The use of glass wool as an attachment surface for studying phenotypic changes inPseudomonas aeruginosa biofilms by two-dimensional gel electrophoresis

Abstract

Two-dimensional polyacrylamide gel electrophoresis was used to demonstrate phenotypic differences between Pseudomonas aeruginosa biofilm cells and the planktonic counterpart cells under defined culture conditions. Glass wool was used as a substratum for cell attachment as it affords a large surface-to-volume ratio (1 g with a mean diameter of 15 microns = 1300 cm2), supports the growth of biofilms, allows for free movement of cells between the inter-strand spaces, and it facilitates the exchange of nutrients and oxygen. It also allows for the separation of the biofilm biomass from the surrounding surface influenced planktonic (SIP) cells for further characterization. Comparative analysis of the respective proteomes indicated striking differences in the protein patterns of planktonic, biofilm and SIP cells. We selected 41 proteins, the levels of which varied in a significant and reproducible way in the respective protein profiles. In the biofilm cells, a general up-regulation of the spots was seen, but in SIP cells expression of these spots were generally down-regulated. Altogether six unique proteins were seen in the planktonic cells, while the biofilm and SIP cells contained five and two unique proteins, respectively. Glass wool, therefore, appears to be an ideal attachment surface for the study of biofilm development.

Multivariate Approach to Comparing Whole-Cell Proteomes of Bacillus cereus Indicates a Biofilm-Specific Proteome

“…An increase in the PGW population would be ascribed either to growth or detachment of cells from the biofilm, while a proportional increase in the attached population implies a net biofilm gain, due either to growth or recruitment from the suspended population. This restricted propensity to form biofilm is similar to the distribution observed for Pseudomonas aeruginosa , but much lower than reported previously for B. cereus DL5 isolated from a dairy cleaning solution . The average biofilm density corresponded to 3 × 10 6 CFU/cm 2 (Figure ).…”

“…The third component discriminated between the PGW proteome and all others (Figure C) . B. cereus DL5 and P. aeruginosa studied under similar conditions also displayed a PGW proteome distinct from its planktonic and biofilm counterparts by expressing some unique protein spots. , Component 3 of this PCA offered evidence that the PGW phenotype was not the result of a mixture of released biofilm and stationary phase planktonic cells. Rather, these planktonic cells constituted a unique phenotype, expressed as response to either the proximal biofilm or periodic contact with the glass surface.…”

“…Steyn et al . reported that PGW (SIP) populations of P. aeruginosa grown for 18 h in a minimal medium in the presence of glass wool differed from the planktonic counterpart. The proteome of a PGW population of B. cereus DL5 grown in rich medium was distinct, but differed to a lesser degree from its planktonic counterpart.…”

“…One approach entails using Tygon or silicone tubing fed with a continuous stream of sterile medium, yielding a biofilm grown under quasi steady-state conditions and devoid of any surrounding suspended cells. Glass wool in batch systems has been used to study P. aeruginosa , , Escherichia coli , Bacillus subtilis , and B. cereus . , The glass wool is placed into Erlenmeyer flasks, so that the inoculated population grows as a batch culture, distributing into attached and free-living sub-populations. The planktonic population is therefore a temporal mixture of sub-populations of free-living cells, cells that were recently attached reversibly, and cells detached from the biofilm.…”

Studies on Development and Antigen Expression of Biofilm Cells of Vibrio anguillarum for Oral Vaccination in Aquaculture

“…Earlier studies have shown that as many as 30-40% of the proteins present in bacterial cell walls differ between sessile and planktonic bacteria (Coghlan, 1996). Furthermore, BF of P. aeruginosa developed on glass wool revealed changes in protein profile (Steym et al, 2001). Studies on starved cells of A. hydrophila have shown that about five outer membrane proteins were lost with the expression of three new proteins compared to FC (Rahman et al, 1998).…”