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

Abstract

The production of biomolecules can provide new functionalities to the synthesizing organism. One important example is the secretion of extracellular polymeric substances (EPS) by biofilm forming bacteria. This biofilm matrix protects the individual bacteria within the biofilm from external stressors such as antibiotics, chemicals and shear flow. Previous studies have determined several main matrix components of biofilms formed by Bacillus subtilis. However, how these matrix components influence the growth behavior and final dimensions of B. subtilis biofilms has not yet been determined. Here, we combine different experimental techniques with theoretical modeling to assess this relation. In particular, we quantify the area covered by the biofilm and the biofilm height by performing time-lapse microscopy and light profilometry, respectively. We study the development of biofilms formed by two wild-type strains (B-1 and NCIB 3610) differing in their matrix composition and NCIB 3610 mutant strains lacking the ability to produce specific EPS. Based on the experimentally obtained growth dynamics, we develop a mathematical model that allows us to quantify the influence of three key biofilm matrix components on the final NCIB 3610 biofilm colony dimensions. In detail, we show that two matrix components, the exopolysaccharide produced by the epsA-O operon and the surface layer protein BslA control the area covered by the biofilm colony. The height of these mature biofilm colonies is mostly affected by BslA.Together, our results emphasize the importance of the biofilm matrix composition for biofilm growth and the final dimensions of mature B. subtilis NCIB 3610 biofilm colonies.

Topography quantifications allow for identifying the contribution of parental strains to physical properties of co-cultured biofilms

“…Here, we tested the contribution of this hydrophobin BslA to the surface hydrophobicity of co-cultured colonies by employing a NCIB 3610 mutant strain unable to produce BslA ( ΔBslA ). Indeed, in full agreement with results described in the literature [ 12 , 21 , 22 , [54] , [55] , [56] , [57] , [58] ], co-cultivation of ΔBslA with one of the other strains results in hydrophilic daughter colonies ( Fig. S7 ).…”

“…Also, the susceptibility of biofilms towards antibiotic solutions, their resilience to other chemical stresses and the erosion resistance of biofilms were shown to be related to the surface topography of those soft materials [ 9 , 10 ]. In fact, in recent years, quantitative topographical analyses of biofilms have become a well-established and useful tool in biofilm research [ 8 , [10] , [11] , [12] , [13] , [14] , [15] ].…”

Metal ions weaken the hydrophobicity and antibiotic resistance of Bacillus subtilis NCIB 3610 biofilms

“…4,6,13,14 The exopolysaccharide, whose synthesis is required for the BslA proteins to localize to the biofilm matrix, bundles the cells and contributes—together with BslA—to the final roughness and height of B. subtilis biofilms. 4,15,16 Finally, the fibers formed by TasA protein connect the cells within the matrix, thus rendering the structure of the biofilm more compact and stable. 15,17 Hence, a combination of chemical and physical contributions, that is, hydrophobic molecules and surface roughness features (the latter of which are established by a complex combination of different biofilm matrix components), gives rise to the highly efficient wetting resistance of those biofilms.…”

“…4,15,16 Finally, the fibers formed by TasA protein connect the cells within the matrix, thus rendering the structure of the biofilm more compact and stable. 15,17 Hence, a combination of chemical and physical contributions, that is, hydrophobic molecules and surface roughness features (the latter of which are established by a complex combination of different biofilm matrix components), gives rise to the highly efficient wetting resistance of those biofilms.…”

Importance of the biofilm matrix for the erosion stability of Bacillus subtilis NCIB 3610 biofilms

“…31,[33][34][35][36] However, only recently direct correlations between the biolm matrix composition and specic biolm properties could be determined. 31,34,35,37 Importantly, it was shown that the surface layer protein BslA is important for the surface stiffness and surface roughness 31 of NCIB 3610 biolms, and this protein also strongly affects the biolm wettability. 29,33,38 Both for B. subtilis B-1 bio-lms and biolms generated by Pseudomonas aeruginosa 39 it was shown that multi-valent ions protect the biolms from erosion.…”