Direct Comparison of Physical Properties of Bacillus subtilis NCIB 3610 and B-1 Biofilms

Abstract

cMany bacteria form surface-attached communities known as biofilms. Due to the extreme resistance of these bacterial biofilms to antibiotics and mechanical stresses, biofilms are of growing interest not only in microbiology but also in medicine and industry. Previous studies have determined the extracellular polymeric substances present in the matrix of biofilms formed by Bacillus subtilis NCIB 3610. However, studies on the physical properties of biofilms formed by this strain are just emerging. In particular, quantitative data on the contributions of biofilm matrix biopolymers to these physical properties are lacking. Here, we quantitatively investigated three physical properties of B. subtilis NCIB 3610 biofilms: the surface roughness and stiffness and the bulk viscoelasticity of these biofilms. We show how specific biomolecules constituting the biofilm matrix formed by this strain contribute to those biofilm properties. In particular, we demonstrate that the surface roughness and surface elasticity of 1-day-old NCIB 3610 biofilms are strongly affected by the surface layer protein BslA. For a second strain, B. subtilis B-1, which forms biofilms containing mainly ␥-polyglutamate, we found significantly different physical biofilm properties that are also differently affected by the commonly used antibacterial agent ethanol. We show that B-1 biofilms are protected from ethanol-induced changes in the biofilm's stiffness and that this protective effect can be transferred to NCIB 3610 biofilms by the sole addition of ␥-polyglutamate to growing NCIB 3610 biofilms. Together, our results demonstrate the importance of specific biofilm matrix components for the distinct physical properties of B. subtilis biofilms. Bacteria embed themselves with secreted biopolymers (1-3), building a community that is referred to as a biofilm. Such biofilms can be formed by a variety of Gram-positive as well as Gram-negative bacteria (4). The composition of the biofilm matrix is dependent on the biofilm-forming bacterium and environmental conditions such as shear forces experienced, temperature, and nutrient availability (5); the matrix can consist of different extracellular polymeric substances (EPSs), such as polysaccharides, proteins, lipids, and nucleic acids (6). Biofilms can grow on various surfaces (7-9). While Bacillus subtilis biofilms are formed on solid nutrient surfaces or at liquid-air interfaces (10, 11), other bacteria can produce biofilms on surfaces under water-saturated conditions (in liquid) (12). Due to their high mechanical stability (13,14) and their resistance to antibiotic or chemical treatment (15-18), such biofilms present significant problems in both industry and health care (19)(20)(21)(22). Although the compositions of many biofilm matrices are known, the biomolecular reason for the outstanding resistance of bacterial biofilms is not well understood. Only a few studies have investigated the influence of specific matrix components on the mechanical properties of biofilms (23, 24). The majority of recent studies...

Elongational rheology of bacterial biofilmsin situ

“…As a benchmark for our testing setup, we performed stretching experiments with agar samples of different thickness and obtain similar Young's moduli for those samples (Fig. S2 [28]) which also agree well with independent measurements of this agar performed with atomic force microscopy [30]. This confirms that our stretching device indeed determines material properties which are independent from the amount of material tested.…”

“…2(B)] and the samples could be used for mechanical testing. With this method, we are able to produce biofilm samples with relatively low variations in material properties, as already reported previously [8,30]. However, for other biofilm variants grown in a liquid environment, a chemostat [31] might be required to achieve growth of biofilm samples with comparable properties.…”

Evolution of exploitative interactions during diversification in Bacillus subtilis biofilms

“…Since previous experiments revealed dramatic differences in colony surface complexity and wetting behavior, we further hypothesized that these differences could be linked to different levels of extracellular matrix (ECM) production by the morphotypes (Kobayashi and Iwano 2012;Kesel et al 2016). In order to test this, the matrix-genes reporters P tapA -gfp and P eps -gfp were introduced into the four morphotypes and the ancestor strain, and changes in matrix genes expression were determined (Fig.…”

Matrix composition determines the dimensions of Bacillus subtilis NCIB 3610 biofilm colonies grown on LB agar

“…During this expansion, the biolm colony is not only expected to increase its thickness but also to develop a rough surface. 36 Hence, a parallel analysis of the biolm colony surface roughness can serve as an indicator for accurate biolm colony formation. To quantify biolm height development and alterations in biolm surface roughness, we performed light prolometry (Fig.…”

“…The use of deletion mutants of B. subtilis has enabled to demonstrate that the protein BslA contributes to the strong wetting resistance 35 as well as the surface stiffness and roughness of B. subtilis biolms. 36 Here, we employ a similar approach and quantify the formation of biolm colonies grown on solid agar surfaces of two B. subtilis wild type strains (B-1 and NCIB 3610) that differ in their biolm matrix composition, 35,[37][38][39] and a biolm defective B. subtilis strain (BD630) which is unable to produce a biolm on solid LB agar surfaces. Using a combination of time-lapse microscopy and light prolometry, we show that for the wellstudied B. subtilis strain NCIB 3610, for which the main components of the biolm matrix are known, 35,38,39 specic matrix components control the dimensions of mature biolm colonies.…”