The Gre factors affect the expression of SPI-1 effector proteins. (A) Contact haemolysis assays with cell-free supernatants from cultures of the WT and Δ greA Δ greB derivative strains. The haemolytic activity of LB bacterial cultures of WT and Δ greA Δ greB strains was monitored as an increase in the OD 550nm of the supernatant of a blood suspension with serial dilutions of cell-free supernatants. U: undiluted supernatant. Three independent bacterial cultures were tested. (B) Cell-free supernatants of two independent LB cultures of WT (SV5015) and its Δ greA , Δ greB and Δ greA Δ greB derivatives. Extracts were analyzed by Coomassie blue stained 12.5% SDS-PAGE. Lane M: molecular mass markers (size in kDa indicated). The bands labelled were identified as SipA (1), FliD (2) and SipC (3) by LC-MS/MS. (C) Immunodetection (lower panel) of the SPI-1 encoded SipA-FLAG protein in whole culture extracts from two independent cultures of WT and Δ greA Δ greB derivative strains. The upper panel is a section of a Coomassie stained gel as a loading control. (D) Immunodetection of SopE protein was performed in extracts from WT and Δ greA Δ greB strains obtained from cell-free supernatants of two independent LB cultures. (E) Semiquantitative RT-PCR of sipA in total RNA samples from LB cultures of the WT (SV5015) and Δ greA Δ greB strains. 16S RNA was used as endogenous control to confirm that equivalent quantities of templates were used. (F) sipC transcriptional expression was tested in cultures of WT and Δ greA Δ greB derivative strains carrying a chromosomal sipC :: lacZ fusion and either pBR322 or pBR greAB . A bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation from three biological replicates. All cultures were grown in LB at 37°C with vigorous shaking (200 rpm) up to an OD 600nm of 2.0.