by decorating lipid a with phosphoethanolamine accepted · lipopolysaccharide (lps) forms the outer...

PmrAC confers pmrHFIJKL-dependent EGTA and polymyxin resistance to msbB

Salmonella by decorating lipid A with phosphoethanolamine

Running title: EGTA and polymyxin resistance in msbB Salmonella

Key words: PmrA, PmrH, PmrF, PmrI, PmrJ, PmrK, PmrL, PagP, MsbB, Salmonella, EGTA,

polymyxin, phosphoethanolamine, lipid A

Sean R. Murray1+

, Robert K. Ernst2, David Bermudes

3/, Samuel I. Miller

2 and K.

Brooks Low4*

1 Department of Biology, Yale University, New Haven, CT

2 Department of Microbiology, University of Washington, Seattle, WA

3 Vion Pharmaceuticals, New Haven, CT

4 Department of Therapeutic Radiology, Yale University School of Medicine, New

Haven, CT

+ Present address: Department of Developmental Biology, Stanford, CA

/ Present address: Celator Pharmaceuticals, Vancouver, BC, Canada

* 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: [email protected].

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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.01969-06 JB Accepts, published online ahead of print on 20 April 2007

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ABSTRACT

Mutations in pmrA were recombined into msbB Salmonella ATCC 14028 to

determine if pmrA-regulated modifications of lipopolysaccharide could suppress msbB

growth defects. A mutation that functions to constitutively activate pmrA (pmrAC)

suppresses msbB growth defects on EGTA-containing media. Lipid A structural analysis

showed that msbB pmrAC Salmonella, as compared to msbB Salmonella, 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 msbB pmrAC Salmonella. These blocks in aminoarabinose 10

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 pmrAC pagP::Tn10, which contains phosphoethanolamine but no

palmitoylated lipid A, suggests that phosphoethanolamine addition is sufficient to confer

EGTA and polymyxin resistance to msbB Salmonella. Additionally, palmitoylated lipid

A was only observed 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 20

affect lipid A palmitate addition in msbB Salmonella.

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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 destination 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 30

outer membrane (1). One of the cytoplasmic lipid A biosynthetic enzymes, MsbB (=LpxM), adds

myristate to lipid IVA (see filled arrow pointing to the myristate moiety in Figure 1).

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, msbB Salmonella enterica serovar Typhimurium 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 effect 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 40

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, SDS-PAGE 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

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EGTA, which preferentially binds calcium. msbB Salmonella are EGTA sensitive (i.e., dependent 50

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

(Figure 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 60

previously identified (9, 11). Since that time, Raetz and colleagues have elucidated the

biochemical functions of PmrH (also called ArnB) (3), PmrI (ArnA) (36), PmrF (ArnC, 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 70

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 msbB Salmonella. As described below, a

constitutive mutation in pmrA (pmrA505) (10) results in an msbB suppressor phenotype. We also

confirm our hypothesis that the addition of phosphoethanolamine to lipid A can confer an EGTA

resistant phenotype to msbB Salmonella and demonstrate that the aminoarabinose biosynthetic

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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. P22 mutant HT105/1int201 (obtained from the Salmonella Genetic Stock Center, Calgary,

Canada) was used for Salmonella transductions. Salmonella Typhimurium strains were grown on

LB-0 or MSB agar or in MSB broth. MSB media consists of LB (Luria-Bertani media) (21) with no

NaCl and supplemented with 2mM MgSO4 and 2mM CaCl2. LB-0 is LB media with no NaCl.

These media were used for the growth of strains under non-selective conditions. LB-0 agar was

used when performing genetic selections with antibiotics. 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), ethylene glycol-bis(ß-Aminoethyl Ether)- N,N,N’,N’-tetraacetic acid (EGTA) (Sigma, St. 90

Louis, MO) (6mM or 6.5mM), 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 to 45 degrees Celsius.

MacConkey Agar Base (Difco) was used to prepare Galactose MacConkey agar.

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). 100

Transduction and Transformation. Salmonella 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).

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Polymyxin survival. 10 mls 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 O.D.600 of 0.4 and held on ice. Once all cultures reached the appropriate O.D. and sat on ice

for at least 15 minutes, dilutions were made in ice-cold plastic eppendorf tubes containing MSB 110

broth. Cells were diluted so that approximately 300 cells would be present in a 500 µl aliquot.

Polymyxin B sulfate was added to media achieve a final concentration of 0.1 µg/ml (a 500 µl

volume of 2x polymyxin B sulfate in MSB broth was added to 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

hours in a 37˚C incubator without shaking. 500 µl from each tube was spread onto MSB agar.

Plates were incubated overnight at 37˚C and CFUs 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. Strains were either grown in 500 ml cultures 120

of MSB broth to an O.D.600 of 0.10 or 100 ml cultures of MSB broth without MgSO4 (pH 8.0), were

grown to an O.D.600 of 1.0. (Cultures were stopped when grown to an O.D.600 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 lack the second mutation for

faster division. Later, we found out that this was unnecessary because the frequency of

suppressors is relatively constant in MSB broth between an O.D.600 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 1mM EGTA was added after 40 minutes of incubation at 37°C. 130

MSB broth cultures were grown with 100 rpm of translational movement and MSB broth lacking

MgSO4 (pH 8.0) cultures 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

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hydrolysis in 1% SDS 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 MALDI matrix in chloroform-

methanol (1:1). Negative-ion MALDI-TOF was performed as described previously (8).

Thin-layer chromatography of lipid A. Lipid A was labeled with P-33, purified, and analyzed by

thin-layer chromatography as previously described (33), except that cells were grown in MSB

broth to an O.D.600 of 0.60 instead of 1.0. 140

Gas Chromatographic Analysis of Lipopolysaccharide (LPS) Fatty Acids. Strains were

grown as described under “Mass spectrometry of lipid A: lipid A purification for MALDI-TOF

anaylsis” section of Materials and Methods. 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).

RESULTS AND DISCUSSION

pmrAC 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 msbB 150

Salmonella ATCC 14028. As we hypothesized, a constitutive mutation (pmrA505, referred to as

pmrAC), but not a loss-of-function mutation, in pmrA partially suppresses EGTA sensitivity in an

msbB background (Figure 2). In contrast, it does not rescue growth on galactose-MacConkey

media.

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+

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Aminoarabinose biosynthetic genes are required for pmrAC to suppress msbB growth

defects. Salmonella’s aminoarabinose operon has previously been described (9, 11). To

determine if the aminoarabinose biosynthetic genes are necessary for the suppression of msbB

growth defects in the pmrAC strain, we moved our msbB::Ωtet marker into pmrA

C strains with non-

polar deletions in pmrH, pmrI, pmrJ, pmrK, and pmrL. As shown in Figure 3, these non-polar

deletions in pmrH, pmrI, pmrJ, pmrK, and pmrL have an unsuppressed msbB phenotype despite

the presence of the pmrAC mutation. In contrast, loss of pmrM, which is not believed to play a

role in the aminoarabinose biosynthetic pathway (11), does not alter the EGTA resistant

phenotype of the msbB pmrAC strain (Figure 3). The rpsL allele, which yields resistance to 170

streptomycin, present in some of these triple mutants does not alter the EGTA or galactose

MacConkey phenotypes (data not shown).

The pmrAC mutation confers polymyxin resistance to wild type and msbB Salmonella

ATCC 14028 grown in MSB broth. Our results (see below) demonstrate that the msbB mutation

confers polymyxin sensitivity to Salmonella ATCC 14028 grown in MSB broth, which is essential

to avoid enrichment for suppressor mutations. Another study, using LB broth, in comparison, also

reported that msbB conferred polymyxin sensitivity to Salmonella strain C5 (31). The pmrAC

mutation was isolated from a screen for polymyxin resistant Salmonella in LB medium (26). To

confirm that the pmrAC mutation also increases polymyxin resistance in msbB Salmonella ATCC 180

14028 grown in MSB medium, polymyxin survival for the various msbB strains tested are shown

in Figure 4. Our results show that the pmrAC mutation confers polymyxin resistance to msbB

Salmonella ATCC 14028 grown in MSB broth. In a wild type background, the pmrAC mutation

allows for ~73% of the cells to survive 1.5 hours 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 pmrAC

mutation allows for ~53% of cells to survive a 1.5 hour exposure to 0.1 µg/ml polymyxin B sulfate

whereas ~0% of msbB Salmonella ATCC 14028 survive this treatment. Thus, although the msbB

mutation confers polymyxin sensitivity (31), the pmrAC allele significantly reduces this sensitivity.

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Non-polar deletions in pmrHFIJKL restored both EGTA (Figure 3) and polymyxin (Figure

4) sensitivity to msbB pmrAC Salmonella ATCC 14028. Loss of pmrM, which has not been shown 190

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 ATCC

14028. Previous reports of lipid A structure in wild type and pmrAC Salmonella were from

cultures grown in LB broth (9, 11, 18, 31, 32, 39). However, msbB Salmonella 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 matrix-assisted laser desorption

ionization-time of flight (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 200

distinct chemical structures (12, 38, 39). As shown in Figure 5A, ATCC 14028 has both

hexaacylated (m/z 1797) and heptaacylated lipid A (m/z 2036) when grown in LB broth.

However, when grown in LB-no-salt broth (Figure 5B) or MSB broth (not shown), lipid A lacks

heptaacylated lipid A (m/z 2036), showing that palmitoylation is upregulated by the presence of

1% NaCl.

Gas chromatography (Figure 5E - under C16 column) confirms that there is an increase

(~2-3-fold) in palmitate addition in both 14028 and 14028 pmrAC grown in LB broth compared to

LB-no-salt broth. Thus, we see that palmitoylation, in both 14028 and 14028 pmrAC, is stimulated

by the addition of 1% NaCl to LB-no-salt broth.

210

Myristoylation of lipid A by MsbB is necessary for decoration with aminoarabinose, but

not phosphoethanolamine. Similar investigations were carried out with ATCC 14028 pmrAC.

As shown in Figure 5C, the pmrAC mutation, in an msbB

+ genetic background, confers

aminoarabinose and phosphoethanolamine addition to lipid A. Peaks corresponding to

hexaacylated lipid A (m/z 1797) with phosphoethanolamine (m/z 1920) or aminoarabinose (m/z

1928) and heptaacylated lipid A (m/z 2036) with phosphoethanolamine (m/z 2159) or

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aminoarabinose (m/z 2167) are apparent. Phosphoethanolamine addition shifts peaks m/z 123

units while aminoarabinose addition shifts peaks m/z 131 units. Figure 5D shows that the effect

of the pmrAC mutation on lipid A structure in rich media lacking salt (LB-no-salt broth). Under

these growth conditions, the pmrAC mutation confers phosphoethanolamine (m/z 1920) and 220

aminoarabinose addition (m/z 1928) to hexaacylated lipid A and very small amounts of

heptaacylated lipid A are present, either in LB-no-salt broth (Figure 5D) or MSB broth (not

shown).

To see if the pmrAC mutation has the same effects on both wild type and MsbB lipid A,

we isolated lipid A from msbB and msbB pmrAC Salmonella ATCC 14028. As seen in Figure 6A,

msbB lipid A contains a pentaacylated peak (m/z 1588) as its major lipid A species (expected

since its lipid A lacks myristate, which yields a m/z shift of 210) and contains a minor amount of

hexaacylated lipid A (m/z 1826 due to palmitate addition). In contrast to the effects of the pmrAC

mutation on wild type (Figure 5D), the pmrAC mutation confers only phosphoethanolamine but not

aminoarabinose addition to msbB lipid A (Figure 6B). In addition to pentaacylated lipid A (m/z 230

1588) decorated with phosphoethanolamine (m/z 1711), there is a large increase in palmitate

addition (m/z 1826) and some hexaacylated lipid A with phosphoethanolamine is observed (m/z

1949). Since the pmrAC mutation results in lipid A aminoarabinose addition in a wild type

(Figures 5C and 5D) but not in a msbB genetic background (Figure 6B), we conclude that

aminoarabinose is not be added to lipid A lacking the myristate residue added by MsbB in MSB

medium. A related finding was reported (31) from experiments using msbB Salmonella C5,

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.

240

Lipid A from EGTA resistant (msbB pmrAC) strains grown in MSB broth have increased

levels of palmitate and phosphoethanolamine addition compared to EGTA sensitive

strains (msbB or msbB pmrA

C-aminoarabinose mutants). MALDI-TOF mass spectrometry

was then used to analyze overall lipid A structure in the EGTA sensitive (EGTAS) and EGTA

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resistant (EGTAR) strains. As seen in Figure 6A, msbB Salmonella ATCC 14028 (EGTA

S) have a

higher pentaacylated lipid A peak (at m/z 1588) whereas msbB pmrAC Salmonella ATCC 14028

(EGTAR, Figure 7B) have a higher hexaacylated lipid A peak (at m/z 1826), resulting from the

addition of palmitate, as well as phosphoethanolamine peaks (pentaacylated lipid A with

phosphoethanolamine at m/z 1711 and hexaacylated lipid A with phosphoethanolamine at m/z

1949). Our mass spectra suggest that there are differences in terms of relative amounts of lipid A 250

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 (Figure 6E) suggests msbB

pmrAC Salmonella ATCC 14028 have more hexaacylated (palmitoylated) lipid A (marked by

arrow; assignment based on previously published data) (38, 39) than msbB somA (Figure 6E) or

msbB Salmonella ATCC 14028 (not shown, msbB somA and msbB Salmonella ATCC 14028

have indistinguishable lipid A TLC profiles). Gas chromatography (Figure 6F, compare C16 bars

1 and 3) shows that the pmrAC mutation results in an ~7-fold increase in lipid A palmitate

incorporation in msbB Salmonella ATCC 14028. Thus, three independent techniques indicate

that the pmrAC mutation, in an msbB background, increases lipid A palmitoylation. We also 260

challenged msbB Salmonella ATCC 14028 with 1 mM EGTA to see if its lipid A structure became

modified. As shown in Figure 6F (C16 column, 2nd

bar), EGTA challenge results in an

approximately 2-fold increase in lipid A palmitoylation in EGTAS msbB Salmonella ATCC 14028.

Mass spectrometry of lipid A from EGTA-challenged msbB Salmonella ATCC 14028 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 msbB pmrAC Salmonella ATCC 14028. Since loss of the

aminoarabinose genes conferred EGTA sensitivity to msbB pmrAC Salmonella ATCC 14028 270

(Figure 3) and we concluded that aminoarabinose can not be added to msbB lipid A (compare

Figures 5C, 5D, and 6B), we investigated lipid A structure in msbB pmrAC strains with loss-of-

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function mutations in pmrH, pmrI, pmrJ, pmrK, pmrL and pmrM to learn what affect these

mutations have on lipid A structure in the msbB pmrAC genetic background. The mutations in

pmrH, pmrF, pmrI, pmrJ, pmrK, and pmrL are non-polar deletions (11).

Figure 6C shows that a non-polar deletion mutation in pmrF, which restores EGTAS to

msbB pmrAC Salmonella ATCC 14028 (Figure 3), also restores pentaacylated 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 loss of phosphoethanolamine

incorporation was found consistently in the triple mutants. Lipid A from msbB pmrAC strains with 280

non-polar 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 pmrAC (not shown). Likewise, a loss-of-function mutation in pmrM does

not make msbB pmrAC EGTA

S (Figure 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 2-fold

increase in palmitoylation. Gas chromatography confirmed that loss-of-function mutations in 290

pmrH and pmrF (Figure 6F – under C16 column) and pmrIJKL (not shown) reduce the levels of

palmitate addition to those observed in unsuppressed msbB strains, despite the presence of the

pmrAC 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 msbB pmrAC Salmonella ATCC 14028).

Lipid A phosphoethanolamine addition is sufficient to confer EGTA and polymyxin

resistance to msbB Salmonella ATCC 14028. Lipid A from msbB pmrAC Salmonella ATCC

14028 has two distinct molecular differences from lipid A harvested from msbB Salmonella ATCC

14028: increased palmitoylation and phosphoethanolamine addition. Either or both could be 300

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responsible for the EGTA and polymyxin resistance phenotypes. In order to address this, we

transduced a pagP loss-of-function mutation into msbB pmrAC Salmonella ATCC 14028. The

msbB pmrAC pagP transductants had EGTA (not shown) and polymyxin (Figure 4) resistant

phenotypes that were indistinguishable from msbB pmrAC strains, suggesting that palmitate

addition is not necessary for EGTA (not shown) or polymyxin resistance (Figure 4). To confirm

that lipid A phosphoethanolamine addition and no palmitoylation is occurring in these strains, we

performed lipid A structural analyses.

Figure 6D shows the lipid A profile from a msbB pmrAC 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 Figure 6D, the mutational block in pagP blocks hexaacylation (no peak at 310

m/z 1806) but not phosphoethanolamine (peak at m/z 1693) addition. The loss-of-function

mutation in pagP greatly reduces lipid A palmitoylation (Figure 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 pmrAC pagP strains is pentaacylated 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.

320

Summary. The msbB mutation is of clinical interest because it allows for Salmonella to be safely

administered to mammals, which is essential for live-vaccine or attenuated-bacterial-vector

development. Suppressor mutations for msbB allow for 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 non-toxic and retaining tumor-targeting and tumor-inhibition,

attenuated bacterial delivery vectors or live-vaccine strains can be genetically optimized.

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In this study, we have shown that the aminoarabinose biosynthetic genes are required for

the pmrAC mutation to suppress msbB growth defects. Lipid structural analysis of these mutants

provided an unexpected result, suggesting that the addition of phosphoethanolamine and 330

palmitate to lipid A, and not aminoarabinose, was responsible for the suppressed phenotype in

msbB pmrAC Salmonella ATCC 14028. To determine if the addition of phosphoethanolamine or

palmitate is responsible for the EGTA and polymyxin resistant phenotypes, we created a msbB

pmrAC pagP strain, which had EGTA and polymyxin-resistant phenotypes indistinguishable from

msbB pmrAC Salmonella ATCC 14028. 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 of how the

aminoarabinose biosynthetic proteins affect lipid A phosphoethanolamine addition. One

possibility is that the aminoarabinose genes are multi-functional, affecting both the

aminoarabinose and phosphoethanolamine branches of the pmrA pathway. An alternative 340

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.

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 Institute of

Health predoctoral training grant in genetics (5 TM32 BM07499) and a Yale University 350

Fellowship.

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REFERENCES

1. Bishop, R. E., H. S. Gibbons, T. Guina, M. S. Trent, S. I. Miller, and C. R.

Raetz. 2000. Transfer of palmitate from phospholipids to lipid A in outer

membranes of gram-negative bacteria. Embo J 19:5071-80.

2. Breazeale, S. D., A. A. Ribeiro, A. L. McClerren, and C. R. Raetz. 2005. A

formyltransferase required for polymyxin resistance in Escherichia coli and the

modification of lipid A with 4-Amino-4-deoxy-L-arabinose. Identification and

function oF UDP-4-deoxy-4-formamido-L-arabinose. J Biol Chem 280:14154-67.

3. Breazeale, S. D., A. A. Ribeiro, and C. R. Raetz. 2003. Origin of lipid A species 360

modified with 4-amino-4-deoxy-L-arabinose in polymyxin-resistant mutants of

Escherichia coli. An aminotransferase (ArnB) that generates UDP-4-deoxyl-L-

arabinose. J Biol Chem 278:24731-9.

4. Caroff, M., A. Tacken, and L. Szabo. 1988. Detergent-accelerated hydrolysis of

bacterial endotoxins and determination of the anomeric configuration of the

glycosyl phosphate present in the "isolated lipid A" fragment of the Bordetella

pertussis endotoxin. Carbohydr Res 175:273-82.

5. Darveau, R. P., and R. E. Hancock. 1983. Procedure for isolation of bacterial

lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and

Salmonella typhimurium strains. J Bacteriol 155:831-8. 370

6. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of

chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad

Sci U S A 97:6640-5.

7. Davis, R. W., D. Botstein, and J. R. Roth. 1980. Advanced bacterial genetics.

Cold Spring Harbor, Cold Spring Harbor, N.Y.

8. Ernst, R. K., E. C. Yi, L. Guo, K. B. Lim, J. L. Burns, M. Hackett, and S. I.

Miller. 1999. Specific lipopolysaccharide found in cystic fibrosis airway

Pseudomonas aeruginosa. Science 286:1561-5.

9. Gunn, J. S., K. B. Lim, J. Krueger, K. Kim, L. Guo, M. Hackett, and S. I.

Miller. 1998. PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid 380

A modification and polymyxin resistance. Mol Microbiol 27:1171-82.

10. Gunn, J. S., and S. I. Miller. 1996. PhoP-PhoQ activates transcription of

pmrAB, encoding a two-component regulatory system involved in Salmonella

typhimurium antimicrobial peptide resistance. J Bacteriol 178:6857-64.

11. Gunn, J. S., S. S. Ryan, J. C. Van Velkinburgh, R. K. Ernst, and S. I. Miller.

2000. Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary

for lipopolysaccharide modification, antimicrobial peptide resistance, and oral

virulence of Salmonella enterica serovar typhimurium. Infect Immun 68:6139-46.

12. Guo, L., K. B. Lim, J. S. Gunn, B. Bainbridge, R. P. Darveau, M. Hackett,

and S. I. Miller. 1997. Regulation of lipid A modifications by Salmonella 390

typhimurium virulence genes phoP-phoQ. Science 276:250-3.

13. Helander, I. M., I. Kilpelainen, and M. Vaara. 1994. Increased substitution of

phosphate groups in lipopolysaccharides and lipid A of the polymyxin-resistant

pmrA mutants of Salmonella typhimurium: a 31P-NMR study. Mol Microbiol

11:481-7.

ACCEPTED


.org/D

ownloaded from

http://jb.asm.org/

16

14. Jia, W., A. E. Zoeiby, T. N. Petruzziello, B. Jayabalasingham, S.

Seyedirashti, and R. E. Bishop. 2004. Lipid trafficking controls endotoxin

acylation in outer membranes of Escherichia coli. J Biol Chem 279:44966-75.

15. Kato, A., and E. A. Groisman. 2004. Connecting two-component regulatory

systems by a protein that protects a response regulator from dephosphorylation by 400

its cognate sensor. Genes Dev 18:2302-13.

16. Khan, S. A., P. Everest, S. Servos, N. Foxwell, U. Zahringer, H. Brade, E. T.

Rietschel, G. Dougan, I. G. Charles, and D. J. Maskell. 1998. A lethal role for

lipid A in Salmonella infections. Mol Microbiol 29:571-9.

17. Kolodkin, A. L., M. A. Capage, E. I. Golub, and K. B. Low. 1983. F sex factor

of Escherichia coli K-12 codes for a single-stranded DNA binding protein. Proc

Natl Acad Sci U S A 80:4422-6.

18. Lee, H., F. F. Hsu, J. Turk, and E. A. Groisman. 2004. The PmrA-regulated

pmrC gene mediates phosphoethanolamine modification of lipid A and

polymyxin resistance in Salmonella enterica. J Bacteriol 186:4124-33. 410

19. Low, K. B., M. Ittensohn, T. Le, J. Platt, S. Sodi, M. Amoss, O. Ash, E.

Carmichael, A. Chakraborty, J. Fischer, S. L. Lin, X. Luo, S. I. Miller, L.

Zheng, I. King, J. M. Pawelek, and D. Bermudes. 1999. Lipid A mutant

Salmonella with suppressed virulence and TNFalpha induction retain tumor-

targeting in vivo. Nat Biotechnol 17:37-41.

20. Low, K. B., M. Ittensohn, X. Luo, L. M. Zheng, I. King, J. M. Pawelek, and

D. Bermudes. 2004. Construction of VNP20009: a novel, genetically stable

antibiotic-sensitive strain of tumor-targeting Salmonella for parenteral

administration in humans. Methods Mol Med 90:47-60.

21. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor, Cold 420

Spring Harbor, N.Y.

22. Murray, S. R., D. Bermudes, K. S. de Felipe, and K. B. Low. 2001. Extragenic

suppressors of growth defects in msbB Salmonella. J Bacteriol 183:5554-61.

23. Murray, S. R., K. S. de Felipe, P. L. Obuchowski, J. Pike, D. Bermudes, and

K. B. Low. 2004. Hot spot for a large deletion in the 18- to 19-centisome region

confers a multiple phenotype in Salmonella enterica serovar Typhimurium strain

ATCC 14028. J Bacteriol 186:8516-23.

24. Nikaido, H. 1996. Outer membrane., p. 29-47. In F. Neidhardt, R. Curtis, J.

Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. Reznikoff, M. Riley, M.

Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: Cellular 430

and molecular biology. ASM Press, Washington, D.C.

25. O'Callaghan, D., and A. Charbit. 1990. High efficiency transformation of

Salmonella typhimurium and Salmonella typhi by electroporation. Mol Gen Genet

223:156-8.

26. Roland, K. L., L. E. Martin, C. R. Esther, and J. K. Spitznagel. 1993.

Spontaneous pmrA mutants of Salmonella typhimurium LT2 define a new two-

component regulatory system with a possible role in virulence. J Bacteriol

175:4154-64.

27. Somerville, J. E., Jr., L. Cassiano, B. Bainbridge, M. D. Cunningham, and R.

P. Darveau. 1996. A novel Escherichia coli lipid A mutant that produces an 440

antiinflammatory lipopolysaccharide. J Clin Invest 97:359-65.

ACCEPTED


.org/D

ownloaded from

http://jb.asm.org/

17

28. Soncini, F. C., and E. A. Groisman. 1996. Two-component regulatory systems

can interact to process multiple environmental signals. J Bacteriol 178:6796-801.

29. Tamayo, R., B. Choudhury, A. Septer, M. Merighi, R. Carlson, and J. S.

Gunn. 2005. Identification of cptA, a PmrA-regulated locus required for

phosphoethanolamine modification of the Salmonella enterica serovar

typhimurium lipopolysaccharide core. J Bacteriol 187:3391-9.

30. Tamayo, R., S. S. Ryan, A. J. McCoy, and J. S. Gunn. 2002. Identification and

genetic characterization of PmrA-regulated genes and genes involved in

polymyxin B resistance in Salmonella enterica serovar typhimurium. Infect 450

Immun 70:6770-8.

31. Tran, A. X., M. E. Lester, C. M. Stead, C. R. Raetz, D. J. Maskell, S. C.

McGrath, R. J. Cotter, and M. S. Trent. 2005. Resistance to the antimicrobial

peptide polymyxin requires myristoylation of Escherichia coli and Salmonella

typhimurium lipid A. J Biol Chem 280:28186-94.

32. Trent, M. S., A. A. Ribeiro, W. T. Doerrler, S. Lin, R. J. Cotter, and C. R.

Raetz. 2001. Accumulation of a polyisoprene-linked amino sugar in polymyxin-

resistant Salmonella typhimurium and Escherichia coli: structural characterization

and transfer to lipid A in the periplasm. J Biol Chem 276:43132-44.

33. Trent, M. S., A. A. Ribeiro, S. Lin, R. J. Cotter, and C. R. Raetz. 2001. An 460

inner membrane enzyme in Salmonella and Escherichia coli that transfers 4-

amino-4-deoxy-L-arabinose to lipid A: induction on polymyxin-resistant mutants

and role of a novel lipid-linked donor. J Biol Chem 276:43122-31.

34. Vaara, M. 1981. Increased outer membrane resistance to

ethylenediaminetetraacetate and cations in novel lipid A mutants. J Bacteriol

148:426-34.

35. Vaara, M., T. Vaara, M. Jensen, I. Helander, M. Nurminen, E. T. Rietschel,

and P. H. Makela. 1981. Characterization of the lipopolysaccharide from the

polymyxin-resistant pmrA mutants of Salmonella typhimurium. FEBS Lett

129:145-9. 470

36. Williams, G. J., S. D. Breazeale, C. R. Raetz, and J. H. Naismith. 2005.

Structure and function of both domains of ArnA, a dual function decarboxylase

and a formyltransferase, involved in 4-amino-4-deoxy-L-arabinose biosynthesis. J

Biol Chem 280:23000-8.

37. Wosten, M. M., L. F. Kox, S. Chamnongpol, F. C. Soncini, and E. A.

Groisman. 2000. A signal transduction system that responds to extracellular iron.

Cell 103:113-25.

38. Zhou, Z., S. Lin, R. J. Cotter, and C. R. Raetz. 1999. Lipid A modifications

characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia

coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and 480

palmitate. J Biol Chem 274:18503-14.

39. Zhou, Z., A. A. Ribeiro, S. Lin, R. J. Cotter, S. I. Miller, and C. R. Raetz.

2001. Lipid A modifications in polymyxin-resistant Salmonella typhimurium:

PMRA-dependent 4-amino-4-deoxy-L-arabinose, and phosphoethanolamine

incorporation. J Biol Chem 276:43111-21.

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Figure and Table Legends

Figure 1. Lipid A structure. Core lipid A structure is shown in black; in msbB strains, no myristoyl 490

group is added (marked with filled arrow); PmrA/B - and PhoP/Q - regulated modifications are

shown in grey. 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 only occurs on lipid A molecules lacking aminoarabinose),

decorated with palmitate (grey 16 carbon chain) and hydroxylated at the myristoyl residue

(marked by a grey X).

Figure 2. Replica plate series showing the effect of PmrA/B mutations on the growth of msbB

Salmonella ATCC 14028. Single colonies were patched onto an LB-no salt agar plate (not 500

shown) and incubated overnight at 37°C. Replica plates were incubated for 10 hours at 37°C.

(A) MSB agar; (B) EGTA agar; (C) Galactose MacConkey agar. 14028 grows well on all 3

media. msbB is sensitive to EGTA and Galactose MacConkey agar. msbB pmrA has a similar

phenotype to unsuppressed msbB Salmonella ATCC 14028. msbB pmrAC Salmonella ATCC

14028 is EGTA resistant but Galactose MacConkey sensitive. All strains grew patches on the

master plate (not shown).

Figure 3. Aminoarabinose biosynthetic genes are required for pmrAC to suppress msbB growth

defects. Replica plates were incubated for 10 hours at 37°C. All strains in this figure carry the

rpsL mutation (for streptomycin resistance). The pmrAC allele confers EGTA resistance to msbB 510

Salmonella ATCC 14028. Non-polar deletions in pmrHFIJKL restore EGTA sensitivity to msbB

pmrAC Salmonella ATCC 14028. 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 msbB

pmrAC Salmonella ATCC 14028.

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Figure 4. The effect of loss-of-function mutations in the aminoarabinose biosynthetic genes on

polymyxin resistance in an msbB pmrAC 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 hours at 37°C and then were plated to determine colony forming units (CFUs). Y-error bars

show variation in CFUs between 3 independent clones for each strain. The pmrAC mutation 520

confers polymyxin resistance and loss-of-function mutations in pmrHFIJKL restore polymyxin

sensitivity to msbB pmrAC Salmonella ATCC 14028. Loss-of-function mutations in pagP and

pmrM do not restore polymyxin sensitivity. PagP and PmrM are not believed to play a role in

aminoarabinose biosynthesis.

Figure 5. Mass spectra and gas chromatography of lipid A from ATCC 14028 and ATCC 14028

pmrAC 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. Y-axis units represent relative intensity and X-axis units (m/z) represent the

mass-to-charge ratio of different lipid A species. (A) ATCC 14028 grown in LB broth; (B) ATCC 530

14028 grown in LB-no-salt broth; (C) 14028 pmrAC (SM2206) grown in LB broth; (D) ATCC

14028 pmrAC (SM2206) grown in LB-no-salt broth. Hexaacylated lipid A (m/z 1797),

heptaacylated lipid A (m/z 2036), hexaacylated lipid A with phosphoethanolamine (m/z 1920) or

aminoarabinose (m/z 1928), and heptaacylated 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.

Figure 6. Lipid A structure in msbB, msbB pmrAC, msbB pmrA

C pmrF, and msbB pmrA

C

pagP strains reveals pmrAC and pmrHFIJKL-dependent phosphoethanolamine addition

and increased palmitoylation. Lipid A was purified from at least two independent 540

cultures for each strain and the resulting mass spectra were nearly identical. Y-axis units

represent relative intensity and X-axis units (m/z) represent the mass-to-charge ratio of

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different lipid A species. (A) msbB (YS1) in MSB broth; (B) msbB pmrAC rpsL

(SM2192) in MSB broth; (C) msbB pmrAC pmrF rpsL (SM2103) in MSB broth; (D)

msbB pmrAC pagP (SM2035) in MSB broth lacking MgSO4. Peaks representing

pentaacylated lipid A (m/z 1588 [A, B, C] and m/z 1570 [D]), hexaacylated lipid A (m/z

1826), pentaacylated lipid A with phosphoethanolamine (m/z 1711 [A,B, C] or m/z 1693

[D]), and hexaacylated lipid A with phosphoethanolamine (m/z 1949) are shown. (E)

Thin-layer chromatography confirms that msbB pmrAC

has a modified lipid A structure.

msbB and msbB somA have indistinguishable lipid A TLC profiles (data not shown) but 550

msbB pmrAC has a distinct profile which 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 TLC plate likely represent different forms

of lipid A containing phosphoethanolamine. (F) Gas chromatographic analysis of LPS

fatty acids from msbB, msbB pmrAC, msbB pmrA

C pmrH and msbB pmrA

C pmrF

Salmonella ATCC 14028 grown in MSB broth without MgSO4. msbB strains have

greatly reduced amounts of C14 incorporation compared to wild type (as shown in Figure

5E). msbB pmrAC strains have increased levels of palmitate (C16) addition and EGTA

challenge also leads to increased palmitate addition.

560

Table 1: Bacterial Strains

Genotypes/phenotypes and derivation/source are shown for each strain of Salmonella

Typhimurium ATCC 14028 used in this study.

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Table 1. Bacterial strains.

STRAIN GENOTYPE OR PHENOTYPE DERIVATION OR SOURCE

Salmonella Typhimurium

ATCC 14028 wild type From ATCC

BE34 pmrA505, zjd::Tn10d-Cam, pmrM::luc S.I. Miller, U. of Washington

CS338 (also JSG421)

pmrA::Tn10d (10)

CS339 (also JSG435)

pmrA505, zjd::Tn10d-Cam (10)

JSG868 pmrA505, zjd::Tn10d-Cam, ∆pmrI, rpsL (11)

JSG883 pmrA505, zjd::Tn10d-Cam, ∆pmrK,

rpsL (11)

JSG884 pmrA505, zjd::Tn10d-Cam, ∆pmrL,

rpsL (11)

JSG911 pmrA505, zjd::Tn10d-Cam, ∆pmrF,

rpsL (11)

JSG1097 pmrA505, zjd::Tn10d-Cam, ∆pmrH,

rpsL (11)

JSG1098 pmrA505, zjd::Tn10d-Cam, ∆pmrJ,

rpsL (11)

SM1792 msbB1::Ωtet, pmrA::Tn10d P22•CS338 x YS1 Tet20R

SM1797 msbB1:: Ωtet , pmrA505, zjd::Tn10d-Cam

P22•CS339 x YS1 Cam20

R

SM2035 msbB1:: Ωtet, pmrA505, zjd::Tn10d-Cam, pagP2::Tn10d

P22•CS435 x SM1797 Tet20

R

SM2103 msbB1:: Ωtet, pmrA505, zjd::Tn10d-Cam, , ∆pmrF, rpsL

P22•YS8211 x JSG991

Tet5R

SM2104 msbB1:: Ωtet, pmrA505, zjd::Tn10d-Cam, ∆pmrL, rpsL

P22•YS8211 x JSG884

Tet5R

SM2105 msbB1:: Ωtet, pmrA505, zjd::Tn10d-Cam, , pmrM::luc

P22•YS8211 x BE34

Tet5R

SM2168 msbB1:: Ωtet, pmrA505, zjd::Tn10d-Cam, ∆pmrI, rpsL

transformed msbB1:: Ωtet into JSG868 using lambda red system (6), Tet5

R

selection

SM2177 msbB1:: Ωtet, pmrA505, zjd::Tn10d-Cam, , ∆pmrK, rpsL

P22•YS8211 x JSG883

Tet5R

SM2178 msbB1:: Ωtet, pmrA505, zjd::Tn10d-Cam, ∆pmrH, rpsL

P22•YS8211 x JSG1097

Tet5R

SM2180 msbB1:: Ωtet, pmrA505, zjd::Tn10d-Cam, ∆pmrJ, rpsL

transformed msbB1:: Ωtet into JSG1098 using lambda red system (6), Tet5

R

selection

SM2190 rpsL P22•JSG884 x 14028 Str200

R

SM2191 msbB1:: Ωtet, rpsL P22•JSG884 x YS1 Str200

R

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SM2192 msbB1:: Ωtet, pmrA505, zjd::Tn10d-Cam, rpsL

P22•JSG884 x SM1797 Str200

R

SM2193 msbB1:: Ωtet, pmrA505, zjd::Tn10d-Cam, pmrM::luc, rpsL

P22•JSG884 x SM2105 Str200

R

SM2206 pmrA505, zjd::Tn10d-Cam P22•CS339 x 14028 Cam20

R

YS1 msbB1:: Ωtet (22)

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Figure 1.

aminoarabinose

phosphoethanolamine

O

OO

O

OH

O

OH

O

orHO

NH2

H2NP

OH

OH

OH

HOHO

HO

HO

HO

O

O

OO

O

O

O

O

O

or

NH2

NH2

P

OH

OHHO

P

HOO

O

OO

OO

O O

O

O

O

O

XX OH

or H

O

O

HO

OH

HO

NH O

ONH

OP

OH

O

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Figure 2.

ATCC 14028

msbB (YS1)

msbB pmrA::Tn10 (SM1792)

msbB pmrAC (SM1797)

A. MSB B. EGTA C. gal.-Mac.

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Figure 3.

ATCC 14028 rpsL (SM2190)

msbB rpsL (SM2191)

msbB pmrAC rpsL (SM2192)

msbB pmrAC pmrH rpsL(SM2178)

msbB pmrAC pmrF rpsL (SM2103)

msbB pmrAC pmrI rpsL (SM2168)

msbB pmrAC pmrJ rpsL (SM2180)

msbB pmrAC pmrK rpsL (SM2177)

msbB pmrAC pmrL rpsL (SM2104)

msbB pmrAC pmrM rpsL (SM2193)

A. MSB B. EGTA

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Figure 4.

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Figure 5.

E.

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Figure 6.

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by decorating lipid a with phosphoethanolamine accepted · lipopolysaccharide (lps) forms the outer...

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