The formation of multicellular microbial communities, called biofilms, starts from the adhesion of a few planktonic cells to the surface. The transition from a free-living planktonic lifestyle to a sessile, attached state is a multifactorial process that is determined by biological, chemical and physical properties of the environment, the surface and the bacterial cell. The initial weak, reversible interactions between a bacterium and a surface strengthen to yield irreversible adhesion. In this Review, we summarize our understanding of the mechanisms governing bacterial adhesion at the single-cell level, including the physical forces experienced by a cell before reaching the surface, the first contact with a surface and the transition from reversible to permanent adhesion.

Bacterial adhesion at the single-cell level

Bacteria within communities can interact to organize their behavior. It has been unclear whether such interactions can extend beyond a single community to coordinate the behavior of distant populations. We discovered that two Bacillus subtilis biofilm communities undergoing metabolic oscillations can become coupled through electrical signaling and synchronize their growth dynamics. Coupling increases competition by also synchronizing demand for limited nutrients. As predicted by mathematical modeling, we confirm that biofilms resolve this conflict by switching from in-phase to antiphase oscillations. This results in time-sharing behavior, where each community takes turns consuming nutrients. Time-sharing enables biofilms to counterintuitively increase growth under reduced nutrient supply. Distant biofilms can thus coordinate their behavior to resolve nutrient competition through time-sharing, a strategy used in engineered systems to allocate limited resources.

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Coupling between distant biofilms and emergence of nutrient time-sharing.

Combining membrane impermeable DNA-binding stain propidium iodide (PI) with membrane-permeable DNA-binding counterstains is a widely used approach for bacterial viability staining. In this paper we show that PI staining of adherent cells in biofilms may significantly underestimate bacterial viability due to the presence of extracellular nucleic acids (eNA). We demonstrate that gram-positive Staphylococcus epidermidis and gram-negative Escherichia coli 24-hour initial biofilms on glass consist of 76 and 96% PI-positive red cells in situ, respectively, even though 68% the cells of either species in these aggregates are metabolically active. Furthermore, 82% of E. coli and 89% S. epidermidis are cultivable after harvesting. Confocal laser scanning microscopy (CLSM) revealed that this false dead layer of red cells is due to a subpopulation of double-stained cells that have green interiors under red coating layer which hints at eNA being stained outside intact membranes. Therefore, viability staining results of adherent cells should always be validated by an alternative method for estimating viability, preferably by cultivation.

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Propidium iodide staining underestimates viability of adherent bacterial cells

Natural biological materials exhibit remarkable properties: self-assembly from simple raw materials, precise control of morphology, diverse physical and chemical properties, self-repair, and the ability to sense-and-respond to environmental stimuli. Despite having found numerous uses in human industry and society, the utility of natural biological materials is limited. But, could it be possible to genetically program microbes to create entirely new and useful biological materials? At the intersection between microbiology, material science, and synthetic biology, the emerging field of biological engineered living materials (ELMs) aims to answer this question. Here we review recent efforts to program cells to produce living materials with novel functional properties, focusing on microbial systems that can be engineered to grow materials and on new genetic circuits for pattern formation that could be used to produce the more complex systems of the future.

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Biological Engineered Living Materials: Growing Functional Materials with Genetically Programmable Properties.

Biofilm formation is a process in which microbial cells aggregate to form collectives that are embedded in a self-produced extracellular matrix. Bacillus subtilis is a Gram-positive bacterium that is used to dissect the mechanisms controlling matrix production and the subsequent transition from a motile planktonic cell state to a sessile biofilm state. The collective nature of life in a biofilm allows emergent properties to manifest, and B. subtilis biofilms are linked with novel industrial uses as well as probiotic and biocontrol processes. In this Review, we outline the molecular details of the biofilm matrix and the regulatory pathways and external factors that control its production. We explore the beneficial outcomes associated with biofilms. Finally, we highlight major advances in our understanding of concepts of microbial evolution and community behaviour that have resulted from studies of the innate heterogeneity of biofilms.

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Fang Yuan

Papers

Species-Independent Attraction to Biofilms through Electrical Signaling