Fig 4.
srfJ is regulated by PhoP, SsrB, and RcsB. (A) β-Galactosidase activities were measured from cultures in LPM of several S. enterica strains: 14028 (wild type [wt]), null mutants (phoP, ssrB, hilA, and rcsB), mutants with constitutive activation of the PhoP/PhoQ or the Rcs system (pho-24 and rcsC55, respectively), and double mutants (pho-24 ssrB and pho-24 rcsC55), all of them carrying a chromosomal srfJ::lacZ transcriptional fusion. Means and standard deviations from duplicate experiments are represented. (B) Expression at the protein level was studied by Western blotting using strains expressing SrfJ-3×FLAG. Extracts from these strains were resolved by SDS-PAGE (4 to 15% gradient), and a monoclonal anti-FLAG antibody was used for immunoblotting (upper panel). A polyclonal anti-GroEL antibody was used to get a loading control (lower panel). (C) A plasmid expressing wild-type phoP (phoP+) under a PBAD promoter was used to complement the effect of a null phoP mutation on srfJ transcription. The empty vector (pBAD18) was used as a control. Means and standard deviations of β-galactosidase activities obtained from strains carrying a chromosomal srfJ::lacZ transcriptional fusion grown in LPM supplemented with 0.2% arabinose are represented. (D) To study the role of RcsB and RcsA in the regulation of srfJ, β-galactosidase activities were measured from cultures in LPM of wild-type (wt) S. enterica or mutants, as indicated, all of them carrying a chromosomal srfJ::lacZ transcriptional fusion. Means and standard deviations from duplicate experiments are represented.