Dataset Information

Site-directed mutagenesis of the quorum-sensing transcriptional regulator SinR affects the biosynthesis of menaquinone in Bacillus subtilis.

ABSTRACT:

Background

Menaquinone (MK-7) is a highly valuable vitamin K₂ produced by Bacillus subtilis. Common static metabolic engineering approaches for promoting the production of MK-7 have been studied previously. However, these approaches caused an accumulation of toxic substances and reduced product yield. Hence, dynamic regulation by the quorum sensing (QS) system is a promising method for achieving a balance between product synthesis and cell growth.

Results

In this study, the QS transcriptional regulator SinR, which plays a significant role in biofilm formation and MK production simultaneously, was selected, and its site-directed mutants were constructed. Among these mutants, sinR knock out strain (KO-SinR) increased the biofilm biomass by 2.8-fold compared to the wild-type. SinR^quad maximized the yield of MK-7 (102.56 ± 2.84 mg/L). To decipher the mechanism of how this mutant regulates MK-7 synthesis and to find additional potential regulators that enhance MK-7 synthesis, RNA-seq was used to analyze expression changes in the QS system, biofilm formation, and MK-7 synthesis pathway. The results showed that the expressions of tapA, tasA and epsE were up-regulated 9.79-, 0.95-, and 4.42-fold, respectively. Therefore, SinR^quad formed more wrinkly and smoother biofilms than BS168. The upregulated expressions of glpF, glpk, and glpD in this biofilm morphology facilitated the flow of glycerol through the biofilm. In addition, NADH dehydrogenases especially sdhA, sdhB, sdhC and glpD, increased 1.01-, 3.93-, 1.87-, and 1.11-fold, respectively. The increased expression levels of NADH dehydrogenases indicated that more electrons were produced for the electron transport system. Electrical hyperpolarization stimulated the synthesis of the electron transport chain components, such as cytochrome c and MK, to ensure the efficiency of electron transfer. Wrinkly and smooth biofilms formed a network of interconnected channels with a low resistance to liquid flow, which was beneficial for the uptake of glycerol, and facilitated the metabolic flux of four modules of the MK-7 synthesis pathway.

Conclusions

In this study, we report for the first time that SinR^quad has significant effects on MK-7 synthesis by forming wrinkly and smooth biofilms, upregulating the expression level of most NADH dehydrogenases, and providing higher membrane potential to stimulate the accumulation of the components in the electron transport system.

SUBMITTER: Wu J

PROVIDER: S-EPMC8183045 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Biofilm formation by Bacillus subtilis requires the expression of genes encoding enzymes for extracellular polysaccharide synthesis and for an amyloid-like protein. The master regulator SinR represses all the corresponding genes, and repression of these key biofilm genes is lifted when SinR interacts with its cognate antagonist proteins. The YmdB phosphodiesterase is a recently discovered factor that is involved in the control of SinR activity: cells lacking YmdB exhibit hyperactive SinR and are unable to relieve the repression of the biofilm genes. In this study, we have examined the dynamics of gene expression patterns in wild-type and ymdB mutant cells by microfluidic analysis coupled to time-lapse microscopy. Our results confirm the bistable expression pattern for motility and biofilm genes in the wild-type strain and the loss of biofilm gene expression in the mutant. Moreover, we demonstrated dynamic behavior in subpopulations of the wild-type strain that is characterized by switches in sets of the expressed genes. In order to gain further insights into the role of YmdB, we isolated a set of spontaneous suppressor mutants derived from ymdB mutants that had regained the ability to form complex colonies and biofilms. Interestingly, all of the mutations affected SinR. In some mutants, large genomic regions encompassing sinR were deleted, whereas others had alleles encoding SinR variants. Functional and biochemical studies with these SinR variants revealed how these proteins allowed biofilm gene expression in the ymdB mutant strains.IMPORTANCE Many bacteria are able to choose between two mutually exclusive lifestyles: biofilm formation and motility. In the model bacterium Bacillus subtilis, this choice is made by each individual cell rather than at the population level. The transcriptional repressor SinR is the master regulator in this decision-making process. The regulation of SinR activity involves complex control of its own expression and of its interaction with antagonist proteins. We show that the YmdB phosphodiesterase is required to allow the expression of SinR-repressed genes in a subpopulation of cells and that such subpopulations can switch between different SinR activity states. Suppressor analyses revealed that ymdB mutants readily acquire mutations affecting SinR, thus restoring biofilm formation. These findings suggest that B. subtilis cells experience selective pressure to form the extracellular matrix that is characteristic of biofilms and that YmdB is required for the homeostasis of SinR and/or its antagonists.