Surface-attached bacterial biofilms are self-replicating active liquid crystals and the dominant form of bacterial life on earth ( 1 – 4 ). In conventional liquid crystals and solid-state materials, the interaction potentials between the molecules that comprise the system determine the material properties. However, for growth-active biofilms it is unclear whether potential-based descriptions can account for the experimentally observed morphologies, and which potentials would be relevant. Here, we overcame previous limitations of single-cell imaging techniques ( 5 , 6 ) to reconstruct and track all individual cells inside growing three-dimensional (3D) biofilms with up to 10,000 individuals. Based on these data, we identify, constrain, and provide a microscopic basis for an effective cell-cell interaction potential, which captures and predicts the growth dynamics, emergent architecture, and local liquid crystalline order of Vibrio cholerae biofilms. Furthermore, we show how external fluid flows control the microscopic structure and 3D morphology of biofilms. Our analysis implies that local cellular order and global biofilm architecture in these active bacterial communities can arise from mechanical cell-cell interactions, which cells can modulate by regulating the production of particular matrix components. These results establish an experimentally validated foundation for improved continuum theories of active matter and thereby contribute to solving the important problem of controlling biofilm growth.
Biofilms are microbial communities that represent a highly abundant form of microbial life on Earth. Inside biofilms, phenotypic and genotypic variations occur in three-dimensional space and time; microscopy and quantitative image analysis are therefore crucial for elucidating their functions. Here, we present BiofilmQ—a comprehensive image cytometry software tool for the automated and high-throughput quantification, analysis and visualization of numerous biofilm-internal and whole-biofilm properties in three-dimensional space and time.
Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default “OFF” state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.
SignificanceMost living systems, from individual cells to tissues and swarms, display collective self-organization on length scales that are much larger than those of the individual units that drive this organization. A fundamental challenge is to understand how properties of microscopic components determine macroscopic, multicellular biological function. Our study connects intracellular physiology to macroscale collective behaviors during multicellular development, spanning five orders of magnitude in length and six orders of magnitude in time, using bacterial swarming as a model system. This work is enabled by a high-throughput adaptive microscopy technique, which we combined with genetics, machine learning, and mathematical modeling to reveal the phase diagram of bacterial swarming and that cell–cell interactions within each swarming phase are dominated by mechanical interactions.
The SKN7 two-component response regulator gene of Candida albicans was deleted, and the phenotype of the mutant was established. This mutant exhibited impaired growth on Spider agar and 10% serum agar compared to wild-type and gene-reconstituted strains. The skn7 mutant was sensitive to H2O2 in vitro, but its virulence was only mildly attenuated. A comparison of the Skn7p and Ssk1p response regulators of C. albicans is discussed
SummaryBacteria can generate benefits for themselves and their kin by living in multicellular, matrix-enclosed communities, termed biofilms, which are fundamental to microbial ecology and the impact bacteria have on the environment, infections, and industry [1, 2, 3, 4, 5, 6]. The advantages of the biofilm mode of life include increased stress resistance and access to concentrated nutrient sources [3, 7, 8]. However, there are also costs associated with biofilm growth, including the metabolic burden of biofilm matrix production, increased resource competition, and limited mobility inside the community [9, 10, 11]. The decision-making strategies used by bacteria to weigh the costs between remaining in a biofilm or actively dispersing are largely unclear, even though the dispersal transition is a central aspect of the biofilm life cycle and critical for infection transmission [12, 13, 14]. Using a combination of genetic and novel single-cell imaging approaches, we show that Vibrio cholerae integrates dual sensory inputs to control the dispersal response: cells use the general stress response, which can be induced via starvation, and they also integrate information about the local cell density and molecular transport conditions in the environment via the quorum sensing apparatus. By combining information from individual (stress response) and collective (quorum sensing) avenues of sensory input, biofilm-dwelling bacteria can make robust decisions to disperse from large biofilms under distress, while preventing premature dispersal when biofilm populations are small. These insights into triggers and regulators of biofilm dispersal are a key step toward actively inducing biofilm dispersal for technological and medical applications, and for environmental control of biofilms.
Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity.
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