Control of glucose- and NaCl-induced biofilm formation by rbf in Staphylococcus aureus

Abstract

Both Staphylococcus aureus and S. epidermidis are capable of forming biofilm on biomaterials. We used Tn917 mutagenesis to identify a gene, rbf, affecting biofilm formation in S. aureus NCTC8325-4. Sequencing revealed that Rbf contained a consensus region signature of the AraC/XylS family of regulators, suggesting that Rbf is a transcriptional regulator. Insertional duplication inactivation of the rbf gene confirmed that the gene was involved in biofilm formation on polystyrene and glass. Phenotypic analysis of the wild type and the mutant suggested that the rbf gene mediates the biofilm formation of S. aureus at the multicellular aggregation stage rather than at initial attachment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the mutation resulted in the loss of an approximately 190-kDa protein. Biofilm production by the mutant could be restored by complementation with a 2.5-kb DNA fragment containing the rbf gene. The rbf-specific mutation affected the induction of biofilm formation by glucose and a high concentration of NaCl but not by ethanol. The mutation did not affect the transcription of the ica genes previously shown to be required for biofilm formation. Taken together, our results suggest that the rbf gene is involved in the regulation of the multicellular aggregation step of S. aureus biofilm formation in response to glucose and salt and that this regulation may be mediated through the 190-kDa protein.

FIG. 1.

Comparison of Rbf sequence and AraC family consensus sequence. The helix-turn-helix (HTH) motifs are indicated. Colons and periods indicate identical and similar residues, respectively.

FIG. 2.

Biofilm formation of NCTC8325-4, its isogenic rbf mutant, and a complemented strain on a glass side (A) and a polystyrene microtiter plate (B).

FIG. 3.

SDS-PAGE of whole-cell extracts from 8325-4(pLI50) (lane 1), rbf mutant CYL1097(pLI50) (lane 2), and CYL1097(pYL8565) (lane 3). Samples were run on a 10% gel. The arrow indicates the 190-kDa protein. Lane M, standards.

FIG. 4.

Biofilm formation in response to glucose. The bacterial strains were tested for the effect of glucose on biofilm formation as described in Materials and Methods. Results represent the averages of at least three independent experiments. Error bars indicate the standard error of the mean. OD₄₉₀, optical density at 490 nm.

FIG. 5.

Biofilm formation in response to NaCl. The bacterial strains were tested for the effect of NaCl on biofilm formation as described in Materials and Methods. Results represent the averages of at least three independent experiments. Error bars indicate the standard error of the mean. OD₄₉₀, optical density at 490 nm.

FIG. 6.

Effect of the rbf mutation on biofilm formation of a clinical isolate. The wild-type strain, its rbf mutant, and a complemented strain were tested for polystyrene binding in wells of a microtiter plate. The results represent the averages of three independent experiments. Error bars indicate the standard error of the mean. OD₄₉₀, optical density at 490 nm.