Pseudomonas aeruginosa can form robust pellicles at the air–liquid interface of a standing liquid culture. The addition of 342 mM sucrose to LB led to the production of a markedly thicker pellicle (Fig. 1a, LBS), which was not observed when adding 171 mM NaCl instead of sucrose (Fig. 1a, LBN). Similar results were observed when biofilm was grown in microtitre wells and stained with crystal violet (Fig. 1b). This suggests that sucrose rather than osmolarity triggers the increase of biofilm formation in our conditions. Using maltose instead of sucrose at the same concentration (342 mM) did not increase the pellicle, suggesting that it was the sucrose that led to the increased pellicle formation in our conditions (Fig. 1a and b, LBM). As pellicle formation has been associated with pel and psl gene cluster expression in P. aeruginosa PAO1 (Friedman & Kolter, 2004b), the transcription levels of pelB and pslB were compared using qRT-PCR in P. aeruginosa H103 grown in LB and LBS (Fig. 1c). The pelB mRNA level was increased almost 3.5-fold in the presence of 342 mM sucrose, suggesting that sucrose promotes pel transcription (Fig. 1c). Conversely, the pslB mRNA levels were found to be very similar in LB and LBS, suggesting that the psl gene cluster was not mainly involved in the sucrose-related biofilm production increase (Fig. 1c). As a control, the effect of sucrose on pellicle production was assayed in a pelA mutant strain (ratio LBS/LB). As shown on Fig. 1d, the pelA mutant strain produced about twofold more pellicle in LBS than LB, and the isogenic parental strain produced about fourfold more pellicle in LBS than LB. These data suggest that Pel polysaccharide was partly involved in the sucrose-mediated pellicle production. However, this also indicates that another part of the sucrose effect on biofilm production remained pel-independent. Biofilm-enhanced production by exogenous polysaccharides has been described previously. Sucrose is the most cariogenic carbohydrate, leading to elevated matrix exopolysaccharides production by oral bacteria and accumulation on the tooth surface (Paes Leme et al., 2006). One of these bacteria, Streptococcus mutans, was shown to form well-defined and tightly adherent biofilms in the presence of 1% of sucrose (Duarte et al., 2008), but not in high salinity media (Kawarai et al., 2009). In the plant growth-promoting bacterium Bacillus subtilis, biofilm formation is triggered by plant-produced polysaccharides such as arabinogalactan, pectin or xylan at very low concentrations (0.5% or even 0.05% of plant arabinogalactan) (Kearns et al., 2005; Beauregard et al., 2013). Plant polysaccharides stimulate biofilm formation in B. subtilis by acting as environmental signals that induce matrix gene expression, and as substrates that are processed and incorporated into the exopolysaccharides biofilm matrix (Beauregard et al., 2013). Interestingly, it was shown previously that addition of glucose at a concentration of 1–5% in LB medium increased production of the biofilm-related polysaccharide alginate in P. aeruginosa PAO1 (Ma et al., 1997). In our study, we assayed sucrose at a very high concentration, that is 342 mM, corresponding to 11.7%. We further assayed lower sucrose concentrations to investigate the putative signalling effect of sucrose on biofilm formation. As shown on Fig. 2a, the sucrose-related effect on biofilm formation occurred only with elevated sucrose concentrations (higher than 5%), suggesting that sucrose did not act as a signal in our conditions. Pseudomonas aeruginosa is furthermore unable to metabolize sucrose due to the lack of the sucrose-hydrolyzing corresponding enzymes (Fig. 2b), suggesting that sucrose is thus unlikely to be used as a carbohydrate source for the building of P. aeruginosa matrix. The mechanisms by which sucrose might increase P. aeruginosa biofilm formation will be further discussed below.
Figure 1. Sucrose-increased biofilm formation of Pseudomonas aeruginosa H103. (a) Pellicle and crystal violet (CV)-stained biofilms formed in microtitre wells by P. aeruginosa H103 grown in LB, LBS (LB + 342 mM sucrose), LBN (LB + 171 mM NaCl) or LBM (LB + 342 mM maltose) media. (b) Measurement of the biofilm produced in LBS (grey bar), LBN (white bar) and LBM (light grey bar) relative to that formed in LB (dotted line). (c) qRT-PCR assays on pelB and pslB in P. aeruginosa H103 grown in LB and LBS media. PCR reactions were performed in triplicate and the standard deviations were lower than 0.15 Ct. (d) Quantification of CV-stained biofilms formed by the wild-type strain PAO1 (grey bar) and its isogenic pelA mutant strain (white bar) grown in LBS compared with LB. Results are given as the ratio LBS/LB. Statistical analysis used pairwise strain comparisons (t-test). *P < 0.05; **P < 0.01; ns, non-significant.
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Figure 2. Effect of sucrose concentration on Pseudomonas aeruginosa biofilm formation. (a) Pellicle at the air–liquid interface and quantification of CV-stained biofilms by P. aeruginosa H103 grown in LB or in LB supplemented with 1, 2, 5 or 11.7% sucrose, relative to that formed in LB. (b) Growth of P. aeruginosa H103 in mineral medium ARJ containing sucrose or glucose as a carbon source. Statistical analysis used pairwise strain comparisons (t-test). *P < 0.05; **P < 0.01; ns, non-significant.
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