APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2007, p. 524–534
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 2
Functional Analysis of Burkholderia cepacia Genes bceD and bceF,
Encoding a Phosphotyrosine Phosphatase and a Tyrosine
Autokinase, Respectively: Role in Exopolysaccharide
Biosynthesis and Biofilm Formation?
Ana S. Ferreira, Jorge H. Leita ˜o, Sı ´lvia A. Sousa, Ana M. Cosme,
Isabel Sa ´-Correia, and Leonilde M. Moreira*
Institute for Biotechnology and Bioengineering, Centro de Engenharia Biolo ´gica e Quı ´mica, Instituto Superior Te ´cnico, Av. Rovisco
Pais, 1049-001 Lisboa, Portugal
Received 23 June 2006/Accepted 6 November 2006
The biosynthesis of the exopolysaccharide (EPS) cepacian by Burkholderia cepacia complex strains requires
the 16.2-kb bce cluster of genes. Two of the clustered genes, bceD and bceF, code for two proteins homologous
to phosphotyrosine phosphatases and tyrosine kinases, respectively. We show experimental evidence indicating
that BceF is phosphorylated on tyrosine and that the conserved lysine residue present at position 563 in the
Walker A ATP-binding motif is required for this autophosphorylation. It was also proved that BceD is capable
of dephosphorylating the phosphorylated BceF. Using the artificial substrate p-nitrophenyl phosphate (PNPP),
BceD exhibited a Vmaxof 8.8 ?mol of PNPP min?1mg?1and a Kmof 3.7 mM PNPP at 30°C. The disruption
of bceF resulted in the abolishment of cepacian accumulation in the culture medium, but 75% of the parental
strain’s EPS production yield was still registered for the bceD mutant. The exopolysaccharide produced by the
bceD mutant led to less viscous solutions and exhibited the same degree of acetylation as the wild-type cepacian,
suggesting a lower molecular mass for this mutant biopolymer. The size of the biofilm produced in vitro by bceD
and bceF mutant strains is smaller than the size of the biofilm formed by the parental strain, and this
phenotype was confirmed by complementation assays, indicating that BceD and BceF play a role in the
establishment of biofilms of maximal size.
Bacteria of the Burkholderia cepacia complex (Bcc) have
emerged as opportunistic pathogens in patients with cystic
fibrosis (CF) and immunocompromised individuals (23, 24, 27,
36). Furthermore, the number of human infections caused by
Bcc strains has increased over the last 2 decades (24, 35, 36).
Approximately 80% of the Bcc isolates recovered from the
sputum of CF patients produce large amounts of exopolysac-
charide (EPS) (16, 50), suggesting a possible role for this EPS
in Bcc pathogenesis, as described for Pseudomonas aeruginosa
alginate (24). In fact, the Bcc mucoid phenotype was found to
affect cell-surface interactions and clearance in an animal
model, which could enhance the persistence and virulence of
Bcc in CF (13). Moreover, the EPS produced by Bcc interferes
with the function of human neutrophils in vitro, inhibiting
chemotaxis and the production of reactive oxygen species (8).
Moreover, the size of the biofilm formed in vitro by mutants
derived from a mucoid CF isolate correlates with their ability
to produce exopolysaccharide (17).
Cepacian is the main exopolysaccharide produced by Bcc
isolates, and it is composed of a branched acetylated heptasac-
charide repeat unit made of D-glucose, D-rhamnose, D-man-
nose, D-galactose, and D-glucuronic acid at the molar ratio
1:1:1:3:1 (9–11, 55). The pathway leading to the nucleotide
sugar precursors necessary for cepacian biosynthesis was pro-
posed previously (51), and the bce cluster of genes directing its
biosynthesis was identified (41). The bce genes also encode
proteins putatively involved in repeat unit assembly, polymer-
ization, and export. Among these genes are the bceD and bceF
genes coding for proteins homologous to protein tyrosine
phosphatases (PTPs) and protein tyrosine kinases (PTKs), re-
spectively. The present study is focused on their functional
Protein phosphorylation on tyrosine has long been consid-
ered specific to eukaryotes, but in the last two decades, several
evidences clearly indicate that it also occurs in bacteria (15).
One of the best-studied examples of tyrosine phosphorylation/
dephosphorylation in bacteria is related to polysaccharide pro-
duction. Indeed, many clusters of genes directing the synthesis
and regulation of exopolysaccharides or capsules encode a PTP
and a PTK (15). In general, PTK homologues from gram-
negative bacteria are integral membrane proteins harboring
two transmembrane domains flanking a large periplasmic loop
and have a cytoplasmic C-terminal region with Walker A and
Walker B ATP-binding motifs and a tyrosine-rich C terminus
where tyrosine phosphorylation occurs (2, 19, 20, 44, 46, 59,
62). In contrast, in gram-positive bacteria, the PTK homo-
logues are present in two separate proteins, exhibiting signifi-
cant sequence similarity to the two halves of the single peptides
from gram-negative bacteria, and both proteins are required
for phosphorylation (38, 43). This is also the case for the two
proteins GelC and GelE, characterized in the gellan-producing
Sphingomonas elodea ATCC 31461, which constitute the ex-
* Corresponding author. Mailing address: Centro de Engenharia
Biolo ´gica e Quı ´mica, Instituto Superior Te ´cnico, Av. Rovisco Pais,
1049-001 Lisboa, Portugal. Phone: (351) 218419031. Fax: (351)
218419199. E-mail: firstname.lastname@example.org.
?Published ahead of print on 17 November 2006.
ception to the single-peptide PTK from gram-negative bacteria
(40). The precise role of protein tyrosine autokinases in poly-
saccharide biosynthesis is not known. However, their presence
in both gram-positive and gram-negative bacteria suggests that
they may play a role in a conserved step of this biosynthetic
process, presumably in the regulation of polymer chain length
(61). A positive correlation between tyrosine phosphorylation
and high-molecular-mass polysaccharide synthesis has been
observed for Sinorhizobium meliloti succinoglycan and for cap-
sular polysaccharides from Escherichia coli K30 or Streptococ-
cus pneumoniae D39 (3, 46, 47). However, in other bacterial
systems, phosphorylated tyrosine kinases are believed to act
differently as negative regulators in colanic acid biosynthesis by
E. coli K-12, in emulsan biosynthesis by Acinetobacter lwoffii
RAG-1, and in capsular polysaccharide biosynthesis by S.
pneumoniae Rx1 (42–44, 59).
PTP homologues are low-molecular-weight proteins with a
conserved CX4CR active motif in the phosphate-binding loop,
flanked at some distance by an essential aspartate residue (30).
In several bacterial species, PTKs are specific endogenous sub-
strates for the corresponding PTPs. This is the case for the
pairs PTP/PTK, Wzb/WzcCA, and Etk/Etp from E. coli, Ptp/Ptk
from Acinetobacter johnsonii, and AmsI/AmsA from Erwinia
amylovora, among others (6, 29, 59).
In the present study, we report the sequence analysis of a
chromosomal region inside the bce gene cluster from the mu-
coid clinical isolate B. cepacia IST408. This region is required
for cepacian biosynthesis and is where the bceD and bceF genes
map. Results on the functional analysis of these genes, encod-
ing a PTP and a PTK, respectively, are shown, as well as their
involvement in cepacian biosynthesis. The hypothesized in-
volvement of BceD and BceF on the formation of biofilms of
maximal size was also examined.
MATERIALS AND METHODS
Bacterial strains, plasmids, and culture conditions. The strains and plasmids
used in this work are listed in Table 1. The cystic fibrosis isolate Burkholderia
cepacia IST408, previously described as a high exopolysaccharide producer (50),
was used as the parental strain for the mutants constructed in this work. E. coli
strains were grown in Lennox broth (53) at 37°C. B. cepacia strains were grown
in Pseudomonas isolation agar (Difco) plates at 37°C. Mannitol solid medium
(12) or S medium (50) was used to quantify EPS production at 30°C by the B.
cepacia strains. Growth media were supplemented with antibiotics when required
to maintain the selective pressure at the following concentrations (in ?g ml?1):
for B. cepacia, trimethoprim, 100; chloramphenicol, 300; for E. coli, ampicillin,
100; kanamycin, 50; trimethoprim, 100; chloramphenicol, 25.
DNA manipulation techniques. Total DNA was extracted from bacterial cells,
harvested from liquid cultures grown overnight at 37°C, using a cell and tissue kit
(Gentra Systems) following the manufacturer’s instructions. Plasmid DNA iso-
lation and purification, DNA restriction/modification, agarose gel electrophore-
TABLE 1. Strains and plasmids used in this study
Strain or plasmidGenotype or descriptionReference or source
Escherichia coli strains
recA1 lac ?F? proAB lacIqZ?M15 Tn10 (Tcr)? thi
e14?(mcrA) ?(mcrCB-hsdSMR-mrr)171 endA1 supE44 thi-1 gyrA96
relA1 lac recB recJ sbsC umuC:Tn5 (Kmr) uvrC ?F? proAB lacIq
Z?M15 Tn10 (Tcr)?
Burkholderia cepacia strains
Cystic fibrosis isolate, genomovar I, EPS?
IST408 derivative with bceD gene interrupted by a Tprgene cassette
IST408 derivative with bceF gene interrupted by a Tprgene cassette
pUC-GM derivative with a 1.1-kb Tprgene cassette, AprTpr
pQE9 derivative, lacIqApr
4,717-bp broad-host-range cloning vector, Cmr
pBBR1 ori, araC-PBAD, Tprmob?
pMLBAD derivative containing the dhfr gene replaced by the cat
gene from pBBR1MCS
Phagemid derived from pUC19, Apr
pBCSK derivative without the BamHI site
TnMod-KmO derivative carrying a 6,063-bp EcoRI fragment
encompassing the bceD, bceE, bceF, and bceG genes, Kmr
pWH844 derivative containing the bceD gene
pWH844 derivative containing the bceF gene
pLM45-1 derivative expressing His6-BceFK563A
pBCSK derivative containing a 2,056-bp SacII fragment from
plasmid D encompassing the bceD gene and flanking regions
pLM411-4 derivative containing a Tprgene cassette at the SphI site
disrupting the bceD gene
pBCSK?BamHI derivative containing a 2,218-bp HindIII fragment
from plasmid D encompassing the bceF gene
pSF54-1 derivative containing a Tprgene cassette at the BamHI site
disrupting the bceF gene
pMLBAD-Cm derivative carrying a 2,242-bp KpnI/HindIII fragment
with the coding region of the bceF gene
pMLBAD-Cm derivative carrying a 501-bp KpnI/HindIII fragment
with the coding region of the bceD gene
VOL. 73, 2007ROLE OF TYROSINE PHOSPHORYLATION IN EPS BIOSYNTHESIS525
sis, Southern blotting experiments, and E. coli transformation were carried out
using standard procedures (53). A specific mutation in one amino acid from
BceF was constructed in vitro using the QuikChange site-directed mutagenesis
method (Stratagene). Briefly, complementary oligonucleotides K11A-1 (5?-CG
GGCATCGGCGCGAGCTTCCTGACGG; substitutions in the primers are un-
derlined) and K11A-2 (5?-CCGTCAGGAAGCTCGCGCCGATGCCCG) were
designed to contain the desired codon change. The template consisted of plasmid
pLM45-1 coding for the BceF protein. Following PCR amplification, the reaction
products were digested with DpnI to eliminate the template, and the remaining
DNA was introduced into E. coli XL1-Blue by electroporation. The mutated
derivatives were sequenced to confirm the mutation and verify that no other
changes were introduced.
B. cepacia electrocompetent cells, prepared as described by Sa ´-Correia and
Fialho (52), were transformed by electroporation using Bio-Rad Gene Pulser II
(200 ?, 25 ?F, 2.5 kV) and grown overnight before being plated in selective
medium. Triparental conjugation to B. cepacia was performed using the helper
plasmid pRK2013 (22).
Construction of bceD and bceF insertion mutations. The 2,056-bp SacII frag-
ment from plasmid D containing the bceD gene coding region and flanking
regions was cloned into the multiple cloning site of pBCSK. The plasmid ob-
tained, pLM411-4, has an SphI site within the bceD coding region, and it was
used to insert an SphI fragment containing the trimethoprim (Tp) resistance
cassette obtained from plasmid pUC-TP (56). The Tp cassette is transcribed in
the same orientation as the bceD gene, and the resulting plasmid with the
insertion mutation in the bceD gene was designated pSF412-1.
To obtain a construction for bceF disruption, a 2,218-kb HindIII fragment
containing the bceF coding region was amplified using primers BceF-up (5?-CC
CAAGCTTGAACACGCAAGCGAAAC; restriction sites are in italics) and
BceF-low (5?-GGGAAGCTTGGATCAGGCGCTCAGGT) and cloned into the
HindIII site of the previously modified plasmid pBCSK lacking the BamHI site
to allow subsequent manipulations. The plasmid obtained (pSF54-1) was further
digested with BamHI, which has a single recognition site within the bceF coding
region. The Tp resistance cassette was obtained from pUC-TP by restriction with
XbaI. After fill-in, the Tp cassette was cloned into pSF54-1, originating plasmid
pSF57-5, which transcribes the cassette in the opposite orientation of the bceF
gene. Each of the bceD::Tp and bceF::Tp insertion constructs in plasmids
pSF412-1 and pSF57-5, respectively, was introduced into B. cepacia IST408 by
electroporation, and transformants were selected by growth on Pseudomonas
isolation agar medium with trimethoprim. The colonies obtained were then
screened in the presence of chloramphenicol. Colonies that did not grow in the
presence of chloramphenicol but were trimethoprim resistant were considered as
candidates for having an allelic exchange of bceD or bceF by the bceD::Tp or
bceF::Tp construct, respectively. The candidate insertion mutants were further
characterized by Southern hybridization or PCR amplification.
Construction of plasmids for complementation experiments. In order to com-
plement the EPS?phenotype and the biofilm formation ability of the B. cepacia
bceF::Tp mutant, the recombinant plasmid pLM63-1 was constructed. For this,
the bceF coding region was amplified by PCR using primers bceFBAD-up (5?-
ACGGGTACCGAACACGCAAGCGA) and bceFBAD-low (5?-GTGAAGCTT
GGATCAGGCGCTCA) and IST408 genomic DNA as the template. The am-
plified fragment was restricted by KpnI/HindIII and inserted into the same sites
of the pMLBAD-Cm vector, obtained by replacing the dhfr gene, coding for
trimethoprim resistance of pMLBAD, with the cat gene from pBBR1MCS,
coding for chloramphenicol resistance. To complement the biofilm formation
ability of the B. cepacia bceD::Tp mutant, the recombinant plasmid pLM69-1 was
constructed. For this, the bceD coding region was amplified by PCR with primers
bceDBAD-up (5?-AGAGGTACCGTTCCGGAACATCC) and bceDBAD-low
(5?-CAGAAGCTTCGTCAGCGCGAC), using IST408 genomic DNA as the
template. The amplified fragment was restricted by KpnI/HindIII and inserted
into pMLBAD-Cm. The nucleotide sequences of the cloned genes were con-
firmed by sequencing.
In vivo complementation of the EPS?phenotype of IST408 bceF::Tp. Plasmid
pLM63-1, encoding the parental bceF gene, was mobilized into the B. cepacia
IST408 bceF::Tp mutant strain by triparental conjugation. To determine the
ability of this plasmid to restore cepacian synthesis, the complemented strain
IST408 bceF::Tp/pLM63-1 was grown in mannitol solid medium, supplemented
with 1% of L-arabinose, at 30°C for 5 days, and the mucoidy of the corresponding
colonies was observed. Mannitol liquid medium supplemented with 1% of L-
arabinose was also used in order to determine the amount of EPS produced
based on the dry weight of the ethanol precipitates from cell-free culture super-
Construction of bceD and bceF overexpression plasmids. The genes bceD and
bceF were PCR amplified with primers Pho-up (5?-AAAGGATCCTTCCGGA
ACATCCT) and Pho-low (5?-CCCAAGCTTGTTTCAGCATAGTT) or BceF-up
and BceF-low, respectively. The amplified product of bceD was digested with
BamHI/HindIII and cloned into the same sites of linearized pWH844, resulting
in plasmid pLM211-1. The amplified product of bceF was digested with HindIII
and cloned in the HindIII-linearized plasmid pWH844, and the plasmid obtained
was named pLM45-1.
Expression and purification of His6-BceD and His6-BceF. E. coli Sure cells
harboring pLM211-1 or pLM45-1 were grown in Lennox medium supplemented
with 0.1% glucose and containing 300 mg liter?1of ampicillin, at 37°C, to an
optical density at 640 nm of 0.5. Induction was started by adding 0.4 mM IPTG
(isopropyl-?-D-thiogalactopyranoside), followed by incubation for 4 h. The His-
tagged BceD was purified from cell-free lysates of E. coli prepared in sonication
buffer (50 mM Tris-HCl, 300 mM NaCl, 10% [vol/vol] glycerol, pH 7.5). After
removal of cell debris by centrifugation at 19,000 ? g for 30 min at 4°C, His6-
BceD was purified by Ni2?affinity chromatography. The Ni2?-nitrilotriacetic
acid (NTA) matrix was washed with 6 volumes of buffer A (50 mM Tris-HCl, 300
mM NaCl, 10% [vol/vol] glycerol, pH 7.5) containing 20 mM imidazole and 6
volumes of buffer B (100 mM Tris-HCl, 500 mM NaCl, pH 8.9) containing 50
mM imidazole. Bound proteins were eluted with buffer B containing 250 mM
His6-BceF purification was done as described for His6-BceD, except that 1.0%
(vol/vol) Triton X-100 was added to the lysate after sonication and elution was
done with 300 mM imidazole. Both proteins were purified freshly before each
experiment. The protein concentrations of the purified His-tagged protein solu-
tions were determined by the method of Bradford (5), using bovine serum
albumin as the standard.
Phosphatase activity. Phosphatase activity was determined based on the con-
tinuous monitoring, at 405 nm, of the p-nitrophenol (PNP) formed from p-
nitrophenol phosphate (PNPP) at 37°C in a Hitachi UV 2000 double-beam
spectrophotometer. The reaction mixture contained, in a total volume of 1 ml,
100 mM sodium citrate buffer, pH 6.5, 1 mM EDTA, 0.1% (vol/vol) ?-mercapto-
ethanol, and PNPP concentrations ranging from 0.5 to 40 mM. The concentra-
tion of PNP formed was estimated using a molar extinction coefficient of 18,000
M?1cm?1(48). In experiments carried out to determine the optimal pH for
phosphatase activity, the citrate buffer pH was varied from 5.5 to 7.5. All reac-
tions were initiated by the addition of 5 ?l of purified His-tagged phosphatase.
One unit of enzyme activity was defined as the amount of enzyme that catalyzes
the formation of 1 ?mol of PNP per minute under the assay conditions. The
results presented for enzyme assays and for protein concentrations are the means
from at least three independent experiments.
Dephosphorylation of His6-BceF was monitored by Western immunoblot
analysis. For that, 1 ?g of His6-BceF and 1 ?g of His6-BceD were incubated in
30 ?l of buffer consisting of 100 mM sodium citrate (pH 6.5) and 1 mM EDTA
at 37°C for 1 to 22 h. The reaction was stopped by the addition of an equal
volume of 2? sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) sample buffer, and the mixture was heated at 100°C for 5 min and
subsequently analyzed by SDS-PAGE, followed by immunodetection using
antiphosphotyrosine antibodies. The relative signal intensity of the His6-BceF
bands obtained by immunodetection was quantified using Quantity One software
Western immunoblot analysis. Protein samples were separated on 15% SDS-
PAGE gels (33) prior to being transferred onto nitrocellulose membranes (58).
The nitrocellulose filters were incubated with either antiphosphotyrosine anti-
bodies (PT-66; Sigma), at a dilution of 1:2,000, or monoclonal antibodies against
the His tag (QIAGEN), at a dilution of 1:2,000. The membranes were then
incubated with alkaline phosphatase-conjugated rabbit anti-mouse or goat anti-
rabbit (Promega) and reacted with ECL reagent (Amersham Biosciences).
Broad-range prestained SDS-PAGE standard from Bio-Rad was used as a mo-
lecular weight marker.
Production and characterization of bacterial exopolysaccharides. EPS pro-
duction was assessed based on the dry weight of the ethanol-precipitated poly-
saccharide present in 2-ml culture samples of the different strains cultivated in S
liquid medium at 30°C with orbital agitation. The viscosities of solutions pre-
pared with the polysaccharides produced by the different strains under study
were compared using EPS obtained after 72 h of culture at 30°C with orbital
agitation. For this, bacterial cells present in the cultures were separated by
centrifugation at 20,000 ? g for 15 min. The EPSs were precipitated from
cell-free supernatants by the addition of 2.5 volumes of cold ethanol and then air
dried and redissolved in distilled water prior to dialysis (molecular mass cutoff,
12 kDa) against water for 3 days at 4°C, followed by EPS lyophilization. Aqueous
solutions of the lyophilized polymers (5 g liter?1) were prepared, and their
viscosity was measured at 30°C using a Brookfield cone and plate viscometer,
model LVIIT. Results are the mean values for at least two independently pre-
526 FERREIRA ET AL.APPL. ENVIRON. MICROBIOL.
pared EPS solutions. The acetyl content of the EPSs produced was determined
as described by McComb and McCready (37), using glucose pentaacetate as the
standard. Results are the mean values from at least three independent determi-
nations. Glucose and galactose content of the polysaccharide fractions was quan-
tified by enzymatic assays after hydrolysis with HCl and subsequent neutraliza-
tion (4, 32).
Biofilm formation assay. Biofilm formation assays were based on the method
described by Cunha et al. (17). Overnight liquid cultures of the different B.
cepacia strains were used to inoculate S liquid medium or mannitol medium
supplemented with 1% of L-arabinose and grown at 30°C with orbital agitation
until mid-exponential phase. The cultures were subsequently diluted to a stan-
dardized culture optical density at 640 nm of 0.1, and 100 ?l of these cell
suspensions was used to inoculate the wells of a 96-well polystyrene microtiter
plate (Greiner Bio-One) containing 100 ?l of S medium or mannitol medium
supplemented with L-arabinose. Wells containing sterile growth medium were
used as negative controls. Plates were incubated at 30°C for 24 or 48 h without
agitation. The biofilm formed was quantified as follows. Culture media and
unattached bacterial cells were removed from the wells by careful rinsing with
water (three times, 200 ?l for each rinse). Adherent bacteria were stained with
200 ?l of a 1% crystal violet solution for 15 min at room temperature, and after
three gentle rinses with 200 ?l of water each time, the dye associated with the
attached cells was solubilized in 200 ?l of 95% ethanol and the biofilm was
quantified by measuring the absorbance of the solution at 590 nm using a
VersaMax tunable microplate reader (Molecular Devices). Results are the mean
values for at least five repeats from three independent experiments.
Computational analysis of nucleotide and protein sequences. DNA and pro-
tein data were analyzed using the open reading frame (ORF) finder tool resident
at the National Center for Biotechnology Information (NCBI). The algorithm
BLAST (1) was used to compare sequences of the deduced amino acids to
database sequences available at the NCBI. Alignments were performed using the
program CLUSTAL W (57). DNA sequences from the B. cenocepacia J2315
genome were extracted from the database at http://www.sanger.ac.uk.
Nucleotide sequence accession number. The nucleotide sequence reported
here for the bce genes was submitted to GenBank under accession no.
Sequence analysis of a B. cepacia chromosomal region where
bceD and bceF genes map. In a previous study, we used a
mutagenesis strategy, based on the use of the plasposon
pTnMod-KmO, to obtain mutants unable to produce exopoly-
saccharide from the mucoid CF isolate B. cepacia IST408. This
manipulation led to the identification of three EPS-deficient
strains (IST408-SS1, IST408-SS2, and IST408-SS3) (41). After
recovery of the plasposons with the genomic flanking regions
as recombinant plasmids, sequencing reactions were carried
out to determine the place of insertion of the plasposon and
the DNA region involved in EPS biosynthesis was identified by
a homology search against the genome of B. cenocepacia J2315
(41). In this work, we report the sequence determination and
characterization of plasmid D, carrying a chromosomal region
from the B. cepacia IST408-SS1 mutant, obtained after diges-
tion with EcoRI and self-ligation. This plasmid contains an
EcoRI fragment of 6,063 bp where the plasposon insertion site
was identified, followed by the sequencing in both strands of
the DNA insert. Homology searches at the nucleotide level
indicated that this chromosomal region of 6,063 bp is 97%
identical to a Burkholderia sp. strain 383 genomic region rang-
ing from nucleotides 2592997 to 2599051. This environmental
strain is also known as ATCC 17660 and is closely related to
the species B. cepacia. The DNA insertion of plasmid D also
showed 94% identity to a genomic region of the cystic fibrosis
isolate B. cenocepacia J2315 (nucleotides 944407 to 950464).
Moreover, other Burkholderia strains such as B. cenocepacia
AU1054, B. thailandensis E264, B. pseudomallei 1106b, and
B. xenovorans LB400, with genome sequences deposited in
GenBank, also have similar chromosomal regions.
Computer-assisted sequence analysis of the 6,063-bp chro-
mosomal region of B. cepacia IST408 revealed four complete
ORFs designated bceD, bceE, bceF, and bceG and two incom-
plete ORFs (bceC and bceH). The ORFs are in the same
orientation, confirming the previous conclusion based on the
inspection of the available B. cenocepacia J2315 sequence (41).
The ORFs bceE and bceG are homologous to several polysac-
charide export proteins and glycosyl transferases from family 2,
respectively. They are putatively involved in cepacian biosyn-
thesis but will not be further examined here. The two other
complete ORFs, bceD and bceF, are homologous to genes
encoding low-molecular-mass acid PTPs and PTKs, respec-
tively, and are the focus of the present study.
B. cepacia IST408 BceD has over 94% identity, at the amino
acid level, with other putative PTPs from several strains from
Burkholderia species. This is the case for the environmental
strain 383, the cystic fibrosis isolate B. cenocepacia PC184, two
environmental strains from the species B. vietnamiensis and B.
ambifaria, and the epidemic strain B. dolosa AU0158 (Table 2).
For the clinical isolate B. pseudomallei 1106b and the two
environmental strains B. thailandensis E264 and B. xenovorans
LB400, the amino acid identity value with BceD is between 62
and 77% (Table 2). The degree of identity, at the amino acid
level, between BceD and other experimentally characterized
low-molecular-mass PTPs from different genera, such as Wzb
from Escherichia or Salmonella, Ptp from Acinetobacter, AmsI
from Erwinia, and EpsP from Ralstonia, is within the range of
33 to 37% (Table 2).
The proteins most homologous to BceD within each Burk-
holderia genome analyzed are listed in Table 2. However, more
than one gene coding for putative PTPs was found to be
present in all of the genomes examined. For example, in Burk-
holderia sp. strain 383, besides the protein with GenBank ac-
cession number ABB12385 with 100% identity, at the amino
acid level, to BceD, two other proteins (ABB12976 and
ABB06443) with 41 and 35% identity, respectively, to BceD
were found (Table 2). Similar results were obtained for the
other Burkholderia species examined, with one of the putative
PTPs being highly homologous to BceD while the other two or
three encoding regions exhibited an identity below 40% (data
The homology between the protein BceF from B. cepacia
IST408 and genomic regions of the sequenced Burkholderia
strains showed that BceF is homologous to putative protein-
tyrosine kinases from Burkholderia sp. strain 383, B. cenocepa-
cia PC184, B. dolosa AU0158, B. vietnamiensis G4, and B.
ambifaria AMMD, with more than 92% identity at the amino
acid level (Table 2). The homologous putative PTKs from B.
thailandensis E264, B. pseudomallei 1106b, and B. xenovorans
LB400 are within the range of 72 to 80% identity, at the amino
acid level, to BceF. Biochemically characterized PTKs, such as
EpsP, AmsA, Wzc, and Ptk from Ralstonia, Erwinia, Salmo-
nella and Escherichia, and Acinetobacter genera, respectively,
presented an identity to B. cepacia IST408 PTK within the
range of 32 to 41% (Table 2).
The search for other BceF homologues within the sequenced
Burkholderia sp. strain 383 genome revealed the presence of
a strong homologue with 98% identity (GenBank accession
VOL. 73, 2007 ROLE OF TYROSINE PHOSPHORYLATION IN EPS BIOSYNTHESIS 527
number ABB12383) and three others (ABB06442, ABB12977,
and ABB11930) with 33 to 37% identity (Table 2). Similar
conclusions were taken from the inspection of other sequenced
Burkholderia genomes (data not shown).
With respect to the conserved sequence motifs characteristic
of PTPs, BceD exhibits the major signature of this type of
enzyme, C-X4-C-R (10CHANVCR16), but has the tyrosine res-
idue of the conserved motif DPY substituted by a histidine
residue (Fig. 1a). The conserved motifs of PTKs are also
present in BceF, which shows the Walker A (556GPTPGIGK
S564) and Walker B (662VLID665) ATP-binding motifs and six
tyrosine residues at positions 596, 651, 659, 728, 732, and 738
in its C-terminal region (Fig. 1b).
An insertion mutant for bceF, but not for bceD, is impaired
in cepacian biosynthesis. To investigate the hypothesized role
of bceD and bceF genes in cepacian biosynthesis, we con-
structed insertion mutants by using a Tp gene resistance cas-
sette based on the procedure described by Sokol et al. (56).
The mutants obtained, designated B. cepacia IST408 bceD::Tp
and B. cepacia IST408 bceF::Tp, showed growth curves similar
to the wild-type B. cepacia IST408 growth curve (Fig. 2a). To
assess cepacian production phenotype, strains were grown in S
liquid medium and samples were taken during growth and
ethanol precipitated to quantify the polysaccharide present in
the supernatant. No precipitate was obtained with the mutant
strain IST408 bceF::Tp, but the mutant strain IST408 bceD::Tp
was mucoid and showed a reduction of only 25% of the etha-
nol-precipitated polysaccharide produced in the supernatant
compared to the wild-type strain IST408 (Fig. 2b and d). Ap-
parently, the polysaccharides produced by both IST408 and
IST408 bceD::Tp strains are identical with respect to the neu-
tral sugar composition since they contained glucose and galac-
tose at a ratio of approximately 1:3 (Table 3). Moreover, both
polymers exhibited similar degrees of acetylation in the range
of 3.2 to 3.8 acetyl residues/repeat unit (Table 3).
The viscosities of aqueous solutions prepared with the puri-
fied EPSs produced by the two strains were different, with the
solution prepared from the ethanol-precipitated material ob-
tained from IST408 bceD::Tp exhibiting a slightly reduced vis-
cosity for solutions with identical EPS concentrations (Fig. 2c).
The protein contents present in the two EPS solutions derived
from B. cepacia IST408 and B. cepacia IST408 bceD::Tp were
40 ?g ml?1and 45 ?g ml?1, respectively. It is therefore un-
likely that the registered differences in the viscosities of so-
lutions prepared with the ethanol precipitate are due to
different levels of protein contamination of the EPSs syn-
thesized by the two strains. These observations, together
with the previous characterization of the biopolymers, sug-
gest a reduced molecular mass for the biopolymers pro-
duced by IST408 bceD::Tp.
TABLE 2. Features of the bceD and bceF genes from Burkholderia cepacia IST408
Position in sequence
Organism (protein name)
Burkholderia sp. strain 383
Burkholderia sp. strain 383
Burkholderia sp. strain 383
B. cenocepacia PC184
B. vietnamiensis G4
B. ambifaria AMMD
B. dolosa AU0158
B. pseudomallei 1106b
B. thailandensis E264
B. xenovorans LB400
Acinetobacter johnsonii (Ptp)
Escherichia coli (Wzb)
Salmonella enterica serovar
Erwinia amylovora (AmsI)
Ralstonia solanacearum (EpsP)
bceF 2651–4876 741 Protein-tyrosine kinase98/99
Burkholderia sp. strain 383
Burkholderia sp. strain 383
Burkholderia sp. strain 383
Burkholderia sp. strain 383
B. cenocepacia PC184
B. dolosa AU0158
B. vietnamiensis G4
B. ambifaria AMMD
B. pseudomallei 1106b
B. thailandensis E264
B. xenovorans LB400
Ralstonia solanacearum (EpsB)
Erwinia amylovora (AmsA)
Salmonella enterica serovar
Escherichia coli (Wzc)
Acinetobacter johnsonii (Ptk)
528 FERREIRA ET AL.APPL. ENVIRON. MICROBIOL.
To confirm that the insertion of the trimethoprim cassette
into the bceF gene has no polar effect on the expression of the
downstream bce genes, an in trans complementation experi-
ment using the bceF gene cloned in the replicative plasmid
pLM63-1 was performed. Since the expression of the bceF
gene present in pLM63-1 is dependent on induction by arabi-
nose and is repressed by glucose, the standard medium for EPS
production (S medium) could not be used for the complemen-
tation experiment. As an alternative, mannitol medium sup-
plemented with 1% of arabinose was used. Under these con-
ditions, the introduction of pLM63-1 into the bceF mutant led
to the recovery of cepacian biosynthesis, associated with the
recovery of mucoidy of the colonies grown in solid medium
(Fig. 2d). Despite the fact that all of the colonies obtained
from the complementation of B. cepacia IST408 bceF::Tp with
plasmid pLM63-1 were mucoid and that it was possible to
recover EPS by ethanol precipitation of the cell-free culture
supernatant, the EPS production yield was only 15% of the
wild-type strain production level. This could be due to a defi-
cient expression of the bceF gene under the control of the E.
coli arabinose promoter that was tested in this work for the first
time in B. cepacia. It can also be the result of the overexpres-
sion of the BceF protein leading to the alteration of the enzy-
matic activity or optimal relation between the several protein
components involved in the biosynthesis of the polysaccharide.
BceD shows phosphatase activity. In order to prove that
BceD is a phosphatase, the protein was purified and the hy-
pothesized enzymatic activity was determined. For that, the
bceD gene from the IST408 strain lacking the start codon ATG
was amplified and cloned in the expression vector pWH844.
The resulting plasmid pLM211-1 allowed the production of the
BceD protein with an N-terminal addition of 12 amino acid
residues, including 6 histidines. Plasmid pLM211-1 was used to
transform E. coli Sure cells, and after induction with 0.4 mM
IPTG, a protein of approximately 18 kDa was overproduced,
consistent with the calculated molecular mass of the fusion
protein His6-BceD (Fig. 3a, lanes 3 and 4).
The phosphatase activity of His6-BceD was assayed for its
ability to cleave the artificial substrate PNPP. The purified
fusion protein was able to efficiently hydrolyze this synthetic
substrate at an optimum pH value of 6.5 (Fig. 3b). The corre-
sponding kinetic constants, Kmand Vmax, determined at 37°C,
were 3.7 mM and 8.8 ?mol min?1mg?1, respectively. These
values are in the same range as those reported for other pro-
karyotic low-molecular-mass phosphatases (44, 48, 59).
BceF autophosphorylation on tyrosine is prevented by a
mutation in the Walker A motif. To demonstrate the predicted
activity of BceF as a tyrosine-phosphorylatable protein, the His
tag fusion protein was purified to homogeneity by affinity chro-
matography. Efficient expression of an 82-kDa protein was
FIG. 1. Comparison of the partial sequences of B. cepacia IST408 BceD and BceF protein homologues. (a) Alignment of B. cepacia (Bc) BceD
with experimentally confirmed prokaryotic low-molecular-mass PTPs from Acinetobacter johnsonii (Aj) Ptp, Acinetobacter lwoffii (Al) Wzb,
Ralstonia solanacearum (Rs) EpsP, Salmonella enterica serovar Typhimurium (St) Wzb, Escherichia coli (Ec) Wzb, and Erwinia amylovora (Ea)
AmsI. (b) Alignment of B. cepacia (Bc) BceF with experimentally confirmed prokaryotic PTKs from Erwinia amylovora (Ea) AmsA, Klebsiella
pneumoniae (Kb) Yco6, Salmonella enterica serovar Typhimurium (St) Wzc, Escherichia coli (Ec) Wzc, Acinetobacter johnsonii (Aj) Ptk, and
Ralstonia solanacearum (Rs) EpsB. The conserved amino acids within the motifs and the C-terminal tyrosine-rich regions of PTKs are shown in
bold. X indicates any amino acid, h indicates a hydrophobic amino acid, and alternative residues are enclosed in brackets. Asterisks indicate the
amino acid residues that are identical in all proteins; one or two dots indicate semiconserved or conserved substitutions, respectively.
VOL. 73, 2007 ROLE OF TYROSINE PHOSPHORYLATION IN EPS BIOSYNTHESIS529
consistent with the calculated molecular mass of the His6-BceF
fusion protein obtained in the soluble fraction of cell crude
extracts following IPTG induction of E. coli Sure cells harbor-
ing plasmid pLM45-1 (Fig. 4a, lanes 2 and 3). The presence of
phosphorylated tyrosine residues was detected in the purified
His6-BceF fusion protein by Western immunoblot analysis us-
ing antibodies against phosphotyrosine (Fig. 4b, lane 1, upper
The BceF protein has two conserved sequences at the
C-terminal cytoplasmic domain (556GPTPGIGKS564
662VLID665) which are similar to the Walker A and Walker B
motifs, respectively, described for many other organisms as
being involved in ATP binding and hydrolysis (15). To assess
the relevance of the ATP-binding motif in His6-BceF with
respect to protein tyrosine kinase activity, a modified fusion
protein, with a specific substitution of the conserved lysine at
position 563 from the Walker A motif, was constructed by
site-directed mutagenesis. After overexpression and purifica-
tion of His6-BceFK563A, this fusion protein was tested by im-
munoblotting for the presence of phosphorylated tyrosine res-
idues. The results obtained indicate that the mutated protein
did not react with the PT66 antibody (Fig. 4b, lane 2, upper
panel). The stripping of the membrane and reprobing with the
antibody against the His tag confirmed the presence of His6-
BceF and His6-BceFK563A(Fig. 4b, lanes 1 and 2, lower panel).
These results firmly support the idea that the mutation in the
ATP-binding motif blocked BceF autophosphorylation on ty-
BceF is a substrate for BceD phosphatase activity. In other
bacterial systems, such as E. coli, Klebsiella pneumoniae, and
Acinetobacter lwoffii, it was demonstrated that tyrosine autoki-
nases are endogenous substrates for low-molecular-mass PTPs
(44, 48, 59). To elucidate whether BceF is a substrate for BceD
phosphatase activity, the purified proteins His6-BceF and His6-
FIG. 2. (a) Growth curves and (b) cepacian production by B. cepacia IST408 (?), B. cepacia IST408 bceD::Tp (?), and B. cepacia IST408
bceF::Tp (F). The standard deviation in panel a is below 5%. In panel c, the viscosities of the aqueous solutions prepared with 5 g liter?1of the
ethanol precipitate (considered equivalent to the EPS recovered) isolated from the culture supernatants of B. cepacia IST408 (?) and B. cepacia
IST408 bceD::Tp (?) are shown. The data are based on mean values from the results of at least three independent cell cultivations. In panel d,
the mucoid colony morphologies of the following strains are compared: (i) IST408, (ii) IST408 bceD::Tp, (iii) IST408 bceF::Tp, and (iv) IST408
bceF::Tp complemented in trans with pLM63-1, expressing the bceF gene, after 5 days of cultivation in mannitol medium supplemented with 1%
arabinose. OD 640, optical density at 640 nm; cP, centipoise.
TABLE 3. Neutral sugar composition and acetylation degree of the exopolysaccharides produced by B. cepacia strainsa
Amt of Glc
Amt of Gal
Amt of acetyl
Total amt of sugar
No. of acetyl
0.053 ? 0.005
0.038 ? 0.002
0.156 ? 0.002
0.115 ? 0.003
118.9 ? 8.3
117.4 ? 4.0
902.1 ? 63.2
909.1 ? 21.6
aData represent means ? standard deviations. Values were calculated considering the following molecular masses: acetyl group, 43 g mol?1; and heptasaccharide
repeat unit 1,133 g mol?1. Glc, glucose; Gal, galactose; RU, repeat unit.
530 FERREIRA ET AL.APPL. ENVIRON. MICROBIOL.
BceD were incubated together at 37°C in dephosphorylation
buffer. Samples were removed at various incubation times and
analyzed by Western immunoblotting using the PT66 antibody
against phosphotyrosine to follow the phosphatase activity of
BceD. In the presence of His6-BceD, it was demonstrated that
the tyrosine kinase His6-BceF was dephosphorylated over time
(Fig. 5, lanes 1 to 4), while in the absence of His6-BceD, no
His6-BceF dephosphorylation occurred (Fig. 5, lanes 5 to 8).
Estimation of the relative immunoblot signal intensity, using
the lane 1 band as 100%, shows that after 22 h of incubation of
the His6-BceF protein with His6-BceD, the band from lane 4
retained 63% of the signal, while the band from lane 8 showed
similar signal intensity. This result suggests that the enzymatic
activity of the tyrosine kinase His6-BceF may be regulated in
vivo by the dephosphorylating activity of His6-BceD.
bceD and bceF expression is required for maximal biofilm
size. The sizes of the biofilms formed by the mucoid strain
IST408 and the two mutant strains, IST408 bceD::Tp and
IST408 bceF::Tp, were quantified after 24 and 48 h of incuba-
tion in S medium at 30°C. These assay conditions were previ-
ously shown to lead to thicker biofilms and the highest cepa-
cian production yield (17). Although the amounts of the
biofilms formed by the three strains were similar after 24 h of
incubation, the amount of biofilm formed by the two mutant
strains after 48 h was remarkably smaller than the amount of
biofilm formed by IST408 (Fig. 6a). This result is particularly
interesting when we keep in mind that both mutant strains
were derived from the same strain, but while IST408 bceD::Tp
still produces a significant amount of EPS, the isogenic IST408
bceF::Tp strain does not produce detectable levels of cepacian.
Due to the reasons described before, the size of the biofilm
from the bceD and bceF complemented mutants could not be
assessed in S medium, and so, the alternative mannitol me-
dium supplemented with 1% of arabinose was tested (Fig. 6b).
Under these growth conditions, the maximal size of the biofilm
FIG. 3. (a) Coomassie blue-stained SDS-PAGE of the fractions obtained during purification of His6-BceD from E. coli Sure cells harboring
plasmid pLM211-1 containing the bceD gene. Lane 1, molecular mass standards; lane 2, protein cell extract obtained before IPTG induction; lane
3, protein cell extract obtained after IPTG induction; lane 4, His6-BceD protein eluted from a Ni2?-NTA affinity chromatography column. (b) pH
dependence of His6-BceD phosphatase activity using 10 mM of PNPP as substrate. (c) Phosphatase activity of the His6-BceD protein in the
presence of increasing concentrations of the substrate PNPP at pH 6.5.
FIG. 4. (a) Coomassie blue-stained SDS-PAGE of the fractions
obtained during purification of His6-BceF from crude cell extract of E.
coli Sure cells harboring plasmid pLM45-1 expressing His6-BceF. Lane
1, protein cell extract obtained before IPTG induction; lane 2, protein
cell extract obtained after IPTG induction; lane 3, His6-BceF protein
eluted from a Ni2?-NTA affinity chromatography column. (b) Western
blot analysis of His6-BceF (lanes 1) or His6-BceFK563A(lanes 2) to
detect the presence of phosphorylated tyrosine residues, using phos-
photyrosine antibodies (upper panel), or the presence of the histidine
tag, using antibodies against the His tag (lower panel).
FIG. 5. In vitro dephosphorylation of His6-BceF by His6-BceD.
The two purified fusion proteins were incubated as described in Ma-
terials and Methods, and the reaction was stopped at 0, 1, 3, and 22 h.
The samples were analyzed by immunodetection using phosphoty-
rosine antibodies. In lanes 1 to 4, His6-BceF was incubated with His6-
BceD for the indicated times: lane 1, 0 h; lane 2, 1 h; lane 3, 3 h; lane
4, 22 h. In lanes 5 to 8, His6-BceF was incubated under identical
conditions but in the absence of His6-BceD, for the same periods of
time as in lanes 1 to 4, respectively, as a control experiment.
VOL. 73, 2007 ROLE OF TYROSINE PHOSPHORYLATION IN EPS BIOSYNTHESIS531
registered was below 2 units of absorbance and the results
obtained after 24 or 48 h of incubation were similar. Although
this alternative growth medium was not optimized to assess
biofilm formation in vitro, it was possible to observe an in-
crease in the size of the biofilm formed by the IST408 bceD::Tp
and IST408 bceF::Tp mutants when complemented with the
lacking genes expressed from recombinant plasmids (Fig. 6b).
In this study, we report results on the functional analysis of
the BceD and BceF proteins from the opportunistic human
pathogen Burkholderia cepacia as well as their involvement in
cepacian biosynthesis and in the size of the biofilms formed.
The protein BceF was found to be tyrosine phosphorylated,
and the importance of the ATP-binding site for tyrosine phos-
phorylation was demonstrated. The other protein under study,
BceD, is homologous to bacterial low-molecular-mass acid
phosphotyrosine phosphatases and dephosphorylates BceF, in
agreement with the demonstrated activity of Wzb phospha-
tases from A. lwoffii and E. coli (44, 59, 60).
The disruption of the bceF gene resulted in an EPS-defective
strain, as shown for the homologous proteins ExoP from Sino-
rhizobium meliloti, WzcCAfrom E. coli K-12, Wzccpsfrom E.
coli K30, and GelC/GelE from Sphingomonas elodea, whose
lack of expression resulted in strong reduction of the amount
of the polysaccharides produced (2, 21, 40, 46, 60). The dis-
ruption of the bceD gene reduced wild-type cepacian produc-
tion by about 25% and led to a slight reduction of the polymer
viscosity, while in E. coli K30, E. coli K-12, Erwinia amylovora
Ea1/79, and Acinetobacter lwoffii RAG-1, the production of
capsular polysaccharides or exopolysaccharides is dramatically
abrogated following disruption of the wzbCPS, wzbCA, amsI,
and wzb phosphatase genes, respectively (6, 44, 60, 62). How-
ever, in agreement with our observation, Minic et al. (39)
recently described that the lack of the EpsB phosphatase from
Streptococcus thermophilus implicates only a slight reduction in
the amount of the exopolysaccharide produced.
A hypothetical model proposed before to explain the poly-
merization and export process of bacterial polysaccharide syn-
thesis predicts the existence of a multienzyme complex, com-
posed of several proteins acting together, including a flippase,
a polymerase, an outer membrane lipoprotein complex, a ty-
rosine kinase, and a phosphotyrosine phosphatase (44, 45, 49).
It has been hypothesized that the presence of all of these
protein components, with the right stoichiometry, is required
for the activity of this multienzyme complex. This model is
consistent with the phenotypes registered with all of the mu-
tants lacking the tyrosine kinase from all of the species tested
(20, 25, 43, 44, 46, 59, 62), including the IST408 bceF mutant
examined here. Indeed, this mutant does not produce cepa-
cian. On the other hand, it appears that the mode of action of
the phosphotyrosine phosphatase, another member of the
polymerization/export complex in gram-negative bacteria, is
strain specific. In fact, in Burkholderia cepacia, a mutant strain
for the BceD phosphatase still retains 75% of wild-type cepa-
cian production ability. However, the biopolymer produced by
the mutant exhibits different rheological properties, suggestive
of a lower molecular mass. The production of a significant
amount of cepacian in the absence of the phosphatase BceD
and the fact that BceF is phosphorylated on tyrosine and re-
quires an active Walker A for autophosphorylation suggest
that BceF has to be phosphorylated to allow cepacian produc-
tion. This contrasts with the requirements for K30 capsular
polysaccharide production in E. coli K30, where although the
kinase WzcCPShas to be phosphorylated for CPS production,
the mutation in the WzbCPSphosphatase resulted in trace
amounts of CPS on the cell surface (62).
The ability of bacteria to form biofilms has been associated
with their capacity to cause disease in the human host (14). It
is well documented that the EPSs produced by E. coli and
Vibrio cholerae are essential for the development of mature
FIG. 6. Size of the biofilm formed after 24 h (open bars) or 48 h (solid bars) of cultivation, without shaking, of (a) the CF isolate B. cepacia
IST408 and the insertion mutants B. cepacia IST408 bceD::Tp and B. cepacia IST408 bceF::Tp in the wells of polystyrene microtiter dishes
containing S medium at 30°C and (b) B. cepacia IST408/pMLBAD-Cm, B. cepacia IST408 bceD::Tp/pMLBAD-Cm, B. cepacia IST408
bceD::Tp/pLM69-1, B. cepacia IST408 bceF::Tp/pMLBAD-Cm, and B. cepacia IST408 bceF::Tp/pLM63-1 in the wells of polystyrene microtiter
dishes containing mannitol medium supplemented with 1% of arabinose at 30°C. The microtiter dishes were rinsed to remove planktonic cells, and
the biofilms were stained with crystal violet. Absorbance at 590 nm (A590) quantifies the amount of crystal violet associated with the biofilm after
532FERREIRA ET AL.APPL. ENVIRON. MICROBIOL.
biofilms, as strains producing null or small amounts of EPS
produce only thin biofilms that are devoid of normal architec-
ture (18, 63). Alginate production in Pseudomonas aeruginosa
has also been correlated with the ability of this bacteria to form
thick and mature biofilms, where the bacteria exhibit higher
resistance to antimicrobials (26, 28) and to host phagocyte
killing. Therefore, it is considered that biofilm formation con-
tributes to persistent infections that may lead to the character-
istic chronic deterioration of CF patients’ airways (24). In a
previous work, we compared the abilities of the CF isolate B.
cepacia IST408 and three isogenic mutants to form biofilms
(17, 41). Two of these plasposon insertion mutants had the
plasposon inserted in the tyrosine kinase bceF gene, and in the
third mutant, the transposon was in the bceI gene, putatively
coding for a polysaccharide polymerase. The amount of biofilm
formed by the three EPS-defective mutants was strongly re-
duced compared with that formed by the mucoid wild-type
strain (17). Since the two bceF plasposon mutants were polar,
it was not possible to be sure whether the EPS-deficient phe-
notype is due to impaired expression of the bce downstream
genes or to the absence of a functional tyrosine kinase. The
results obtained in this work, using the nonpolar bceF mutant
strain constructed here, allowed the confirmation of the crucial
role that a functional tyrosine kinase has in EPS biosynthesis.
The inability of the B. cepacia bceF mutant to form, in vitro,
biofilms of the same size as those produced by the parental
strain IST408 is consistent with the notion that EPS biosynthe-
sis is an important factor in obtaining biofilms with the maxi-
mal size. However, although it produced about 75% of the
EPS of the parental strain IST408, the bceD mutant also
formed biofilms with a reduced size, close to the size of bceF
mutant biofilms. Since it is believed that alterations in EPS
structure significantly change its physicochemical proper-
ties, it is possible that the less viscous EPS produced by the
bceD mutant strain may prevent the stable development of a
biofilm of larger size. Although the development of thick
biofilms in the B. cepacia complex certainly involves other
strain-dependent factors besides the biosynthesis of EPS
(17), this study reinforces the notion of EPS involvement in
the development of biofilms with a potential role in the
persistence and virulence of respiratory infections caused by
these opportunistic bacteria.
The kind supply of the trimethoprim resistance cassette by P. A.
Sokol (University of Calgari, Canada) is gratefully acknowledged.
This work was partially supported by FEDER, POCTI, and the
POCI Programmes from Fundac ¸a ˜o para a Cie ˆncia e a Tecnologia,
Portugal (contracts POCTI/BME/44441/2002, POCTI/AGG/39533/
2001, POCTI/BIO/38273/2001, and POCI/BIO/58401/2004 and Ph.D.
grants to A.S.F., S.A.S., and A.M.C.).
1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,
and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation
of protein database search programs. Nucleic Acids Res. 25:3389–3402.
2. Becker, A., K. Niehaus, and A. Puhler. 1995. Low-molecular-weight succi-
noglycan is predominantly produced by Rhizobium meliloti strains carrying a
mutated ExoP protein characterized by a periplasmic N-terminal domain
and a missing C-terminal domain. Mol. Microbiol. 16:191–203.
3. Bender, M. H., R. T. Cartee, and J. Yother. 2003. Positive correlation be-
tween tyrosine phosphorylation of CpsD and capsular polysaccharide pro-
duction in Streptococcus pneumoniae. J. Bacteriol. 185:6057–6066.
4. Beutler, H. O. 1984. Lactose and D-galactose: UV method, p. 104–112. In
H. U. Bergemeyer (ed.), Methods of enzymatic analysis, 3rd ed., vol. 6.
Verlag Chemie, Weinheim, Germany.
5. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye bind-
ing. Anal. Biochem. 72:248–254.
6. Bugert, P., and K. Geider. 1997. Characterization of the amsI gene product
as a low molecular weight acid phosphatase controlling exopolysaccharide
synthesis of Erwinia amylovora. FEBS Lett. 400:252–256.
7. Bullock, W. C., J. M. Fernandez, and J. M. Short. 1987. XL1-Blue: a high
efficiency plasmid transforming recA Escherichia coli strain with beta-galac-
tosidase selection. BioTechniques 5:376–379.
8. Bylund, J., L. A. Burgess, P. Cescutti, R. K. Ernst, and D. P. Speert. 2006.
Exopolysaccharides from Burkholderia cenocepacia inhibit neutrophil che-
motaxis and scavenge reactive oxygen species. J. Biol. Chem. 281:2526–2532.
9. Ce ´rantola, S., J. Bounery, C. Segonds, N. Marty, and H. Montrozier. 2000.
Exopolysaccharide production by mucoid and non-mucoid strains of Burk-
holderia cepacia. FEMS Microbiol. Lett. 185:243–246.
10. Cerantola, S., A. Lemassu-Jacquier, and H. Montrozier. 1999. Structural
elucidation of a novel exopolysaccharide produced by a mucoid clinical
isolate of Burkholderia cepacia. Characterization of a trisubstituted gluc-
uronic acid residue in a heptasaccharide repeating unit. Eur. J. Biochem.
11. Cescutti, P., M. Bosco, F. Picotti, G. Impallomeni, J. H. Leitão, J. A. Richau,
and I. Sá-Correia. 2000. Structural study of the exopolysaccharide produced
by a clinical isolate of Burkholderia cepacia. Biochem. Biophys. Res. Com-
12. Chiarini, L., P. Cescutti, L. Drigo, G. Impallomeni, Y. Herasimenka, A.
Bevivino, C. Dalmastri, S. Tabacchioni, G. Manno, F. Zanetti, and R. Rizzo.
2004. Exopolysaccharides produced by Burkholderia cenocepacia recA lineages
IIIA and IIIB. J. Cyst. Fibros. 3:165–172.
13. Conway, B. A., K. K. Chu, J. Bylund, E. Altman, and D. P. Speert. 2004.
Production of exopolysaccharide by Burkholderia cenocepacia results in al-
tered cell-surface interactions and altered bacterial clearance in mice. J. In-
fect. Dis. 190:957–966.
14. Costerton, J. W., P. S. Stewart, and E. P. Greenberg. 1999. Bacterial biofilms:
a common cause of persistent infections. Science 284:1318–1322.
15. Cozzone, A. J., C. Grangeasse, P. Doublet, and B. Duclos. 2004. Protein
phosphorylation on tyrosine in bacteria. Arch. Microbiol. 181:171–181.
16. Cunha, M. V., J. H. Leita ˜o, E. Mahenthiralingam, P. Vandamme, L. Lito, C.
Barreto, M. J. Salgado, and I. Sa ´-Correia. 2003. Molecular analysis of
Burkholderia cepacia complex isolates from a Portuguese cystic fibrosis cen-
ter: a 7-year study. J. Clin. Microbiol. 41:4113–4120.
17. Cunha, M. V., S. A. Sousa, J. H. Leita ˜o, L. M. Moreira, P. A. Videira, and I.
Sa ´-Correia. 2004. Studies on the involvement of the exopolysaccharide pro-
duced by cystic fibrosis-associated isolates of the Burkholderia cepacia com-
plex in biofilm formation and in persistence of respiratory infections. J. Clin.
18. Danese, P. N., L. A. Pratt, and R. Kolter. 2000. Exopolysaccharide produc-
tion is required for development of Escherichia coli K-12 biofilm architec-
ture. J. Bacteriol. 182:3593–3596.
19. Doublet, P., C. Grangeasse, B. Obadia, E. Vaganay, and A. J. Cozzone. 2002.
Structural organization of the protein-tyrosine autokinase Wzc within Esch-
erichia coli cells. J. Biol. Chem. 277:37339–37348.
20. Doublet, P., C. Vincent, C. Grangeasse, A. J. Cozzone, and B. Duclos. 1999.
On the binding of ATP to the autophosphorylating protein, Ptk, of the
bacterium Acinetobacter johnsonii. FEBS Lett. 445:137–143.
21. Drummelsmith, J., and C. Whitfield. 1999. Gene products required for
surface expression of the capsular form of the group 1 K antigen in Esche-
richia coli (O9a:K30). Mol. Microbiol. 31:1321–1332.
22. Figurski, D. H., and D. R. Helinski. 1979. Replication of an origin-containing
derivative of plasmid RK2 dependent on a plasmid function provided in
trans. Proc. Natl. Acad. Sci. USA 76:1648–1652.
23. Govan, J. R., P. H. Brown, J. Maddison, C. J. Doherty, J. W. Nelson, M.
Dodd, A. P. Greening, and A. K. Webb. 1993. Evidence for transmission of
Pseudomonas cepacia by social contact in cystic fibrosis. Lancet 342:15–19.
24. Govan, J. R., and V. Deretic. 1996. Microbial pathogenesis in cystic fibrosis:
mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol. Rev.
25. Grangeasse, C., P. Doublet, and A. J. Cozzone. 2002. Tyrosine phosphory-
lation of protein kinase Wzc from Escherichia coli K12 occurs through a
two-step process. J. Biol. Chem. 277:7127–7135.
26. Hentzer, M., G. M. Teitzel, G. J. Balzer, A. Heydorn, S. Molin, M. Givskov,
and M. R. Parsek. 2001. Alginate overproduction affects Pseudomonas
aeruginosa biofilm structure and function. J. Bacteriol. 183:5395–5401.
27. Holmes, A., R. Nolan, R. Taylor, R. Finley, M. Riley, R. Z. Jiang, S. Stein-
bach, and R. Goldstein. 1999. An epidemic of Burkholderia cepacia trans-
mitted between patients with and without cystic fibrosis. J. Infect. Dis. 179:
28. Hoyle, B. D., and J. W. Costerton. 1991. Bacterial resistance to antibiotics:
the role of biofilms. Prog. Drug Res. 37:91–105.
29. Ilan, O., Y. Bloch, G. Frankel, H. Ullrich, K. Geider, and I. Rosenshine.
VOL. 73, 2007ROLE OF TYROSINE PHOSPHORYLATION IN EPS BIOSYNTHESIS533
1999. Protein tyrosine kinases in bacterial pathogens are associated with Download full-text
virulence and production of exopolysaccharide. EMBO J. 18:3241–3248.
30. Kennelly, P. J., and M. Potts. 1999. Life among the primitives: protein
O-phosphatases in prokaryotes. Front. Biosci. 4:D372–D385.
31. Kovach, M. E., R. W. Phillips, P. H. Elzer, R. M. Roop II, and K. M.
Peterson. 1994. pBBR1MCS: a broad-host-range cloning vector. BioTech-
32. Kunst, A., B. Draeger, and J. Zoegenhorn. 1984. UV methods with hexoki-
nase and glucose-6-phosphate dehydrogenase, p. 163–172. In H. U. Berge-
meyer (ed.), Methods of enzymatic analysis, 3rd ed., vol. 6. Verlag Chemie,
33. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227:680–685.
34. Lefebre, M. D., and M. A. Valvano. 2002. Construction and evaluation of
plasmid vectors optimized for constitutive and regulated gene expression in
Burkholderia cepacia complex isolates. Appl. Environ. Microbiol. 68:5956–
35. LiPuma, J. J. 2003. Burkholderia and emerging pathogens in cystic fibrosis.
Semin. Respir. Crit. Care Med. 24:681–692.
36. Mahenthiralingam, E., T. A. Urban, and J. B. Goldberg. 2005. The multi-
farious, multireplicon Burkholderia cepacia complex. Nat. Rev. Microbiol.
37. McComb, E. A., and R. M. McCready. 1957. Determination of acetyl in
pectin and acetylated carbohydrate polymers. Anal. Chem. 29:819–821.
38. Mijakovic, I., S. Poncet, G. Boel, A. Maze, S. Gillet, E. Jamet, P. Decottig-
nies, C. Grangeasse, P. Doublet, P. Le Marechal, and J. Deutscher. 2003.
Transmembrane modulator-dependent bacterial tyrosine kinase activates
UDP-glucose dehydrogenases. EMBO J. 22:4709–4718.
39. Minic, Z., C. Marie, C. Delorme, J.-M. Faurie, G. Mercier, D. Ehrlich, and
P. Renault. 15 September 2006. Control of EpsE, the phosphoglycosyltrans-
ferase initiating exopolysaccharide synthesis in Streptococcus thermophilus,
by EpsD tyrosine-kinase. J. Bacteriol. doi:10.1128/JB.01122-06.
40. Moreira, L. M., K. Hoffmann, H. Albano, A. Becker, K. Niehaus, and I.
Sá-Correia. 2004. The gellan gum biosynthetic genes gelC and gelE encode
two separate polypeptides homologous to the activator and the kinase do-
mains of tyrosine autokinases. J. Mol. Microbiol. Biotechnol. 8:43–57.
41. Moreira, L. M., P. A. Videira, S. A. Sousa, J. H. Leitão, M. V. Cunha, and I.
Sá-Correia. 2003. Identification and physical organization of the gene cluster
involved in the biosynthesis of Burkholderia cepacia complex exopolysaccha-
ride. Biochem. Biophys. Res. Commun. 312:323–333.
42. Morona, J. K., R. Morona, D. C. Miller, and J. C. Paton. 2003. Mutational
analysis of the carboxy-terminal (YGX)4repeat domain of CpsD, an auto-
phosphorylating tyrosine kinase required for capsule biosynthesis in Strep-
tococcus pneumoniae. J. Bacteriol. 185:3009–3019.
43. Morona, J. K., J. C. Paton, D. C. Miller, and R. Morona. 2000. Tyrosine
phosphorylation of CpsD negatively regulates capsular polysaccharide bio-
synthesis in Streptococcus pneumoniae. Mol. Microbiol. 35:1431–1442.
44. Nakar, D., and D. L. Gutnick. 2003. Involvement of a protein tyrosine kinase
in production of the polymeric bioemulsifier emulsan from the oil-degrading
strain Acinetobacter lwoffii RAG-1. J. Bacteriol. 185:1001–1009.
45. Nesper, J., C. M. Hill, A. Paiment, G. Harauz, K. Beis, J. H. Naismith, and
C. Whitfield. 2003. Translocation of group 1 capsular polysaccharide in
Escherichia coli serotype K30. Structural and functional analysis of the outer
membrane lipoprotein Wza. J. Biol. Chem. 278:49763–49772.
46. Niemeyer, D., and A. Becker. 2001. The molecular weight distribution of
succinoglycan produced by Sinorhizobium meliloti is influenced by specific
tyrosine phosphorylation and ATPase activity of the cytoplasmic domain of
the ExoP protein. J. Bacteriol. 183:5163–5170.
47. Paiment, A., J. Hocking, and C. Whitfield. 2002. Impact of phosphorylation
of specific residues in the tyrosine autokinase, Wzc, on its activity in assembly
of group 1 capsules in Escherichia coli. J. Bacteriol. 184:6437–6447.
48. Preneta, R., S. Jarraud, C. Vincent, P. Doublet, B. Duclos, J. Etienne, and
A. J. Cozzone. 2002. Isolation and characterization of a protein-tyrosine
kinase and a phosphotyrosine-protein phosphatase from Klebsiella pneu-
moniae. Comp. Biochem. Physiol. Part B 131:103–112.
49. Reid, A. N., and C. Whitfield. 2005. Functional analysis of conserved gene
products involved in assembly of Escherichia coli capsules and exopolysac-
charides: evidence for molecular recognition between Wza and Wzc for
colanic acid biosynthesis. J. Bacteriol. 187:5470–5481.
50. Richau, J. A., J. H. Leita ˜o, M. Correia, L. Lito, M. J. Salgado, C. Barreto, P.
Cescutti, and I. Sa ´-Correia. 2000. Molecular typing and exopolysaccharide
biosynthesis of Burkholderia cepacia isolates from a Portuguese cystic fibrosis
center. J. Clin. Microbiol. 38:1651–1655.
51. Richau, J. A., J. H. Leitão, and I. Sá-Correia. 2000. Enzymes leading to the
nucleotide sugar precursors for exopolysaccharide synthesis in Burkholderia
cepacia. Biochem. Biophys. Res. Commun. 276:71–76.
52. Sa ´-Correia, I., and A. M. Fialho. 2000. Electrotansformation of Sphingomo-
nas spp., p. 108–118. In N. Eynard and J. Teissie (ed.), Electrotransformation
of bacteria: lab manual. Springer, Heidelberg, Germany.
53. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a
laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,
54. Schirmer, F., S. Ehrt, and W. Hillen. 1997. Expression, inducer spectrum,
domain structure, and function of MopR, the regulator of phenol degrada-
tion in Acinetobacter calcoaceticus NCIB8250. J. Bacteriol. 179:1329–1336.
55. Sist, P., P. Cescutti, S. Skerlavaj, R. Urbani, J. H. Leitão, I. Sá-Correia, and
R. Rizzo. 2003. Macromolecular and solution properties of Cepacian: the
exopolysaccharide produced by a strain of Burkholderia cepacia isolated from
a cystic fibrosis patient. Carbohydr. Res. 338:1861–1867.
56. Sokol, P. A., P. Darling, D. E. Woods, E. Mahenthiralingam, and C. Kooi.
1999. Role of ornibactin biosynthesis in the virulence of Burkholderia cepa-
cia: characterization of pvdA, the gene encoding L-ornithine N5-oxygenase.
Infect. Immun. 67:4443–4455.
57. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment through
sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res. 22:4673–4680.
58. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: procedure and
some applications. Proc. Natl. Acad. Sci. USA 76:4350–4354.
59. Vincent, C., P. Doublet, C. Grangeasse, E. Vaganay, A. J. Cozzone, and B.
Duclos. 1999. Cells of Escherichia coli contain a protein-tyrosine kinase, Wzc,
and a phosphotyrosine-protein phosphatase, Wzb. J. Bacteriol. 181:3472–
60. Vincent, C., B. Duclos, C. Grangeasse, E. Vaganay, M. Riberty, A. J. Coz-
zone, and P. Doublet. 2000. Relationship between exopolysaccharide pro-
duction and protein-tyrosine phosphorylation in gram-negative bacteria. J.
Mol. Biol. 304:311–321.
61. Whitfield, C., and A. Paiment. 2003. Biosynthesis and assembly of Group 1
capsular polysaccharides in Escherichia coli and related extracellular poly-
saccharides in other bacteria. Carbohydr. Res. 338:2491–2502.
62. Wugeditsch, T., A. Paiment, J. Hocking, J. Drummelsmith, C. Forrester, and
C. Whitfield. 2001. Phosphorylation of Wzc, a tyrosine autokinase, is essen-
tial for assembly of group 1 capsular polysaccharides in Escherichia coli.
J. Biol. Chem. 276:2361–2371.
63. Yildiz, F. H., and G. K. Schoolnik. 1999. Vibrio cholerae O1 El Tor: identi-
fication of a gene cluster required for the rugose colony type, exopolysac-
charide production, chlorine resistance, and biofilm formation. Proc. Natl.
Acad. Sci. USA 96:4028–4033.
534 FERREIRA ET AL.APPL. ENVIRON. MICROBIOL.