Probing the growth and mechanical properties of Bacillus subtilis biofilms through genetic mutation strategies

Abstract

Bacterial communities form biofilms on various surfaces by synthesizing a cohesive and protective extracellular matrix, and these biofilms protect microorganisms against harsh environmental conditions. Bacillus subtilis is a widely used experimental species, and its biofilms are used as representative models of beneficial biofilms. Specifically, B. subtilis biofilms are known to be rich in extracellular polymeric substances (EPS) and other biopolymers such as DNA and proteins like the amyloid protein TasA and the hydrophobic protein BslA. These materials, which form an interconnected, cohesive, three-dimensional polymer network, provide the mechanical stability of biofilms and mediate their adherence to surfaces among other functional contributions. Here, we explored how genetically-encoded components specifically contribute to regulate the growth status, mechanical properties, and antibiotic resistance of B. subtilis biofilms, thereby establishing a solid empirical basis for understanding how various genetic engineering efforts are likely to affect the structure and function of biofilms. We noted discrete contributions to biofilm morphology, mechanical properties, and survival from major biofilm components such as EPS, TasA and BslA. For example, EPS plays an important role in maintaining the stability of the mechanical properties and the antibiotic resistance of biofilms, whereas BslA has a significant impact on the resolution that can be obtained for printing applications. This work provides a deeper understanding of the internal interactions of biofilm components through systematic genetic manipulations. It thus not only broadens the application prospects of beneficial biofilms, but also serves as the basis of future strategies for targeting and effectively removing harmful biofilms.

Keywords: Bacillus subtilis; Biofilms; Environmental tolerance; Living materials; Synthetic biology.

Fig. 1

Bacillus subtilis biofilms formation. (a) Digital camera image of the wild-type B. subtilis biofilm. The mature biofilm exhibits a complex network of intertwined wrinkles and ridges and is highly hydrophobic. (b) A schematic of wild-type B. subtilis biofilm. The biofilm matrix composition is complex and can contain self-produced molecules, including TasA fibers, EPS, BslA and bacteria.

Fig. 2

Macroscopic and microscopic structure of B. subtilis biofilms. (a) Digital camera image of WT, single mutant, and multiple knockout B. subtilis biofilms. Water contact assay was also shown (insets). WT biofilm had a typical wrinkled pellicle that exhibits strong hydrophobicity. Microscale observations including (b) FE-SEM and (c) TEM. There was no amyloid fiber in the tasA knockout sample, no extracellular polysaccharide in the epsàO knockout sample, and no extracellular matrix in the triple knockout sample. Note: all the biofilms used in the above experiments were cultured for two days. Scale bar: 5 mm in a, 500 nm in b and c.

Fig. 3

Growth status and antibiotic resistance of B. subtilis biofilms. (a) The growth status of WT and mutant B. subtilis biofilms over a 6-day culture. (b) Water contact angle and (c) weight of WT and mutant B. subtilis biofilms. (d) Antibiotic resistance of solid cultured biofilms. (e) Biomass of biofilms in solid cultured with different concentrations of chloramphenicol. The ΔEPS biofilm exhibited a significantly lower biomass than other single mutant biofilm, demonstrating that EPS contributed to antibiotic resistance. Note: each of the circles in a has a diameter of 1.25 cm. Experiments in b, c and e were repeated three times with similar results based on biologically independent biofilm cultures (n = 3). Experiments in e were repeated three times (n = 3). **P < 0.01, ***P < 0.001, two-sided t-test.

Fig. 4

Rheological properties of B. subtilis biofilms. (a) The steady-state flow behavior of biofilms measured by viscosity curves at changing shear rates (from 0.001 to 100 s-1). The biofilms exhibited shear-thinning behavior. (b) The measured storage modulus (G′) and loss modulus (G″) of different biofilm variants as a function of strain amplitude from 0.01 to 100% angular frequency, 1 rad/s. The biofilms all exhibited predominantly viscous behavior. (c) The measured storage modulus (G′) and loss modulus (G″) of different biofilm variants as a function of angular frequency from 0.1 to 100 rad/s（strain amplitude, 1%). All biofilms had a gel-like structure. (d) Shear viscosity of biofilms at a selected shear rate of 0.316 s–1. (e) Storage modulus of biofilms at a selected strain amplitude of 1%. (f) Storage modulus of biofilms at a selected angular frequency of 10 rad/s. The WT biofilm exhibited relatively strong viscoelasticity. All experiments were repeated three times (n = 3). ***P < 0.001, two-sided t-test.

Fig. 5

Printability of B. subtilis biofilms. (a) Digital images of 3D printed B. subtilis biofilm variants. The printed lines of the WT biofilm were substantially expanded compared to the mutant biofilms. (b) Instantaneous recovery of the viscoelastic network of biofilms shown by a sudden shear process in a steady state (100 s⁻¹), followed by an oscillatory sweep measurement. All the biofilms showed an immediate recovery in viscosity. (c) The extents of viscosity recovery of biofilm variants were measured (t1 = 60 s, t2 = 300 s). The ΔbslA biofilm had the strongest instantaneous recovery capability among other mutant biofilms. Experiments in b and c were repeated three times (n = 3). *P < 0.05, ***P < 0.001, two-sided t-test.