www.fems-microbiology.org

FEMS Microbiology Letters 245 (2005) 337–344

Conservation of a novel protein associated with an antibioticefflux operon in Burkholderia cenocepacia

Bindu M. Nair a,1, Lukasz A. Joachimiak b, Sujay Chattopadhyay c, Idalia Montano a,2,Jane L. Burns a,*

a Department of Pediatrics, Division of Infectious Diseases, Immunology and Rheumatology, University of Washington, Seattle, WA, United Statesb Department of Biochemistry and Program in Biomolecular Structure and Design, University of Washington, Seattle, WA, United States

c Department of Microbiology, University of Washington, Seattle, WA, United States

Received 2 March 2005; received in revised form 13 March 2005; accepted 15 March 2005

First published online 25 March 2005

Edited by A.M. George

Abstract

Burkholderia cenocepacia is a significant problem in individuals with cystic fibrosis and is a member of the B. cepacia complex of

closely related antibiotic resistant bacteria. A salicylate-regulated antibiotic efflux operon has been identified in B. cenocepacia and

one of its four genes, llpE, is without parallel in previously reported efflux operons. PCR amplification and sequencing of llpE from

B. cepacia complex isolates demonstrated the highest prevalence in B. cenocepacia with a high degree of sequence conservation.

While at least one non-synonymous mutation was identified between isolates from different genomovars, only synonymous differ-

ences were identified within the IIIA and IIIB sub-groups of B. cenocepacia. Structural modeling suggests that LlpE is a member of

the a/b hydrolase enzyme family. Identification of strong structural homology to hydrolases and a high degree of conservation in B.

cenocepacia suggests an enzymatic function for LlpE, benefiting survival in the cystic fibrosis lung.

� 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Burkholderia cenocepacia; Antibiotic efflux; Hydrolytic enzymes

1. Introduction

Burkholderia cepacia complex strains are important

pulmonary pathogens in patients with cystic fibrosis.

Although isolated from only 3.1% of patients over the

age of five [1], this organism may be associated with se-

0378-1097/$22.00 � 2005 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2005.03.027

* Corresponding author. Children�s Hospital and Regional Medical

Center, 307 Westlake Ave N, Suite 300, Seattle, WA 98109, United

States. Tel.: +206 987 2073; fax: +206 987 7311.

E-mail address: jane.burns@seattlechildrens.org (J.L. Burns).1 Present address: Department of Radiation Medicine, Roswell Park

Cancer Institute, Buffalo, NY, United States.2 Present address: Laboratory of Receptor Biology and Gene

Expression, National Cancer Institute, Bethesda, MD, United States.

vere morbidity and mortality in the cystic fibrosis popu-

lation [2]. The complex is a group of at least ten related

genomovars [3–7]. Although virtually all genomovars

have been isolated from individuals with cystic fibrosis,

most cystic fibrosis isolates are genomovars II and III,

now called B. multivorans and B. cenocepacia, respec-tively [8,9]. Of these, B. cenocepacia has been most com-

monly associated with epidemic spread and increased

clinical virulence [3,10]. Based on recA typing, B. ceno-

cepacia has subsequently been divided into four sub-

groups, of which genomovars IIIA and IIIB are most

prevalent in cystic fibrosis [5].

Multiple antibiotic resistance is a characteristic of all

B. cepacia complex isolates, particularly those from

. Published by Elsevier B.V. All rights reserved.

338 B.M. Nair et al. / FEMS Microbiology Letters 245 (2005) 337–344

individuals with cystic fibrosis. Antibiotic efflux is a

common mechanism of resistance in Gram-negative

bacteria [11]. Efflux pumps have been implicated in resis-

tance to diverse antibiotics [12–19] and other toxic com-

pounds including dyes, detergents, disinfectants, and

fatty acids [11,19].We previously identified efflux-mediated multidrug

resistance to chloramphenicol, trimethoprim, and cip-

rofloxacin in B. cenocepacia [20,21]. The ceo efflux op-

eron responds to salicylate as an inducer and as a

substrate of efflux [21]. The genes ceoA, ceoB, and

opcM have significant homologies to the resistance/

nodulation/cell division family of proteins, members

of which are components of multidrug efflux systems.A fourth gene, llpE, has been identified in the cluster

and is co-transcribed with ceoA, ceoB, and opcM [21],

but its role in efflux is unclear. There is no counterpart

of LlpE in any other efflux operons characterized to

date. In this study, we elaborate on the potential role

of LlpE in efflux, examining its prevalence in the B.

cepacia complex, establishing a putative structural

model for LlpE based on homologous proteins, andspeculating on a possible enzymatic function in cystic

fibrosis.

2. Materials and methods

2.1. Culture conditions

Strains used in this study are listed in Table 1. B.

cepacia complex strains were grown in L-broth at

37 �C, 250 rpm.

2.2. PCR conditions

Genomic DNA for amplification of llpE was isolated

from B. cepacia complex isolates using the DNeasy tis-sue Kit (Qiagen, Valencia, CA). For assessing the prev-

alence of llpE, genomic DNA was PCR amplified using

primers llpE-F1 (5 0GGCCTGGAAGCTTGCTTCG-

G3 0) and llpE-R (5 0GCATTAGTCCATGGTTATTC-

GGGACGGTTCGGC 3 0) [21]. PCR used the GC Rich

PCR system (Roche Applied Science, Indianapolis,

IN), 100 ng of template DNA, 20 pmol of each primer,

and 200 lM deoxynucleotides. An initial denaturationat 95 �C for 4 min was followed by 30 cycles of denatur-

ation at 95 �C for 30 s, annealing at 66 �C for 30 s and

elongation at 72 �C for 60 s, with a 5 s increment in elon-

gation temperature per cycle for the last 20 cycles. After

a final extension at 72 �C for 7 min, the samples were

examined by agarose gel electrophoresis. PCR bands

from selected strains were gel-purified and sequenced

using the ABI PRISM BigDye Terminator CycleSequencing Ready Reaction Kit (Applied Biosystems/

Perkin–Elmer, Foster City, CA).

2.3. Phylogenetic analysis

Based on zonal analysis, a recent tool for visualizing

gene evolution [22], an unrooted protein phylogram was

constructed based on llpE coding sequences from 12

strains of the B. cepacia complex. This phylogram wascreated to give information on inter- and intra-nodal

synonymous variations, where each node represents a

structural variant. ClustalX 1.83 was used to align the

various DNA and protein sequences. The aligned

DNA dataset was the input in the PAUP*4.0b program

to generate a maximum likelihood unrooted DNA

phylogram, which was finally converted to a protein

phylogram incorporating the synonymous and non-syn-onymous changes from the aligned protein sequence

dataset.

2.4. Homology modeling

Comparative models for LlpE were built with the

help of the Robetta server [23,24]. Robetta is a fully

automated full-chain protein structure prediction serverand the LlpE model was built from a homologous pro-

tein (E-value 2E-28, sequence identity 38%) detected

with BLAST [25] and aligned by the K*Sync alignment

method [23]. Loop regions were assembled ab initio by

the Rosetta program fragments and optimized to fit

the aligned template structure [26].

3. Results

3.1. Sequence analysis

The region upstream of ceoABopcM from K61-3 was

sequenced and shown to code for a 286 amino acid pro-

tein. This gene, termed llpE, is the first gene in the ceo

antibiotic efflux operon [21]. The N-terminal 70 aminoacids of LlpE were analyzed for presence of signal pep-

tide, using SignalP V2.0 (http://www.cbs.dtu.dk/services/

SignalP-2.0/) [27]. LlpE is predicted to be a non-secre-

tory protein and to have a signal peptide that is cleaved

between amino acids 22 and 23. Analysis of LlpE by

PSORT (http://psort.nibb.ac.jp/) [28], a program for

prediction of protein localization, predicts a periplasmic

or outer membrane localization.

3.2. Identification of homologs

Subjecting LlpE to a BLAST search [25] did not re-

veal homology to previously characterized B. cepacia

lipases, none of which are associated with antibiotic

resistance [29,30]. However, the search did reveal

homology to various eukaryotic and prokaryotic lipasesand esterases belonging to the a/b hydrolase family of

proteins. These proteins have diverse functions, but

Table 1

Strains used in this study

Strain Phenotypea Source

K61-3 B. cenocepacia, IIIA, CF clinical isolate, original source for ceo efflux operon [20]

ATCC 25416 B. cepacia, onion isolate [36]

ATCC 17759 B. cepacia, soil isolate [36]

CEP509 B. cepacia, CF isolate [36]

LMG 17997 B. cepacia, UTI isolate [36]

C5393 B. multivorans, CF isolate [36]

LMG 13010 B. multivorans, CF isolate [36]

C1576 B. multivorans, CF isolate [36]

CP-A1-1 B. multivorans, CF isolate [36]

JTC B. multivorans, CGD isolate [36]

C1962 B. multivorans, abscess isolate [36]

249-2 B. multivorans, laboratory isolate [36]

ATCC 17616 B. multivorans, soil isolate [36]

J2315 B. cenocepacia, IIIA, CF isolate [36]

BC7 B. cenocepacia, IIIA, CF isolate [36]

K56-2 B. cenocepacia, IIIA, CF isolate [36]

C5424 B. cenocepacia, IIIA, CF isolate [36]

C6433 B. cenocepacia, IIIA, CF isolate [36]

C1394 B. cenocepacia, IIIB, CF isolate [36]

PC184 B. cenocepacia, IIIB, CF isolate [36]

CEP511 B. cenocepacia, IIIB, CF isolate [36]

J415 B. cenocepacia, IIIB, CF isolate [36]

ATCC 17765 B. cenocepacia, IIIB, UTI isolate [36]

LMG 14294 B. stabilis, CF isolate [36]

C7322 B. stabilis, CF isolate [36]

LMG 18888 B. stabilis, non-CF clinical isolate [36]

LMG 14086 B. stabilis, ventilator isolate [36]

PC259 B. vietnamiensis, CF isolate [36]

LMG 16232 B. vietnamiensis, CF isolate [36]

FC441 B. vietnamiensis, CGD isolate [36]

LMG 10929 B. vietnamiensis, rice isolate [36]

CEP021 B. dolosa, CF isolate [37]

FC353 B. dolosa, CF isolate J. LiPuma

ATCC53266 B. ambifaria, soil isolate [37]

FC876 B. ambifaria, soil isolate T. Heulin

ATCC17760 B. ambifaria, soil isolate ATCC

a Abbreviations: CF, cystic fibrosis; UTI, urinary tract infection; CGD, chronic granulomatous disease.

B.M. Nair et al. / FEMS Microbiology Letters 245 (2005) 337–344 339

share a common three-dimensional structure, including

a/b hydrolase folds, often in the absence of detectable

homology [31–34]. Multiple sequence alignment of nine

of the most homologous proteins using the BCM SearchLauncher [35] and subsequent BOXSHADE analysis re-

veals alignment of a consensus sequence Sm-X-Nu-X-

Sm-Sm, where Sm is a small amino acid like glycine,

X is any amino acid and Nu is the nucleophilic residue

(G-H-D132-A-G-G in LlpE). This sequence is conserved

in the GXD/SXG family of lipolytic enzymes and the

Type B family of carboxylesterases. The most homolo-

gous proteins included: 1JJIA, a novel hyper-thermo-philic carboxylesterase from the archaeon A. fulgidus;

AaaD, human arylacetamide deacetylase; Aes, an Esch-

erichia coli acetyl esterase, Est, an esterase from Acineto-

bacter lwoffii; Bah, an acetyl hydrolase from

Streptomyces hygroscopicus; LipS, a human hormone

sensitive lipase (HSL); VSH5, Dictyostelium discoideum

vegetative specific protein H5; Est-P, Drosophila melano-

gaster esterase P precursor; and Est-1, a human liver

carboxylesterase 1 precursor. All of these proteins con-

tain a catalytic triad in the conserved order of nucleo-

philic residue-acidic residue-histidine, which in LlpE is

predicted to be D132, D226 and H256. In all the solvedstructures of these proteins, the triad is accommodated

on loops that are the best-conserved features of these

proteins.

3.3. Prevalence among genomovars

Genomic DNA was harvested from 37 strains from

seven genomovars belonging to the B. cepacia complex[36,37] and subjected to PCR amplification for llpE.

PCR conditions were optimized using genomic DNA

from B. cenocepacia K61-3, to show amplification of a

single 630 bp band. Agarose gel analysis revealed a sin-

gle 630 bp band for all five B. cepacia (genomovar I)

strains, all 11 B. cenocepacia strains, and four of five

B. stabilis (genomovar IV) strains (Fig. 1). Multiple

bands or a single band of the wrong size were amplified

Fig. 1. PCR amplification of llpE from B. cepacia complex experimental panel strains. III: B. cenocepacia, I: B. cepacia, II: B. multivorans, IV: B.

stabilis, V: B. vietnamiensis, VI: B. dolosa, VII: B. ambifaria, mw: molecular weight markers (identical in all gels) with sizes shown in Kb. Lanes 1:

J2315, 2: C5424, 3: PC184, 4: J415, 5: BC7, 6: C6433, 7: CEP511, 8: ATCC 17765, 9: K56-2, 10: C1394, 11: K61-3 (source of cloned llpE), 12: ATCC

25416, 13: ATCC 17759, 14: CEP509, 15: LMG 17997, 16: K61-3, 17: C5393, 18: LMG 13010, 19: C1576, 20: CP-A1-1, 21: JTC, 22: C1962, 23: 249-2,

24: ATCC 17616, 25: LMG 14294, 26: C7322, 27: LMG 18888, 28: LMG 14086, 29: K61-3, 30: PC259, 31: LMG 16232, 32: FC441, 33: LMG 10929,

34: CEP21, 35: FC353, 36: ATCC17760, 37: FC876, 38: ATCC53266.

340 B.M. Nair et al. / FEMS Microbiology Letters 245 (2005) 337–344

from all strains belonging to B. multivorans, B. vietnami-

ensis, B. dolosa and B. ambifaria, as well as from the

remaining B. stabilis strain.

3.4. Phylogenetic analysis

The 630 bp PCR bands from all 11 B. cenocepacia

strains and one representative strain each from B. cepa-

cia and B. stabilis were gel-purified and sequenced to

examine genetic relatedness. In Fig. 2, each node is rep-

resented by a single genomovar group (example, genom-

ovar I and IV) or sub-group (example, IIIA and IIIB).

Phylogenetic analysis of B. cepacia and B. stabilis re-

vealed 19 variations in the 2 llpE gene sequences, ofwhich 15 are synonymous and 4 are non-synonymous.

1

11

11

CCTA61452

481CP

115PEC

CCTA56771

514J4931C

8

01

5 )12(

2K

L702I

V451A

1A

Fig. 2. Zonal analysis diagram. From the unrooted maximum likelihood tree

single node. The branch(es) connecting any two nodes corresponds to the n

positions are shown along the branches in italics. The values along the branch

the two respective nodes. The number of alleles in each of the two multiple-a

changes in parentheses. Since each node (i.e., each structural variant) repres

denoted beside the nodes.

B. cenocepacia isolates, including distinct IIIA and IIIB

subgroups, were also separated from B. cepacia and

B. stabilis and from each other by synonymous and

non-synonymous mutations However, within these two

subgroups there were no non-synonymous mutations.

Analysis of genomovar IIIB reveals that the nodeconsists of five strains differentiated by 21 changes in

the llpE gene, all of which are synonymous, whereas in

genomovar IIIA, strain C6433 differs from the five other

genomovar IIIA strains by two synonymous mutations.

Based on the extent of sequence differences, ATCC25416

(genomovar I) and LMG14294 (genomovar IV) appear

as outgroups compared to genomovar III subgroups.

Sequence analysis of selected non-630 bp PCR bandsdid not show homology to llpE (data not shown).

15

GML49241

7CB2-65K3-16K5132J4245C

3346C3

5

6 )2(

N43

S732G:I002M:V21

, the alleles differing only in synonymous variations are collapsed into a

umber of non-synonymous changes and the amino acid changes and

es represent the numbers of inter-nodal synonymous mutations between

llele nodes is shown along with the number of intra-nodal synonymous

ents a particular genomovar group or subgroup, the genomovars are

Fig. 3. Putative model of LlpE. (a) Topology map of the LlpE model generated by the Robetta server. The N-terminal cap region is truncated

relative to the parent 1JJI but the remainder of the core structure is nearly identical to the Afest fold. (b) Surface representation of the LlpE model in

teal; in orange are the nucleophilic elbow residues(130–134, including D132); in yellow are the active site residues D132, D226 and H256; in red are

external loops R175–I180, D164–R169 and L193–Q195; and in white are the internal loops H58–V63 and G160–L163. At the bottom is a zoom of the

active site residues, colored as before.

B.M. Nair et al. / FEMS Microbiology Letters 245 (2005) 337–344 341

3.5. Putative model of LlpE

Conservation of the catalytic triad has been corrob-

orated in LlpE by homology modeling of the LlpEstructure using the Robetta server [23,24]. The template

for the LlpE model was detected by BLAST and de-

rived from the structure of a novel hyper-thermophilic

carboxylesterase from the archaeon, Archaeoglobus ful-

gidus (Protein data bank ID:1JJI) [38]. This protein

demonstrates a canonical a/b hydrolase core that con-tains a central-sheet composed of eight strands. The

core is shielded on the C-terminal side by a cap region

constituted of two separate regions (amino acid resi-

dues 1–54 and 188–246) at the carboxyl edge of the

342 B.M. Nair et al. / FEMS Microbiology Letters 245 (2005) 337–344

central b-sheet. The putative LlpE model has a near

identical core region to the parent structure, but has

a simplified cap (Fig. 3(a)). The cap region is com-

prised of the equivalent region on the parent structure

(188–246), with a truncated and unstructured N-termi-

nal portion, which is consistent with it being a signalsequence for export into the periplasm [21]. Residues

in the putative catalytic triad in LlpE (D132, D226,

and H256) occupy canonical positions, where the nucle-

ophile aspartic acid (132) is located within a sharp turn

known as the nucleophilic elbow (Fig. 3(b)). At its

apex, this turn contains the signature motif GXD/

SXG, which characterizes all members of this family

of proteins. Analysis using JEvTrace [39] revealed thatthe highly conserved GXD/SXG motif fits nicely in a

pocket at the base of the b-sheet and leads to a large

ovoidal tunnel that could accommodate a large sub-

strate with approximate dimensions of 10 A · 12 A

(Fig. 3(b)). It is conceivable that the almost planar sub-

strates of the ceo efflux system including the antibiotics

chloramphenicol, trimethoprim, and ciprofloxacin, and

the siderophore, salicylate, with dimensions of9 · 3 · 4 A, 9 · 7 · 4 A, 11 · 6 · 1 A, and 4 · 3 A,

respectively, can be accommodated in this tunnel dur-

ing efflux.

The close structural and sequence similarity of the

LlpE model to the parent structures allows definition

of equivalent regions. As in the A. fulgidus structure,

the loop regions R175–I180, D164–R169 and L193–Q195

define the entrance to the tunnel whereas the loop re-gions H58–V63 and G160–L163 define the internal bor-

ders. Further, the homologous crystal structure

revealed the presence of a covalent adduct to a pipera-

zine-ethane moiety at the active site S160, which has

been speculated to be the acyl-binding pocket [38].

The location of the two other catalytic residues, D226

and H256, coordinate the proximal function of these

residues as a charge relay network and proton carrier,respectively.

4. Discussion

Originally known as B. cepacia genomovar III [6], B.

cenocepacia can be a life-threatening multidrug resistant

pathogen and is the most commonly encounteredgenomovar in patients with cystic fibrosis [3,8]. We pre-

viously demonstrated salicylate (and iron) regulated

antibiotic efflux in B. cenocepacia strain K61-3 [20,21].

The current study investigated a novel protein, LlpE,

that is a component of the ceo antibiotic efflux operon.

Prevalence of the gene within the B. cepacia complex

and phylogenetic relatedness within and among genom-

ovars was examined. A structural model was generatedbased on sequence homologies with other a/b hydrolase

family proteins.

Of the seven genomovars examined, llpE homologs

were found only in representatives of genomovars I,

III, IV and VI. All B. cepacia and B. cenocepacia isolates

tested by PCR amplification, demonstrated llpE. Sub-

jecting llpE sequences from these strains to phylogenetic

analysis, revealed the presence of at least one non-syn-onymous variation between genomovar groups I, IIIA,

IIIB, and VI but none within individual B. cenocepacia

subgroups. LlpE from genomovars IIIA and IIIB differ

in the amino acid residue at LlpE position 234. An

asparagine (N) occupies this position in genomovar IIIA

strains, while a lysine (K) occupies it in genomovar IIIB

strains. Based on the LlpE model, N234 is in helix 8

immediately after the putative active residue D226. Themutation N234K (in genomovar IIIB) might result in salt

bridges between K234 and either D164 or E208. It is also

noteworthy that D164 belongs to a loop that could

potentially interact with the substrate and, if a lysine

at residue 234 could commit D164 to a salt bridge, this

might result in restricted movement of the loop itself

and impact subsequent interactions with substrate.

Although many synonymous variations were demon-strated among different strains within B. cenocepacia

subgroups IIIA and IIIB, phylogenetic analysis demon-

strated that B. cepacia and B. stabilis each have a

unique structural variant of llpE differing from each

other and B. cenocepacia subgroups by at least a single

non-synonymous variation. It appears that DNA

sequence conservation of llpE within a genomovar or

subgroup is not maintained, as we observed that thewithin-group variation is more diverse than the be-

tween-group variation. Rather, it is the corresponding

protein sequence that is conserved within a genomovar

or subgroup, with at least one non-synonymous muta-

tion between any two genomovar groups or subgroups,

but none within each of them. Synonymous changes are

considered to be accumulating at random in a species,

although at a constant rate for a particular gene, there-by acting as a molecular clock. The IIIB node (5

strains) has accumulated a much higher number of

synonymous mutations (21 substitutions), than the IIIA

node (6 strains) that has only two such changes. Thus,

the naıve expectation would be that IIIB is evolution-

arily older than IIIA. This may not be true, however.

In addition to a low sample size that precludes a

definitive resolution of age, the IIIA strains, which werechosen from the B. cepacia experimental panel [36,37],

might be more closely associated as they are all

epidemic strains.

The model structure for LlpE was based on the crys-

tal structure of a novel hyper-thermophilic carboxylest-

erase from the archaeon A. fulgidus. This structure

presents typical features of the a/b hydrolase fold

including positioning of the putative catalytic triadresidues as well as the GXD/SXG signature motif.

This family of proteins includes lipases, esterases,

B.M. Nair et al. / FEMS Microbiology Letters 245 (2005) 337–344 343

carboxypeptidases and haloperoxidases that show a

strong structural similarity, sometimes even in the ab-

sence of sequence similarity [40,41]. These proteins have

the potential to hydrolyze several different types of

bonds such as those seen in amides, esters and even car-

bon–carbon bonds. Among the physiologically impor-tant enzymes in the a/b hydrolase superfamily, the

human hormone sensitive lipase, an LlpE homolog,

has a broad substrate specificity that includes acylglyce-

rols, cholesteryl esters, retinyl esters, steroid esters and

p-nitrophenyl esters [42]. Hydrolytic functions of some

of these proteins with pathogenic implications include

esterase activity of EstA from Aspergillus niger, an

opportunistic fungal pathogen [43], which also hasstrong homologs in several pathogenic fungal species.

Another member of the a/b hydrolase fold family of en-

zymes, XylF, catalyzes hydrolytic cleavage of a carbon–

carbon bond of meta-cleavage products of aromatic

compounds [44]. Human epoxide hydrolases convert

epoxides to the more water-soluble and less toxic diols,

thereby protecting us from harmful xenobiotic com-

pounds [45]. BfaE, a detoxifying enzyme isolated fromBacillus subtilis, hydrolyzes and inactivates Brefeldin

A, a potent fungal inhibitor of intracellular vesicle-

dependent secretory transport and poliovirus RNA rep-

lication [46], while the haloalkane dehalogenase DhlA

from Xanthobacter autotrophicus hydrolyzes 1-haloalk-

anes to corresponding alcohols [47,37]. Similar to DhlA,

which has a catalytic triad of D124, D260, H289, LlpE is

predicted to possess a catalytic triad composed of D132,D226, H256. Instead of a serine, which usually acts as the

catalytic nucleophile, DhlA D124 and, presumably, LlpE

D124 act as catalytic nucleophiles. All of the above men-

tioned members of the a/b hydrolase family of proteins

participate in detoxification of exogenous or endoge-

nous substrates. This potential for broad substrate spec-

ificity of LlpE may not be unusual for a component of a

multidrug efflux operon.Given the overall high rate of recombination in B.

cepacia complex, llpE is remarkably conserved among

B. cenocepacia isolates, suggesting the importance of

its sequence conservation on some functional activities

of the species. Independent of llpE, the other three com-

ponents of the ceo operon, ceoABopcM, were sufficient

to impart chloramphenicol and salicylate efflux to a pre-

viously susceptible strain of B. cepacia 249–2 [21]. LlpEis clearly not essential for antibiotic efflux, because llpE

deletion does not adversely influence antibiotic suscepti-

bility and homologues have not been identified associ-

ated with other efflux operons. Its conservation among

cystic fibrosis isolates suggests a role for LlpE in the

unique airway milieu; perhaps its presence imparts an

advantage to B. cenocepacia allowing it to thrive in

conditions that would otherwise prove toxic. As suchit may prove to be a novel target for antimicrobial

development.

Acknowledgments

This work was supported by awards to J.L.B. from

the Cystic Fibrosis Foundation (BURNS00P0) and the

Royalty Research Fund at the University of Washing-

ton. S.C. is supported by NIH Grants GM60731 andAI45820.

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