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. 2007 Oct;189(20):7213-22.
doi: 10.1128/JB.00973-07. Epub 2007 Aug 10.

PhoPQ-mediated regulation produces a more robust permeability barrier in the outer membrane of Salmonella enterica serovar typhimurium

Takeshi Murata  1 , Will Tseng, Tina Guina, Samuel I Miller, Hiroshi Nikaido
Affiliations

PhoPQ-mediated regulation produces a more robust permeability barrier in the outer membrane of Salmonella enterica serovar typhimurium

Takeshi Murata et al. J Bacteriol. 2007 Oct.

Abstract

The PhoPQ two-component system of Salmonella enterica serovar Typhimurium produces a remodeling of the lipid A domain of the lipopolysaccharide, including the PagP-catalyzed addition of palmitoyl residue, the PmrAB-regulated addition of the cationic sugar 4-aminoarabinose and phosphoethanolamine, and the LpxO-catalyzed addition of a 2-OH group onto one of the fatty acids. By using the diffusion rates of the dyes ethidium, Nile red, and eosin Y across the outer membrane, as well as the susceptibility of cells to large, lipophilic agents, we evaluated the function of this membrane as a permeability barrier. We found that the remodeling process in PhoP-constitutive strains produces an outer membrane that serves as a very effective permeability barrier in an environment that is poor in divalent cations or that contains cationic peptides, whereas its absence in phoP null mutants produces an outer membrane severely compromised in its barrier function under these conditions. Removing combinations of the lipid A-remodeling functions from a PhoP-constitutive strain showed that the known modification reactions explain a major part of the PhoPQ-regulated changes in permeability. We believe that the increased barrier property of the remodeled bilayer is important in making the pathogen more resistant to the stresses that it encounters in the host, including attack by the cationic antimicrobial peptides. On the other hand, drug-induced killing assays suggest that the outer membrane containing unmodified lipid A may serve as a better barrier in the presence of high concentrations (e. g VSports手机版. , 5 mM) of Mg(2+). .

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"V体育平台登录" Figures

FIG. 1.
FIG. 1.
Modification of lipid A structure by the PhoPQ regulatory system. The modified structures are indicated by bold type and thick lines.
FIG. 2.
FIG. 2.
Ethidium influx into tolC null mutants. HN1138 (phoP+ tolC) (wt), HN1139 (phoP null, tolC), and HN1140 (PhoP constitutive [PhoPC], tolC) were grown in LB broth with aeration by shaking, and cells were harvested at the beginning of the stationary phase (at an OD600 between 2.3 and 2.4). Cells were washed and resuspended in 50 mM potassium phosphate buffer (pH 7.0), and the ethidium influx was assayed in the absence and presence of 50 μM CCCP. AU, arbitrary units.
FIG. 3.
FIG. 3.
Effect of Mg2+ on the rates of entry of ethidium in tolC mutants. The initial rates of entry were measured with HN1140 (PhoP constitutive, tolC) (▪) and HN1139 (phoP null, tolC) (▴) in potassium phosphate buffer (pH 7.0). The rates are expressed as the change in fluorescence intensity (in arbitrary units) per second. EtBr, ethidium bromide.
FIG. 4.
FIG. 4.
Penetration of a neutral dye and an acidic dye across the OM of tolC mutants. Isogenic strains HN1139 (phoP null, tolC) and HN1140 (PhoP constitutive [PhoPc], tolC) were grown and harvested as described in Materials and Methods. For the influx of a neutral dye, Nile red, the cells were resuspended in 50 mM potassium phosphate buffer (pH 7.0), and the dye was added to a final concentration of 2 μM at the beginning of the experiment. Fluorescence was monitored at 630 nm with excitation at 540 nm. For the assay of the penetration of an acidic dye, eosin Y, cells were washed and resuspended in 50 mM morpholineethanesulfonic acid (MES)-KOH buffer (pH 5.5) at a concentration of 40 OD600 units/ml. Eosin Y (Na salt) was added to a concentration of 1 mM, and the mixtures were kept at 37°C for 2 h to allow for passive accumulation of this weakly acidic dye in the cytosol. For the assay, 5 μl of the mixture was added, at about 20 s, to 2 ml of 50 mM phosphate buffer (pH 7.0), and the fluorescence was recorded at 540 nm with excitation at 520 nm.
FIG. 5.
FIG. 5.
Disk diffusion drug susceptibility assay. Either the phoP null strain HN1139 (left panels) or the PhoP-constitutive strain HN1140 (right panels) was spread on plates of modified M9 minimal medium containing 0.1 mM MgCl2 and 0.4% glucose. Disks contained the indicated amounts of erythromycin (EM), apramycin (AP), fusidic acid (FA), and gentamicin (GM).
FIG. 6.
FIG. 6.
Killing rates in the modified M9 medium. Early-exponential-phase cultures of HN1139 and HN1140 were diluted 100-fold into modified M9 medium containing the indicated concentrations of erythromycin, novobiocin, or rifampin. The numbers of surviving cells after 3 h of incubation were determined by plating onto LB agar plates. Circles, HN1139 (phoP null, tolC); triangles, HN1140 (PhoP constitutive, tolC); open symbols, M9 medium containing 0.1 mM MgCl2; filled symbols, M9 medium containing 5 mM MgCl2.
FIG. 7.
FIG. 7.
Ethidium influx into PhoP-constitutive strains carrying additional mutations in lipid A remodeling genes. Ethidium entry was measured as described in Materials and Methods. The strains used were an isogenic series, all containing an rpsL mutation and the same tolC null mutation, and included HN1147 (phoP null), HN1145 (PhoP constitutive [PhoPc], lpxO pagP pmrA), HN1144 (PhoP constitutive, lpxO pagP), HN1142 (PhoP constitutive, lpxO), HN1143 (PhoP constitutive, pagP), and HN1140 (PhoP constitutive). a.u., arbitrary units.
FIG. 8.
FIG. 8.
Rate of entry of ethidium into CS093 cells with various concentrations of CCCP. Wild-type S. enterica serovar Typhimurium strain CS093 was grown in LB broth with aeration by shaking and was harvested when the OD600 reached 1.8. The influx of ethidium into cells, which generates fluorescence due to binding of the dye with nucleic acids, was measured as described in Materials and Methods, in 50 mM potassium phosphate buffer (pH 7.0). Cells were preincubated with the indicated concentrations of CCCP for 5 min, before addition of 6 μM (final concentration) ethidium. The ordinate indicates the entry rate of ethidium expressed as the change in the fluorescence emission intensity (in arbitrary units) per second. EtBr, ethidium bromide.
FIG. 9.
FIG. 9.
Influx of ethidium into CS015 (phoP null) and CS022 (PhoP-constitutive [PhoPc]) mutant cells. The strains were grown in LB broth with shaking, and the cells were harvested when the OD600 reached 1.8; then the cells were washed and resuspended in 50 mM potassium phosphate buffer with or without 2 mM MgCl2. The ethidium influx was assayed in the same buffer with 100 μM CCCP, as described in Materials and Methods. AU, arbitrary units.
FIG. 10.
FIG. 10.
Rate of entry of ethidium into wild-type (D21) and deep rough LPS mutant (D21f2) cells of E. coli K-12 in the presence of 50 μM CCCP. Cells were grown in LB broth with aeration by shaking and were harvested when the OD600 reached 1.8. The influx of ethidium into cells, which generates fluorescence due to binding of the dye with nucleic acids, was measured as described in Materials and Methods, in 50 mM potassium phosphate buffer (pH 7.0) containing 5 mM MgCl2. Cells were preincubated with CCCP for 5 min before addition of 2 μM (final concentration) ethidium at 50 s.
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