1 - 5 of 5
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    An Engineered B. subtilis Inducible Promoter System with over 10 000-Fold Dynamic Range
    (American Chemical Society, 2019) Castillo-Hair, Sebastian M.; Fujita, Masaya; Igoshin, Oleg A.; Tabor, Jeffrey J.; Center for Theoretical Biological Physics
    Bacillus subtilis is the leading model Gram-positive bacterium, and a widely used chassis for industrial protein production. However, B. subtilis research is limited by a lack of inducible promoter systems with low leakiness and high dynamic range. Here, we engineer an inducible promoter system based on the T7 RNA Polymerase (T7 RNAP), the lactose repressor LacI, and the chimeric promoter PT7lac, integrated as a single copy in the B. subtilis genome. In the absence of IPTG, LacI strongly represses T7 RNAP and PT7lac and minimizes leakiness. Addition of IPTG derepresses PT7lac and simultaneously induces expression of T7RNAP, which results in very high output expression. Using green fluorescent and β-galactosidase reporter proteins, we estimate that this LacI-T7 system can regulate expression with a dynamic range of over 10 000, by far the largest reported for an inducible B. subtilis promoter system. Furthermore, LacI-T7 responds to similar IPTG concentrations and with similar kinetics as the widely used Phy-spank IPTG-inducible system, which we show has a dynamic range of at most 300 in a similar genetic context. Due to its superior performance, our LacI-T7 system should have broad applications in fundamental B. subtilis biology studies and biotechnology.
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    Chromosomal Arrangement of Phosphorelay Genes Couples Sporulation and DNA Replication
    (Elsevier, 2015) Narula, Jatin; Kuchina, Anna; Lee, Dong-yeon; Fujita, Masaya; Süel, Gürol M.; Igoshin, Oleg A.
    Genes encoding proteins in a common regulatory network are frequently located close to one another on the chromosome to facilitate co-regulation or couple gene expression to growth rate. Contrasting with these observations, here, we demonstrate a functional role for the arrangement of Bacillus subtilis sporulation network genes on opposite sides of the chromosome. We show that the arrangement of two sporulation network genes, one located close to the origin and the other close to the terminus, leads to a transient gene dosage imbalance during chromosome replication. This imbalance is detected by the sporulation network to produce cell-cycle coordinated pulses of the sporulation master regulator Spo0A∼P. This pulsed response allows cells to decide between sporulation and continued vegetative growth during each cell cycle spent in starvation. The simplicity of this coordination mechanism suggests that it may be widely applicable in a variety of gene regulatory and stress-response settings.
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    Optogenetic control of Bacillus subtilis gene expression
    (Springer Nature, 2019) Castillo-Hair, Sebastian M.; Baerman, Elliot A.; Fujita, Masaya; Igoshin, Oleg A.; Tabor, Jeffrey J.
    The Gram-positive bacterium Bacillus subtilis exhibits complex spatial and temporal gene expression signals. Although optogenetic tools are ideal for studying such processes, none has been engineered for this organism. Here, we port a cyanobacterial light sensor pathway comprising the green/red photoreversible two-component system CcaSR, two metabolic enzymes for production of the chromophore phycocyanobilin (PCB), and an output promoter to control transcription of a gene of interest into B. subtilis. Following an initial non-functional design, we optimize expression of pathway genes, enhance PCB production via a translational fusion of the biosynthetic enzymes, engineer a strong chimeric output promoter, and increase dynamic range with a miniaturized photosensor kinase. Our final design exhibits over 70-fold activation and rapid response dynamics, making it well-suited to studying a wide range of gene regulatory processes. In addition, the synthetic biology methods we develop to port this pathway should make B. subtilis easier to engineer in the future.
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    Slowdown of growth controls cellular differentiation
    (EMBO Press, 2016) Narula, Jatin; Kuchina, Anna; Zhang, Fang; Fujita, Masaya; Süel, Gürol M.; Igoshin, Oleg A.
    How can changes in growth rate affect the regulatory networks behavior and the outcomes of cellular differentiation? We address this question by focusing on starvation response in sporulating Bacillus subtilis. We show that the activity of sporulation master regulator Spo0A increases with decreasing cellular growth rate. Using a mathematical model of the phosphorelay—the network controlling Spo0A—we predict that this increase in Spo0A activity can be explained by the phosphorelay protein accumulation and lengthening of the period between chromosomal replication events caused by growth slowdown. As a result, only cells growing slower than a certain rate reach threshold Spo0A activity necessary for sporulation. This growth threshold model accurately predicts cell fates and explains the distribution of sporulation deferral times. We confirm our predictions experimentally and show that the concentration rather than activity of phosphorelay proteins is affected by the growth slowdown. We conclude that sensing the growth rates enables cells to indirectly detect starvation without the need for evaluating specific stress signals.
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    Ultrasensitivity of the Bacillus subtilis sporulation decision
    (National Academy of Sciences, 2012) Narula, Jatin; Devi, Seram N.; Fujita, Masaya; Igoshin, Oleg A.
    Starving Bacillus subtilis cells execute a gene expression program resulting in the formation of stress-resistant spores. Sporulation master regulator, Spo0A, is activated by a phosphorelay and controls the expression of a multitude of genes, including the forespore- specific sigma factor σF and the mother cell-specific sigma factor σE. Identification of the system-level mechanism of the sporulation decision is hindered by a lack of direct control over Spo0A activity. This limitation can be overcome by using a synthetic system in which Spo0A activation is controlled by inducing expression of phosphorelay kinase KinA. This induction results in a switch-like increase in the number of sporulating cells at a threshold of KinA. Using a combination of mathematical modeling and single-cell microscopy, we investigate the origin and physiological significance of this ultrasensitive threshold. The results indicate that the phosphorelay is unable to achieve a sufficiently fast and ultrasensitive response via its positive feedback architecture, suggesting that the sporulation decision is made downstream. In contrast, activation of σF in the forespore and of σE in the mother cell compartments occurs via a cascade of coherent feed-forward loops, and thereby can produce fast and ultrasensitive responses as a result of KinA induction. Unlike σF activation, σE activation in the mother cell compartment only occurs above the KinA threshold, resulting in completion of sporulation. Thus, ultrasensitive σE activation explains the KinA threshold for sporulation induction. We therefore infer that under uncertain conditions, cells initiate sporulation but postpone making the sporulation decision to average stochastic fluctuations and to achieve a robust population response.