Biofilm formation phenotype of the double ΔmgrA ΔsrtA mutants of RN6390 and MW2. Significant differences in biofilm formation capacity were found between the mgrA mutant and the ΔmgrA ΔsrtA double mutant. The significance data obtained with the Student t test were as follows: the mgrA mutant versus RN6390, P < 0.0001; the mgrA srtA mutant versus the mgrA mutant of RN6390, P < 0.0012; the mgrA mutant versus MW2, P < 0.0002; and the mgrA srtA mutant versus the mgrA mutant of MW2, P < 0.001. The microtiter plate results are shown below.

The proposed model of biofilm formation in mgrA mutants. The inactivation of mgrA would reduce agr expression, enhancing surface protein expression, reducing nuclease secretion (23), and hence augmenting biofilm formation. A mutation in mgrA also increases CidA expression as well as reduces LrgA expression (23), thus promoting autolysis and extracellular DNA release to facilitate biofilm formation. SrtA may also affect surface protein expression. The mode of interaction between MgrA and SarA in biofilm formation remains to be defined.

(A) Analysis of biofilm formation in wild-type strains RN6390 and MW2, isogenic mgrA mutants, sarA mutants, and the double ΔmgrA ΔsarA mutants. Significant differences were found between the mgrA mutants and the corresponding ΔmgrA ΔsarA mutants. P values were as follows: RN6390 versus the mgrA mutant, P < 0.002; the mgrA mutant of RN6390 versus the double mgrA sarA mutant, P < 0.02; MW2 versus the mgrA mutant, P < 0.004; the mgrA mutant of MW2 versus the double mgrA sarA mutant, P < 0.003. The results of the microtiter plates are shown below. (B) Biofilm formation in microtiter plates of mgrA mutants, sarA mutants, ΔmgrA ΔsarA mutants, and the ΔmgrA ΔsarA Δaur and ΔmgrA ΔsarA sspA triple mutants (top panels). The corresponding protease activity by these strains on skimmed-milk agar plates is shown in the bottom panels.

Detachment of preformed biofilms by proteinase K. The biofilms of RN6390 and MW2 and their isogenic ΔmgrA mutant strains grown in TSB-glucose for 16 h at 37°C were treated for 2 h at 37°C with 100 μg of proteinase K/ml (striped bars). The bacteria that remained attached in the microtiter plate were stained with crystal violet. The dye was dissolved with 30% glacial acetic acid (200 μl per well) and measured by OD₅₆₂. The untreated controls are represented by filled bars. The microtiter views are shown below.

Disruption of the early biofilm formation of mgrA mutants treated with DNase I. The biofilms of RN6390 and MW2 and their corresponding ΔmgrA mutant strains grown in 200 μl of TSB-glucose in microtiter wells for 16 h at 37°C in the presence of DNase I (0.7 units per ml) are represented by the striped bars. Biofilm formation was quantified by washing the biofilms three times with water, staining with crystal violet, and measuring the OD₅₆₂. The filled bars are the untreated controls. The corresponding views of the microtiter plates are shown below.

Biofilm formation by mgrA mutants. Top panel, biofilm formation of the mgrA mutants, wild types (wt), and their respective complemented strains (c) was quantified by solubilizing the crystal violet stain in 30% glacial acetic acid and measuring the absorbance at 562 nm. As assessed by the Student t test, the significance of the data was as follows: the mgrA mutant of RN6300 versus the parent and complement, P < 0.0004; the mgrA mutant of SH1000 versus the parent and complement, P < 0.03; the mgrA mutant of MW2 versus the parent and mutant, P < 0.0001. Bottom panel, biofilm formation of mgrA mutants and their complemented strains on microtiter plates grown in TSB supplemented with glucose (0.25%) at 37°C over 16 h. A characterization of the colony morphology of different mgrA mutants and their respective complemented strains on CRA plates is presented below.

(A) Dot blot analysis of PIA/PNAG accumulation in mgrA mutants and wild-type strains. Cell surface extracts from overnight cultures of S. aureus wild-type strains RN6390 and MW2, their mgrA mutants, and the positive control SA113, treated as described in Materials and Methods, were placed onto nitrocellulose membranes. PIA/PNAG production was detected with an anti-PIA/PNAG polyclonal antibody. (B) Quantification of the activity of the 164-bp ica promoter (19) in the mgrA mutants and their corresponding wild types by transcriptional fusion studies. The expression of green fluorescent protein driven by the ica promoter was measured as a fluorescence/OD₅₉₅ ratio using average values of triplicate readings. This experiment was repeated at least three times, with one representative experiment shown.

Effect of RNAIII on biofilm formation in mgrA mutants of RN6390 and MW2. (A) agr deletion mutants were compared with mgrA mutants and double mgrA agr mutants for biofilm formation. Biofilms were quantitated by solubilizing the crystal violet in 30% glacial acetic acid and measuring the OD₅₆₂. Statistical values as determined by the Student t test were as follows: RN6390 versus the mgrA mutant, P < 0.0002; RN6390 versus the agr mutant, P < 0.000001; RN6390 versus the mgrA agr mutant, P < 0.00015; MW2 versus the mgrA mutant, P < 0.0001; MW2 versus the agr mutant, P < 0.004; and MW2 versus the mgrA agr mutant, P < 0.00002. There were no statistical differences among the mgrA, agr, and mgrA agr mutants in both RN6390 and MW2. (B) The plasmid pRN6735 with the blaZ promoter driving RNAIII was introduced into mgrA mutants of RN6390 and MW2 followed by quantitation of the biofilm formation as above, using parents and the mgrA mutant as controls. The P values were as follows: the mgrA mutant of RN6390 versus the mgrA mutant with RNAIII, P < 0.0004; RN6390 versus the mgrA mutant with RNAIII, P < 0.02; the mgrA mutant of MW2 versus the mgrA mutant with RNAIII, P < 0.005; MW2 versus the mgrA mutant with RNAIII, P < 0.003. (C) The plasmid pALC1743 with the RNAIII promoter driving the expression of GFP_uvr was introduced into MW2 and its isogenic mgrA mutant. Promoter activity was measured as fluorescence units per OD₅₆₂ over a 10-h period and then overnight. Each value represents the mean of three distinct clones from each genetic background. * and **, statistical significance by the Student t test when comparing fluorescence at 10 h (P < 0.03) and overnight (P < 0.05), respectively, between MW2 and the isogenic mgrA mutant.