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Journal of Bacteriology, July 2007, p. 5161-5169, Vol. 189, No. 14
0021-9193/07/$08.00+0     doi:10.1128/JB.01969-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

PmrA(Con) Confers pmrHFIJKL-Dependent EGTA and Polymyxin Resistance on msbB Salmonella by Decorating Lipid A with Phosphoethanolamine{triangledown}

Sean R. Murray,1,{dagger} Robert K. Ernst,2 David Bermudes,3,{ddagger} Samuel I. Miller,2 and K. Brooks Low4*

Department of Biology, Yale University, New Haven, Connecticut,1 Department of Microbiology, University of Washington, Seattle, Washington,2 Vion Pharmaceuticals Inc., New Haven, Connecticut,3 Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut4

Received 29 December 2006/ Accepted 6 April 2007


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ABSTRACT
 
Mutations in pmrA were recombined into Salmonella strain ATCC 14028 msbB to determine if pmrA-regulated modifications of lipopolysaccharide could suppress msbB growth defects. A mutation that functions to constitutively activate pmrA [pmrA(Con)] suppresses msbB growth defects on EGTA-containing media. Lipid A structural analysis showed that Salmonella msbB pmrA(Con) strains, compared to Salmonella msbB strains, have increased amounts of palmitate and phosphoethanolamine but no aminoarabinose addition, suggesting that aminoarabinose is not incorporated into msbB lipid A. Surprisingly, loss-of-function mutations in the aminoarabinose biosynthetic genes restored EGTA and polymyxin sensitivity to Salmonella msbB pmrA(Con) strains. These blocks in aminoarabinose biosynthesis also prevented lipid A phosphoethanolamine incorporation and reduced the levels of palmitate addition, indicating previously unknown roles for the aminoarabinose biosynthetic enzymes. Lipid A structural analysis of the EGTA- and polymyxin-resistant triple mutant msbB pmrA(Con) pagP::Tn10, which contains phosphoethanolamine but no palmitoylated lipid A, suggests that phosphoethanolamine addition is sufficient to confer EGTA and polymyxin resistance on Salmonella msbB strains. Additionally, palmitoylated lipid A was observed only in wild-type Salmonella grown in the presence of salt in rich media. Thus, we correlate EGTA resistance and polymyxin resistance with phosphoethanolamine-decorated lipid A and demonstrate that the aminoarabinose biosynthetic proteins play an essential role in lipid A phosphoethanolamine addition and affect lipid A palmitate addition in Salmonella msbB strains.


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INTRODUCTION
 
Lipopolysaccharide (LPS) forms the outer leaflet of the gram-negative bacterial outer membrane. It consists of three molecular domains: lipid A, core oligosaccharide, and O antigen. The endotoxic portion, lipid A, anchors LPS into the asymmetric outer membrane and is essential for outer membrane barrier function and cell viability. LPS biosynthesis initiates on the cytoplasmic side of the inner membrane before intermediates in LPS biosynthesis are transported to their final destinations in the outer membrane. 4-Amino-4-deoxy-L-arabinose (hereafter simply referred to as aminoarabinose) can be added to lipid A on the periplasmic side of the inner membrane (32), and palmitate can be added to the lipid A portion of LPS once it arrives in the outer membrane (1). One of the cytoplasmic lipid A biosynthetic enzymes, MsbB (LpxM), adds myristate to lipid IVA (Fig. 1).


Figure 1
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  		FIG. 1. Lipid A structure. Core lipid A structure is shown in black; in msbB strains, no myristoyl group is added (marked with filled arrow); PmrA/B- and PhoP/Q-regulated modifications are shown in gray. Activation of PmrA/B results in modification of the phosphates of lipid A with phosphoethanolamine and aminoarabinose. The PmrA/B two-component system can be turned on by PhoP via the PmrD protein (15). Activation of PhoP/Q results in lipid A lacking one acyl group (marked by open arrow; this occurs only on lipid A molecules lacking aminoarabinose), decorated with palmitate (gray 16-carbon chain), and hydroxylated at the myristoyl residue (marked by a gray X).

MsbB mutants are of particular interest in microbial pathogenesis and vaccine development research because lipid A lacking myristate no longer has strong endotoxic activity (16, 19, 27). Furthermore, Salmonella enterica serovar Typhimurium msbB strains have severe growth defects in LB, galactose-MacConkey, or EGTA-containing media that can be suppressed by extragenic compensatory mutations that arise at high frequency (22). Thus, an understanding of the different suppressors of msbB and their effects on cell growth and virulence is essential for their application in the engineering of attenuated live-bacterial vaccines or bacterial vectors. Our group has thus far identified two spontaneous suppressor mutations, somA (22) and the Suwwan deletion (23). During our investigations, we hypothesized that modification of lipid A could suppress msbB growth defects. However, our analyses of lipid A from spontaneous msbB suppressor strains YS1456 (msbB somA1) and YS1170 (msbB Suwwan) revealed no structural changes in lipid A from unsuppressed msbB strains (23). In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of these strains showed no changes in LPS carbohydrate structure.

EDTA, a general chelator of divalent cations, has been proposed to disrupt the outer membrane by increasing electrostatic repulsion between neighboring LPS molecules, leading to the presence of phospholipid domains within the outer leaflet of the outer membrane and impairing outer membrane barrier function (24). A more specific chelator of divalent cations is EGTA, which preferentially binds calcium. Salmonella msbB strains are EGTA sensitive (i.e., dependent on high calcium levels for growth), and many suppressor mutations confer EGTA-resistant phenotypes (22, 23). We hypothesized that the modification of the phosphate groups of lipid A with either phosphoethanolamine or aminoarabinose would yield an EGTA-resistant phenotype, since the mutants would no longer be as dependent on [Ca2+] because these modifications could reduce the amount of electrostatic repulsion between neighboring lipid A molecules.

The addition of aminoarabinose and phosphoethanolamine to the lipid A portion of LPS (Fig. 1), mediated by the two-component system PmrA/B, contributes to virulence (11) and resistance to polymyxin (35), which is a cyclic antimicrobial lipopeptide. The PmrA/B two-component system is activated under either mildly acidic, low-[Mg2+/Ca2+] (28), or high-ferric-chloride growth conditions (37). The Salmonella aminoarabinose biosynthetic genes were previously identified (9, 11). Since that time, Breazeale et al., Williams et al., and Trent and colleagues have elucidated the biochemical functions of PmrH (also called ArnB) (3), PmrI (ArnA) (36), PmrF (ArnC and PbgP) (2), and PmrK (ArnT) (33) in aminoarabinose biosynthesis. The pmrE (ugd) gene is physically separated from the other genes in this pathway (pmrHFIJKLM), which form a transcriptional regulon in Salmonella. Strains carrying loss-of-function mutations in pmrM, in contrast to strains with loss-of-function mutations in pmrE and pmrHFIJKL, can still modify lipid A with aminoarabinose (11). Thus far, the only lipid A phosphoethanolamine biosynthetic protein identified in Salmonella is PmrC (18). pmrC lies directly in front of pmrAB in a transcriptional regulon, and PmrC is an inner membrane protein with a large periplasmic domain. The protein responsible for adding phosphoethanolamine to the LPS core, CptA, has been recently identified and is PmrA regulated (29).

To determine whether the addition of aminoarabinose and/or phosphoethanolamine to lipid A could suppress msbB growth defects, we recombined constitutive and loss-of-function mutations in the PmrA/B two-component system into a Salmonella msbB strain. As described below, a constitutive mutation in pmrA (pmrA505) (10) resulted in an msbB suppressor phenotype. We also confirm our hypothesis that the addition of phosphoethanolamine to lipid A can confer an EGTA-resistant phenotype on Salmonella msbB strains and demonstrate that the aminoarabinose biosynthetic proteins are required for lipid A phosphoethanolamine incorporation and affect palmitate addition in an msbB genetic background.


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MATERIALS AND METHODS
 
Bacterial strains, phage, and media. The bacterial strains used in this study are listed in Table 1. The P22 mutant HT105/1int201 (obtained from the Salmonella Genetic Stock Center, Calgary, Canada) was used for Salmonella transductions. Salmonella enterica serovar Typhimurium strains were grown on LB-0 or MSB agar or in MSB broth. MSB medium consists of LB (21) with no NaCl and supplemented with 2 mM MgSO4 and 2 mM CaCl2. LB-0 is LB medium with no NaCl. These media were used for the growth of strains under nonselective conditions. LB-0 agar was used when genetic selections with antibiotics were performed. Plates were solidified with 1.5% agar. LB-0 agar and MSB broth were supplemented with chloramphenicol (15 µg/ml in broth; 25 µg/ml in agar), EGTA (Sigma, St. Louis, MO; 6 mM or 6.5 mM), streptomycin (200 µg/ml), or tetracycline (3, 5, or 20 µg/ml) as needed. A 350 mM stock of EGTA at pH 8.0 (adjusted with NaOH) was dissolved and then autoclaved. Antibiotics were added to LB-0 agar after cooling it to 45°C. MacConkey Agar Base (Difco) was used to prepare galactose MacConkey agar.


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  		TABLE 1. Bacterial strainsa

Growth analysis. Phenotypes of strains were confirmed by replica plating. Replica plating was performed using the double-velvet technique (17).

Preparation of electroporation-competent cells. A standard protocol for making electrocompetent cells (25) was modified as described previously (22).

Transduction and transformation. Salmonella sp. strain P22 transductions were carried out as previously described (7), except that EGTA was not added to the medium. A Bio-Rad Gene Pulser was used for transformation, following the unique electroporation protocol described previously (22).

Polymyxin survival. Ten milliliters of MSB broth was inoculated from a patch on a master plate, with phenotypes confirmed by replica plating. Cultures were grown in 2.5-cm-diameter glass tubes to an optical density at 600 nm (OD600) of 0.4 and held on ice. Once all cultures reached the appropriate OD and sat on ice for at least 15 min, dilutions were made in ice-cold plastic Eppendorf tubes containing MSB broth. The cells were diluted so that approximately 300 cells would be present in a 500-µl aliquot. Polymyxin B sulfate was added to media to achieve a final concentration of 0.1 µg/ml (a 500-µl volume of 2x polymyxin B sulfate in MSB broth was added to a 500-µl aliquot of cells, giving a total of 1 ml in each tube) in a plastic Eppendorf tube for each strain, and they were incubated for 1.5 h in a 37°C incubator without shaking; 500 µl from each tube was spread onto MSB agar. The plates were incubated overnight at 37°C, and CFU were determined.

Mass spectrometry of lipid A: lipid A purification for MALDI-TOF analysis. Lipid A samples were purified from at least two independent cultures, and all independent cultures from a given strain yielded nearly identical lipid A mass spectra. The strains were grown in 500-ml cultures of MSB broth to an OD600 of 0.10 or 100 ml cultures of MSB broth without MgSO4 (pH 8.0) were grown to an OD600 of 1.0. (Cultures were stopped when they had grown to an OD600 of 0.1 in order to decrease the chance of jackpots with derivatives. Jackpots arise when a suppressor mutation that confers a growth advantage spontaneously occurs early in the growth of a culture and cells with this secondary mutation overgrow the original clone, which lacks the second mutation for faster division. Later, we found that this was unnecessary because the frequency of suppressors is relatively constant in MSB broth between OD600s of 0.1 and 1.0. Furthermore, we detected no changes in lipid A structure in our strains grown under both conditions.) Cultures in MSB broth lacking MgSO4 (pH 8.0) were inoculated with 1:500 dilutions of overnight cultures grown in the same broth, and 1 mM EGTA was added after 40 min of incubation at 37°C. MSB broth cultures were grown with 100 rpm of translational movement, and MSB broth cultures lacking MgSO4 (pH 8.0) were grown at 125 rpm of translational movement. LPS was purified by the Mg2+-ethanol precipitation method as previously described (5). Lipid A was purified by hydrolysis in 1% sodium dodecyl sulfate at pH 4.5 (4). Before being applied on a sample plate, the lyophilized lipid A was dissolved in 20 µl of 5-chloro-2-mercaptobenzothiazole matrix-assisted laser desorption ionization (MALDI) matrix in chloroform-methanol (1:1). Negative-ion MALDI-time of flight (TOF) was performed as described previously (8).

Thin-layer chromatography of lipid A. Lipid A was labeled with [33P]orthophosphate, purified, and analyzed by thin-layer chromatography as previously described (33), except that cells were grown in MSB broth to an OD600 of 0.60 instead of 1.0.

Gas chromatographic analysis of LPS fatty acids. Strains were grown as described above. The rapid LPS purification technique for gas chromatography was performed as previously described (27). Fatty acid components of LPS were converted to methyl esters by methanolysis (27).


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RESULTS AND DISCUSSION
 
pmrA(Con) suppresses msbB growth defects. To determine if mutations in pmrA could suppress msbB growth defects, we transduced loss-of-function and constitutive alleles of pmrA into Salmonella strain ATCC 14028 msbB. As we hypothesized, a constitutive mutation [pmrA505, referred to as pmrA(Con)], but not a loss-of-function mutation, in pmrA partially suppressed EGTA sensitivity in an msbB background (Fig. 2). In contrast, it did not rescue growth on galactose-MacConkey medium.


Figure 2
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  		FIG. 2. Replica plate series showing the effect of PmrA/B mutations on the growth of Salmonella strain ATTC 14028 msbB. Single colonies were patched onto an LB-no-salt agar plate (not shown) and incubated overnight at 37°C. Replica plates were incubated for 10 h at 37°C. (A) MSB agar. (B) EGTA agar. (C) galactose-MacConkey agar. 14028 grew well on all three media. msbB is sensitive to EGTA and galactose-MacConkey agar. msbB pmrA has a phenotype similar to that of unsuppressed Salmonella strain ATTC 14028 msbB. Salmonella strain ATTC 14028 msbB pmrA(Con) is EGTA resistant but galactose-MacConkey sensitive. All strains grew patches on the master plate (not shown).

Polymyxin resistance has previously been correlated with EDTA resistance in pmrA mutants (34). Constitutive activation of pmrA has been shown to result in the modification of the phosphate groups of lipid A with aminoarabinose and/or phosphoethanolamine (9, 13, 30, 39). We propose that these lipid A structural modifications suppress msbB growth defects by decreasing the electrostatic repulsion between neighboring phosphate groups, which would make the cells less dependent on divalent cations, like Mg2+ and Ca2+.

Aminoarabinose biosynthetic genes are required for pmrA(Con) to suppress msbB growth defects. The Salmonella aminoarabinose operon has been described previously (9, 11). To determine if the aminoarabinose biosynthetic genes are necessary for the suppression of msbB growth defects in the pmrA(Con) strain, we moved our msbB::{Omega}tet marker into pmrA(Con) strains with nonpolar deletions in pmrH, pmrI, pmrJ, pmrK, and pmrL. As shown in Fig. 3, these nonpolar deletions in pmrH, pmrI, pmrJ, pmrK, and pmrL have an unsuppressed msbB phenotype despite the presence of the pmrA(Con) mutation. In contrast, loss of pmrM, which is not believed to play a role in the aminoarabinose biosynthetic pathway (11), did not alter the EGTA-resistant phenotype of the msbB pmrA(Con) strain (Fig. 3). The rpsL allele present in some of these triple mutants, which yields resistance to streptomycin, does not alter the EGTA or galactose-MacConkey phenotypes (data not shown).


Figure 3
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  		FIG. 3. Aminoarabinose biosynthetic genes are required for pmrA(Con) to suppress msbB growth defects. Replica plates were incubated for 10 h at 37°C. All strains in this figure carry the rpsL mutation (for streptomycin resistance). The pmrA(Con) allele confers EGTA resistance on Salmonella strain ATTC 14028 msbB. Nonpolar deletions in pmrHFIJKL restore EGTA sensitivity to Salmonella strain ATTC 14028 msbB pmrA(Con). However, a loss-of-function mutation in pmrM (which is not believed to play a role in aminoarabinose biosynthesis) did not restore EGTA sensitivity to Salmonella strain ATTC 14028 msbB pmrA(Con).

The pmrA(Con) mutation confers polymyxin resistance on the wild type and Salmonella strain ATCC 14028 msbB grown in MSB broth. Our results (see below) demonstrate that the msbB mutation confers polymyxin sensitivity on Salmonella strain ATCC 14028 grown in MSB broth, which is essential to avoid enrichment for suppressor mutations. In comparison, another study, using LB broth, also reported that msbB conferred polymyxin sensitivity on Salmonella strain C5 (31). The pmrA(Con) mutation was isolated from a screen for polymyxin-resistant Salmonella in LB medium (26). To confirm that the pmrA(Con) mutation also increases polymyxin resistance in Salmonella strain ATCC 14028 msbB grown in MSB medium, polymyxin survival rates for the various msbB strains tested are shown in Fig. 4. Our results show that the pmrA(Con) mutation confers polymyxin resistance on Salmonella strain ATCC 14028 msbB grown in MSB broth. In a wild-type background, the pmrA(Con) mutation allows ~73% of the cells to survive 1.5 h of exposure to 3.0 µg/ml polymyxin B sulfate in comparison to ~1.3% survival in ATCC 14028 (not shown). In an msbB background, the pmrA(Con) mutation allows ~53% of cells to survive a 1.5-h exposure to 0.1 µg/ml polymyxin B sulfate, whereas ~0% of Salmonella strain ATCC 14028 msbB bacteria survive this treatment. Thus, although the msbB mutation confers polymyxin sensitivity (31), the pmrA(Con) allele significantly reduces this sensitivity.


Figure 4
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  		FIG. 4. Effects of loss-of-function mutations in the aminoarabinose biosynthetic genes on polymyxin resistance in an msbB pmrA(Con) genetic background. Polymyxin sensitivity was measured in terms of percent survival. Cells were exposed to 0.1 µg/ml polymyxin B sulfate for 1.5 h at 37°C and then were plated to determine CFU. The error bars show variations in CFU between three independent clones for each strain. The pmrA(Con) mutation confers polymyxin resistance, and loss-of-function mutations in pmrHFIJKL restore polymyxin sensitivity to Salmonella strain ATTC 14028 msbB pmrA(Con). Loss-of-function mutations in pagP and pmrM do not restore polymyxin sensitivity. PagP and PmrM are not believed to play roles in aminoarabinose biosynthesis.

Nonpolar deletions in pmrHFIJKL restored both EGTA (Fig. 3) and polymyxin (Fig. 4) sensitivity to Salmonella strain ATCC 14028 msbB pmrA(Con). Loss of pmrM, which has not been shown to play a role in lipid A modification, does not affect either EGTA or polymyxin resistance.

Salt in LB broth stimulates the palmitoylation of lipid A in wild-type Salmonella strain ATCC 14028. Previous reports of lipid A structure in the wild type and Salmonella pmrA(Con) were from cultures grown in LB broth (9, 11, 18, 31, 32, 39). However, Salmonella msbB must be grown in MSB broth to prevent rapid overgrowth by suppressor mutants (22). To determine if there were any differences in lipid A structure based on growth conditions, we analyzed lipid A from strains grown in LB, LB-no-salt, and MSB broth using negative-ion MALDI-TOF mass spectrometry. The lipid A portion of LPS has been well characterized by mass spectrometry, with published mass-to-charge ratios correlated with distinct chemical structures (12, 38, 39). As shown in Fig. 5A, ATCC 14028 has both hexa-acylated (m/z 1797) and hepta-acylated (m/z 2036) lipid A when grown in LB broth. However, when grown in LB-no-salt broth (Fig. 5B) or MSB broth (not shown), lipid A lacks hepta-acylated lipid A (m/z 2036), showing that palmitoylation is upregulated by the presence of 1% NaCl.


Figure 5
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  		FIG. 5. Mass spectra and gas chromatography of lipid A from ATCC 14028 and ATCC 14028 pmrA(Con) show increased palmitate addition when grown in the presence of salt. Lipid A was purified from at least two independent cultures for each strain, and the resulting mass spectra were nearly identical. Units on the y axis represent relative intensity, and units on the x axis (m/z) represent the mass-to-charge ratios of different lipid A species. (A) ATCC 14028 grown in LB broth. (B) ATCC 14028 grown in LB-no-salt broth. (C) 14028 pmrA(Con) (SM2206) grown in LB broth. (D) ATCC 14028 pmrA(Con) (SM2206) grown in LB-no-salt broth. Hexa-acylated lipid A (m/z 1797), hepta-acylated lipid A (m/z 2036), hexa-acylated lipid A with phosphoethanolamine (m/z 1920) or aminoarabinose (m/z 1928), and hepta-acylated lipid A with phosphoethanolamine (m/z 2159) or aminoarabinose (m/z 2167) peaks are shown. (E) Gas chromatography results for lipid A from these strains grown in LB and LB-no-salt broth.

Gas chromatography (Fig. 5E, C16 column) confirmed that there was an increase (~2 to 3-fold) in palmitate addition in both 14028 and 14028 pmrA(Con) grown in LB broth compared to LB-no-salt broth. Thus, we see that palmitoylation, in both 14028 and 14028 pmrA(Con), is stimulated by the addition of 1% NaCl to LB-no-salt broth.

Myristoylation of lipid A by MsbB is necessary for decoration with aminoarabinose, but not phosphoethanolamine. Similar investigations were carried out with ATCC 14028 pmrA(Con). As shown in Fig. 5C, the pmrA(Con) mutation, in an msbB+ genetic background, conferred aminoarabinose and phosphoethanolamine addition on lipid A. Peaks corresponding to hexa-acylated lipid A (m/z 1797) with phosphoethanolamine (m/z 1920) or aminoarabinose (m/z 1928) and hepta-acylated lipid A (m/z 2036) with phosphoethanolamine (m/z 2159) or aminoarabinose (m/z 2167) were apparent. Phosphoethanolamine addition shifted peaks 123 m/z units, while aminoarabinose addition shifted peaks 131 m/z units. Figure 5D shows the effect of the pmrA(Con) mutation on lipid A structure in rich medium lacking salt (LB-no-salt broth). Under these growth conditions, the pmrA(Con) mutation conferred phosphoethanolamine (m/z 1920) and aminoarabinose (m/z 1928) addition on hexa-acylated lipid A, and very small amounts of heptaacylated lipid A were present, either in LB-no-salt broth (Fig. 5D) or MSB broth (not shown).

To see if the pmrA(Con) mutation has the same effect on both wild-type and MsbB lipid A, we isolated lipid A from Salmonella strain ATCC 14028 msbB and msbB pmrA(Con). As seen in Fig. 6A, msbB lipid A contained a penta-acylated peak (m/z 1588) as its major lipid A species (as expected, since its lipid A lacks myristate, which yields an m/z shift of 210) and contained a minor amount of hexa-acylated lipid A (m/z 1826 due to palmitate addition). In contrast to the effects of the pmrA(Con) mutation on the wild type (Fig. 5D), the pmrA(Con) mutation conferred only phosphoethanolamine but not aminoarabinose addition on msbB lipid A (Fig. 6B). In addition to penta-acylated lipid A (m/z 1588) decorated with phosphoethanolamine (m/z 1711), there was a large increase in palmitate addition (m/z 1826) and some hexa-acylated lipid A with phosphoethanolamine was observed (m/z 1949). Since the pmrA(Con) mutation resulted in lipid A aminoarabinose addition in a wild-type (Fig. 5C and D) but not in an msbB (Fig. 6B) genetic background, we conclude that aminoarabinose is not added to lipid A lacking the myristate residue added by MsbB in MSB medium. A related finding was reported (31) from experiments using Salmonella C5 msbB, showing that aminoarabinose is not added to MsbB lipid A in LB broth. Perhaps the myristoyl group added by MsbB is required for the localization or proper positioning of one of the aminoarabinose biosynthetic enzymes, thereby conferring substrate specificity.


Figure 6
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  		FIG. 6. Lipid A structures in msbB, msbB pmrA(Con), msbB pmrA(Con) pmrF, and msbB pmrA(Con) pagP strains revealed pmrA(Con) and pmrHFIJKL-dependent phosphoethanolamine addition and increased palmitoylation. Lipid A was purified from at least two independent cultures for each strain, and the resulting mass spectra were nearly identical. Units on the y axis represent relative intensity, and units on the x axis (m/z) represent the mass-to-charge ratios of different lipid A species. (A) msbB (YS1) in MSB broth. (B) msbB pmrA(Con) rpsL (SM2192) in MSB broth. (C) msbB pmrA(Con) pmrF rpsL (SM2103) in MSB broth. (D) msbB pmrA(Con) pagP (SM2035) in MSB broth lacking MgSO4. Peaks representing penta-acylated lipid A (m/z 1588 [A, B, and C] and m/z 1570 [D]), hexa-acylated lipid A (m/z 1826), penta-acylated lipid A with phosphoethanolamine (m/z 1711 [A, B, and C] or m/z 1693 [D]), and hexa-acylated lipid A with phosphoethanolamine (m/z 1949) are shown. (E) Thin-layer chromatography confirmed that msbB pmrA(Con) has a modified lipid A structure. msbB and msbB somA have indistinguishable lipid A thin-layer chromatography profiles (data not shown), but msbB pmrA(Con) has a distinct profile that includes phosphoethanolamine and palmitoylated lipid A. Palmitoylated lipid A has been putatively marked with an arrow, and the additional spots near the bottom of the thin-layer chromatography plate likely represent different forms of lipid A containing phosphoethanolamine. (F) Gas chromatographic analysis of LPS fatty acids from Salmonella strain ATTC 14028 msbB, msbB pmrA(Con), msbB pmrA(Con) pmrH, and msbB pmrA(Con) pmrF grown in MSB broth without MgSO4. msbB strains have greatly reduced amounts of C14 incorporation compared to the wild type (as shown in Fig. 5E). msbB pmrA(Con) strains have increased levels of palmitate (C16) addition, and EGTA challenge also leads to increased palmitate addition.

Lipid A from EGTA-resistant [msbB pmrA(Con)] strains grown in MSB broth has increased levels of palmitate and phosphoethanolamine addition compared to EGTA-sensitive strains [msbB or msbB pmrA(Con)-aminoarabinose mutants]. MALDI-TOF mass spectrometry was used to analyze overall lipid A structure in the EGTA-sensitive and EGTA-resistant strains. As seen in Fig. 6A, Salmonella strain ATCC 14028 msbB (EGTA sensitive) had a higher penta-acylated lipid A peak (at m/z 1588), whereas Salmonella strain ATCC 14028 msbB pmrA(Con) (EGTA resistant) (Fig. 6B) had a higher hexa-acylated lipid A peak (at m/z 1826), resulting from the addition of palmitate, as well as phosphoethanolamine peaks (penta-acylated lipid A with phosphoethanolamine at m/z 1711 and hexa-acylated lipid A with phosphoethanolamine at m/z 1949). Our mass spectra suggest that there are differences in terms of relative amounts of lipid A species between the strains. Since mass spectrometry is qualitative, we used thin-layer chromatography and gas chromatography to demonstrate quantitative differences between the amounts of lipid A species. Thin-layer chromatography of lipid A (Fig. 6E) suggested Salmonella strain ATCC 14028 msbB pmrA(Con) has more hexa-acylated (palmitoylated) lipid A (assignment was based on previously published data) (38, 39) than Salmonella strain ATCC 14028 msbB somA (Fig. 6E) or msbB (not shown) (Salmonella strain ATCC 14028 msbB somA and msbB strains have indistinguishable lipid A thin-layer chromatography profiles). Gas chromatography (Fig. 6F, compare C16 bars 1 and 3) showed that the pmrA(Con) mutation results in an ~7-fold increase in lipid A palmitate incorporation in msbB Salmonella strain ATCC 14028. Thus, three independent techniques indicated that the pmrA(Con) mutation, in an msbB background, increases lipid A palmitoylation. We also challenged Salmonella strain ATCC 14028 msbB with 1 mM EGTA to see if its lipid A structure became modified. As shown in Fig. 6F (C16 column, second bar), EGTA challenge resulted in an ~2-fold increase in lipid A palmitoylation in EGTA-sensitive Salmonella strain ATCC 14028 msbB. Mass spectrometry of lipid A from EGTA-challenged Salmonella strain ATCC 14028 msbB did not suggest any additional changes (not shown). Escherichia coli responds to EDTA similarly, by palmitoylating its lipid A molecules (14).

The aminoarabinose genes are essential for lipid A phosphoethanolamine addition and affect palmitate addition in Salmonella strain ATCC 14028 msbB pmrA(Con). Since loss of the aminoarabinose genes conferred EGTA sensitivity on Salmonella strain ATCC 14028 msbB pmrA(Con) (Fig. 3) and we concluded that aminoarabinose cannot be added to msbB lipid A (compare Fig. 5C and D and 6B), we investigated lipid A structures in msbB pmrA(Con) strains with loss-of-function mutations in pmrH, pmrI, pmrJ, pmrK, pmrL, and pmrM to learn what affect these mutations have on lipid A structure in the msbB pmrA(Con) genetic background. The mutations in pmrH, pmrF, pmrI, pmrJ, pmrK, and pmrL are nonpolar deletions (11).

Figure 6C shows that a nonpolar-deletion mutation in pmrF, which restores EGTA sensitivity to Salmonella strain ATCC 14028 msbB pmrA(Con) (Fig. 3), also restores penta-acylated lipid A (peak at m/z 1588) as the dominant form and loses lipid A phosphoethanolamine incorporation (loss of peaks at m/z 1711 and m/z 1949). This decrease in palmitoylation and a loss of phosphoethanolamine incorporation were found consistently in the triple mutants. Lipid A from msbB pmrA(Con) strains with nonpolar deletions in pmrH, pmrI, pmrJ, pmrK, and pmrL reproducibly produce similar mass spectra, and the rpsL mutation in these triple mutants has no obvious effects on lipid A mass spectra (not shown). pmrM, although part of the transcriptional regulon pmrHFIJKLM, is not believed to be part of the aminoarabinose biosynthetic pathway and has a lipid A structural profile similar to that of msbB pmrA(Con) (not shown). Likewise, a loss-of-function mutation in pmrM does not make msbB pmrA(Con) EGTA sensitive (Fig. 3).

Similar lipid A profiles were observed for these strains grown in MSB broth lacking MgSO4 and in strains grown in this medium challenged with EGTA after entering exponential growth. As mentioned above, the only difference noticed in EGTA-challenged cells was a twofold increase in palmitoylation. Gas chromatography confirmed that loss-of-function mutations in pmrH and pmrF (Fig. 6F, C16 column) and pmrIJKL (not shown) reduce the levels of palmitate addition to those observed in unsuppressed msbB strains, despite the presence of the pmrA(Con) allele in the triple mutants. These data demonstrate that the aminoarabinose genes affect palmitate incorporation [i.e., as described above, loss of the aminoarabinose biosynthetic genes results in decreased palmitoylation of lipid A in Salmonella strain ATCC 14028 msbB pmrA(Con)].

Lipid A phosphoethanolamine addition is sufficient to confer EGTA and polymyxin resistance on msbB Salmonella strain ATCC 14028. Lipid A from Salmonella strain ATCC 14028 msbB pmrA(Con) has two distinct molecular differences from lipid A harvested from Salmonella strain ATTC 14028 msbB: increased palmitoylation and phosphoethanolamine addition. Either or both could be responsible for the EGTA and polymyxin resistance phenotypes. In order to address this, we transduced a pagP loss-of-function mutation into Salmonella strain ATTC 14028 msbB pmrA(Con). The msbB pmrA(Con) pagP transductants had EGTA-resistant (not shown) and polymyxin-resistant (Fig. 4) phenotypes that were indistinguishable from those of msbB pmrA(Con) strains, suggesting that palmitate addition is not necessary for EGTA (not shown) or polymyxin (Fig. 4) resistance. To confirm that lipid A phosphoethanolamine addition and no palmitoylation was occurring in these strains, we performed lipid A structural analyses.

Figure 6D shows the lipid A profile from an msbB pmrA(Con) pagP2::Tn10d strain. The spectra in this sample were calibrated differently and are shifted to the left by approximately 18 m/z units. As seen in Fig. 6D, the mutational block in pagP blocks hexa-acylation (no peak at m/z 1806) but not phosphoethanolamine (peak at m/z 1693) addition. The loss-of-function mutation in pagP greatly reduces lipid A palmitoylation (Fig. 6F, last bar). In the pagP mutant, ~1.5% of total lipid A fatty acids were C16. This suggests that another acyl transferase may be catalyzing the addition of palmitate at a low level or that our lipid A samples were contaminated with palmitate derived from phospholipids. In any case, our mass spectrometry data demonstrate that the dominant form of lipid A in msbB pmrA(Con) pagP strains is penta-acylated lipid A decorated with phosphoethanolamine. Since this strain's lipid A seems to be enriched for phosphoethanolamine, it raises the possibility that palmitoylation may inhibit phosphoethanolamine addition.

Summary. The msbB mutation is of clinical interest because it allows Salmonella to be safely administered to mammals, which is essential for live-vaccine or attenuated-bacterial-vector development. Suppressor mutations for msbB allow Salmonella to avoid the septic shock response in a stable genetic background and thus are clinically useful. By choosing a suppressor mutation that confers the desired characteristics of a given product, such as tumor-targeting Salmonella VNP2009 (20, 23) being nontoxic and retaining tumor targeting and tumor inhibition, attenuated bacterial delivery vectors or live-vaccine strains can be genetically optimized.

In this study, we have shown that the aminoarabinose biosynthetic genes are required for the pmrA(Con) mutation to suppress msbB growth defects. Lipid structural analysis of these mutants provided an unexpected result, suggesting that the addition of phosphoethanolamine and palmitate to lipid A, and not aminoarabinose, was responsible for the suppressed phenotype in Salmonella strain ATTC 14028 msbB pmrA(Con). To determine if the addition of phosphoethanolamine or palmitate is responsible for the EGTA- and polymyxin-resistant phenotypes, we created an msbB pmrA(Con) pagP strain that had EGTA- and polymyxin-resistant phenotypes indistinguishable from that of Salmonella strain ATTC 14028 msbB pmrA(Con). This suggests that lipid A phosphoethanolamine, and not palmitate, addition is sufficient and necessary for both EGTA and polymyxin B resistance.

Additional experiments will be needed to determine the mechanism by which the aminoarabinose biosynthetic proteins affect lipid A phosphoethanolamine addition. One possibility is that the aminoarabinose genes are multifunctional, affecting both the aminoarabinose and phosphoethanolamine branches of the pmrA pathway. An alternative hypothesis is that both aminoarabinose and phosphoethanolamine biosynthetic proteins form a complex, and when essential proteins of the complex are not present, the complex may dissociate, resulting in a loss of proper protein localization and loss of activity for both pathways.


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ACKNOWLEDGMENTS
 
We thank Hiroshi Nikaido, Pamela Obuchowski, and Karim Suwwan de Felipe for their critical reading of the manuscript and Chris Murray for helping with the digital images.

This work was supported by a grant from Vion Pharmaceuticals, Inc. (K.B.L.), NIH SBIR grant 1 R43 CA97595-01 (K.B.L.), and NIH grant AI30479 (S.I.M.). S.R.M. was supported by a National Institutes of Health predoctoral training grant in genetics (5 TM32 BM07499) and a Yale University Fellowship.


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FOOTNOTES
 
* Corresponding author. Mailing address: Radiobiology Laboratories, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520. Phone: (203) 785-2976. Fax: (203) 785-6309. E-mail: brooks.low{at}yale.edu Back

{triangledown} Published ahead of print on 20 April 2007. Back

{dagger} Present address: Department of Biology, California State University, Northridge, Northridge, CA. Back

{ddagger} Present address: Celator Pharmaceuticals Corp., Vancouver, BC, Canada. Back


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Journal of Bacteriology, July 2007, p. 5161-5169, Vol. 189, No. 14
0021-9193/07/$08.00+0     doi:10.1128/JB.01969-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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