PmrA(Con) Confers pmrhfijkl-dependent EGTA and Polymyxin Resistance on msbb Salmonella by Decorating Lipid A with Phosphoethanolamine

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1 JOURNAL OF BACTERIOLOGY, July 2007, p Vol. 189, No /07/$ doi: /jb Copyight 2007, Ameican Society fo Micobiology. All Rights Reseved. PmA(Con) Confes pmhfijkl-dependent EGTA and Polymyxin Resistance on msbb Salmonella by Decoating Lipid A with Phosphoethanolamine Sean R. Muay, 1 Robet K. Enst, 2 David Bemudes, 3 Samuel I. Mille, 2 and K. Books Low 4 * Depatment of Biology, Yale Univesity, New Haven, Connecticut 1 ; Depatment of Micobiology, Univesity of Washington, Seattle, Washington 2 ; Vion Phamaceuticals Inc., New Haven, Connecticut 3 ; and Depatment of Theapeutic Radiology, Yale Univesity School of Medicine, New Haven, Connecticut 4 Received 29 Decembe 2006/Accepted 6 Apil 2007 Mutations in pma wee ecombined into Salmonella stain ATCC msbb to detemine if pmaegulated modifications of lipopolysacchaide could suppess msbb gowth defects. A mutation that functions to constitutively activate pma [pma(con)] suppesses msbb gowth defects on EGTA-containing media. Lipid A stuctual analysis showed that Salmonella msbb pma(con) stains, compaed to Salmonella msbb stains, have inceased amounts of palmitate and phosphoethanolamine but no aminoaabinose addition, suggesting that aminoaabinose is not incopoated into msbb lipid A. Supisingly, loss-of-function mutations in the aminoaabinose biosynthetic genes estoed EGTA and polymyxin sensitivity to Salmonella msbb pma(con) stains. These blocks in aminoaabinose biosynthesis also pevented lipid A phosphoethanolamine incopoation and educed the levels of palmitate addition, indicating peviously unknown oles fo the aminoaabinose biosynthetic enzymes. Lipid A stuctual analysis of the EGTA- and polymyxin-esistant tiple mutant msbb pma(con) pagp::tn10, which contains phosphoethanolamine but no palmitoylated lipid A, suggests that phosphoethanolamine addition is sufficient to confe EGTA and polymyxin esistance on Salmonella msbb stains. Additionally, palmitoylated lipid A was obseved only in wild-type Salmonella gown in the pesence of salt in ich media. Thus, we coelate EGTA esistance and polymyxin esistance with phosphoethanolamine-decoated lipid A and demonstate that the aminoaabinose biosynthetic poteins play an essential ole in lipid A phosphoethanolamine addition and affect lipid A palmitate addition in Salmonella msbb stains. Downloaded fom * Coesponding autho. Mailing addess: Radiobiology Laboatoies, Yale Univesity School of Medicine, 333 Ceda St., New Haven, CT Phone: (203) Fax: (203) books.low@yale.edu. Pesent addess: Depatment of Biology, Califonia State Univesity, Nothidge, Nothidge, CA. Pesent addess: Celato Phamaceuticals Cop., Vancouve, BC, Canada. Published ahead of pint on 20 Apil Lipopolysacchaide (LPS) foms the oute leaflet of the gam-negative bacteial oute membane. It consists of thee molecula domains: lipid A, coe oligosacchaide, and O antigen. The endotoxic potion, lipid A, anchos LPS into the asymmetic oute membane and is essential fo oute membane baie function and cell viability. LPS biosynthesis initiates on the cytoplasmic side of the inne membane befoe intemediates in LPS biosynthesis ae tanspoted to thei final destinations in the oute membane. 4-Amino-4-deoxy-L-aabinose (heeafte simply efeed to as aminoaabinose) can be added to lipid A on the peiplasmic side of the inne membane (32), and palmitate can be added to the lipid A potion of LPS once it aives in the oute membane (1). One of the cytoplasmic lipid A biosynthetic enzymes, MsbB (LpxM), adds myistate to lipid IVA (Fig. 1). MsbB mutants ae of paticula inteest in micobial pathogenesis and vaccine development eseach because lipid A lacking myistate no longe has stong endotoxic activity (16, 19, 27). Futhemoe, Salmonella enteica seova Typhimuium msbb stains have sevee gowth defects in LB, galactose-macconkey, o EGTA-containing media that can be suppessed by extagenic compensatoy mutations that aise at high fequency (22). Thus, an undestanding of the diffeent suppessos of msbb and thei effects on cell gowth and viulence is essential fo thei application in the engineeing of attenuated live-bacteial vaccines o bacteial vectos. Ou goup has thus fa identified two spontaneous suppesso mutations, soma (22) and the Suwwan deletion (23). Duing ou investigations, we hypothesized that modification of lipid A could suppess msbb gowth defects. Howeve, ou analyses of lipid A fom spontaneous msbb suppesso stains YS1456 (msbb soma1) and YS1170 (msbb Suwwan) evealed no stuctual changes in lipid A fom unsuppessed msbb stains (23). In addition, sodium dodecyl sulfate-polyacylamide gel electophoesis analysis of these stains showed no changes in LPS cabohydate stuctue. EDTA, a geneal chelato of divalent cations, has been poposed to disupt the oute membane by inceasing electostatic epulsion between neighboing LPS molecules, leading to the pesence of phospholipid domains within the oute leaflet of the oute membane and impaiing oute membane baie function (24). A moe specific chelato of divalent cations is EGTA, which pefeentially binds calcium. Salmonella msbb stains ae EGTA sensitive (i.e., dependent on high calcium levels fo gowth), and many suppesso mutations confe EGTA-esistant phenotypes (22, 23). We hypothesized that the modification of the phosphate goups of lipid A with on Febuay 26, 2019 by guest 5161

2 5162 MURRAY ET AL. J. BACTERIOL. FIG. 1. Lipid A stuctue. Coe lipid A stuctue is shown in black; in msbb stains, no myistoyl goup is added (maked with filled aow); PmA/B- and PhoP/Q-egulated modifications ae shown in gay. Activation of PmA/B esults in modification of the phosphates of lipid A with phosphoethanolamine and aminoaabinose. The PmA/B two-component system can be tuned on by PhoP via the PmD potein (15). Activation of PhoP/Q esults in lipid A lacking one acyl goup (maked by open aow; this occus only on lipid A molecules lacking aminoaabinose), decoated with palmitate (gay 16-cabon chain), and hydoxylated at the myistoyl esidue (maked by a gay X). eithe phosphoethanolamine o aminoaabinose would yield an EGTA-esistant phenotype, since the mutants would no longe be as dependent on [Ca 2 ] because these modifications could educe the amount of electostatic epulsion between neighboing lipid A molecules. The addition of aminoaabinose and phosphoethanolamine to the lipid A potion of LPS (Fig. 1), mediated by the twocomponent system PmA/B, contibutes to viulence (11) and esistance to polymyxin (35), which is a cyclic antimicobial lipopeptide. The PmA/B two-component system is activated unde eithe mildly acidic, low-[mg 2 /Ca 2 ] (28), o highfeic-chloide gowth conditions (37). The Salmonella aminoaabinose biosynthetic genes wee peviously identified (9, 11). Since that time, Beazeale et al., Williams et al., and Tent and colleagues have elucidated the biochemical functions of PmH (also called AnB) (3), PmI (AnA) (36), PmF (AnC and PbgP) (2), and PmK (AnT) (33) in aminoaabinose biosynthesis. The pme (ugd) gene is physically sepaated fom the othe genes in this pathway (pmhfijklm), which fom a tansciptional egulon in Salmonella. Stains caying loss-offunction mutations in pmm, in contast to stains with lossof-function mutations in pme and pmhfijkl, can still modify lipid A with aminoaabinose (11). Thus fa, the only lipid A phosphoethanolamine biosynthetic potein identified in Salmonella is PmC (18). pmc lies diectly in font of pmab in a tansciptional egulon, and PmC is an inne membane potein with a lage peiplasmic domain. The potein esponsible fo adding phosphoethanolamine to the LPS coe, CptA, has been ecently identified and is PmA egulated (29). To detemine whethe the addition of aminoaabinose and/o phosphoethanolamine to lipid A could suppess msbb gowth defects, we ecombined constitutive and loss-of-function mutations in the PmA/B two-component system into a Salmonella msbb stain. As descibed below, a constitutive mutation in pma (pma505) (10) esulted in an msbb suppesso phenotype. We also confim ou hypothesis that the addition of phosphoethanolamine to lipid A can confe an EGTA-esistant phenotype on Salmonella msbb stains and demonstate that the aminoaabinose biosynthetic poteins ae equied fo lipid A phosphoethanolamine incopoation and affect palmitate addition in an msbb genetic backgound. MATERIALS AND METHODS Bacteial stains, phage, and media. The bacteial stains used in this study ae listed in Table 1. The P22 mutant HT105/1int201 (obtained fom the Salmonella Genetic Stock Cente, Calgay, Canada) was used fo Salmonella tansductions. Salmonella enteica seova Typhimuium stains wee gown on LB-0 o MSB aga o in MSB both. MSB medium consists of LB (21) with no NaCl and supplemented with 2 mm MgSO 4 and 2 mm CaCl 2. LB-0 is LB medium with no NaCl. These media wee used fo the gowth of stains unde nonselective conditions. LB-0 aga was used when genetic selections with antibiotics wee pefomed. Plates wee solidified with 1.5% aga. LB-0 aga and MSB both wee supplemented with chloamphenicol (15 g/ml in both; 25 g/ml in aga), EGTA (Sigma, St. Louis, MO; 6 mm o 6.5 mm), steptomycin (200 g/ml), o tetacycline (3, 5, o 20 g/ml) as needed. A 350 mm stock of EGTA at ph 8.0 (adjusted with NaOH) was dissolved and then autoclaved. Antibiotics wee added to LB-0 aga afte cooling it to 45 C. MacConkey Aga Base (Difco) was used to pepae galactose MacConkey aga. Gowth analysis. Phenotypes of stains wee confimed by eplica plating. Replica plating was pefomed using the double-velvet technique (17). Pepaation of electopoation-competent cells. A standad potocol fo making electocompetent cells (25) was modified as descibed peviously (22). Tansduction and tansfomation. Salmonella sp. stain P22 tansductions wee caied out as peviously descibed (7), except that EGTA was not added to the medium. A Bio-Rad Gene Pulse was used fo tansfomation, following the unique electopoation potocol descibed peviously (22). Polymyxin suvival. Ten millilites of MSB both was inoculated fom a patch on a maste plate, with phenotypes confimed by eplica plating. Cultues wee gown in 2.5-cm-diamete glass tubes to an optical density at 600 nm (OD 600 )of 0.4 and held on ice. Once all cultues eached the appopiate OD and sat on ice fo at least 15 min, dilutions wee made in ice-cold plastic Eppendof tubes containing MSB both. The cells wee diluted so that appoximately 300 cells would be pesent in a 500- l aliquot. Polymyxin B sulfate was added to media to achieve a final concentation of 0.1 g/ml (a 500- l volume of 2 polymyxin B sulfate in MSB both was added to a 500- l aliquot of cells, giving a total of 1 ml

3 VOL. 189, 2007 EGTA AND POLYMYXIN RESISTANCE IN SALMONELLA msbb STRAINS 5163 TABLE 1. Bacteial stains a S. enteica seova Typhimuium stain Genotype o phenotype Refeence, deivation, o souce b ATCC Wild type ATCC BE34 pma505 zjd::tn10d-cam pmm::luc S. I. Mille, U. of Washington CS338 (also JSG421) pma::tn10d 10 LG069/CS339 (also JSG435) pho-24 pagp2::tn10d-tet phon2 zxx6251::tn10d-cam 110 pma505 zjd::tn10d-cam JSG868 pma505 zjd::tn10d-cam pmi psl 11 JSG883 pma505 zjd::tn10d-cam pmk psl 11 JSG884 pma505 zjd::tn10d-cam pml psl 11 JSG911 pma505 zjd::tn10d-cam pmf psl 11 JSG1097 pma505 zjd::tn10d-cam pmh psl 11 JSG1098 pma505 zjd::tn10d-cam pmj psl 11 SM1792 msbb1:: tet pma::tn10d P22 CS338 YS1 3 Tet 20 SM1797 msbb1:: tet pma505 zjd::tn10d-cam P22 CS339 YS1 3 Cam 20 SM2035 msbb1:: tet pma505 zjd::tn10d-cam pagp2::tn10d P22 LG069 SM Tet 20 SM2103 msbb1:: tet pma505 zjd::tn10d-cam pmf psl P22 YS1 JSG991 3 Tet 5 SM2104 msbb1:: tet pma505 zjd::tn10d-cam pml psl P22 YS1 JSG884 3 Tet 5 SM2105 msbb1:: tet pma505 zjd::tn10d-cam pmm::luc P22 YS1 BE34 3 Tet 5 SM2168 msbb:: tet pma505 zjd::tn10d-cam pmi psl Tansfomed msbb1:: tet into JSG868 using lambda ed system (6); Tet 5 selection SM2177 msbb1:: tet pma505 zjd::tn10d-cam pmk psl P22 YS1 JSG883 3 Tet 5 SM2178 msbb1:: tet pma505 zjd::tn10d-cam pmh psl P22 YS1 JSG Tet 5 SM2180 msbb1:: tet pma505 zjd::tn10d-cam pmj psl Tansfomed msbb1:: tet into JSG1098 using lambda ed system (6); Tet 5 selection SM2190 psl P22 JSG St 200 SM2191 msbb1:: tet psl P22 JSG884 YS1 3 St 200 SM2192 msbb1:: tet pma505 zjd::tn10d-cam psl P22 JSG884 SM St 200 SM2193 msbb1:: tet pma505 zjd::tn10d-cam pmm::luc psl P22 JSG884 SM St 200 SM2206 pma505 zjd::tn10d-cam P22 CS Cam 20 YS1 msbb1:: tet 22 a Genotype/phenotyps and deivation/souce ae shown fo each stain of S. enteica seova Typhimuium ATCC used in this study. b Tet 20, esistance to 20 g/ml tetacycline. Simila fo Cam (chloamphenicol) and St (steptomycin sulfate). Downloaded fom in each tube) in a plastic Eppendof tube fo each stain, and they wee incubated fo 1.5 h in a 37 C incubato without shaking; 500 l fom each tube was spead onto MSB aga. The plates wee incubated ovenight at 37 C, and CFU wee detemined. Mass spectomety of lipid A: lipid A puification fo MALDI-TOF analysis. Lipid A samples wee puified fom at least two independent cultues, and all independent cultues fom a given stain yielded nealy identical lipid A mass specta. The stains wee gown in 500-ml cultues of MSB both to an OD 600 of 0.10 o 100 ml cultues of MSB both without MgSO 4 (ph 8.0) wee gown to an OD 600 of 1.0. (Cultues wee stopped when they had gown to an OD 600 of 0.1 in ode to decease the chance of jackpots with deivatives. Jackpots aise when a suppesso mutation that confes a gowth advantage spontaneously occus ealy in the gowth of a cultue and cells with this seconday mutation ovegow the oiginal clone, which lacks the second mutation fo faste division. Late, we found that this was unnecessay because the fequency of suppessos is elatively constant in MSB both between OD 600 s of 0.1 and 1.0. Futhemoe, we detected no changes in lipid A stuctue in ou stains gown unde both conditions.) Cultues in MSB both lacking MgSO 4 (ph 8.0) wee inoculated with 1:500 dilutions of ovenight cultues gown in the same both, and 1 mm EGTA was added afte 40 min of incubation at 37 C. MSB both cultues wee gown with 100 pm of tanslational movement, and MSB both cultues lacking MgSO 4 (ph 8.0) wee gown at 125 pm of tanslational movement. LPS was puified by the Mg 2 -ethanol pecipitation method as peviously descibed (5). Lipid A was puified by hydolysis in 1% sodium dodecyl sulfate at ph 4.5 (4). Befoe being applied on a sample plate, the lyophilized lipid A was dissolved in 20 l of 5-chloo-2-mecaptobenzothiazole matix-assisted lase desoption ionization (MALDI) matix in chloofom-methanol (1:1). Negative-ion MALDI-time of flight (TOF) was pefomed as descibed peviously (8). Thin-laye chomatogaphy of lipid A. Lipid A was labeled with [ 33 P]othophosphate, puified, and analyzed by thin-laye chomatogaphy as peviously descibed (33), except that cells wee gown in MSB both to an OD 600 of 0.60 instead of 1.0. Gas chomatogaphic analysis of LPS fatty acids. Stains wee gown as descibed above. The apid LPS puification technique fo gas chomatogaphy was pefomed as peviously descibed (27). Fatty acid components of LPS wee conveted to methyl estes by methanolysis (27). RESULTS AND DISCUSSION pma(con) suppesses msbb gowth defects. To detemine if mutations in pma could suppess msbb gowth defects, we tansduced loss-of-function and constitutive alleles of pma into Salmonella stain ATCC msbb. As we hypothesized, a constitutive mutation [pma505, efeed to as pma(con)], but not a loss-of-function mutation, in pma patially suppessed EGTA sensitivity in an msbb backgound (Fig. 2). In FIG. 2. Replica plate seies showing the effect of PmA/B mutations on the gowth of Salmonella stain ATTC msbb. Single colonies wee patched onto an LB-no-salt aga plate (not shown) and incubated ovenight at 37 C. Replica plates wee incubated fo 10 h at 37 C. (A) MSB aga. (B) EGTA aga. (C) galactose-macconkey aga gew well on all thee media. msbb is sensitive to EGTA and galactose-macconkey aga. msbb pma has a phenotype simila to that of unsuppessed Salmonella stain ATTC msbb. Salmonella stain ATTC msbb pma(con) is EGTA esistant but galactose- MacConkey sensitive. All stains gew patches on the maste plate (not shown). on Febuay 26, 2019 by guest

4 5164 MURRAY ET AL. J. BACTERIOL. FIG. 3. Aminoaabinose biosynthetic genes ae equied fo pma- (Con) to suppess msbb gowth defects. Replica plates wee incubated fo 10 h at 37 C. All stains in this figue cay the psl mutation (fo steptomycin esistance). The pma(con) allele confes EGTA esistance on Salmonella stain ATTC msbb. Nonpola deletions in pmhfijkl estoe EGTA sensitivity to Salmonella stain ATTC msbb pma(con). Howeve, a loss-of-function mutation in pmm (which is not believed to play a ole in aminoaabinose biosynthesis) did not estoe EGTA sensitivity to Salmonella stain ATTC msbb pma(con). FIG. 4. Effects of loss-of-function mutations in the aminoaabinose biosynthetic genes on polymyxin esistance in an msbb pma(con) genetic backgound. Polymyxin sensitivity was measued in tems of pecent suvival. Cells wee exposed to 0.1 g/ml polymyxin B sulfate fo 1.5 h at 37 C and then wee plated to detemine CFU. The eo bas show vaiations in CFU between thee independent clones fo each stain. The pma(con) mutation confes polymyxin esistance, and loss-of-function mutations in pmhfijkl estoe polymyxin sensitivity to Salmonella stain ATTC msbb pma(con). Loss-offunction mutations in pagp and pmm do not estoe polymyxin sensitivity. PagP and PmM ae not believed to play oles in aminoaabinose biosynthesis. contast, it did not escue gowth on galactose-macconkey medium. Polymyxin esistance has peviously been coelated with EDTA esistance in pma mutants (34). Constitutive activation of pma has been shown to esult in the modification of the phosphate goups of lipid A with aminoaabinose and/o phosphoethanolamine (9, 13, 30, 39). We popose that these lipid A stuctual modifications suppess msbb gowth defects by deceasing the electostatic epulsion between neighboing phosphate goups, which would make the cells less dependent on divalent cations, like Mg 2 and Ca 2. Aminoaabinose biosynthetic genes ae equied fo pma- (Con) to suppess msbb gowth defects. The Salmonella aminoaabinose opeon has been descibed peviously (9, 11). To detemine if the aminoaabinose biosynthetic genes ae necessay fo the suppession of msbb gowth defects in the pma- (Con) stain, we moved ou msbb:: tet make into pma- (Con) stains with nonpola deletions in pmh, pmi, pmj, pmk, and pml. As shown in Fig. 3, these nonpola deletions in pmh, pmi, pmj, pmk, and pml have an unsuppessed msbb phenotype despite the pesence of the pma(con) mutation. In contast, loss of pmm, which is not believed to play a ole in the aminoaabinose biosynthetic pathway (11), did not alte the EGTA-esistant phenotype of the msbb pma(con) stain (Fig. 3). The psl allele pesent in some of these tiple mutants, which yields esistance to steptomycin, does not alte the EGTA o galactose-macconkey phenotypes (data not shown). The pma(con) mutation confes polymyxin esistance on the wild type and Salmonella stain ATCC msbb gown in MSB both. Ou esults (see below) demonstate that the msbb mutation confes polymyxin sensitivity on Salmonella stain ATCC gown in MSB both, which is essential to avoid enichment fo suppesso mutations. In compaison, anothe study, using LB both, also epoted that msbb confeed polymyxin sensitivity on Salmonella stain C5 (31). The pma(con) mutation was isolated fom a sceen fo polymyxinesistant Salmonella in LB medium (26). To confim that the pma(con) mutation also inceases polymyxin esistance in Salmonella stain ATCC msbb gown in MSB medium, polymyxin suvival ates fo the vaious msbb stains tested ae shown in Fig. 4. Ou esults show that the pma(con) mutation confes polymyxin esistance on Salmonella stain ATCC msbb gown in MSB both. In a wild-type backgound, the pma(con) mutation allows 73% of the cells to suvive 1.5 h of exposue to 3.0 g/ml polymyxin B sulfate in compaison to 1.3% suvival in ATCC (not shown). In an msbb backgound, the pma(con) mutation allows 53% of cells to suvive a 1.5-h exposue to 0.1 g/ml polymyxin B sulfate, wheeas 0% of Salmonella stain ATCC msbb bacteia suvive this teatment. Thus, although the msbb mutation confes polymyxin sensitivity (31), the pma(con) allele significantly educes this sensitivity. Nonpola deletions in pmhfijkl estoed both EGTA (Fig. 3) and polymyxin (Fig. 4) sensitivity to Salmonella stain ATCC msbb pma(con). Loss of pmm, which has not been shown to play a ole in lipid A modification, does not affect eithe EGTA o polymyxin esistance. Salt in LB both stimulates the palmitoylation of lipid A in wild-type Salmonella stain ATCC Pevious epots of lipid A stuctue in the wild type and Salmonella pma(con) wee fom cultues gown in LB both (9, 11, 18, 31, 32, 39). Howeve, Salmonella msbb must be gown in MSB both to pevent apid ovegowth by suppesso mutants (22). To detemine if thee wee any diffeences in lipid A stuctue based on gowth conditions, we analyzed lipid A fom stains gown

5 VOL. 189, 2007 EGTA AND POLYMYXIN RESISTANCE IN SALMONELLA msbb STRAINS 5165 Downloaded fom FIG. 5. Mass specta and gas chomatogaphy of lipid A fom ATCC and ATCC pma(con) show inceased palmitate addition when gown in the pesence of salt. Lipid A was puified fom at least two independent cultues fo each stain, and the esulting mass specta wee nealy identical. Units on the y axis epesent elative intensity, and units on the x axis (m/z) epesent the mass-to-chage atios of diffeent lipid A species. (A) ATCC gown in LB both. (B) ATCC gown in LB-no-salt both. (C) pma(con) (SM2206) gown in LB both. (D) ATCC pma(con) (SM2206) gown in LB-no-salt both. Hexa-acylated lipid A (m/z 1797), hepta-acylated lipid A (m/z 2036), hexa-acylated lipid A with phosphoethanolamine (m/z 1920) o aminoaabinose (m/z 1928), and hepta-acylated lipid A with phosphoethanolamine (m/z 2159) o aminoaabinose (m/z 2167) peaks ae shown. (E) Gas chomatogaphy esults fo lipid A fom these stains gown in LB and LB-no-salt both. on Febuay 26, 2019 by guest in LB, LB-no-salt, and MSB both using negative-ion MALDI- TOF mass spectomety. The lipid A potion of LPS has been well chaacteized by mass spectomety, with published massto-chage atios coelated with distinct chemical stuctues (12, 38, 39). As shown in Fig. 5A, ATCC has both hexa-acylated (m/z 1797) and hepta-acylated (m/z 2036) lipid A when gown in LB both. Howeve, when gown in LB-no-salt both (Fig. 5B) o MSB both (not shown), lipid A lacks heptaacylated lipid A (m/z 2036), showing that palmitoylation is upegulated by the pesence of 1% NaCl. Gas chomatogaphy (Fig. 5E, C 16 column) confimed that thee was an incease ( 2 to 3-fold) in palmitate addition in both and pma(con) gown in LB both compaed to LB-no-salt both. Thus, we see that palmitoylation, in both and pma(con), is stimulated by the addition of 1% NaCl to LB-no-salt both. Myistoylation of lipid A by MsbB is necessay fo decoation with aminoaabinose, but not phosphoethanolamine. Simila investigations wee caied out with ATCC pma- (Con). As shown in Fig. 5C, the pma(con) mutation, in an

6 5166 MURRAY ET AL. J. BACTERIOL. msbb genetic backgound, confeed aminoaabinose and phosphoethanolamine addition on lipid A. Peaks coesponding to hexa-acylated lipid A (m/z 1797) with phosphoethanolamine (m/z 1920) o aminoaabinose (m/z 1928) and heptaacylated lipid A (m/z 2036) with phosphoethanolamine (m/z 2159) o aminoaabinose (m/z 2167) wee appaent. Phosphoethanolamine addition shifted peaks 123 m/z units, while aminoaabinose addition shifted peaks 131 m/z units. Figue 5D shows the effect of the pma(con) mutation on lipid A stuctue in ich medium lacking salt (LB-no-salt both). Unde these gowth conditions, the pma(con) mutation confeed phosphoethanolamine (m/z 1920) and aminoaabinose (m/z 1928) addition on hexa-acylated lipid A, and vey small amounts of heptaacylated lipid A wee pesent, eithe in LBno-salt both (Fig. 5D) o MSB both (not shown). To see if the pma(con) mutation has the same effect on both wild-type and MsbB lipid A, we isolated lipid A fom Salmonella stain ATCC msbb and msbb pma(con). As seen in Fig. 6A, msbb lipid A contained a penta-acylated peak (m/z 1588) as its majo lipid A species (as expected, since its lipid A lacks myistate, which yields an m/z shift of 210) and contained a mino amount of hexa-acylated lipid A (m/z 1826 due to palmitate addition). In contast to the effects of the pma(con) mutation on the wild type (Fig. 5D), the pma- (Con) mutation confeed only phosphoethanolamine but not aminoaabinose addition on msbb lipid A (Fig. 6B). In addition to penta-acylated lipid A (m/z 1588) decoated with phosphoethanolamine (m/z 1711), thee was a lage incease in palmitate addition (m/z 1826) and some hexa-acylated lipid A with phosphoethanolamine was obseved (m/z 1949). Since the pma(con) mutation esulted in lipid A aminoaabinose addition in a wild-type (Fig. 5C and D) but not in an msbb (Fig. 6B) genetic backgound, we conclude that aminoaabinose is not added to lipid A lacking the myistate esidue added by MsbB in MSB medium. A elated finding was epoted (31) fom expeiments using Salmonella C5 msbb, showing that aminoaabinose is not added to MsbB lipid A in LB both. Pehaps the myistoyl goup added by MsbB is equied fo the localization o pope positioning of one of the aminoaabinose biosynthetic enzymes, theeby confeing substate specificity. Lipid A fom EGTA-esistant [msbb pma(con)] stains gown in MSB both has inceased levels of palmitate and phosphoethanolamine addition compaed to EGTA-sensitive stains [msbb o msbb pma(con)-aminoaabinose mutants]. MALDI-TOF mass spectomety was used to analyze oveall lipid A stuctue in the EGTA-sensitive and EGTA-esistant stains. As seen in Fig. 6A, Salmonella stain ATCC msbb (EGTA sensitive) had a highe penta-acylated lipid A peak (at m/z 1588), wheeas Salmonella stain ATCC msbb pma(con) (EGTA esistant) (Fig. 6B) had a highe hexa-acylated lipid A peak (at m/z 1826), esulting fom 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). Ou mass specta suggest that thee ae diffeences in tems of elative amounts of lipid A species between the stains. Since mass spectomety is qualitative, we used thinlaye chomatogaphy and gas chomatogaphy to demonstate quantitative diffeences between the amounts of lipid A species. Thin-laye chomatogaphy of lipid A (Fig. 6E) suggested Salmonella stain ATCC msbb pma(con) has moe hexa-acylated (palmitoylated) lipid A (assignment was based on peviously published data) (38, 39) than Salmonella stain ATCC msbb soma (Fig. 6E) o msbb (not shown) (Salmonella stain ATCC msbb soma and msbb stains have indistinguishable lipid A thin-laye chomatogaphy pofiles). Gas chomatogaphy (Fig. 6F, compae C 16 bas 1 and 3) showed that the pma(con) mutation esults in an 7-fold incease in lipid A palmitate incopoation in msbb Salmonella stain ATCC Thus, thee independent techniques indicated that the pma(con) mutation, in an msbb backgound, inceases lipid A palmitoylation. We also challenged Salmonella stain ATCC msbb with 1 mm EGTA to see if its lipid A stuctue became modified. As shown in Fig. 6F (C 16 column, second ba), EGTA challenge esulted in an 2-fold incease in lipid A palmitoylation in EGTA-sensitive Salmonella stain ATCC msbb. Mass spectomety of lipid A fom EGTA-challenged Salmonella stain ATCC msbb did not suggest any additional changes (not shown). Escheichia coli esponds to EDTA similaly, by palmitoylating its lipid A molecules (14). The aminoaabinose genes ae essential fo lipid A phosphoethanolamine addition and affect palmitate addition in Salmonella stain ATCC msbb pma(con). Since loss of the aminoaabinose genes confeed EGTA sensitivity on Salmonella stain ATCC msbb pma(con) (Fig. 3) and we concluded that aminoaabinose cannot be added to msbb lipid A (compae Fig. 5C and D and 6B), we investigated lipid A stuctues in msbb pma(con) stains with loss-of-function mutations in pmh, pmi, pmj, pmk, pml, and pmm to lean what affect these mutations have on lipid A stuctue in the msbb pma(con) genetic backgound. The mutations in pmh, pmf, pmi, pmj, pmk, and pml ae nonpola deletions (11). Figue 6C shows that a nonpola-deletion mutation in pmf, which estoes EGTA sensitivity to Salmonella stain ATCC msbb pma(con) (Fig. 3), also estoes penta-acylated lipid A (peak at m/z 1588) as the dominant fom and loses lipid A phosphoethanolamine incopoation (loss of peaks at m/z 1711 and m/z 1949). This decease in palmitoylation and a loss of phosphoethanolamine incopoation wee found consistently in the tiple mutants. Lipid A fom msbb pma(con) stains with nonpola deletions in pmh, pmi, pmj, pmk, and pml epoducibly poduce simila mass specta, and the psl mutation in these tiple mutants has no obvious effects on lipid A mass specta (not shown). pmm, although pat of the tansciptional egulon pmhfijklm, is not believed to be pat of the aminoaabinose biosynthetic pathway and has a lipid A stuctual pofile simila to that of msbb pma(con) (not shown). Likewise, a loss-of-function mutation in pmm does not make msbb pma(con) EGTA sensitive (Fig. 3). Simila lipid A pofiles wee obseved fo these stains gown in MSB both lacking MgSO 4 and in stains gown in this medium challenged with EGTA afte enteing exponential gowth. As mentioned above, the only diffeence noticed in EGTA-challenged cells was a twofold incease in palmitoylation. Gas chomatogaphy confimed that loss-of-function mutations in pmh and pmf (Fig. 6F, C 16 column) and pmijkl (not shown) educe the levels of palmitate addition to

7 VOL. 189, 2007 EGTA AND POLYMYXIN RESISTANCE IN SALMONELLA msbb STRAINS 5167 FIG. 6. Lipid A stuctues in msbb, msbb pma(con), msbb pma(con) pmf, and msbb pma(con) pagp stains evealed pma(con) and pmhfijkl-dependent phosphoethanolamine addition and inceased palmitoylation. Lipid A was puified fom at least two independent cultues fo each stain, and the esulting mass specta wee nealy identical. Units on the y axis epesent elative intensity, and units on the x axis (m/z) epesent the mass-to-chage atios of diffeent lipid A species. (A) msbb (YS1) in MSB both. (B) msbb pma(con) psl (SM2192) in MSB both. (C) msbb pma(con) pmf psl (SM2103) in MSB both. (D) msbb pma(con) pagp (SM2035) in MSB both lacking MgSO 4. Peaks epesenting 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] o m/z 1693 [D]), and hexa-acylated lipid A with phosphoethanolamine (m/z 1949) ae shown. (E) Thin-laye chomatogaphy confimed that msbb pma(con) has a modified lipid A stuctue. msbb and msbb soma have indistinguishable lipid A thin-laye chomatogaphy pofiles (data not shown), but msbb pma(con) has a distinct pofile that includes phosphoethanolamine and palmitoylated lipid A. Palmitoylated lipid A has been putatively maked with an aow, and the additional spots nea the bottom of the thin-laye chomatogaphy plate likely epesent diffeent foms of lipid A containing phosphoethanolamine. (F) Gas chomatogaphic analysis of LPS fatty acids fom Salmonella stain ATTC msbb, msbb pma(con), msbb pma(con) pmh, and msbb pma(con) pmf gown in MSB both without MgSO 4. msbb stains have geatly educed amounts of C 14 incopoation compaed to the wild type (as shown in Fig. 5E). msbb pma(con) stains have inceased levels of palmitate (C 16 ) addition, and EGTA challenge also leads to inceased palmitate addition.

8 5168 MURRAY ET AL. J. BACTERIOL. those obseved in unsuppessed msbb stains, despite the pesence of the pma(con) allele in the tiple mutants. These data demonstate that the aminoaabinose genes affect palmitate incopoation [i.e., as descibed above, loss of the aminoaabinose biosynthetic genes esults in deceased palmitoylation of lipid A in Salmonella stain ATCC msbb pma(con)]. Lipid A phosphoethanolamine addition is sufficient to confe EGTA and polymyxin esistance on msbb Salmonella stain ATCC Lipid A fom Salmonella stain ATCC msbb pma(con) has two distinct molecula diffeences fom lipid A havested fom Salmonella stain ATTC msbb: inceased palmitoylation and phosphoethanolamine addition. Eithe o both could be esponsible fo the EGTA and polymyxin esistance phenotypes. In ode to addess this, we tansduced a pagp loss-of-function mutation into Salmonella stain ATTC msbb pma(con). The msbb pma(con) pagp tansductants had EGTA-esistant (not shown) and polymyxinesistant (Fig. 4) phenotypes that wee indistinguishable fom those of msbb pma(con) stains, suggesting that palmitate addition is not necessay fo EGTA (not shown) o polymyxin (Fig. 4) esistance. To confim that lipid A phosphoethanolamine addition and no palmitoylation was occuing in these stains, we pefomed lipid A stuctual analyses. Figue 6D shows the lipid A pofile fom an msbb pma- (Con) pagp2::tn10d stain. The specta in this sample wee calibated diffeently and ae shifted to the left by appoximately 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 lossof-function mutation in pagp geatly educes lipid A palmitoylation (Fig. 6F, last ba). In the pagp mutant, 1.5% of total lipid A fatty acids wee C 16. This suggests that anothe acyl tansfease may be catalyzing the addition of palmitate at a low level o that ou lipid A samples wee contaminated with palmitate deived fom phospholipids. In any case, ou mass spectomety data demonstate that the dominant fom of lipid A in msbb pma(con) pagp stains is penta-acylated lipid A decoated with phosphoethanolamine. Since this stain s lipid A seems to be eniched fo phosphoethanolamine, it aises the possibility that palmitoylation may inhibit phosphoethanolamine addition. Summay. The msbb mutation is of clinical inteest because it allows Salmonella to be safely administeed to mammals, which is essential fo live-vaccine o attenuated-bacteial-vecto development. Suppesso mutations fo msbb allow Salmonella to avoid the septic shock esponse in a stable genetic backgound and thus ae clinically useful. By choosing a suppesso mutation that confes the desied chaacteistics of a given poduct, such as tumo-tageting Salmonella VNP2009 (20, 23) being nontoxic and etaining tumo tageting and tumo inhibition, attenuated bacteial delivey vectos o livevaccine stains can be genetically optimized. In this study, we have shown that the aminoaabinose biosynthetic genes ae equied fo the pma(con) mutation to suppess msbb gowth defects. Lipid stuctual analysis of these mutants povided an unexpected esult, suggesting that the addition of phosphoethanolamine and palmitate to lipid A, and not aminoaabinose, was esponsible fo the suppessed phenotype in Salmonella stain ATTC msbb pma- (Con). To detemine if the addition of phosphoethanolamine o palmitate is esponsible fo the EGTA- and polymyxinesistant phenotypes, we ceated an msbb pma(con) pagp stain that had EGTA- and polymyxin-esistant phenotypes indistinguishable fom that of Salmonella stain ATTC msbb pma(con). This suggests that lipid A phosphoethanolamine, and not palmitate, addition is sufficient and necessay fo both EGTA and polymyxin B esistance. Additional expeiments will be needed to detemine the mechanism by which the aminoaabinose biosynthetic poteins affect lipid A phosphoethanolamine addition. One possibility is that the aminoaabinose genes ae multifunctional, affecting both the aminoaabinose and phosphoethanolamine banches of the pma pathway. An altenative hypothesis is that both aminoaabinose and phosphoethanolamine biosynthetic poteins fom a complex, and when essential poteins of the complex ae not pesent, the complex may dissociate, esulting in a loss of pope potein localization and loss of activity fo both pathways. ACKNOWLEDGMENTS We thank Hioshi Nikaido, Pamela Obuchowski, and Kaim Suwwan de Felipe fo thei citical eading of the manuscipt and Chis Muay fo helping with the digital images. This wok was suppoted by a gant fom Vion Phamaceuticals, Inc. (K.B.L.), NIH SBIR gant 1 R43 CA (K.B.L.), and NIH gant AI30479 (S.I.M.). S.R.M. was suppoted by a National Institutes of Health pedoctoal taining gant in genetics (5 TM32 BM07499) and a Yale Univesity Fellowship. REFERENCES 1. Bishop, R. E., H. S. Gibbons, T. Guina, M. S. Tent, S. I. Mille, and C. R. Raetz Tansfe of palmitate fom phospholipids to lipid A in oute membanes of gam-negative bacteia. EMBO J. 19: Beazeale, S. D., A. A. Ribeio, A. L. McCleen, and C. R. Raetz A fomyltansfease equied fo polymyxin esistance in Escheichia coli and the modification of lipid A with 4-amino-4-deoxy-L-aabinose. Identification and function of UDP-4-deoxy-4-fomamido-L-aabinose. J. Biol. Chem. 280: Beazeale, S. D., A. A. Ribeio, and C. R. Raetz Oigin of lipid A species modified with 4-amino-4-deoxy-L-aabinose in polymyxin-esistant mutants of Escheichia coli. An aminotansfease (AnB) that geneates UDP-4-deoxyl-L-aabinose. J. Biol. Chem. 278: Caoff, M., A. Tacken, and L. 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