Exopolymer Diversity and the Role of Levan in <i>Bacillus subtilis</i> Biofilms

<div><p>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 <i>Bacillus subtilis</i> 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 <i>eps</i> knockout were significantly thinner than those of the <i>tasA</i> knockout in all media. The biofilm defective phenotypes of <i>tasA</i> and <i>eps</i> 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 <i>B. subtilis</i> floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.</p></div

Biofilm (pellicle) thickness in µm.

<p>The error bars represent standard deviation of the mean (n≥4).</p>a<p>Not detectable</p>*,<p>^†Among these pairs of values the difference is insignificant (p>0.05)</p

Size exclusion liquid chromatography (HPSEC) representative chromatograms of EPS isolated from biofilm (A) and from spent medium below the biofilms (B) grown in three media.

<p>SYM (—––); Czapek (– – –), and MSgg (- - - - - -). The molecular weight (M) distributions were graphically split into five size fractions. Their chemical composition and relative abundance of polysaccharides (▪), proteins (▪) and nucleic acids (▪) in these size fractions is graphically represented in A1 and B1. Chemical composition of EPS size fractions was determined from RI (refractive index) chromatograms shown in A and B and from UV 280 and 260 nm chromatograms (not shown).</p

Expression of the <i>epsA-O-gfp</i> operon in biofilms grown in SYM (▪), Czapek (▪) and MSgg (▪) media given as weighted average of mean normalized fluorescence intensities of individual objects determined by fluorescence microscopy (n≥5).

<p>Expression of the <i>epsA-O-gfp</i> operon in biofilms grown in SYM (▪), Czapek (▪) and MSgg (▪) media given as weighted average of mean normalized fluorescence intensities of individual objects determined by fluorescence microscopy (n≥5).</p

Biofilm (pellicle) thickness at different time points during growth in (A) Czapek (———), Czapek + sucrose (▪ ▪ ▪ ▪ ▪); (B) MSgg (———), MSgg + sucrose (▪ ▪ ▪ ▪ ▪).

<p>The errors represent standard deviation of the mean (n≥4).</p

Fractions of polysaccharides (▪), proteins (▪) and nucleic acids (▪) in EPS isolated from biofilms (A) and corresponding spent medium below the biofilm (B) grown in three different media.

<p>SYM, Czapek, MSgg. The error bars represent standard deviation of the mean (n≥4).</p

Images of 9-day old biofilms disintegrated by vortex stirring as observed under stereomicroscope.

<p>Scale bar corresponds to 1 mm. Biofilms from (A) MSgg + sucrose, (B) Czapek + sucrose, (C) SYM, (D) MSgg and (E) Czapek growth medium.</p

Optical densities (OD₆₅₀) of standing cultures of <i>B. subtilis</i> grown in SYM (▴), Czapek (▪) and MSgg (♦).

<p>One of the four replicates is shown, with error bars representing standard deviations.</p