Research ArticleMicrobiology

Gene expression kinetics governs stimulus-specific decoration of the Salmonella outer membrane

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Science Signaling  08 May 2018:
Vol. 11, Issue 529, eaar7921
DOI: 10.1126/scisignal.aar7921
  • Fig. 1 Signal-dependent control of lipid A modifications.

    (A) Schematic representation of the lipid A moiety of the LPS and the lipid A modifications that alter its negative charge. In Salmonella, the predominant lipid A species is hexa-acylated and phosphorylated at the 1 and 4′ positions. LpxT adds a second phosphate group to the 1 position, resulting in a 1-diphosphorylated (1-PP) species. Alternatively, the lipid A 1-phosphate can be modified with pEtN by PmrC. The 4′-phosphate can be modified with l-Ara4N by Ugd and PbgP. Alternatively, but to a lesser extent, the 4′-phosphate may be modified by the addition of pEtN and the 1-phosphate modified by the addition of l-Ara4N. PmrR binds to LpxT, inhibiting its activity. (B) Model for activation of the PhoP/PhoQ and PmrA/PmrB two-component systems and regulation by the PhoP and PmrA proteins. Transcription of PhoP-activated genes is promoted when the sensor PhoQ experiences low cytoplasmic pH (31), low periplasmic Mg2+, or the presence of particular antimicrobial peptides in the periplasm. Transcription of PmrA-activated genes is promoted when the sensor PmrB experiences Fe3+, Al3+, or a mildly acidic pH in the periplasm (4). PmrA is also activated in low Mg2+ by the PhoP-activated pmrD gene product. Phosphorylated PhoP promotes transcription of the pmrD and lpxT genes. Phosphorylated PmrA promotes transcription of the pbgP, pmrC, pmrR, and ugd genes. The pbgP, ugd, and pmrC genes encode enzymes that compete with LpxT for the modification of the lipid A 1-phosphate. The lipid A modifications catalyzed by PbgP, Ugd, and PmrC reduce the negative charge of lipid A. (C) Schematic of the lipid A structure over time after bacteria encounter low Mg2+ or a mildly acidic pH. Under low Mg2+, lpxT is activated by PhoP within 5 min, resulting in 1-PP lipid A. The additional phosphate group at the 1-phosphate prevents the addition of pEtN at this position. By 30 min, lipid A is preferentially modified at the 4′-phosphate with l-Ara4N. PmrR later inhibits LpxT, thereby preventing the incorporation of a phosphate at the 1 position. pEtN is not detected at later times despite inhibition of LpxT by PmrR, suggesting that l-Ara4N–modified lipid A is not a good substrate for PmrC. In a mildly acidic pH, transcription of lpxT gene occurs after that of genes encoding Ugd, PbgP, PmrC, and PmrR, resulting in lipid A modified with both l-Ara4N and pEtN.

  • Fig. 2 PhoP promotes lpxT transcription directly by binding to the lpxT promoter.

    (A) Quantification of lpxT transcripts by quantitative polymerase chain reaction (qPCR) before and 60 min after switching wild-type (WT; 14028s) and pmrA (EG7139), phoP (MS7953s), and phoP pmrA (EG12443) mutant Salmonella from high-Mg2+ medium (10 mM MgCl2) to low-Mg2+ medium (10 μM MgCl2) at pH 7.7, normalized to the rrs (16S ribosomal RNA) transcript. (B) Genomic structure and partial nucleotide sequence of the Salmonella yeiR-lpxT chromosomal region. The sequence in red corresponds to the PhoP box in the lpxT promoter, the underlined sequence to the yeiR stop codon, +1 to the lpxT transcription start site (TSS), and lowercase sequence (atg) to the lpxT start codon. (C) Primer extension analysis of lpxT in WT (14028s) and phoP mutant (MS7953s) Salmonella grown in N-minimal medium (pH 7.7) containing high Mg2+ or low Mg2+. Lanes C, G, A, and T indicate the sequence of the lpxT promoter. The boxed “G” indicates the TSS of lpxT. (D) Deoxyribonuclease I (DNase I) footprinting analysis of the lpxT promoter region using the indicated amounts of purified phosphorylated PhoP-6×His protein. Lanes A, T, C, and G indicate the sequence of the lpxT promoter. The sequence highlighted in red corresponds to the PhoP binding site. (E) Filter assay for binding of the purified PhoP protein to the WT lpxT promoter and to the mutant lpxTB promoter, which contains substitutions in the PhoP binding site. The sequence of the WT lpxT promoter and the nucleotide substitutions in the mutant lpxTB promoter are shown in the box above the graph. The sequence highlighted in red corresponds to the PhoP binding site. The graph shows the percentage of 32P signal compared to the maximum for binding of the indicated amount of PhoP protein to 32P-labeled WT and promoter fragments. Data are representative of three independent experiments. (F) Abundance of lpxT transcript before and 60 min after switching WT (14028s), phoP (MS7953s), and lpxTB (XH16) Salmonella from N-minimal medium (pH 7.7) containing high Mg2+ to low Mg2+, normalized to the rrs transcript. Mean and SD of three independent experiments are shown.

  • Fig. 3 The lpxT gene is expressed earlier relative to other lipid A–modifying genes in low Mg2+.

    Quantification of the indicated transcripts at different times after switching WT Salmonella (14028s) from high Mg2+ to low Mg2+ at pH 7.7, normalized to the rrs transcript. Data are means ± SD of three independent experiments.

  • Fig. 4 PhoP-dependent lpxT expression promotes lipid A modification with l-Ara4N in low Mg2+ but not in a mildly acidic pH.

    (A) Autoradiogram of a thin-layer chromatography (TLC) plate showing lipid A from WT Salmonella (14028s) at different times after they were switched from N-minimal medium (pH 7.7) containing high Mg2+ to low Mg2+ in the presence of radiolabeled phosphate. Data are representative of three independent experiments. The graph below the autoradiogram shows the quantification of lipid A 1-PP from the image, representing mean and SD (normalized to the total signal of each lane). (B) TLC showing lipid A from WT (14028s), lpxTB (XH16), and lpxT (DC72) Salmonella grown in N-minimal medium (pH 7.7) containing high Mg2+, N-minimal medium (pH 7.7) containing low Mg2+, or N-minimal medium (pH 4.9) containing high Mg2+ in the presence of radiolabeled phosphate. (C) TLC showing lipid A from WT (14028s), lpxT (DC72), pbgP (EG9241), pbgP lpxT (XH246), ugd (EG17898), and ugd lpxT (XH251) Salmonella grown in N-minimal medium (pH 7.7) containing high Mg2+ or low Mg2+. Data are representative of two independent experiments. The 1-PP band shows different mobility in each of the images because the samples in each panel were run for different amounts of time. Asterisks (*) indicate positions of lipid A containing l-Ara4N, and open circles (o) indicate positions of lipid A containing pEtN.

  • Fig. 5 Effects of low Mg2+, acidic pH, and PhoP-dependent transcription of the lpxT gene on polymyxin B resistance.

    (A) Quantification of the indicated transcripts at different times after switching WT Salmonella (14028s) from high Mg2+ at pH 7.7 to high Mg2+ at pH 4.9, normalized to the rrs transcript. Data are means ± SD of three independent experiments. (B) Percent survival of WT (14028s), pmrA (EG7139), lpxTB (XH16), and lpxT (DC72) Salmonella exposed to polymyxin B after growth in N-minimal medium (pH 7.7) with low Mg2+ or that (pH 4.9) with high Mg2+ (acidic pH). Survival values were calculated as relative to the original inoculum. Mean and SD of three independent experiments are shown. Unpaired Student’s t tests were performed comparing mutant strains with the WT under the same growth condition. *P < 0.05, **P < 0.01, and ****P < 0.0001; n.s. indicates P > 0.05 (not significant).

  • Fig. 6 PhoP-dependent transcriptional activation of the lpxT gene is conserved in a subset of Gram-negative species.

    (A) Alignment of the nucleotide sequence of the lpxT promoter region in selected enteric bacterial species reveals conservation of the PhoP binding site (underlined). The boxed “G” indicates the transcription start site of lpxT mapped in Salmonella. Asterisks (*) denote positions that are conserved in all four species: STM, S. enterica serovar Typhimurium; ECO, E. coli; CFD, C. freundii; KPN, K. pneumoniae. (B) Abundance of lpxT mRNA before and 60 min after WT (MG1655) and phoP (EG12976) E. coli were switched from N-minimal medium (pH 7.7) containing high Mg2+ to low Mg2+, normalized to the rrs transcript. Mean and SD of three independent experiments are shown.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/529/eaar7921/DC1

    Fig. S1. LpxT protein amounts in wild-type Salmonella.

    Fig. S2. yeiR and lpxT transcript abundances in wild-type Salmonella in low Mg2+.

    Fig. S3. Abundance of lpxT transcript in wild-type, phoP, and lpxT promoter mutant Salmonella.

    Fig. S4. Lipid A profiles of wild-type Salmonella in low Mg2+.

    Fig. S5. Lipid A profiles of wild-type, lpxT promoter, pmrC, and double pmrC lpxT promoter mutant Salmonella.

    Fig. S6. Lipid A profiles of wild-type, lpxT, pmrA, and pmrA lpxT Salmonella.

    Fig. S7. Resistance of wild-type, pmrA, pbgP, pmrC, and lpxT Salmonella to polymyxin B.

    Table S1. Lipid A profiles of isogenic Salmonella strains determined by mass spectrometry.

    Table S2. Coexistence of lipid A–modifying genes in the genomes of seven Gram-negative bacterial species.

    Table S3. Bacterial strains and plasmids used in this study.

    Table S4. Primers used in the construction of strains and plasmids.

    Table S5. Primers used in this study for the quantification of transcripts in Salmonella.

  • Supplementary Materials for:

    Gene expression kinetics governs stimulus-specific decoration of the Salmonella outer membrane

    Xinyu Hong, H. Deborah Chen, Eduardo A. Groisman*

    *Corresponding author. Email: eduardo.groisman{at}yale.edu

    This PDF file includes:

    • Fig. S1. LpxT protein amounts in wild-type Salmonella.
    • Fig. S2. yeiR and lpxT transcript abundances in wild-type Salmonella in low Mg2+.
    • Fig. S3. Abundance of lpxT transcript in wild-type, phoP, and lpxT promoter mutant Salmonella.
    • Fig. S4. Lipid A profiles of wild-type Salmonella in low Mg2+.
    • Fig. S5. Lipid A profiles of wild-type, lpxT promoter, pmrC, and double pmrC lpxT promoter mutant Salmonella.
    • Fig. S6. Lipid A profiles of wild-type, lpxT, pmrA, and pmrA lpxT Salmonella.
    • Fig. S7. Resistance of wild-type, pmrA, pbgP, pmrC, and lpxT Salmonella to polymyxin B.
    • Legend for table S1
    • Table S2. Coexistence of lipid A–modifying genes in the genomes of seven Gram-negative bacterial species.
    • Table S3. Bacterial strains and plasmids used in this study.
    • Table S4. Primers used in the construction of strains and plasmids.
    • Table S5. Primers used in this study for the quantification of transcripts in Salmonella.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Lipid A profiles of isogenic Salmonella strains determined by mass spectrometry.

    © 2018 American Association for the Advancement of Science

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