Figure 3

Analysis of LuxS localization. (A) Growth of S. Typhimurium wildtype and luxSβla with ampicillin. The minimal inhibitory concentration (MIC) for sensitivity to ampicillin (μg ml^-1) in liquid culture was determined for each strain as described in the Methods section. These data are representative for three biological repeats. (B) Strains were grown on LB plates containing the chromogenic alkaline phosphatase substrate BCIP. Active alkaline phosphatase converts this substrate into a blue product. Negative and positive control strains express PhoA either without or with signal peptide (SP) from a constitutive promoter (pCMPG5748 and pCMPG5734); pCMPG5730 expresses a LuxS-PhoA fusion protein. All strains carry a ΔphoN mutation (CMPG5726). (C) Strains were grown to mid-exponential phase (OD₅₉₅ 1) and a PhoA activity test was performed. Average results of at least 3 biological replicates are shown with standard deviations. (D) Cellular fractionation of LuxS-PhoA fusion and control strains. (E) Cellular fractionation of S. Typhimurium expressing chromosomally FLAG-tagged LuxS. Total cells (T), grown to OD₅₉₅ 1, were separated into periplasmic (P), cytoplasmic (C) and membrane (M) fractions as described in the Methods section. The proteins maltose binding protein (MBP), alkaline phosphatase without signal peptide (PhoA-SP) and outer membrane protein A (OmpA) were used as periplasmic, cytoplasmic and membrane associated control proteins, respectively. All antibodies used are listed in the Methods section. Remarkably, in both panels D an E, the LuxS-PhoA fusion protein and FLAG-tagged LuxS protein respectively, seem to differ in molecular weight between the different fractions. This might be related to the unknown translocation mechanism.