Pfs promotes autolysis-dependent release of eDNA and biofilm formation in Staphylococcus aureus

Yan Bao1#, Xu Zhang1#, Qiu Jiang1, Ting Xue2and Baolin Sun1*

1. Department of Microbiology and Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China

2. School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, China

# These authors contributed equally to this work.

* Correspondence: Baolin Sun; Tel: (86) 551 6360 6748; Fax: (86) 551 6360 7438; E-mail: ; Postal address: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China

Supplementary information:

Fig. S1. The biofilm formation of the wild-type (SBY2), the pfs mutant strain (SBY3) and the complementationstrain(SBY4) in the flow cell system was imaged using a macroscopic camera at different time point.The data represents two independent analyses.

Fig. S2. The degree of initial attachment during biofilm formation in the wild-type (SBY2), the pfs mutant strain (SBY3) and the complementationstrain(SBY4) was compared. Primary attachment was expressed as a percentage of the CFU remaining on the wells after washing for 60 min post-incubation. The data represent at least three independent analyses; error bars indicate SEM of three replicates.

Fig. S3.DNase I treatment has little effect on bacterial growth. The growth of cultures was determined through the measurement of the optical density at 600 nm of each well at 24 h after biofilm incubation. The data represent at least three independent analyses, and the error bars indicate the SEM of at least five replicates.

Fig. S4. Autolysis-related genes not affected by pfs deletion. The transcriptional profile of lytN (A), sle1(B), lytH (C), lytA (D), cidAB (E), lrgAB (F), SarV (G), SarA (H), ArlRS (I), LytSR (J), and MgrA (K)of the pfs mutant strain (SBY3) was compared with that of the wild-type (SBY2) and complementation (SBY4) strains using RNA isolated from the cultures at different growth phases (3-6/8 h). The data represent at least two independent analyses, and the error bars indicate the SEM of three replicates.

Supplementary methods

Initial attachment assay on polystyrene

The attachment of the cells to polystyrene was determined as previously described [23]. Briefly, overnight cultures grown in TSB were diluted 2.5 × 106-fold in fresh TSB supplemented with 3% NaCl, and 1 ml of the dilution cultures was added to each well of 24-well plates. After incubation at 37 °C for 1 h without shaking, the plates were gently rinsed with water six times and covered with 700 l of molten 0.8% TSA maintained at 45 °C for each well. In parallel, the cells were plated on TSA plates to determine the original number of cells used in the microtiter plates. The primary attachment was expressed as a percentage of the CFU remaining on the wells after washing. Each experiment was repeated three times.

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