Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Oligonucleotides primers used in this study.
Figure 1.
LPS analysis of S. Enteritidis strains overexpressing RcsB (A) or PmrA (B) protein.
Equal amount of LPS was loaded in each lane and analyzed by Tricine/SDS-PAGE on a 14% (w/v) acrylamide gel followed by silver staining. The concentration of LPS was determined by measuring KDO using the purpald assay. A. Lanes 1–4: bacteria grown in N-minimal medium containing 10 mM MgCl2; lanes 5–8: bacteria grown in N-minimal medium containing 10 µM MgCl2 100 µM FeSO4. B. Lanes 1–4: bacteria grown in N-minimal medium containing 10 mM MgCl2; lanes 5–7: bacteria grown in N-minimal medium containing 10 µM MgCl2 100 µM FeSO4. Plasmids pIZ833, prcsB and ppmrA bears the dam, rcsB and pmrA genes respectively.
Figure 2.
LPS analysis of rcsB (A) and pmrA (B) mutants of S. Enteritidis strains.
Equal amount of LPS was loaded in each lane and analyzed by Tricine/SDS-PAGE on a 14% (w/v) acrylamide gel followed by silver staining. The concentration of LPS was determined by measuring KDO using the purpald assay. Plasmids prcsB and ppmrA bears the rcsB and pmrA genes respectively.
Figure 3.
Relative expression of pmrA rcsB, and wzz mRNA determined by real-time quantitative PCR.
Total mRNA was harvested from cultures of SEΔdam, S. Enteritidis #5694 (wild type) and complemented strains. The relative mRNA amount was determined by reverse transcription real-time quantitative PCR and related to mRNA levels in wild type strain, set as 1. Values are means ± SD of five independent mRNA extractions performed in triplicates. Plasmid pIZ833 bears the dam gen. * significant difference p<0.01 with respect to wild type strain.
Figure 4.
Synthesis of RcsB (A) and PmrA (B) protein in S. Enteritidis dam mutant.
Western blot analysis of total proteins from S. Enteritidis #5694 wild type strain and dam mutant strains harboring an rcsB::3×FLAG (A) or pmrA::3×FLAG (B) transcriptional fusion in the chromosome grown in LB medium and harvested at an OD600 of 0.6. Protein loading was normalized to 106 CFU. Blots were probed with anti-FLAG antibodies. Band intensity was determined by densitometry; relative intensities are presented in arbitrary units (a.u.). Panel A. wt: wild type strain #5694 SErcsB::3×FLAG; dam: dam mutant strain SErcsB::3×FLAG. Panel B. wt: wild type strain #5694 SEpmrA::3×FLAG; dam: dam mutant strain SEpmrA::3×FLAG. Plasmid pIZ833 bears the dam gene. * Significant difference p<0.05. Data are expressed as means ± SD of percent change in band intensity relative to wild type of five independent experiments performed in duplicates.
Figure 5.
Relative expression of wzz, rcsB and pmrA mRNA in pmrA and rcsB mutant by real-time quantitative PCR.
Total mRNA was harvested from cultures of SEΔrcsB, SEΔpmrA and S. Enteritidis wild type #5694 (wild type) grown in LB medium (A,B,C) or grown in low Mg2+, low Mg2++Fe3+ and high Mg2+ (D). The relative amount of wzz mRNA was determined by reverse transcription real-time quantitative PCR and related to mRNA levels in wild type strain #5694 (A,B,C) or in wild type strain #5694 grown in low Mg2+ (D), set as 1. Values are means ± SD of five independent mRNA extractions performed in triplicates. * significant difference p<0.01 with respect to wild type strain #5694 grown in the same media; § significant difference p<0.01 with respect to the same strain grown in pmrA-inducing conditions (low Mg2+ and low Mg2++Fe3+ ); ¥ significant difference p<0.05 with respect to the same strain grown in pmrA-inducing conditions (low Mg2+ and low Mg2++Fe3+).
Figure 6.
Relative expression of wzz mRNA (A and B) and LPS analysis (C) of rcsB pmrA double mutant.
A,B. Total mRNA was harvested from cultures of SEΔrcsBΔpmrA double mutant and S. Enteritidis wild type #5694 grown in LB media (A) or grown in low Mg2+, low Mg2++Fe3+ and high Mg2+ (B). The relative mRNA amount was determined by reverse transcription real-time quantitative PCR and related to mRNA levels in wild type strain (A) or in wild type strain grown in low Mg2+ (B), set as 1. Values are means ± SD of five independent mRNA extractions performed in triplicates. * significant difference p<0.01 with respect to wild type strain grown in the same media; † significant difference p<0.05 with respect to same strain grown in pmrA-inducing conditions (low Mg2+ and low Mg2++Fe3+). C. Equal amount of LPS was loaded in each lane and analyzed by Tricine/SDS-PAGE on a 14% (w/v) acrylamide gel followed by silver staining. The concentration of LPS was determined by measuring KDO using the purpald assay.
Figure 7.
Schematic diagram of the proposed regulatory network of wzz gene expression in S. Enteritidis.
The regulatory cascade for wzz gene expression involves Dam methylation, PmrB/PmrA and RcsC/RcsD/RcsB two-component regulatory system and a putative third regulator (X). Proteins are indicated by ovals, whereas genes are symbolized by block arrows. Black dots indicate methylation sites (5′-GATC-3′ sequences). Dashed lines indicate direct interactions demonstrated in S. Typhimurium. Positive regulation (induction) is labeled with ↑ and (+), whereas negative regulation (repression) is labeled with ⊥ and (−). The question mark indicates a putative regulation.