Transcriptional profiling of S. aureus mutants

The Newman wild-type strain, V149A, and D119A mutants were grown at 37 °C overnight in TSB. Cell cultures were then 1:100 diluted into fresh TSB supplemented with 50 μM DHBP when indicated and incubated at 37 °C for 2 h until the cultures reached an OD₆₀₀ of∼0.6. Bacteria were collected by centrifugation and washed with PBS. The cells were lysed by a bead beater and the total RNA was extracted by using an RNeasy minikit (Qiagen) with DNase I treatment step. The rRNA was removed by a MICROBExpress kit (Ambion) and the remaining RNA was utilized to construct the cDNA library by a TruSeq RNA sample prep kit (Illumina). The library was sequenced by next-generation sequencing (HiSeq 2000 (Illumina)). The sequencing reads were mapped to the S. aureus Newman strain genome by TopHat (version 2.0.0) with two mismatches allowed42 (link). Cutffdiff software (version 2.0.0) was used to perform gene differential expression analysis43 (link). Genes were annotated by BLAST and the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 (http://david.abcc.ncifcrf.gov). The experiments were performed in duplicate.