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.
Highlights
It is generally accepted that the extracellular matrix, which holds constituent cells of the biofilm together, is composed of polysaccharides, proteins and nucleic acids [1,2,3,4,5,6]
Both TasA and polysaccharides encoded by epsA-O operon are considered to be essential for the formation of the B. subtilis NCIB
Growth media and statistical analysis Strains used in this study are the wild Bacillus subtilis NCIB 3610 [42] and its derivatives: the eps mutant ((epsA-O)::tet), the tasA mutant and the tasA eps double mutant (tasA::spc::tet)
Summary
It is generally accepted that the extracellular matrix, which holds constituent cells of the biofilm together, is composed of polysaccharides, proteins and nucleic acids [1,2,3,4,5,6] These polymers, collectively known as exopolymeric substances (EPS), are only partially characterized in B. subtilis [2], [7,8,9,10], a bacterium, which is a model organism for the study of biofilm formation [11,12,13,14]. It is linked to the cell wall by TapA, a member of the tapAsipW-tasA operon, which is a minor extracellular component and is presumably incorporated into the TasA fibers [16], [23] Both TasA and polysaccharides encoded by epsA-O operon are considered to be essential for the formation of the B. subtilis NCIB
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