Sticking together: building a biofilm the Bacillus subtilis way

Abstract

Biofilms are ubiquitous communities of tightly associated bacteria encased in an extracellular matrix. Bacillus subtilis has long served as a robust model organism to examine the molecular mechanisms of biofilm formation, and a number of studies have revealed that this process is regulated by several integrated pathways. In this Review, we focus on the molecular mechanisms that control B. subtilis biofilm assembly, and then briefly summarize the current state of knowledge regarding biofilm disassembly. We also discuss recent progress that has expanded our understanding of B. subtilis biofilm formation on plant roots, which are a natural habitat for this soil bacterium.

Figure 1

The life-cycle of a Bacillus subtilis biofilm. This process occurs in stages which comprise the development, maturation and disassembly of the community. Motile cells with flagella differentiate into non-motile matrix-producing cells that become organized in chains and are surrounded by extracellular matrix (orange). In mature biofilms matrix-producing cells sporulate (dark brown spores). In aged biofilms, some cells secrete small molecules such as D-amino acids and polyamines that break down the extracellular matrix and the cells disperse in the environment.

Figure 2

A) Top-down view of a colony grown at room temperature on biofilm-inducing medium (MSgg) for 7 days. Scale bar is 2 mm. A time-lapse movie of the growth of this colony can be viewed in the supplemental material (H. Vlamakis, unpublished movie). B) Top-down view of a pellicle grown at room temperature for 5 days (image from ).

Figure 3

Simplified schematic of the regulatory network that controls biofilm formation in B. subtilis. A) Several sub-networks (I-IV) are integrated to activate (arrows) or repress (T-bars) matrix gene expression depending on the environmental condition. Details are discussed in the text. The genes encoding components of the extracellular matrix are coloured in blue and encode: EPS (epsA-O), TasA (tapA-sipW-tasA), BslA (bslA), and PGA (pgs). Red and pink lines indicate gene regulation whereas yellow lines indicate protein-protein interactions. Dashed lines indicate indirect activity. B) The double negative feedback loop (involving the slrR gene, the SlrR protein and the SinR protein) exists in SlrR low (left side of figure) and SlrR high (right side of figure) states. The SinR–SlrR switch regulates matrix genes (epsA-O and tapA-sipW-tasA), autolysin (lytABC and lytF) and motility genes (hag), as well as the gene (slrR) for SlrR itself. In the SlrR low state (left) the SinR protein represses the slrR gene, keeping the levels of the SlrR protein low. In the SlrR high state (right), SlrR binds to SinR, trapping it in the heteromeric SinR•SlrR complex. This titrates SinR, resulting in derepression of matrix genes and slrR, setting up a self-reinforcing switch leading to high SlrR levels. At the same time, SlrR repurposes SinR in that the SinR•SlrR complex represses autolysin and motility genes (right). Image adapted from. The double negative loop is epigenetic in that both the SlrR low and SlrR high states self-reinforcing and are stable for many generations. During biofilm formation, SinI produced under the control of Spo0A~P (see text) drives the switch into the high state by binding to and inhibiting SinR.

Figure

A) Wild-type B. subtlis cells or an eps mutant constitutively expressing YFP were inoculated with 6 day-old seedlings of A. thaliana. Colonization of the root was observed after 24h. Overlays of fluorescence (false-coloured green) and transmitted light images (gray) are shown. Bars are 50 μm. Images in A) by P. Beauregard (unpublished). B) Schematic illustration of B. subtilis colonizing a plant root. B. subtilis secretes the lipopetide surfactin, which is required for B. subtilis (green rods) biofilm formation on the root. A second trigger for B. subtilis matrix gene expression is malic acid, which is constitutively secreted in the rhizosphere by tomato plants but secreted by A. thaliana only when the plant is infected with the pathogen P. syringae. B. subtilis exerts beneficial effects on the plant by promoting its growth and helping to fight of pathogens (such as P. syringae), directly via the secretion of surfactin and other antimicrobials and indirectly by eliciting induced systemic resistance in the plant.

Figure

FloT-YFP localizes in a punctate pattern in the cell membrane and is disrupted by inhibitors of squalene synthase. Fluorescence image (false-coloured red) is overlayed on the transmitted light image. An untreated cell is shown (top image) and a series of images taken over a period of 8 h after treatment with zaragozic acid. Scale bar is 2 μm. Images from .