skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.


Title: Negative Interplay between Biofilm Formation and Competence in the Environmental Strains of Bacillus subtilis
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.  more » « less
Award ID(s):
1651732
PAR ID:
10199526
Author(s) / Creator(s):
; ; ; ; ; ; ;
Editor(s):
Methe, Barbara
Date Published:
Journal Name:
mSystems
Volume:
5
Issue:
5
ISSN:
2379-5077
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. 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. 
    more » « less
  2. Kovács, Ákos T. (Ed.)
    ABSTRACT In Bacillus subtilis , master regulator Spo0A controls several cell-differentiation pathways. Under moderate starvation, phosphorylated Spo0A (Spo0A~P) induces biofilm formation by indirectly activating genes controlling matrix production in a subpopulation of cells via an SinI-SinR-SlrR network. Under severe starvation, Spo0A~P induces sporulation by directly and indirectly regulating sporulation gene expression. However, what determines the heterogeneity of individual cell fates is not fully understood. In particular, it is still unclear why, despite being controlled by a single master regulator, biofilm matrix production and sporulation seem mutually exclusive on a single-cell level. In this work, with mathematical modeling, we showed that the fluctuations in the growth rate and the intrinsic noise amplified by the bistability in the SinI-SinR-SlrR network could explain the single-cell distribution of matrix production. Moreover, we predicted an incoherent feed-forward loop; the decrease in the cellular growth rate first activates matrix production by increasing in Spo0A phosphorylation level but then represses it via changing the relative concentrations of SinR and SlrR. Experimental data provide evidence to support model predictions. In particular, we demonstrate how the degree to which matrix production and sporulation appear mutually exclusive is affected by genetic perturbations. IMPORTANCE The mechanisms of cell-fate decisions are fundamental to our understanding of multicellular organisms and bacterial communities. However, even for the best-studied model systems we still lack a complete picture of how phenotypic heterogeneity of genetically identical cells is controlled. Here, using B. subtilis as a model system, we employ a combination of mathematical modeling and experiments to explain the population-level dynamics and single-cell level heterogeneity of matrix gene expression. The results demonstrate how the two cell fates, biofilm matrix production and sporulation, can appear mutually exclusive without explicitly inhibiting one another. Such a mechanism could be used in a wide range of other biological systems. 
    more » « less
  3. Abstract Pulcherrimin is an iron-binding reddish pigment produced by various bacterial and yeast species. In the soil bacteriumBacillus subtilis, this pigment is synthesized intracellularly as the colorless pulcherriminic acid by using two molecules of tRNA-charged leucine as the substrate; pulcherriminic acid molecules are then secreted and bind to ferric iron extracellularly to form the red-colored pigment pulcherrimin. The biological importance of pulcherrimin is not well understood. A previous study showed that secretion of pulcherrimin caused iron depletion in the surroundings and growth arrest on cells located at the edge of aB. subtiliscolony biofilm. In this study, we identified that pulcherrimin is primarily produced under biofilm conditions and provides protection to cells in the biofilm against oxidative stress. We presented molecular evidence on how pulcherrimin lowers the level of reactive oxygen species (ROS) and alleviates oxidative stress and DNA damage caused by ROS accumulation in a mature biofilm. We also performed global transcriptome profiling to identify differentially expressed genes in the pulcherrimin-deficient mutant compared with the wild type, and further characterized the regulation of genes by pulcherrimin that are related to iron homeostasis, DNA damage response (DDR), and oxidative stress response. Based on our findings, we propose pulcherrimin as an important antioxidant that modulatesB. subtilisbiofilm development. 
    more » « less
  4. Parsek, Matthew (Ed.)
    ABSTRACT Many bacterial species typically live in complex three-dimensional biofilms, yet much remains unknown about differences in essential processes between nonbiofilm and biofilm lifestyles. Here, we created a CRISPR interference (CRISPRi) library of knockdown strains covering all known essential genes in the biofilm-forming Bacillus subtilis strain NCIB 3610 and investigated growth, biofilm colony wrinkling, and sporulation phenotypes of the knockdown library. First, we showed that gene essentiality is largely conserved between liquid and surface growth and between two media. Second, we quantified biofilm colony wrinkling using a custom image analysis algorithm and found that fatty acid synthesis and DNA gyrase knockdown strains exhibited increased wrinkling independent of biofilm matrix gene expression. Third, we designed a high-throughput screen to quantify sporulation efficiency after essential gene knockdown; we found that partial knockdowns of essential genes remained competent for sporulation in a sporulation-inducing medium, but knockdown of essential genes involved in fatty acid synthesis exhibited reduced sporulation efficiency in LB, a medium with generally lower levels of sporulation. We conclude that a subset of essential genes are particularly important for biofilm structure and sporulation/germination and suggest a previously unappreciated and multifaceted role for fatty acid synthesis in bacterial lifestyles and developmental processes. IMPORTANCE For many bacteria, life typically involves growth in dense, three-dimensional communities called biofilms that contain cells with differentiated roles held together by extracellular matrix. To examine how essential gene function varies between vegetative growth and the developmental states of biofilm formation and sporulation, we created and screened a comprehensive library of strains using CRISPRi to knockdown expression of each essential gene in the biofilm-capable Bacillus subtilis strain 3610. High-throughput assays and computational algorithms identified a subset of essential genes involved in biofilm wrinkling and sporulation and indicated that fatty acid synthesis plays important and multifaceted roles in bacterial development. 
    more » « less
  5. 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. 
    more » « less