Amino acid addition to Vibrio cholerae LPS establishesa link between surface remodeling in Gram-positiveand Gram-negative bacteriaJessica V. Hankinsa, James A. Madsenb, David K. Gilesa, Jennifer S. Brodbeltb, and M. Stephen Trenta,c,1

aSection of Molecular Genetics and Microbiology, bDepartment of Chemistry and Biochemistry, and cInstitute of Cellular and Molecular Biology, University ofTexas, Austin, TX 78712

Edited by Hiroshi Nikaido, University of California, Berkeley, CA, and approved April 20, 2012 (received for review January 23, 2012)

Historically, the O1 El Tor and classical biotypes of Vibrio choleraehave been differentiated by their resistance to the antimicrobialpeptide polymyxin B. However, the molecular mechanisms associ-ated with this phenotypic distinction have remained a mystery for50 y. Both Gram-negative and Gram-positive bacteria modify theircell wall components with amine-containing substituents to re-duce the net negative charge of the bacterial surface, thereby pro-moting cationic antimicrobial peptide resistance. In the presentstudy, we demonstrate that V. cholerae modify the lipid A anchorof LPS with glycine and diglycine residues. This previously unchar-acterized lipid A modification confers polymyxin resistance inV. cholerae El Tor, requiring three V. cholerae proteins: Vc1577(AlmG), Vc1578 (AlmF), and Vc1579 (AlmE). Interestingly, the pro-tein machinery required for glycine addition is reminiscent of theGram-positive system responsible for D-alanylation of teichoicacids. Such machinery was not thought to be used by Gram-neg-ative organisms. V. cholerae O1 El Tor mutants lacking genes in-volved in transferring glycine to LPS showed a 100-fold increase insensitivity to polymyxin B. This work reveals a unique lipid A mod-ification and demonstrates a charge-based remodeling strategyshared between Gram-positive and Gram-negative organisms.

outer membrane | cell envelope | endotoxin | ultraviolet photodissociation

The Gram-negative pathogen Vibrio cholerae is the causativeagent responsible for ∼300,000 reported cases annually of the

severe diarrheal disease cholera. Since 1961, resistance to poly-myxin B, a cationic antimicrobial peptide (CAMP), has beenused to clinically differentiate between the two V. cholerae O1biotypes, El Tor and classical (1). Interestingly the O1 classicalbiotype, which is polymyxin-sensitive, caused the first six cholerapandemics; however, polymyxin-resistant O1 El Tor strains areresponsible for the current, seventh pandemic. Despite its im-portance, the molecular mechanisms accounting for this phe-notypic difference have remained unknown for nearly 50 y.During infection, the cells of the innate immune system se-

crete CAMPs, which are small, positively charged proteins.Much like polymyxin, these peptides bind to and disrupt thebacterial cell membrane, ultimately resulting in death of the in-vading bacterial cell. Although Gram-negative and Gram-posi-tive bacteria possess different cell wall structures, both haveevolved mechanisms to remodel their cell envelope in responseto CAMPs. These mechanisms often involve a common theme:neutralizing the negative charge of major cell wall components.The major surface component of Gram-negative bacteria is

LPS, which is composed of three distinct regions: lipid A, coreoligosaccharide, and O-antigen polysaccharide (2). Lipid A is thebioactive portion of LPS, which activates the human innate im-mune system through the Toll-like receptor 4 (TLR4)/myeloiddifferentiation factor 2 complex (3). The negatively charged lipidA domain is synthesized through a well-conserved pathway, TheRaetz pathway, that results in a β-1′,6-linked disaccharide ofglucosamine that is hexa-acylated and bis-phosphorylated (2,4, 5). Some Gram-negative bacteria (e.g., Salmonella enterica)modify their lipid A domain in response to CAMPs by masking

the 1- or 4′-phosphate groups with either phosphoethanolamineor aminoarabinose (Fig. 1) (5–8). The addition of an amine-containing substituent to the lipid A anchor of LPS disrupts thebinding of CAMPs to the bacterial surface.Bacteria also modify other essential cell wall components in

response to CAMPs. For example, many Gram-positive bacteriaundergo D-alanylation of both wall teichoic acids and lip-oteichoic acids (Fig. 1). Wall teichoic acids, which are linked topeptidoglycan, typically consist of a disaccharide linkage unitattached to a polyribitol phosphate or polyglycerol phosphatechain (9). Lipoteichoic acids are anchored to the membraneglycolipids of Gram-positive bacteria and consist of a (poly)glycerolphosphate backbone (9). Transfer of D-alanine to theribitol phosphate or glycerolphosphate units of these polymersreduces the net negative charge of the Gram-positive cell wall.Modified forms of glycerophospholipids have also been de-scribed in bacteria. Aminoacyl esters of phosphatidylglycerolinvolving L-alanine and L-lysine (Fig. 1) represent major mem-brane lipids in several Gram-positives (10, 11) and have beenimplicated in conferring resistance to CAMPs, as well as en-hancing survival in acidic conditions (10, 11).The mechanism of V. cholerae polymyxin B resistance has been

poorly understood. In these findings, we identify the addition ofa glycine and a diglycine residue to the V. cholerae O1 El Torlipid A species, which is required for polymyxin B resistance.Three V. cholerae proteins, Vc1577 (AlmG), Vc1578 (AlmF),and Vc1579 (AlmE), were found to be essential for this uniquelipid A modification. Furthermore, these proteins share se-quence homology with machinery involved in D-alanylation oflipoteichoic acid in Gram-positive bacteria. Discovery of anamino acid modification of the lipid A of V. cholerae illuminatesan interesting link between Gram-negative and Gram-positivecell wall modification systems. Additionally, our findings providea well-defined mechanism for the different polymyxin-resistantphenotypes observed in V. cholerae classical and El Tor biotypesfor 50 y.

ResultsV. cholerae O1 El Tor Synthesize Glycine-Modified Lipid A Species.V. cholerae O1 classical and El Tor biotypes synthesize a com-mon lipid A species (Fig. 2A) that can be detected at m/z 1,756using MALDI-TOF MS in the negative-ion mode (Fig. 2 Band C). In a previous report from our laboratory (12), theO1 classical lipid A structure was elucidated by interrogatingthe singly deprotonated species (m/z 1,756) by tandem massspectrometry (MS/MS). MALDI-TOF MS results also showed

Author contributions: J.V.H., J.A.M., D.K.G., J.S.B., and M.S.T. designed research; J.V.H.,J.A.M., and D.K.G. performed research; J.V.H., J.A.M., D.K.G., J.S.B., and M.S.T. analyzeddata; and J.V.H., J.A.M., D.K.G., J.S.B., and M.S.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed: E-mail: strent@mail.utexas.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1201313109/-/DCSupplemental.

8722–8727 | PNAS | May 29, 2012 | vol. 109 | no. 22 www.pnas.org/cgi/doi/10.1073/pnas.1201313109

that V. cholerae O1 El Tor biotypes synthesize additional lipidA species (m/z 1,813.9 and 1,870.9) (Fig. 2B), which are notproduced by the classical biotype (Fig. 2C).First, to verify that both classical and El Tor strains have

a common lipid A species on their surface, MS/MS was per-formed on the ion of m/z 1,756 peak produced by the El Torbiotype. The 193-nm UV photodissociation (UVPD) methodwas used because it has been shown to increase the depth ofstructural information for lipid A species over standard MS/MStechniques (13). Fig. 3A shows the UVPD spectrum of the [M–H]− ion (m/z 1,756) of the V. cholerae El Tor lipid A, as well as itsassociated fragmentation map, which matches m/z values of ob-served fragment ions to distinctive cleavage sites along the lipidA structure (Fig. 3A and Fig. S1). The inter-ring glucosaminecleavage that generated the product ion of m/z 682.42 confirmsthe acyl chain configuration: the distal side of the moleculecontains four acyl chains, whereas the proximal side containsonly two. The combination of C-O bond cleavages at the phos-phate groups (cleavage 1) and at the 3′- and 3-linked primaryacyl chains (cleavages 4, 5, 6, and 7), and 2′- and 3′-linked sec-ondary acyl chains (cleavages 8 and 10) allow identification ofthe basic structure of V. cholerae O1 El Tor lipid A. More spe-cifically, cleavage at the 3′-linked secondary acyl chain, denotedas cleavage 8, confirms the general position of hydroxylation.Importantly, the abundant ion of m/z 1,627.83 (cleavage 23)identifies the specific position of the hydroxyl group on the 3′-linked secondary acyl chain. This preferential cleavage by UVPDhas been used previously to locate the same hydroxyl group inthe classical biotype (12). For a comparison with UVPD, colli-sion-induced dissociation (CID) was also used to interrogate thesame singly deprotonated ion of m/z 1,756 (Fig. S2) and yieldedessentially the same C-O bond cleavages seen for UVPD. Thecombination of the more rich UVPD patterns and the simplerCID fragmentation patterns work well as complementarymethods and confirm the structural assignment of lipid A fromthe El Tor biotype.Fig. 3B shows the UVPD spectrum of the singly deprotonated

species of m/z 1,813, which is presumably lipid A with a 57-Damodification. Several of the product ions observed for the un-modified lipid A in Fig. 3A are also seen in Fig. 3B, confirmingthe general V. cholerae lipid A structure. Cleavages 3, 4, 6, and 8localize the modification to the 3′-linked primary acyl chain;more specifically, cleavages 3 (neutral loss of the 57-Da modifi-cation) and 8 (loss of 3′-linked secondary acyl chain) confirm the

general location of the 57-Da modification on the 3′-linkedsecondary acyl chain. The product ion of m/z 1,685.83 (cleavage23) also provides further evidence concerning the position of the57-Da modification; however, this product is isobaric with thepotential ion produced from C-C cleavage adjacent to the hy-droxyl group on the 3-linked acyl chain, thus making it somewhatambiguous. The abundant ions of m/z 1,738.00, 1,755.75,1,785.00, and 1,796.00 that correspond to cleavages 3, 11, 17, and18 (labeled in red in Fig. 3B; the latter three all uniquely ob-served upon upon UVPD yet not by CID), respectively, confirmthat the 57 Da modification is consistent with a glycine moiety.The rich level of detail in the region of m/z 1,650–1,800 of theUVPD spectrum is essential for mapping the glycine modifica-tion. The simpler CID spectrum seen in Fig. S2, which yields thegeneral construction of the lipid A and complements the moredetailed structural information obtained by UVPD, also aids inthe confident structural assignment of the V. cholerae m/z1,813 peak.Fig. 3C displays the UVPD spectrum of the singly deprotonated

ion of m/z 1,870 of the V. cholerae lipid A that contains a 114-Dadiglycine modification. UVPD again yielded the same types ofcomplementary product ions (Fig. 3C and Fig. S1) for the digly-cine-modified lipid A as were observed for the glycine-modifiedspecies in Fig. 3B, with considerable fragmentation detail related

Lipid A

Salmonella enterica

Lysylphosphatidyl-glycerol

Staphylococcus aureus

D-alanyl

Lipoteichoic acid

Staphylococcus aureus

40

Fig. 1. Gram-negative and Gram-positive bacteria modify their cell wallcomponents with amine-containing substituents (shown in red). Some Gram-negative bacteria (e.g., Salmonella enterica) modify their lipid A phosphategroups with phosphoethanolamine and aminoarabinose. Additionally,glycerophospholipids modified with amino acids, such as lysine, representmajor membrane lipids in Gram-positive bacteria (e.g., S. aureus). Gram-positives can further modify lipoteichoic acids and wall teichoic acids withD-alanine. The addition of amine-containing substituents to these cell wallcomponents helps decrease the overall negative charge of the bacterial cellwall and protects the bacterium against attack by CAMPs.

A

Exact Mass:

1757.2

1560 1820 2080 2340

0

10

20

30

40

50

60

70

80

90

100%

In

ten

sit

y

Voyager Spe c #1[BP = 1756.1, 29506 ]

1756.1

Classical

m/z

1782.1

1756.1

1729.1

0

40

20

60

80

100

1300 1560 1820 2080 2340 2600

[M-H]-

1300 1560 1820 2080 2340

Mass (m/z )

0

10

20

30

40

50

60

70

80

90

100

Voyager Spe c #1[BP = 1813.9, 5249]

1813.92

Re

la

tiv

e a

bu

nd

an

ce

0

2206.0

1870.9

[M-H]-

El Tor 1755.93

40

20

60

80

100

m/z

1300 1560 1820 2080 2340 2600

1813.9

B

12

12

14

14 12 14

Re

la

tiv

e a

bu

nd

an

ce

C

Fig. 2. MALDI-TOF MS of V. cholerae O1 El Tor and O1 classical lipid Aspecies. Previously, V. cholerae O1 El Tor and O1 classical strains were shownto synthesize a similar hexa-acylated lipid A species (12) (A). This commonlipid A species is shown at m/z 1,755.93 and m/z 1,756.1 for the El Tor (B) andclassical (C) biotypes, respectively. The El Tor biotype produces additionallipid A species with peaks at m/z 1,813.9 and m/z 1,870.9.

Hankins et al. PNAS | May 29, 2012 | vol. 109 | no. 22 | 8723

MICRO

BIOLO

GY

to the diglycine modification. The primary structure and site-specificity of the diglycine modification at the same 3′-linkedsecondary acyl chain were confirmed based on the abundant ionslabeled in red of m/z 1,741.92, 1,738.17, 1,756.17, 1,812.17,1,842.17, 1,853.17, and 1,825.17 that correspond to cleavages 23,3, 11, 20, 21, and 22. Interestingly, the ion of m/z 1,812.17 is themost abundant one in the spectrum and is a product from thecleavage of the diglycine backbone between the amine and car-bonyl group, which is the same type of bond (i.e., amide bond)most preferentially cleaved upon UVPD of singly deprotonatedlipid A ions. This product ion corresponds predictably withestablished UVPD fragmentation behavior (12, 13) and supportsthe assignment of the 114-Da modification as a diglycine moiety.The corresponding CID spectrum is shown in Fig. S2.

Vc1577 Is Required for Glycine Modification of V. cholerae O1 El TorLipid A. The Clusters of Orthologous Groups database (14)identifies three putative V. cholerae lipid A late acyltransferasehomologs: Vc0212, Vc0213, and Vc1577. Previous work fromour laboratory (12, 15) identified Vc0212 (LpxN) and Vc0213(LpxL) as functional V. cholerae lipid A late acyltransferases,which are responsible for the addition of myristate (C14:0) and3-hydroxylaurate (3-OH C12:0) to the 2′- and 3′-positions of theglucosamine disaccharide, respectively (Fig. 2A and Fig. S3).Although BLASTp results indicate that Vc1577 is an unlikelylipid A late acyltransferase homolog (E-value 3.9, 22% identity),the protein contains a lysophospholipid acyltransferase (LPLAT)domain. Because proteins of the LPLAT superfamily are typi-cally involved in the synthesis of membrane lipids (e.g., glycer-ophospholipids) catalyzing the transfer of an acyl chain froman acyl donor (e.g., acyl-acyl carrier protein, acyl-ACP) to lipidprecursors, we hypothesized that Vc1577 may play a role in thesynthesis of lipid A.To determine if Vc1577 was involved in transfer of a glycine

residue, a vc1577 mutant was generated in a V. cholerae O1 ElTor strain (Table S1). Purified lipid A from the vc1577 mutant

was analyzed by MALDI-TOF MS and produced a predominantpeak at m/z 1,757.1, consistent with the exact mass of the hexa-acylated V. cholerae lipid A species lacking the glycine modifi-cation (Fig. 4A, Left). Complementation of the vc1577 mutantrestored the addition of glycine and diglycine, producing pre-dominant peaks of m/z 1,812.9 and 1,869.9 (Fig. 4A, Right).

Vc1578 and Vc1579 Also Contribute to Glycine-Modification of V.cholerae Lipid A. Examination of the genomic context vc1577suggests that it is cotranscribed with vc1578 and vc1579. To de-termine if vc1577, vc1578, and vc1579 are in an operon, RT-PCRwas performed using RNA isolated from the O1 El Tor biotype.As shown in Fig. 4B, V. cholerae O1 El Tor synthesize a contig-uous mRNA containing vc1577, vc1578, and vc1579 (Fig. 4B).Vc1579 is annotated as a putative component (EntF) of

enterobactin synthetase and contains an amino acid adenylationdomain found in nonribosomal peptide synthetases involved insiderophore biosynthesis (16). However, amino acid adenylationdomains are also found in proteins involved in modification ofcell wall components of Gram-positives. Vc1579 shares homol-ogy (E-value 10−33, 28% identity) with the Staphylococcus aureusD-alanine-D-alanyl carrier protein ligase (Dcl) (9, 17–19). Dclactivates D-alanine as an adenylate followed by condensation ofthe activated amino acid onto a small carrier protein, termed D-alanine carrier protein (Dcp). D-alanyl-Dcp serves as the donorsubstrate for modification of teichoic acids (9, 17–19).Although no closely related homologs of Vc1578 were iden-

tified, the protein homology/analogY recognition engine (Phyre)(20) revealed that this small acidic protein has structural simi-larity to acyl-ACP (Fig. S4). ACP and its homologs serve ascarrier proteins for the synthesis of a number of biologicallyimportant molecules, including fatty acids, polyketides, andnonribosomal peptides (21). These small acidic proteins sharea conserved serine residue that is subject to posttranslationalmodification by a 4′-phosphopantetheine moiety that “carries”the substrate (e.g., D-alanine) as a thioester. Based on amino

A

B

C

Fig. 3. V. cholerae O1 El Tor syn-thesize glycine-modified lipid Aspecies. UVPD MS was used to ana-lyze the V. cholerae ions ([M – H]−,m/z 1,756; [M – H]−, m/z 1,813; and[M – H]−, m/z 1,870 [A–C]). Cleav-ages are indicated by dashed linesand the m/z values of the resultingfragment ions are shown in Fig. S1.The m/z values and cleavage siteshighlighted in red font representthe unique fingerprints associatedwith the glycine or diglycine mod-ifications. The precursor ion isdenoted by an asterisk. The frag-mentation patterns support thatV. cholerae synthesizes a glycine- anddiglycine-modified lipid A structureat the 3′-position of the glucos-amine disaccharide. “x55,” “x65,”and “x20” denote a section of thespectrum that has been magnified55, 65, or 20 times, respectively, tomore easily visualize product ions.

8724 | www.pnas.org/cgi/doi/10.1073/pnas.1201313109 Hankins et al.

acid sequence alignments (Fig. S4A) and 3D structural pre-dictions (Fig. S4B), Vc1578 shares the conserved serine (residue34) as well as the overall helix bundle structure found in othercharacterized carrier proteins (21).To confirm that Vc1579 and Vc1578 are required for glycine

modification of lipid A, vc1578 and vc1579 transposon insertions(Fig. S5) were obtained from the V. cholerae O1 El Tor non-redundant transposon insertion library (22). MALDI-TOFanalysis of lipid A isolated from vc1578 and vc1579 mutantsshowed a lack of glycine and diglycine modified forms (Fig. S6).Introduction of pVc1579, an arabinose inducible covering plas-mid (Table S1), into vc1579 restored lipid A modification.Complementation of the vc1578 mutant also resulted in glycinemodified species, albeit at lower levels compared with wild-type(Fig. S6). Given their role in aminoacyl lipid modification, wepropose that Vc1577, Vc1578, and Vc1579 be assigned thedesignations, AlmG, AlmF, and AlmE, respectively.

Glycine Modification of Lipid A Is Required for Polymyxin B Resistanceof V. cholerae Strains. V. cholerae O1 El Tor strains were found tobe highly resistant to polymyxin B, exhibiting a minimum in-hibitory concentration (MIC) of ∼100 μg/mL using ETest poly-myxin B strips (Fig. 5A). As a comparison, polymyxin-resistantvariants of E. coli or S. enterica are resistant at ∼10–12 μg/mL(23). Deletion of vc1577 (almG) resulted in nearly a 100-folddecrease in polymyxin resistance compared with wild-type levels;however, the polymyxin-resistant phenotype can be restoredwhen vc1577 (almG) is expressed in trans (Fig. 5A). Additionally,vc1578 (almF) and vc1579 (almE) El Tor mutants were morethan 100-fold more sensitive to polymyxin B compared with theEl Tor wild-type strain (Table S2). Consistent with MS results(Fig. S6), complementation of vc1579 (almE) restored antimi-crobial peptide resistance similar to wild-type levels (MIC, 64 μg/mL), whereas complementation of the vc1578 (almF) mutantwas only partial (MIC, 12 μg/mL). Still, expression of vc1578 intrans yields a 24-fold increase in resistance in comparison with

the vc1578 mutant (MIC, 0.5 μg/mL) (Table S2). This partialcomplementation could arise due to the stoichiometry and fluxof the system.Although the classical strain O395 synthesizes unmodified

hexa-acylated lipid A, expression of vc1577-79 (almEFG) in transresulted in an 85-fold increase in polymyxin resistance comparedwith the wild-type strain (Fig. 5B). MALDI-TOF MS analysis oflipid A isolated from O1 classical expressing AlmEFG showedglycine modified species with major ions at m/z 1,813.5 and1,870.5 (Fig. 5C). Taken together, these results confirm thatAlmEFG confers polymyxin resistance to V. cholerae by glycineaddition to surface-exposed LPS.

Polymyxin Resistance Correlates with Glycine Modification in Clinicaland Environmental V. cholerae Strains. To determine if otherpolymyxin-resistant V. cholerae isolates synthesize glycine-modi-fied LPS, lipid A from various environmental/clinical V. choleraeisolates (O1 El Tor, O1 classical, and non-O1) was analyzed byMALDI-TOF MS (Table S3). Our results indicated glycine-modified lipid A forms in all but one strain, whereas polymyxin-sensitive strains did not contain these lipids (Table S3). Theseresults provide a strong correlation between polymyxin resistanceand glycine modification of the V. cholerae surface.

Effect of Glycine Modification on Activation of TLR4. Previously, wedemonstrated that reduction in the number of acyl chains dras-tically decreased the endotoxicity of V. cholerae LPS (12). Todetermine the impact of glycine modification on Toll activation,we performed reporter cell signaling assays with purified LPSfrom V. cholerae O1 El Tor and vc1577::kan (Fig. S7). Whereasneither strain stimulated TLR2, a TLR activated by bacteriallipoproteins and lipoteichoic acids (24), the absence of glycineon lipid A caused only a modest decrease (P < 0.01) in TLR4activation with 10 ng/mL of LPS and higher (Fig. S7). Thus, itdoes not appear that this particular lipid A modification playsa major role in endotoxicity.

1300 1560 1820 2080 2340 2600Mass (m/z )

00

10

20

30

40

50

60

70

80

90

100

% In

ten

sity

1757.1

Rel

ativ

e ab

unda

nce

2149.3

1757.1

1782.1

[M-H]-

vc1577::kan

40

20

60

80

100

m/z 1300 1560 1820 2080 2340 2600

0

1729.1

1300 1560 1820 2080 2340 2600Mass (m/z )

00

10

20

30

40

50

60

70

80

90

100

% In

ten

sity

Voyager Spe c #1=>BC[BP = 1812.9, 3996]

2206.1

1812.9

1755.9

[M-H]-

vc1577::kan, pVc1577

m/z 1560 1820 2080 2340 2600

1869.9

Rel

ativ

e ab

unda

nce

40

20

60

80

100

0

A

vc1579 (almE)

vc1578 (almF)

vc1577 (almG)

237 bp 1671 bp 822 bp

1

2

3

Product 2

Product 1

Product 3

1300

Exact mass: 1814.2 or 1871.2

B

1757.2 Exact mass: or

1842.0 1784.9

Fig. 4. vc1577 is involved in glycine modification of V. cholerae lipid A and is cotranscribed with vc1578 and vc1579. (A) Lipid A was isolated from O1 El Torvc1577::kan and analyzed by MALDI-TOF MS. A predominant peak at m/z 1,757.1 was consistent with the loss of a glycine. MALDI-TOF analysis of lipid Aisolated from vc1577::kan, vc1577 resulted in major peaks at m/z 1,812.9 and 1,869.9, which is consistent with the addition of glycine and diglycine residues,respectively. Unmodified hexa-acylated lipid A is also present at m/z 1,755.9. (B) A schematic of the genetic organization of vc1577 (almG), vc1578 (almF), andvc1579 (almE) is shown. RT-PCR was done to determine if vc1577, vc1578, and vc1579 are cotranscribed and indicated a read-through transcript (product 1)containing vc1579-77. Additional RT-PCRs confirmed read-through transcripts for vc1579-78 (product 2) and vc1578-77 (product 3). V. cholerae genomic DNAtemplate was used as a positive control for primers and amplified product sizes; however, for the negative control (−RT), cDNA without reverse transcriptaseadded was used as template, verifying that no DNA contamination had occurred.

Hankins et al. PNAS | May 29, 2012 | vol. 109 | no. 22 | 8725

MICRO

BIOLO

GY

DiscussionTo adapt to their surrounding environment, bacteria haveevolved the ability to remodel their cellular envelope. Themodification of bacterial cell wall components (phospholipids,LPS, or techoic acids) has been shown to play a vital role inantimicrobial peptide resistance in Gram-negative and Gram-positive bacteria. Both organisms use amine-containing sub-stituents to alter the overall negative charge of their surface inresponse to CAMP attack (Fig. 1). Our findings herein identifya unique lipid A modification that occurs in the Gram-negativeorganism V. cholerae. The rich level of structural detail affordedby UVPD (13) (Fig. 3 and Fig. S1) proved critical for mappingthe identity and location of the glycine/diglycine modification.The presence of a glycine residue was found to be essential forpolymyxin resistance and presumably decreases the negativecharge of the bacterial surface. However, it is also possible thatglycine modification alters membrane fluidity, thereby influenc-ing antimicrobial peptide resistance.Although prevalent in Gram-positives, aminoacylation of

glycerophospholipids has been observed only in select Gram-negatives (i.e., Pseudomonas and Rhizobium) and depend onaminoacyl-tRNAs for their synthesis (10, 11). However, V.cholerae has evolved an amino acid modification system similarto that found in Gram-positive bacteria for modification of tei-choic acids. Sequence analysis revealed that Vc1579 (AlmE)shares sequence homology to the Gram-positive Dcl (E-value10−47, 33% identity) (9, 19). Based on bioinformatic and struc-tural analysis, Vc1578 was hypothesized to serve as a glycinecarrier protein similar to the D-alanine carrier protein Dcp (Fig.

S4) (9, 17–19). Sequence alignments show Vc1578 (AlmF) pos-sesses a conserved serine at residue 34 that likely acts as the siteof attachment of a 4′-phosphopantetheine group. Although theD-alanine transferase is unknown in Gram-positive bacteria, wehypothesize that Vc1577 (AlmG) catalyzes the transfer of glycineto the unmodified hexa-acylated V. cholerae lipid A. AlmGcontains a conserved LPLAT domain that may provide recog-nition of both the lipid A domain of LPS and the amino acid-charged carrier protein. A model for glycine modification ofV. cholerae lipid A species is shown in Fig. 6.The proteins involved in glycine modification are predicted

soluble proteins by TMHMM (25) and contain no signal peptidesrequired for secretion across the inner membrane. This predictionis consistent with the fact that the amino acid ligase (AlmE) wouldrequire cytoplasmic substrates, such as ATP and glycine, for ac-tivity (Fig. 6). Given that AlmG uses hexa-acylated lipid A asa substrate, one would expect that amino acid addition occur at themembrane. Indeed, a previous report from our laboratory showedthat AlmG (Vc1577) is membrane-associated (15). A periplasmiclocaliziation would require charged AlmF to be transported acrossthe inner membrane to serve as a donor for AlmG. Generally, lipidA modification systems are located in the extracytoplasmic com-partments of the bacterial cell separated from the conserved bio-synthetic pathway known as the Raetz pathway (5). Although ourdata suggests that glycine addition occurs on the cytoplasmic faceof the inner membrane, further biochemical characterization isnecessary to confirm the proposed model (Fig. 6).Earlier reports indicated that the outer membrane porin,

OmpU, contributes toward polymyxin resistance in the O1classical biotype (26). However, as demonstrated here and byothers (1, 27) O1 classical strains are quite sensitive to poly-myxin (MIC of 0.75 μg/mL) (Table S2), making it difficult todetermine phenotypic differences in classical strains. V. choleraeO1 El Tor strains lacking either of the major porins, OmpUor OmpT, show wild-type levels of polymyxin-resistant (MIC96–128 μg/mL) (Table S2), whereas alm mutants are sensitive(MIC 0.5–1.0 μg/mL).Both V. cholerae O1 El Tor and classical biotypes synthesize

a hexa-acylated lipid A species with a 3-hydroxylaurate (3-OHC12:0) acyl chain linked at 3′-position of the molecule (Figs. 2and 3) (12). In a previous report it was shown that the presence

vc1577::

kan

vc1577::kan,

pVc1577

1.0

64

El Tor Classical

96

0.75

C

1300 1560 1820 2080 2340 2600

Mass (m/z )

0

1.7E+4

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sity

Voyager Spe c #1=>BC[BP = 1870.5, 17240]

1870.5415

1757.5

Classical,

pVc1577-79

1813.5

m/z

1560 1820 2080 2340 2600

1870.5

[M-H]-

1898.6

A R

ela

tiv

e a

bu

nd

an

ce

B

Classical,

pVc1577-79

64

Exact mass:

1814.2 or 1871.2

0

40

20

60

80

1300

100

or

Fig. 5. AlmG (Vc1577), AlmF (Vc1578), and AlmE (Vc1579) confer polymyxinresistance to V. cholerae. Polymyxin MIC for various strains was determinedon LB agar using ETest polymyxin B strips. (A) Wild-type V. cholerae O1 El Torare polymyxin-resistant with a MIC of 96 μg/mL However, the vc1577mutant,lacking the glycine modification of lipid A, is nearly 100-fold more sensitiveto polymyxin B. When O1 El Tor vc1577::kan is complemented in trans, thestrain gains polymyxin resistance. (B) Introduction of a plasmid expressingvc1579-77 confers polymyxin resistance to O1 classical strains. (C) MALDI-TOFMS of the lipid A of the classical biotype expressing pW77-79 revealed ions atm/z 1,813.5 and m/z 1,870.5, corresponding to lipid A modified by either oneor two glycine residues, respectively. The minor peak at m/z 1,757.5 repre-sents unmodified hexa-acylated V. cholerae lipid A.

Outer Membrane

Inner Membrane

Lipid A

OH

ATP + Glycine

AlmF

AlmG

AlmE

Cytoplasm

Periplasm

Polymyxin+ + + + +

S GlyAlmFSH

AMP + PPi

Fig. 6. Proposed model for the synthesis of glycine-modified lipid A species inV. cholerae. The model indicates glycine is ligated to the carrier protein, AlmF-SH (Vc1578), as a thioester. The reaction is catalyzed by the amino acid ligase,AlmE (Vc1579), in the cytoplasm. AlmG (Vc1577) then catalyzes the transfer ofglycine from AllmF-S-glycine to the hexa-acylated V. cholerae lipid A species inthe inner membrane. Presumably, diglycine would arise from a second addi-tion to the lipid A molecule. Glycine-modified forms of lipid A are thentransported to the bacterial surface providing resistance to polymyxin.

8726 | www.pnas.org/cgi/doi/10.1073/pnas.1201313109 Hankins et al.

of the 3-hydroxylauroyl group was necessary for antimicrobialpeptide resistance (12, 28). Our laboratory identified an unusualacyl transferase, LpxN, which is selective for hydroxylated acylchains (Fig. S3) (12), and demonstrated that the hydroxyl groupitself promotes polymyxin resistance. Here, we establish that the3-hydroxyl group is actually the site of glycine modification. Ourprevious findings also demonstrated that reduction in the num-ber of acyl chains drastically decreased the endotoxicity ofV. cholerae LPS (12). To determine the impact of glycine modifi-cation on Toll activation, we performed reporter cell-signalingassays with purified LPS from V. cholerae O1 El Tor and vc1577::kan. Whereas neither strain stimulated TLR2, a TLR activatedby bacterial lipoproteins and lipoteichoic acids (24), the absenceof glycine on lipid A caused only a modest decrease (P < 0.01) inTLR4 activation with 10 ng/mL of LPS and higher (Fig. S7).Thus, it does not appear that this particular lipid A modificationplays a major role in endotoxicity.One key question is why classical strains lack glycine-modified

lipid A. Comparison of the genomes of sequenced classical andEl Tor strains showed that both biotypes contain the almEFGlocus. However, sequence alignments comparing the coding re-gion revealed that classical strain O395 has a nonsense mutation,resulting in a truncated almF carrier protein lacking the con-served serine (Fig. S8). When the classical strain is com-plemented with the El Tor almEFG region in trans, thepolymyxin phenotype is restored and glycine-modified lipid Aspecies are synthesized (Fig. 5 B and C). The O139 serogroup(strain MO10) that was associated with outbreaks in the 1990s isalso polymyxin-resistant, synthesizing glycine-modified lipid A(Table S3). O139 arose from a progenitor O1 El Tor strain andhas an intact almEFG locus.The life cycle of V. cholerae is complex, having both human

and environmental stages. Given that the El Tor biotype dis-placed the classical biotype, it is tempting to speculate that

amino acid modification of the cell surface enhances bacterialsurvival. Another consideration is whether glycine addition isregulated in different environments. This work also provides theopportunity to investigate the biochemical mechanisms of cellenvelope modifications aiding in development of new antimi-crobial compounds. On the whole, our findings identify a uniqueLPS modification that mechanistically links Gram-positive andGram-negative cell wall modification systems.

Materials and MethodsBacterial Strains and Growth Conditions. The bacterial strains and plasmidsused in this study are summarized in Tables S1 and S4. V. cholerae strainswere grown routinely at 37 °C in Luria-Bertani (LB) broth or on LB agar or ina modified g56 minimal media, described previously (12). E. coli strains weregrown in LB at 37 °C. Antibiotics were used at the following concentrations:100 μg/mL ampicillin, 60 μg/mL kanamycin, 10 μg/mL streptomycin. For V.cholerae complementation, 0.5 mM isopropyl β-D-1-thiogalactopyranosidewas added to growth media.

MS and UVPD. All MS experiments were undertaken on a Thermo FisherScientific LTQ XL linear ion-trap mass spectrometer equipped with a 193-nmCoherent ExciStar XS excimer laser. The setup was similar to that previouslydescribed (29, 30). An online nanoelectrospray setup was used for directinfusion of lipid A samples as reported previously (13).

Other Methods. Methods describing recombinant DNA and RNA techniques,lipid A isolation, polymyxin B MIC assays, complementation plasmids, andgene deletions are described in SI Materials and Methods.

ACKNOWLEDGMENTS. The authors thank Dr. Shelley Payne (University ofTexas at Austin) and Dr. Karl Klose (University of Texas at San Antonio) forproviding strains. This work was supported by National Institutes of HealthGrants AI064184 and AI76322 (to M.S.T.) and Grants Welch F1155 andNational Science Foundation CHE-1012622 (to J.S.B.).

1. Gangarosa EJ, Bennett JV, Boring JR, 3rd (1967) Differentiation between Vibriocholerae and Vibrio cholerae biotype El Tor by the polymyxin B disc test: Comparativeresults with TCBS, Monsur’s, Mueller-Hinton and nutrient agar media. Bull WorldHealth Organ 36:987–990.

2. Raetz CR, Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71:635–700.

3. Park BS, et al. (2009) The structural basis of lipopolysaccharide recognition by theTLR4-MD-2 complex. Nature 458:1191–1195.

4. Raetz CR, Reynolds CM, Trent MS, Bishop RE (2007) Lipid A modification systems ingram-negative bacteria. Annu Rev Biochem 76:295–329.

5. Trent MS, Stead CM, Tran AX, Hankins JV (2006) Diversity of endotoxin and its impacton pathogenesis. J Endotoxin Res 12:205–223.

6. Zhou Z, Lin S, Cotter RJ, Raetz CR (1999) Lipid A modifications characteristic ofSalmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detectionof 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate. J Biol Chem274:18503–18514.

7. Zhou Z, et al. (2001) Lipid A modifications in polymyxin-resistant Salmonella typhi-murium: PMRA-dependent 4-amino-4-deoxy-L-arabinose, and phosphoethanolamineincorporation. J Biol Chem 276:43111–43121.

8. Trent MS, Ribeiro AA, Lin S, Cotter RJ, Raetz CR (2001) An inner membrane enzyme inSalmonella 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 BiolChem 276:43122–43131.

9. Neuhaus FC, Baddiley J (2003) A continuum of anionic charge: Structures and func-tions of D-alanyl-teichoic acids in gram-positive bacteria. Microbiol Mol Biol Rev 67:686–723.

10. Ernst CM, Peschel A (2011) Broad-spectrum antimicrobial peptide resistance by MprF-mediated aminoacylation and flipping of phospholipids. Mol Microbiol 80:290–299.

11. Roy H (2009) Tuning the properties of the bacterial membrane with aminoacylatedphosphatidylglycerol. IUBMB Life 61:940–953.

12. Hankins JV, et al. (2011) Elucidation of a novel Vibrio cholerae lipid A secondaryhydroxy-acyltransferase and its role in innate immune recognition. Mol Microbiol 81:1313–1329.

13. Madsen JA, Cullen TW, Trent MS, Brodbelt JS (2011) IR and UV photodissociation asanalytical tools for characterizing lipid A structures. Anal Chem 83:5107–5113.

14. Tatusov RL, Galperin MY, Natale DA, Koonin EV (2000) The COG database: A tool forgenome-scale analysis of protein functions and evolution. Nucleic Acids Res 28:33–36.

15. Hankins JV, Trent MS (2009) Secondary acylation of Vibrio cholerae lipopolysaccha-ride requires phosphorylation of Kdo. J Biol Chem 284:25804–25812.

16. Fischbach MA, Walsh CT (2006) Assembly-line enzymology for polyketide and nonribo-somal Peptide antibiotics: Logic, machinery, and mechanisms. Chem Rev 106:3468–3496.

17. Heaton MP, Neuhaus FC (1994) Role of the D-alanyl carrier protein in the biosynthesisof D-alanyl-lipoteichoic acid. J Bacteriol 176:681–690.

18. Debabov DV, et al. (1996) The D-alanyl carrier protein in Lactobacillus casei: Cloning,sequencing, and expression of dltC. J Bacteriol 178:3869–3876.

19. Perego M, et al. (1995) Incorporation of D-alanine into lipoteichoic acid and wallteichoic acid in Bacillus subtilis. Identification of genes and regulation. J Biol Chem270:15598–15606.

20. Kelley LA, Sternberg MJ (2009) Protein structure prediction on the Web: A case studyusing the Phyre server. Nat Protoc 4:363–371.

21. Byers DM, Gong H (2007) Acyl carrier protein: Structure-function relationships ina conserved multifunctional protein family. Biochem Cell Biol 85:649–662.

22. Cameron DE, Urbach JM, Mekalanos JJ (2008) A defined transposon mutant libraryand its use in identifying motility genes in Vibrio cholerae. Proc Natl Acad Sci USA105:8736–8741.

23. Herrera CM, Hankins JV, Trent MS (2010) Activation of PmrA inhibits LpxT-dependentphosphorylation of lipid A promoting resistance to antimicrobial peptides. Mol Mi-crobiol 76:1444–1460.

24. Jin MS, et al. (2007) Crystal structure of the TLR1-TLR2 heterodimer induced bybinding of a tri-acylated lipopeptide. Cell 130:1071–1082.

25. Sonnhammer EL, von Heijne G, Krogh A (1998) A hidden Markov model for predictingtransmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol 6:175–182.

26. Mathur J, Waldor MK (2004) The Vibrio cholerae ToxR-regulated porin OmpU confersresistance to antimicrobial peptides. Infect Immun 72:3577–3583.

27. Han GK, Khie TS (1963) A new method for the differentiation of Vibrio comma andVibrio El Tor. Am J Hyg 77:184–186.

28. Matson JS, Yoo HJ, Hakansson K, Dirita VJ (2010) Polymyxin B resistance in El TorVibrio cholerae requires lipid acylation catalyzed by MsbB. J Bacteriol 192:2044–2052.

29. Gardner MW, Vasicek LA, Shabbir S, Anslyn EV, Brodbelt JS (2008) Chromogenic cross-linker for the characterization of protein structure by infrared multiphoton dissoci-ation mass spectrometry. Anal Chem 80:4807–4819.

30. Madsen JA, Boutz DR, Brodbelt JS (2010) Ultrafast ultraviolet photodissociation at 193nm and its applicability to proteomic workflows. J Proteome Res 9:4205–4214.

Hankins et al. PNAS | May 29, 2012 | vol. 109 | no. 22 | 8727

MICRO

BIOLO

GY