PmrA-regulated loci identified included dgoA and yibD and demonstrated 500-and 2,500-fold activation by PmrA , respectively .
The remaining PMs mutants ( surA and tolB ) , as well as the two PmrA-regulated gene ( yibD and dgoA ) mutants , retained aminoarabinose on lipid-A .
The PmrA-regulated gene mutants JSG897 ( yibD ) and JSG899 ( dgoA ) were also analyzed for sensitivity to PM by MIC assay .
Mutants identified in the MudJ transposon mutagenesis screen Tn insertion site Description Strain PM sensitive imp/surA tolB gnd gnd yibD dgoA pmrC pmrC pmrC pmrC pmrI pmrI 851 895 JSG JSG JSG JSG JSG JSG JSG JSG JSG JSG JSG JSG 1272-1290-897-899 901-918-973-1036 923 925 PmrA regulated PmrA regulated , PM sensitive PmrA-regulated genes identified in the screen in iron resistance , JSG897 ( yibD ) and JSG899 ( dgoA ) were grown on solid N-minimal-medium supplemented with 100 M FeSO4 .
Mutants identified in the MudJ transposon mutagenesis screen Tn insertion site Description Strain PM sensitive imp/surA tolB gnd gnd yibD dgoA pmrC pmrC pmrC pmrC pmrI pmrI 851 895 JSG JSG JSG JSG JSG JSG JSG JSG JSG JSG JSG JSG 1272-1290-897-899 901-918-973-1036 923 925 PmrA regulated PmrA regulated , PM sensitive PmrA-regulated genes identified in the screen in iron resistance , JSG897 ( yibD ) and JSG899 ( dgoA ) were grown on solid N-minimal-medium supplemented with 100 M FeSO4 .
For further characterization of the PmrA-regulated gene mutants , the promoter regions of yibD and dgoA were analyzed for the presence of a putative PmrA-binding site , which was defined by the occurrence of the consensus binding sequence for PmrA , YTTAAK-N5-YTTAAK ( 1 ) .
yibD , dgoA , and gnd ( likely affecting pmrE ) played no role in PmrA-regulated resistance to high iron concentrations , while surA and tolB mutations grew poorly on high iron media .
JSG897 and JSG899 , with mutations in PmrA-regulated genes yibD and dgoA , respectively , also still possessed Ara4N modification of lipid-A in similar amounts to the parental PmrAc strain .
PmrA-regulated gene mutants JSG1525 ( yibD ) and JSG1526 ( dgoA ) were as virulent as wild-type Salmonella serovar Typhimurium , so the genes interrupted have no essential role in the infection process ( Table 4 ) .
All PMs mutants identified in this study demonstrated a defect in virulence compared to wild-type Salmonella serovar Typhimurium when administered orally to mice , while the PmrA-regulated gene ( yibD and dgoA ) mutants showed normal virulence in mice .
While it is possible that these PmrA-regulated genes ( as well as the genes involved in PM resistance ) could affect the other known PmrA-induced LPS modification , pEtN , we feel that this is unlikely based on what is known about the identified genes ( surA , tolB , and pmrE ) and because yibD and dgoA do not affect virulence or PM resistance , which are predicted phenotypes of the loss of pEtN modification from the core ( 59 ) .
A strong match to the consensus PmrA-binding site was identified upstream of yibD , indicating direct regulation by PmrA .
To determine whether PmrA can bind directly to promoter fragments of PmrA-regulated loci that lack the published PmrA consensus binding site , gel mobility-shift assays were performed to compare the ability of purified PmrA to bind the promoters of yibD and dgoR-KAT [ 34 ] .
PmrA was able to bind the yibD promoter .
We also made double mutants deleted in additional PmrA-regulated genes or operons ( i.e. ugd , yibD , and the pbgPE operon ) .