Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms

Abstract

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.

Viscoelastic Properties of Levan-DNA Mixtures Important in Microbial Biofilm Formation as Determined by Micro- and Macrorheology

“…It has been demonstrated that in sucrose-rich growth media, Bacillus subtilis produces copious amounts of levan, resulting in thick floating biofilms (9). Levan is the main component of such biofilms and comprises~95% of the extracellular matrix mass.…”

“…In addition, it may be loosely attached to the cell as an extracellular polymeric substance (EPS), which gives bacterial colonies their typical mucoid morphology (8). Recently, it was reported that levan is an important structural component of microbial biofilms, playing a stabilizing role in their formation and maintenance (9,10). Since levan is an extremely soluble (i.e., up to 60% w/v), low-viscosity polymer, it is surprising that it can stabilize biofilm formation.…”

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

“…Again, this nding was unexpected as tasA biolm pellicles formed on MSgg medium are considerable decreased in biolm height compared to pellicles generated by the wild-type strain. 52 Whereas the absence of the TasA protein leads to an increased biolm height for biolm colonies grown on LB agar plates, the absence of the BslA protein led to a decrease of the nal biolm height by $30%. A similar result was obtained for biolm colonies formed by the epsA-O mutant strain.…”

From fructans to difructose dianhydrides

“…1). Inulin principally occurs in dicot plants, however, levan more commonly occurs in monocot plants and bacteria (Dogsa et al 2013;Valluru and van den Ende 2008). In plants, inulin is biosynthesized from sucrose by a two-step reaction.…”