Differential gene expression profiling of Staphylococcus aureus cultivated under biofilm and planktonic conditions

Abstract

It is well known that biofilm formation by pathogenic staphylococci on implanted medical devices leads to "chronic polymer-associated infections." Bacteria in these biofilms are more resistant to antibiotics and the immune defense system than their planktonic counterparts, which suggests that the cells in a biofilm have altered metabolic activity. To determine which genes are up-regulated in Staphylococcus aureus biofilm cells, we carried out a comparative transcriptome analysis. Biofilm growth was simulated on dialysis membranes laid on agar plates. Staphylococci were cultivated planktonically in Erlenmeyer flasks with shaking. mRNA was isolated at five time points from cells grown under both conditions and used for hybridization with DNA microarrays. The gene expression patterns of several gene groups differed under the two growth conditions. In biofilm cells, the cell envelope appeared to be a very active compartment since genes encoding binding proteins, proteins involved in the synthesis of murein and glucosaminoglycan polysaccharide intercellular adhesin, and other enzymes involved in cell envelope synthesis and function were significantly up-regulated. In addition, evidence was obtained that formate fermentation, urease activity, the response to oxidative stress, and, as a consequence thereof, acid and ammonium production are up-regulated in a biofilm. These factors might contribute to survival, persistence, and growth in a biofilm environment. Interestingly, toxins and proteases were up-regulated under planktonic growth conditions. Physiological and biochemical tests for the up-regulation of urease, formate dehydrogenase, proteases, and the synthesis of staphyloxanthin confirmed the microarray data.

FIG. 1.

Comparison of the expression profiles of selected gene groups in biofilm cells and planktonic cells. Cells were grown as described in the text, and total RNA was extracted from the cells at the five times indicated and used in DNA microarray analyses. The data indicate the fold differences in expression of selected gene groups in biofilm cells compared to the expression in planktonic cells. (A) Expression pattern of genes encoding the biosynthetic enzymes for PIA, icaADBC (SA2459 to SA2462). (B) Expression pattern of genes encoding the binding proteins clumping factor B (SA2423), Ser-Asp-rich fibrinogen-binding or bone sialoprotein (SA0519), fibrinogen-binding protein A or clumping factor (SA0742), immunodominant antigen B (SA2431), and the lipoprotein streptococcal adhesion PsaA homolog (SA0587). (C) Expression pattern of genes with sequence similarity to genes encoding the extracellular, immune dominant protein staphylococcal secretory antigen A (SsaA), including a hypothetical protein similar to SsaA (SA2353), a secretory antigen precursor SsaA homolog (SA2093), and a hypothetical protein similar to SsaA (SA2097). (D) Expression pattern of genes of the staphyloxanthin biosynthesis cluster, encoding proteins similar to acyltransferase (SA2354), a hypothetical protein (SA2352), phytoene dehydrogenase (SA2351), a conserved hypothetical protein (SA2350), squalene synthase (SA2349), squalene desaturase (SA2348), aspartate aminotransferase (SA2347), and a d-specific d-2-hydroxyacid dehydrogenase ddh homolog (SA2346). (E) Expression pattern of genes involved in formate metabolism, encoding NAD-dependent formate dehydrogenase (SA0171), a formate dehydrogenase homolog (SA2102), formate-acyltransferase-activating enzyme (SA0219), formate acyltransferase (SA0218), and a protein similar to formate transporter NirC (SA0293). (F) Expression pattern of genes involved in urease activity, encoding urease accessory protein UreD (SA2088), urease accessory protein UreF (SA2086), urease beta subunit (SA2083), and urease alpha subunit (SA2084). (G) Expression pattern of genes involved in stress response, encoding superoxide dismutase SodA (SA1382), catalase (SA1170), a protein similar to glutathione peroxidase (SA2414), and alkaline shock protein (SA0194). (H) Expression pattern of genes of the arginine deiminase cluster, encoding a hypothetical protein similar to the transcriptional regulator Crp/Fnr family protein (SA2424), carbamate kinase (SA2425), arginine/ornithine antiporter (SA2426), ornithine transcarbamylase (SA2427), and arginine deiminase (SA2428).

FIG. 2.

Production of staphyloxanthin in biofilm and planktonic cultures. Staphyloxanthin was extracted from the culture supernatants with ethanol. After 48 h, the biofilm supernatant was yellow-orange, whereas the planktonic supernatant was almost colorless (not shown). (A) Staphyloxanthin normally shows a typical peak in its spectrum at 460 nm; therefore, the absorption at this wavelength was measured for all samples (B).

FIG. 3.

Comparisonof the expression profiles of genes encoding toxins and proteases in biofilm and planktonic cultures. Microarray analysis was performed and expression levels were determined as described in the legend to Fig. 1. The data indicate the fold differences in expression of genes in planktonic cells compared to the expression in biofilm cells. (A) Expression pattern of genes encoding toxins, including the leukotoxin LukD (SA1637), exotoxin 7 (SA0383), exotoxin 13 (SA0389), exotoxin 8 (SA0384), exotoxin 10 (SA0386), an alpha-hemolysin precursor (SA1007), a protein similar to exotoxin 2 (SA0357), and exotoxin 6 (SA0382). (B) Expression pattern of genes encoding proteases, including protease ClpX (SA1498), a protein similar to protease (SA1440), serine protease HtrA (SA0879), a protein similar to protease (SA1441), a cysteine protease precursor (SA0900), serine protease (SA0901), serine protease SplA (SA1631), serine protease SplB (SA1630), serine protease SplC (SA1629), and serine protease SplD (SA1628).

FIG. 4.

Proteolytic activities of the supernatants of biofilm cells (A) and planktonic cells (B). The supernatants of cells grown for different times were used to test the proteolytic activity on casein agar plates. Proteolytic activity is indicated by halo formation.

FIG. 5.

Urease activity in biofilm cells (A) and planktonic cells (B). Cells were grown as described in the text and harvested after 48 h of growth. Urease activity was determined using urease diagnostic tablets (575-21; Rosco), which leads to a red color if urea is hydrolyzed by urease to form two molecules of ammonia. The red/purple color of the biofilm cells appeared after 30 min of incubation. The planktonic cells remained nearly white.