Synthesis of RcsB (A) and PmrA (B) protein in <i>S.</i> Enteritidis <i>dam</i> mutant.

<p>Western blot analysis of total proteins from <i>S.</i> Enteritidis #5694 wild type strain and <i>dam</i> mutant strains harboring an <i>rcsB</i>::3×FLAG (A) or <i>pmrA::3×</i>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.). <b>Panel A.</b> wt: wild type strain #5694 SE<i>rcsB</i>::3×FLAG; <i>dam</i>: <i>dam</i> mutant strain SE<i>rcsB</i>::3×FLAG. <b>Panel B.</b> wt: wild type strain #5694 SE<i>pmrA</i>::3×FLAG; <i>dam</i>: <i>dam</i> mutant strain SE<i>pmrA</i>::3×FLAG. Plasmid pIZ833 bears the <i>dam</i> 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.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

LPS analysis of <i>rcsB</i> (A) and <i>pmrA</i> (B) mutants of <i>S.</i> Enteritidis strains.

<p>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 p<i>rcsB</i> and p<i>pmrA</i> bears the <i>rcsB</i> and <i>pmrA</i> genes respectively.</p

Relative expression of <i>wzz</i>, <i>rcsB</i> and <i>pmrA</i> mRNA in <i>pmrA</i> and <i>rcsB</i> mutant by real-time quantitative PCR.

<p>Total mRNA was harvested from cultures of SEΔ<i>rcsB</i>, SEΔ<i>pmrA</i> and <i>S.</i> 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 <i>wzz</i> 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 <i>pmrA-</i>inducing conditions (low Mg²⁺ and low Mg²⁺+Fe³⁺ ); ¥ significant difference p<0.05 with respect to the same strain grown in <i>pmrA-</i>inducing conditions (low Mg²⁺ and low Mg²⁺+Fe³⁺).</p

Schematic diagram of the proposed regulatory network of <i>wzz</i> gene expression in <i>S.</i> Enteritidis.

<p>The regulatory cascade for <i>wzz</i> 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 <i>S.</i> Typhimurium. Positive regulation (induction) is labeled with ↑ and (+), whereas negative regulation (repression) is labeled with ⊥ and (−). The question mark indicates a putative regulation.</p

Oligonucleotides primers used in this study.

<p>Oligonucleotides primers used in this study.</p

Relative expression of <i>wzz</i> mRNA (A and B) and LPS analysis (C) of <i>rcsB pmrA</i> double mutant.

<p>A,B. Total mRNA was harvested from cultures of SEΔ<i>rcsB</i>Δ<i>pmrA</i> double mutant and <i>S.</i> 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 <i>pmrA-</i>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.</p

LPS analysis of <i>S.</i> Enteritidis strains overexpressing RcsB (A) or PmrA (B) protein.

<p>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. <b>A.</b> 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>B.</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, p<i>rcsB</i> and p<i>pmrA</i> bears the <i>dam</i>, <i>rcsB</i> and <i>pmrA</i> genes respectively.</p

Dam Methylation Participates in the Regulation of PmrA/PmrB and RcsC/RcsD/RcsB Two Component Regulatory Systems in Salmonella enterica Serovar Enteritidis

The absence of Dam in Salmonella enterica serovar Enteritidis causes a defect in lipopolysaccharide (LPS) pattern associated to a reduced expression of wzz gene. Wzz is the chain length regulator of the LPS O-antigen. Here we investigated whether Dam regulates wzz gene expression through its two known regulators, PmrA and RcsB. Thus, the expression of rcsB and pmrA was monitored by quantitative real-time RT-PCR and Western blotting using fusions with 36FLAG tag in wild type (wt) and dam strains of S. Enteritidis. Dam regulated the expression of both rcsB and pmrA genes; nevertheless, the defect in LPS pattern was only related to a diminished expression of RcsB. Interestingly, regulation of wzz in serovar Enteritidis differed from that reported earlier for serovar Typhimurium; RcsB induces wzz expression in both serovars, whereas PmrA induces wzz in S. Typhimurium but represses it in serovar Enteritidis. Moreover, we found that in S. Enteritidis there is an interaction between both wzz regulators: RcsB stimulates the expression of pmrA and PmrA represses the expression of rcsB. Our results would be an example of differential regulation of orthologous genes expression, providing differences in phenotypic traits between closely related bacterial serovars.Fil: Sarnacki, Sebastian Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas . Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina;Fil: Aya Castañeda, Maria del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas . Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina;Fil: Noto Llana, Mariangeles. Consejo Nacional de Investigaciones Científicas y Técnicas . Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina;Fil: Giacomodonato, Mónica Nancy. Consejo Nacional de Investigaciones Científicas y Técnicas . Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina;Fil: Valvano, Miguel Angel. The Queens University Of Belfast;Fil: Cerquetti, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas . Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina