In vitro characterization of transcriptional-fusions to virulence genes As a first application of the two-colour flow cytometric technique , the in-vivo activities of three promoters that drive the expression of genes with differential roles in virulence were studied : PsicA , which drives the expression of the operon sicA sipBCDA iacP within SPI1 ( Darwin and Miller , 2000 ) , P ( Valdivia and Falkow , 1997 ; called ssaH PssaG by Hensel et al. , 1998 ) , which drives the expression of the operon ssaHIJKLMV within SPI2 ( Cirillo et al. , 1998 ) , and PpagC , which drives the PhoP-activated expression of pagC ( Gunn et al. , 1995 ) .
In order to obtain direct evidence for this novel mode of PhoP regulation , we performed real time PCR analysis of pagC , a previously identified PhoP-activated gene .
We have investigated the transcriptional regulation and the interaction of the response regulator PhoP with the promoter regions of the PhoP-activated loci phoPQ , mgtA , slyB , pmrD , pcgL , phoN , pagC , and mgtCB .
The mgtC and pagC genes are both PhoP-activated genes required for intramacrophage growth [ 2,5 -- 7 ] .
The pagD and pagC genes are PhoP-activated genes ( 9 ) located next to each other in the Salmonella chromosome but transcribed in opposite directions from a shared intergenic region .
The PhoP-activated gene C ( pagC ) promoter of Salmonella is an inducible promoter , which is inhibited by Mg2 + in-vitro and activated after Salmonella phagocytosis by macrophages and dendritic cells in-vivo [ 52,53 ] .
The PhoP-activated gene pagC regulated by the PhoP/PhoQ system was identified as an IVI virulence protein in S. Typhi , and the PagC antibody was detected in sera from 11 of 14 patients , which shows that it has the potential to be developed as a diagnostic antigen ( 12 ) .
Distinct temporal expression of PhoP-activated genes in response to mildly acidic pH and low Mg2 + We examined the mRNA levels of the PhoP-activated genes pagC and pagK at 2 h and 4 h after Salmonella was shifted from non-inducing conditions for the PhoQ protein to media containing either mildly acidic pH or low ( i.e. , 10 μM ) Mg2 + .
Distinct temporal expression of PhoP-activated genes in response to mildly acidic pH and low Mg2 + he MgtA protein is necessary for full transcription of a subset of PhoP-activated genes hen the PhoQ inducing signal is low Mg2 + T w Wild-type and mgtA strains had similar pagC and pagK mRNA levels at 2 h post induction ( Fig. 2A ) ( i.e. , ratio of wild-type/mgtA of 1 ) .
PhoP dephosphorylation subsequently inhibits the PhoPQ to activate the pagC in S. Choleraesuis