Reciprocal Control between a Bacterium's Regulatory System and the Modification Status of Its Lipopolysaccharide  Akinori Kato, H. Deborah Chen, Tammy Latifi, Eduardo A. Groisman  Molecular Cell  Volume 47, Issue 6, Pages 897-908 (September 2012) DOI: 10.1016/j.molcel.2012.07.017 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 47, 897-908DOI: (10.1016/j.molcel.2012.07.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 PmrA-Regulated Lipid A Modifications, Regulation of and by the PmrA/PmrB System, and Control of Fe3+ Availability by PmrA-Dependent LPS Modifications (A) Schematic representation of the lipid A moiety of the LPS and of the lipid A modifications discussed in the paper. 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 species. The lipid A phosphates can be modified with phosphoethanolamine (pEtN) by PmrC or with L-4-aminoarabinose (L-Ara4N) by Ugd and PbgP. PmrR inhibits LpxT activity. (B) Model for activation of the PmrA/PmrB system and selected targets of PmrA control. Transcription of PmrA-activated genes is promoted during growth in low Mg2+ via the PhoP/PhoQ system, the PmrD protein, and the PmrA/PmrB system, and in the presence of Fe3+ via the PmrA/PmrB system and independently of PhoP/PhoQ and PmrD. Phosphorylated PmrA activates transcription of LPS-modification loci (i.e., pbgP, ugd, and pmrC) and controls its own levels by positively regulating the pmrCAB operon and the peptide-specifying pmrR, and by repressing transcription of the pmrD gene. (C) When Salmonella experience noninducing conditions (i.e., high Mg2+ and low Fe3+), the PmrB sensor, which is localized to the inner membrane (IM), functions primarily as a PmrA-P phosphatase, and there is no transcription of PmrA-dependent loci. Iron acquisition systems (green rectangles) are highly expressed. The LPS, which constitutes the outer leaflet of the outer membrane (OM), has a net negative charge (in red). (D) At early times during growth in the presence of Fe3+, high levels of Fe3+ associate with negatively charged LPS, leading to a corresponding increase in periplasmic Fe3+ levels, which are sensed by the PmrB protein. PmrB promotes the phosphorylation of PmrA, resulting in transcription of PmrA-activated genes, including those responsible for lipid A modifications that lower the net negative charge of the LPS. (E) At later times during growth in the presence of Fe3+, PmrA-regulated LPS modifications that reduce the net negative charge on the bacterial surface have taken place (in blue), lowering the amount of Fe3+ bound to the LPS. The expression of iron acquisition systems (green rectangles) has decreased. Less Fe3+ is available for binding to and activating PmrB in the periplasm, and transcription of PmrA-dependent loci is dampened. Molecular Cell 2012 47, 897-908DOI: (10.1016/j.molcel.2012.07.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 A PmrA-Activated Gene that Encodes a Short Membrane Peptide (A) Alignment of the PmrA box-containing DNA region located downstream of the pmrB coding region in the S. enterica (STM), E. coli (ECO), C. koseri (CKO), and K. pneumoniae (KPN) genomes. Asterisks indicate identical nucleotides in the listed bacterial species. The predicted PmrA binding sites (PmrA box) are bolded in gray. The predicted −10 sequences of the RNA polymerase recognition site, the Shine Dalgarno (SD), sequences and the start codon of PmrR are bolded in black. An arrow indicates the PmrA/PmrB-dependent transcription start site for pmrR. (B) Deduced amino acid sequence of PmrR in S. enterica (STM), C. koseri (CKO), E. coli (ECO), and K. pneumoniae (KPN). Asterisks correspond to amino acids that are conserved in the four species. Twin dots correspond to conservative amino acid substitutions. (C) The FLAG-His6-PmrR peptide localizes to the inner membrane. Western blot analyses using an anti-FLAG antibody of the inner and outer membrane fractions were prepared from a Salmonella ΔpmrAB-pmrR strain (EG17955) harboring plasmids pBAD18-pmrR or pBAD18-FLAG-His6-pmrR. NADH oxidase activity (μmol of substrate oxidized/min/μg protein) was used as an indicator of inner membrane purity. OM, outer membrane fraction; IM, inner membrane fraction. See also Figure S1. Molecular Cell 2012 47, 897-908DOI: (10.1016/j.molcel.2012.07.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 PmrR Interacts with LpxT, Hindering LpxT-Mediated Modification of Lipid A (A) β-galactosidase activities (arbitrary units) from E. coli DHP1 Δcya strains harboring plasmids in the combinations indicated in the figure. The plasmids encode adenylate cyclase protein fusions either to the N terminus of PmrR, LpxT, and YbjG (T25-PmrR, T18-LpxT, and T18-YbjG) or to the C terminus of LpxT, YbjG, and PmrC (LpxT-T18, YbjG-T18, and PmrC-T18). Cells harboring the plasmids encoding T25-Zip and Zip-T18, which are known to interact in the BACTH assay, were used as a positive control. Data correspond to the mean of three independent experiments performed in duplicate as described (Camp and Losick, 2009), and error bars represent standard deviation. (B) β-galactosidase activities (Miller units) from E. coli BTH101 cya strains harboring plasmids in the combinations indicated in the figure. The plasmids encode adenylate cyclase protein fusions either to the N terminus of PmrR and PmrR W25A (T25-PmrR and T25-PmrR W25A) or to the C terminus of LpxT (LpxT-T18). Data correspond to the mean of three independent experiments performed in duplicate as described (Miller, 1972), and error bars represent standard deviation. (C) Coimmunoprecipitation analyses from membrane fractions of an lpxT-lacZ+ strain (AK1049) harboring plasmids pBAD18 or pBAD18-FLAG-His6-pmrR and of the wild-type strain (14028s) harboring plasmid pBAD18-FLAG-His6-pmrR. Total membranes (input) and Ni-NTA eluate (pull-down) were analyzed by SDS-PAGE, and the presence of LpxT-LacZ and CorA (indicated by arrows) was determined by western blotting using anti-LacZ or anti-CorA antibodies, respectively. (D) Thin-layer chromatograph (TLC) analysis of 32P-labeled lipid A from wild-type (14028s) and lpxT mutant (AK1032) strains harboring the vector pBAD18 or plasmid pBAD18-pmrR. Cells were grown for 10 hr in N-minimal medium, pH 7.7, with 10 mM Mg2+ and in the presence of 1 mM L-arabinose. The identity of the lipid A species is based on data presented here and in Herrera et al. (2010). (E) TLC analysis of 32P-labeled lipid A from wild-type (14028s), pmrR promoter mutant (EG15437), lpxT mutant (AK1032), and lpxT pmrR promoter double mutant (AK1038); and of wild-type Salmonella (14028s) harboring the vector pBAD18 or plasmid pBAD18-pmrR. Bacteria were grown for 12 hr in N-minimal medium, pH 7.7, with 10 μM Mg2+ and 100 μM Fe3+ or with 10 mM Mg2+ and 1 mM L-arabinose. Molecular Cell 2012 47, 897-908DOI: (10.1016/j.molcel.2012.07.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 PmrR Decreases Fe3+ Binding and Transcription of PmrA-Activated Genes in an LpxT-Dependent Manner (A) Levels of Fe3+ associated with wild-type (14028s), pmrR promoter mutant (EG15437), pmrR start codon mutant (EG15439), lpxT mutant (DC72), lpxT pmrR promoter mutant (DC73), lpxT pmrR start codon mutant (DC82), pmrA null mutant (EG7139), and pmrA505 (EG9242) Salmonella. Data correspond to the mean of at least three independent experiments, and error bars represent standard deviation. Broken line corresponds to Fe3+ levels in the wild-type strain. (B) β-galactosidase activities (arbitrary units) from ugd-lac or pbgP-lac transcriptional fusions were determined in strains with a wild-type pmrR (EG9524 or EG9241), pmrR promoter mutation (EG17203 or EG15433), and pmrR start codon mutation (EG17204 or EG15435). Bacteria were grown for 8 hr in N-minimal medium, pH 7.7, with 10 μM Mg2+ and 100 μM Fe3+. Data correspond to the mean of three independent experiments performed in duplicate as described (Camp and Losick, 2009), and error bars represent standard deviation. (C) β-galactosidase activities (arbitrary units) from a ugd-lac transcriptional fusion determined in the wild-type pmrR+ (EG9524), pmrR promoter mutant (EG17203), and pmrR start codon mutant (EG17204) strains and the isogenic derivatives lacking the lpxT gene (AK1040, AK1041, and AK1042, respectively). Bacteria were grown as described in (B). Data correspond to the mean of three independent experiments performed in duplicate as described in Camp and Losick (2009), and error bars represent standard deviation. (D and E) mRNA levels of the PmrA-activated ugd (D) and pmrC (E) genes produced during growth in the presence of Fe3+ by wild-type (14028s), pmrR promoter mutant (EG15437), pmrR start codon mutant (EG15439), lpxT mutant (DC72), lpxT pmrR promoter double mutant (DC73), and lpxT pmrR start codon double mutant (DC82) Salmonella. RNA samples were prepared from bacteria grown for 1 hr in N-minimal medium at pH 7.7 with 10 μM Mg2+ and 100 μM Fe3+, and mRNA levels of PmrA-activated genes were determined by reverse-transcription qPCR analysis. Data correspond to the mean of three independent experiments, and error bars represent standard deviation. (F) β-galactosidase activities (arbitrary units) from a ugd-lac transcriptional fusion were determined in the wild-type strain (EG9524) harboring the vector pBAD18 and in the pmrR promoter mutant strain (EG17203) harboring plasmids pBAD18, pBAD18-pmrR, pBAD18-FLAG-His6-pmrR, or pBAD18-pmrR-FLAG-His6. Bacteria were grown as described in (B) in the presence of 1 mM L-arabinose. Data correspond to the mean of three independent experiments performed in duplicate as described (Camp and Losick, 2009), and error bars represent standard deviation. (G) β-galactosidase activities (Miller units) from a ugd-lac transcriptional fusion determined in the wild-type pmrR promoter strain (EG17202) harboring vector pBAD18 and in a strain with a pmrR promoter mutation (EG17203) harboring plasmids pBAD18, pBAD18-pmrR, or pBAD18- pmrR W25A. Bacteria were grown as described in (B) in the presence of 1 mM L-arabinose. Data correspond to the mean of three independent experiments performed in duplicate as described (Miller, 1972), and error bars represent standard deviation. See also Figure S2. Molecular Cell 2012 47, 897-908DOI: (10.1016/j.molcel.2012.07.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Preventing Modification of Lipid A with L-Ara4N Enhances Fe3+ Binding and Expression of PmrA-Activated Genes (A and B) The levels of the PmrA-activated pbgP (A) and pmrC (B) mRNAs produced during growth in the presence of Fe3+ were determined in wild-type (14028s), pmrR promoter mutant (EG15437), pmrR promoter deletion mutant (EG15357), ugd mutant (EG17202), ugd pmrR promoter double mutant (EG17203), pmrC mutant (EG9460), pmrC ugd double mutant (DC169), pmrC pmrR promoter deletion double mutant (DC171), and pmrC ugd pmrR promoter deletion triple mutant (DC173) Salmonella. RNA samples were prepared from bacteria grown for 4 hr in N-minimal medium at pH 7.7 with 10 μM Mg2+ and 100 μM Fe3+, and mRNA levels of PmrA-activated genes were determined by reverse-transcription qPCR analysis. Data correspond to the mean of three independent experiments, and error bars represent standard deviation. Broken line corresponds to expression levels in the wild-type strain. (C) Levels of Fe3+ associated with wild-type (14028s), pmrR promoter mutant (EG15437), pmrR promoter deletion mutant (EG15357), ugd mutant (EG17202), ugd pmrR promoter double mutant (EG17203), pmrC mutant (EG9460), pmrC ugd double mutant (DC169), pmrC pmrR promoter deletion double mutant (DC171), and pmrC ugd pmrR promoter deletion triple mutant (DC173) Salmonella. Bacteria were grown as described above. Data correspond to the mean of at least three independent experiments, and error bars represent standard deviation. Broken line corresponds to Fe3+ levels in the wild-type strain. (D) Levels of Fe3+ associated with wild-type (14028s), lpxT mutant (DC72), ugd mutant (EG17898), and lpxT ugd double mutant (DC298) Salmonella. Bacteria were grown as described above. Data correspond to the mean of at least three independent experiments, and error bars represent standard deviation. Broken line corresponds to Fe3+ levels in the wild-type strain. (E and F) The levels of the PmrA-activated pbgP (E) and pmrC (F) mRNAs produced during growth in the presence of Fe3+ were determined in wild-type (14028s), lpxT mutant (DC72), ugd mutant (EG17898), and lpxT ugd double mutant (DC298) Salmonella. RNA was prepared from bacteria grown as described above, and levels of PmrA-activated mRNAs were determined by reverse-transcription qPCR analysis. Data correspond to the mean of three independent experiments, and error bars represent standard deviation. Broken line corresponds to expression levels in the wild-type strain. See also Figure S3. Molecular Cell 2012 47, 897-908DOI: (10.1016/j.molcel.2012.07.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Control of the PmrA/PmrB System by Ugd-Mediated Lipid A Modification Takes Place at Late Times Following Exposure to Fe3+ (A) Levels of Fe3+ associated with wild-type (14028s) and ugd mutant (EG17898) Salmonella. Bacteria were grown in N-minimal medium at pH 7.7 with 10 mM Mg2+ to OD600 ∼0.4, shifted to medium containing 10 μM Mg2+ and 100 μM Fe3+ and harvested at the designated times to determine the total amount of iron. Data correspond to the mean of at least three independent experiments, and error bars represent standard deviation. Broken line corresponds to Fe3+ levels in the wild-type strain. (B) Levels of PmrA-activated pbgP mRNAs produced in wild-type and ugd mutant (EG17898) strains as determined by reverse-transcription qPCR analysis. RNA was prepared from bacteria grown as described in (A). Data correspond to the mean of three independent experiments, and error bars represent standard deviation. (C) TLC analysis of 32P-labeled lipid A from wild-type (14028s) and ugd mutant (EG17898) Salmonella. Bacteria were grown as described in (A) in the presence of 32P-orthophosphate and harvested at the designated times to isolate lipid A. (D) Percentage survival of wild-type (14028s) or ugd mutant (EG17898) Salmonella following incubation in the presence of 4 μg/ml polymyxin B relative to the initial inoculum. Bacteria were grown as described in (A) and harvested at the designated times for polymyxin B treatment. Data correspond to the mean of at least three independent experiments, and error bars represent standard deviation. Note logarithmic scale of y axis. See also Figure S4. Molecular Cell 2012 47, 897-908DOI: (10.1016/j.molcel.2012.07.017) Copyright © 2012 Elsevier Inc. Terms and Conditions