JOURNAL OF BACTERIOLOGY, Jan. 2009, p. 365–374
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 1
Expression and Physiological Relevance of Agrobacterium tumefaciens
Phosphatidylcholine Biosynthesis Genes?
Sonja Klu ¨sener,1# Meriyem Aktas,1# Kai M. Thormann,1,2Mirja Wessel,1and Franz Narberhaus1*
Microbial Biology, Ruhr-University Bochum, Bochum, Germany,1and Department of Ecophysiology, Max Planck Institute for
Terrestrial Microbiology, Marburg, Germany2
Received 22 August 2008/Accepted 23 October 2008
Phosphatidylcholine (PC), or lecithin, is the major phospholipid in eukaryotic membranes, whereas only
10% of all bacteria are predicted to synthesize PC. In Rhizobiaceae, including the phytopathogenic bacterium
Agrobacterium tumefaciens, PC is essential for the establishment of a successful host-microbe interaction. A.
tumefaciens produces PC via two alternative pathways, the methylation pathway and the Pcs pathway. The
responsible genes, pmtA (coding for a phospholipid N-methyltransferase) and pcs (coding for a PC synthase),
are located on the circular chromosome of A. tumefaciens C58. Recombinant expression of pmtA and pcs in
Escherichia coli revealed that the individual proteins carry out the annotated enzyme functions. Both genes and
a putative ABC transporter operon downstream of PC are constitutively expressed in A. tumefaciens. The
amount of PC in A. tumefaciens membranes reaches around 23% of total membrane lipids. We show that PC
is distributed in both the inner and outer membranes. Loss of PC results in reduced motility and increased
biofilm formation, two processes known to be involved in virulence. Our work documents the critical impor-
tance of membrane lipid homeostasis for diverse cellular processes in A. tumefaciens.
Phosphatidylcholine (PC), or lecithin, is the most-abundant
phospholipid in eukaryotic membranes. Apart from its struc-
tural function in membrane bilayers and lipoproteins, PC is
involved in many signal transduction pathways (2). Meanwhile,
PC has also been found in an increasing number of bacteria, in
particular in species that interact with eukaryotic hosts (27). In
bacteria, PC is synthesized either by the methylation pathway
or by the Pcs pathway (Fig. 1). In the first pathway, the pre-
cursor phosphatidylethanolamine (PE) is methylated in three
reactions by one or several phospholipid N-methyltransferases
(Pmt enzymes) via the intermediates monomethylphosphati-
dylethanolamine (MMPE) and dimethylphosphatidylethanol-
amine (DMPE) to form PC. Each reaction step requires S-
adenosylmethionine as the methyl donor. In the Pcs pathway,
PC is produced via a direct condensation of choline and CDP-
diacylglycerol. This reaction is catalyzed by the PC synthase
(Pcs), an enzyme unique to prokaryotes (42).
The gram-negative alphaproteobacterium Agrobacterium tu-
mefaciens is most commonly known for causing crown gall
disease in plants. It synthesizes PC by using both the methyl-
ation pathway and the Pcs pathway (Fig. 1) (21, 23). The latter
pathway requires choline in the medium. During growth in
minimal medium, only marginal amounts of PC were formed
by the Pcs enzyme (45). In Brucella abortus, PC production via
the Pcs pathway has been reported to depend on the presence
of choline provided by the host (5). The plant symbiont Sino-
rhizobium meliloti uses plant-exuded choline for PC biosynthe-
sis (13). The finding that A. tumefaciens cannot produce cho-
line de novo (41) implies that there must be a choline uptake
system to supply the Pcs pathway with choline. The only evi-
dence for a choline uptake system so far is that A. tumefaciens
was shown to take up radiolabeled choline from the cultivation
medium (41). The responsible transporter, however, remains
Several symbiotic and pathogenic bacteria depend on PC to
establish a successful host-microbe interaction. We demon-
strated previously that an A. tumefaciens mutant lacking both
predicted PC biosynthesis pathways did not produce any PC
and was incapable of tumor formation on plant leaves (45).
This virulence defect was caused by the absence of the mem-
brane-spanning type IV secretion system, which delivers the
oncogenic T-DNA from the tumor-inducing (Ti) plasmid and
effector proteins to plant cells (4, 47). In Bradyrhizobium ja-
ponicum, the nitrogen-fixing symbiont of the soybean Glycine
max, PC is required to establish functional root nodules (31).
PC also is necessary for full virulence of the human pathogens
B. abortus (6) and Legionella pneumophila (7). Although this
strongly suggests that PC is a critical determinant in host-
microbe interactions, there still is little understanding re-
garding the exact role that PC might play in these processes.
Moreover, it is largely unclear whether the expression of PC
biosynthesis genes is regulated. The first hints that the ex-
pression of PC biosynthesis genes might be subject to envi-
ronmental control were obtained in B. japonicum. This or-
ganism encodes a pmt multigene family comprised of pmtA
and four additional pmt genes (18). Under normal condi-
tions, only pmtA and pmtX1 are expressed. However, in a
pmtA mutant, pmtX3 and, in particular, pmtX4 are expressed
In this study we examined the expression and physiological
role of A. tumefaciens PC biosynthesis genes pmtA and pcs. The
most important findings are that PC produced by these en-
zymes is distributed in the inner and outer membrane and that
* Corresponding author. Mailing address: Lehrstuhl fu ¨r Biologie der
Mikroorganismen, Fakulta ¨t fu ¨r Biologie und Biotechnologie, Ruhr-
Universita ¨t Bochum, D-44780 Bochum, Germany. Phone: 49 (234) 32
23100. Fax: 49 (234) 32 14620. E-mail: firstname.lastname@example.org.
# S.K. and M.A. contributed equally to this study.
?Published ahead of print on 31 October 2008.
the absence of PC is associated with severe phenotypes in
motility and biofilm formation.
MATERIALS AND METHODS
Bacterial strains and growth conditions. All strains and plasmids used in this
work are listed in Table 1. Oligonucleotides are listed in Table 2. Escherichia coli
cells were grown at 37°C in Luria-Bertani (LB) medium (38), supplemented with
kanamycin, streptomycin, and/or spectinomycin at a final concentration of 50
?g/ml if appropriate. E. coli DH5? was used as host for all cloning procedures.
E. coli BL21(DE3), which contains the phage T7 polymerase gene under the
control of the lacUV5 promoter (43), served as the host for overproduction of
PmtA and Pcs from the corresponding pET24b-based expression plasmids. A.
tumefaciens strain C58 (wild type) and its derivatives (?pmtA, ?pcs, ?pmtA ?pcs,
and ?pmtA ?abc mutants) were routinely grown at 30°C in YEB complex or AB
minimal medium (pH 5.5, 1% [wt/vol] glucose) (40), supplemented with 100
?g/ml ampicillin, 2 ?g/ml tetracycline, 50 ?g/ml kanamycin, 100 ?g/ml strepto-
mycin, and/or 300 ?g/ml spectinomycin if necessary. For ?-galactosidase assays,
choline (Sigma-Aldrich, Mu ¨nchen, Germany) was added to the AB medium in
final concentrations of 0.1 mM, 0.5 mM, or 1 mM. In support of virulence gene
induction, Agrobacterium cells were precultivated to an optical density at 600 nm
(OD600) of 0.2 at 30°C in liquid AB minimal medium (pH 5.5) with 1% (wt/vol)
glucose prior to the addition of acetosyringone. Cells were further incubated
for 16 to 20 h at 23°C. Acetosyringone was always used at a final concentra-
tion of 0.1 mM.
Plasmid and mutant construction. Recombinant DNA work was carried out
according to standard protocols (38). PCR-generated fragments of the promoter
regions of pmtA, pcs, and abc (16, 48) were digested with KpnI and XhoI and
ligated into pAC01 (26) treated with the same enzymes to construct transcrip-
tional fusions to the lacZ gene. For overproduction of the agrobacterial PmtA
and Pcs in E. coli, the expression plasmids pET_PmtA and pET_Pcs were
constructed. The pmtA and pcs genes were amplified by PCR. The fragments
were cleaved with NdeI/SalI and NdeI/HindIII, respectively, and ligated into
pET24b treated with the same enzymes. For overproduction of PmtA and Pcs in
A. tumefaciens, the coding regions, including their own ribosome binding site,
were amplified via PCR. The PCR products were digested with EcoRI/SalI and
EcoRI/HindIII, respectively, and cloned into the corresponding sites of the
vector pVS-BADNco (49), resulting in the plasmids pVS-PmtA and pVS-Pcs.
The markerless abc1-4 deletion was introduced into the A. tumefaciens ?pmtA
strain as described previously (45) by using the suicide vector pK19mobsacB. The
abc1-4 up- and downstream regions were amplified by PCR. The PCR product of
the abc1-4 upstream region was digested with EcoRI/PstI and cloned into the
vector pK19mobsacB, resulting in the plasmid pK19mobsacB_abc_up. The PCR
product of the downstream region was digested with PstI/HindIII and cloned into
pK19mobsacB_abc_up, resulting in the plasmid pK19mobsacB_abc_updo. The
correct nucleotide sequences of all plasmids constructed were confirmed by
automated sequencing. Plasmids were transferred into E. coli via transformation
and into A. tumefaciens via electroporation.
Overproduction of PmtA and Pcs in E. coli. E. coli BL21(DE3) carrying
pET_PmtA or pET_Pcs was cultivated in LB medium at 37°C until the OD600
FIG. 1. PC biosynthesis pathways in A. tumefaciens C58. As indi-
cated by the question marks, the choline uptake system is unknown.
SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; PmtA,
phospholipid N-methyltransferase; Pcs, phosphatidylcholine synthase.
TABLE 1. Strains and plasmids used in this study
Strain or plasmid Relevant characteristic(s)a
Reference or source
C58Wild typeC. Baron, Montreal,
C58 ?pmtA ?pcs
C58 ?pmtA ?abc
Derivative of the wild type with deletion of the pmtA gene
Derivative of the wild type with deletion of the pcs gene
Derivative of the wild type with deletion of the pmtA and pcs genes
Derivative of the wild type with deletion of the pmtA and abc1-4 genes
TcrApr; transcriptional lacZ fusion vector containing promoterless lacZ gene
Kmr; high-copy His-tag expression vector
SprStr; A. tumefaciens expression vector
Kmr; suicide vector
TcrApr; pmtA-lacZ fusion in pAC01
TcrApr; pcs-lacZ fusion in pAC01
TcrApr; abc-lacZ fusion in pAC01
Kmr; derivative of pET24b for overproduction of PmtA with a C-terminal His tag
Kmr; derivative of pET24b for overproduction of Pcs with a C-terminal His tag
SprStr; derivative of pVS-BADNco carrying pmtA
SprStr; derivative of pVS-BADNco carrying pcs
Kmr; derivative of pK19mobsacB carrying the upstream region of abc1-4
Kmr; derivative of pK19mobsacB carrying the up- and downstream regions of
lacIqPtac::gfp-mut2 TcrrepA oriVpVSIoriVp15AoriT
pLacTac-GfpK. Thormann, Marburg
aAp, ampicillin; Km, kanamycin; Sp, spectinomycin; St, streptomycin; Tc, tetracycline.
366KLU ¨SENER ET AL. J. BACTERIOL.
reached a value between 0.5 and 0.8. Then, the synthesis of PmtA or Pcs was
induced by the addition of isopropyl-?-D-thiogalactopyranoside (IPTG) to a final
concentration of 0.4 mM and the cultures were incubated for another 2 h at 30°C.
Subsequently, 1 ml culture was harvested by centrifugation. Cell pellets were
resuspended in 1? sodium dodecyl sulfate (SDS) loading buffer according to the
OD600(OD600of 1 ? 100 ?l 1? SDS loading buffer) and boiled for 10 min. Ten
microliters of each sample was separated on 12.5% SDS-polyacrylamide gels,
and the proteins were stained with Coomassie blue.
Lipid analysis by TLC. The lipid composition of A. tumefaciens and E. coli
strains was determined via thin-layer chromatography (TLC). Cells were culti-
vated as mentioned above, harvested by centrifugation, washed with 500 ?l
water, and resuspended in 100 ?l water. The lipids were extracted according to
the method of Bligh and Dyer (1), separated by one-dimensional thin-layer
chromatography using HPTLC silica gel 60 plates (Merck, Darmstadt, Ger-
many), and stained with molybdenum blue spray reagent (Sigma-Aldrich) or
Cu2SO4solution [300 mM copper(II)-sulfate-pentahydrate, 8.5% (vol/vol) phos-
phoric acid]. In the case of the separated inner and outer membranes, the lipid
extraction was started directly from the collected fractions. PE, MMPE, DMPE,
and PC were used as phospholipid standards (Sigma-Aldrich), and n-propanol–
propionate–chloroform–water (3:2:2:1) as the running solvent.
A. tumefaciens strains were cultivated in YEB complex medium
until exponential phase. Total RNA was isolated by using a Micro-to-Midi total
RNA purification system (Invitrogen, Karlsruhe, Germany). The RNA was fur-
ther treated with DNase I (amplification grade; Invitrogen) as specified by the
manufacturer to remove contaminating chromosomal DNA. Reverse transcrip-
tase (RT)-PCR and subsequent PCRs were performed according to the manu-
facturer’s manual for the ThermoScript RT-PCR system (Invitrogen). The prim-
ers used for these experiments are listed in Table 2 (see also Fig. 3A and C). The
reaction products were separated by electrophoresis in 2% (wt/vol) agarose gels
and visualized by staining with ethidium bromide.
?-Galactosidase assays. The ?-galactosidase activity of A. tumefaciens cells
grown in liquid YEB complex medium or in AB minimal medium was measured
according to standard protocols (30). The plasmid pAC01 (26) containing the
promoterless lacZ gene was used as the negative control.
Separation of inner and outer membrane. Membrane separation was per-
formed as described previously (12), with minor modifications. Four hundred
milliliters YEB cell culture was grown to an OD600of 0.5 to 0.6 at 30°C and
harvested by centrifugation at 10,000 ? g, 4°C, for 10 min. The cells were
resuspended in 24 ml lysis buffer (50 mM Tris-HCl, pH 7.5, 20% [wt/vol] sucrose,
0.2 M KCl, 0.2 mM dithiothreitol, 0.2 mg/ml DNase I, 0.2 mg/ml RNase A, 1 mM
phenylmethylsulfonyl fluoride) and disrupted by two passes through a chilled
French pressure cell at 16,000 lb/in2. The lysate was treated with 0.5 mg/ml
lysozyme for 1 h on ice and centrifuged at 10,000 ? g, 4°C, for 20 min to remove
the unbroken cells. The supernatant was centrifuged at 150,000 ? g (SW40Ti),
4°C, for 1 h in an ultracentrifuge to collect the membranes. The resulting
membrane pellet was carefully resuspended in 2 ml of 20% (wt/vol) sucrose
containing 5 mM EDTA, pH 7.5, and 0.2 mM dithiothreitol. The resuspended
membranes were centrifuged for 5 min at 16,000 ? g to remove the insoluble
membranes. The gradient was prepared by layering 7.5 ml 53% (wt/vol) sucrose
over a cushion of 2.5 ml 70% (wt/vol) sucrose. Both sucrose solutions contained
5 mM EDTA, pH 7.5. The membrane suspension was layered on the top of the
gradient, and sucrose density gradient ultracentrifugation was carried out at
100,000 ? g (SW40Ti), 4°C, for 16 h. After ultracentrifugation, the separated
membranes were fractionated in 500-?l aliquots to analyze the protein concen-
TABLE 2. Oligonucleotides used in this study
PurposeOligonucleotide Sequence (5’33’)a
Transcriptional lacZ fusionspmtA_Fw_KpnI
RT-PCR pmtA_K1 (P1)
Overproduction of PmtA and Pcs
in E. coli
Overproduction of PmtA and Pcs
in A. tumefaciens
Construction of a markerless
abc1–4 deletion mutant in A.
aRestriction sites in the oligonucleotides are underlined.
VOL. 191, 2009PC BIOSYNTHESIS IN AGROBACTERIUM TUMEFACIENS 367
tration, the NADH activity, and the phospholipid pattern. The protein concen-
tration was determined by using a Bradford assay (Bio-Rad Laboratories GmbH,
Mu ¨nchen, Germany). The NADH oxidase activity was detected by the method of
Osborn et al. (32).
Motility assay. Motility assays were carried out on AB minimal medium
solidified with 0.3% (wt/vol) agar (Difco, Lawrence, KS). A single colony from a
YEB agar plate was inoculated onto the surface of the motility plates. The plates
were examined after 48 h of incubation at 23°C. To check whether calcium and/or
magnesium influence motility, MgSO4was added in final concentrations of 0.24
mM, 1.2 mM, or 12 mM and CaCl2was added in final concentrations of 0.014
mM, 0.07 mM, or 0.7 mM to AB medium.
SDS-PAGE and Western blotting. Wild-type and mutant A. tumefaciens cells
were cultivated as mentioned above, and 1 ml of each culture was harvested and
resuspended in 1? SDS loading buffer in relation to the OD600(OD600of 1 ?
100 ?l 1? SDS loading buffer). The samples were incubated for 10 min at 95°C,
separated by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE), and blot-
ted onto polyvinylidene difluoride membranes (Bio-Rad). Detection was per-
formed with a chemiluminescence-based system (Pierce Biotechnology, Rock-
ford, IL) using A. tumefaciens flagellum protein-specific antiserum (1:30,000).
Flow cell biofilms. Biofilms of A. tumefaciens strains carrying plasmid-encoded
green fluorescent protein (GFP) were cultivated at 30°C in three-channel flow
cells with individual channel dimensions of 1 by 4 by 40 mm. Each flow chamber
was prepared by gluing a boron-silicate glass microscope coverslip, which served
as a substratum for microbial attachment, onto the flow chamber with silicone
and leaving it to dry for 24 h at room temperature prior to use. The assembly of
the flow system was carried out essentially as described earlier (44).
The appropriate A. tumefaciens strains were cultivated until reaching the
exponential or the stationary growth phase. The OD600was then adjusted to 0.01
in AB minimal medium with 0.5% (vol/vol) glycerol. One milliliter of the diluted
cell suspension was injected into each channel after the flow of medium was
arrested, and the chambers were turned upside down to facilitate initial attach-
ment. After 30 min of incubation at 30°C, the flow cells were inverted, and the
flow of medium was started at a constant rate of 75 ?l/min per channel, using a
Watson-Marlow Bredel 205S peristaltic pump (Cornwall, United Kingdom). All
biofilm characterizations were conducted in triplicate in at least two independent
Sixty minutes prior to microscopy, gfp expression from plasmid pLacTac-Gfp
was induced by the addition of IPTG (1 mM). Microscopic visualization of
biofilms was carried out using an inverted Zeiss LSM510 confocal laser scanning
microscope (Carl Zeiss, Jena, Germany) equipped with the following objectives:
10?/0.3 W Plan-Neofluar, 20?/0.5 W Achroplan, and 40?/1.2 W C-Apochro-
mat. For quantitative analysis, the image data were further processed using the
IMARIS software package (Bitplane AG, Zu ¨rich, Switzerland) and Adobe Pho-
RESULTS AND DISCUSSION
Functional analysis of pmtA and pcs gene products. We have
previously shown that an A. tumefaciens ?pmtA ?pcs mutant is
no longer able to synthesize PC (45). To provide conclusive
evidence that PmtA and Pcs act as phospholipid N-methyl-
transferase and PC synthase, respectively, we cloned pmtA and
pcs into the expression vector pET24b, resulting in plasmids
pET_PmtA and pET_Pcs. The analysis of crude protein ex-
tracts from E. coli cells containing the first plasmid by SDS-
PAGE revealed an overproduced protein of 22 kDa (Fig. 2A),
which is in close agreement with the calculated mass of the
agrobacterial pmtA gene product. In contrast, overexpression
of Pcs (calculated mass of 29 kDa), which is thought to be a
membrane protein, was not observed (Fig. 2A). Either Pcs was
not synthesized at all or it was synthesized in amounts below
the detection limit.
To obtain proof for the biochemical activity of both recom-
binant proteins, we compared the membrane lipid contents of
E. coli BL21(DE3) cells carrying pET_PmtA, pET_Pcs, or the
empty vector pET24b. E. coli membranes are known to contain
the lipids PE, phosphatidylglycerol, and cardiolipin (8), which
migrate as a single spot in one-dimensional TLC (Fig. 2B). As
expected, E. coli BL21(DE3)/pET24b did not produce PC or
methylated intermediates. The membranes of E. coli cells ex-
pressing pmtA consist of PE, MMPE, DMPE, and PC (Fig.
2B). This is consistent with the results described for a Sinorhi-
zobium meliloti pmtA gene expressed in E. coli BL21(DE3)
(31). Only PC and no methylated intermediates were produced
when the Rhodobacter sphaeroides pmtA gene was expressed in
E. coli (31). The expression of the B. japonicum pmtA gene in
E. coli BL21(DE3) led predominantly to the formation of
MMPE, with some DMPE but only marginal amounts of PC
(18). Apparently, different Pmt enzymes possess different sub-
strate and product specificities which cannot be predicted
solely on the basis of their primary sequence.
As the Pcs enzyme uses choline directly to form PC, the E.
coli strain containing pET_Pcs produces PE and PC but no
mono- or dimethylated intermediates (Fig. 2B). It is notable
that significant amounts of PC accumulated in this strain al-
though the Pcs protein was only poorly expressed (Fig. 2A).
Our results demonstrate that both recombinant proteins are
active when expressed in E. coli and are probably responsible
for the biosynthesis of PC in A. tumefaciens without the re-
quirement of additional proteins.
FIG. 2. Activity of agrobacterial PmtA and Pcs after expression in
E. coli. (A) Detection of agrobacterial phospholipid N-methyltrans-
ferase (PmtA) and PC synthase (Pcs) in crude extracts of E. coli cells
via SDS-PAGE. E. coli BL21(DE3) cells with pET_PmtA, pET_Pcs, or
pET24b were cultivated in LB complex medium, and protein expres-
sion was induced with IPTG. Protein bands were visualized by Coo-
massie blue staining. M, BenchMark protein standard (Invitrogen). ?,
present; ?, absent. (B) Lipid formation after expression of agrobac-
terial PmtA and Pcs in E. coli BL21(DE3). Lipids of BL21(DE3)
derivatives were extracted and separated by one-dimensional TLC.
Phospholipids were specifically stained with molybdenum blue spray
and compared to phospholipid standards in lane M (PE, MMPE,
DMPE, and PC).
368KLU ¨SENER ET AL.J. BACTERIOL.
Genetic organization of the Agrobacterium pmtA and pcs
gene regions. The pmtA gene is flanked by two open reading
frames, namely dnaN and atu0299, which are oriented in the
same direction as pmtA (Fig. 3A). The pmtA gene is separated
from dnaN and atu0299 by 195 bp and 95 bp, respectively. To
analyze whether pmtA is cotranscribed with dnaN and/or
atu0299, RT-PCR experiments were carried out. Total RNA
was isolated from a wild-type A. tumefaciens culture grown in
YEB complex medium. Primers P2, N2, and B were used for
RT reactions to produce cDNA. Primer pairs P1 and P2, N1
and N2, and A and B were applied for PCR amplification (Fig.
3A). As the positive control, PCRs were carried out with chro-
mosomal DNA as template. Reaction mixtures without RT
served as the control for the absence of chromosomal DNA in
our RNA preparations and did not produce amplification
products (Fig. 3B). The expected product of 591 bp featuring
an internal pmtA fragment appeared only in the presence of
RT, showing that the gene was expressed. The presence of a
258-bp PCR product with primer combination A and B sug-
gests cotranscription of pmtA and atu0299. Since atu0299, pyrF,
and atu0297 are separated by only 4 and 19 bp, respectively,
it seems reasonable that these genes are cotranscribed.
Thus, pmtA-atu0299-pyrF-atu0297 might form a tetracis-
tronic operon. No PCR product was detectable with primer
pair N1 by N2, indicating that dnaN and pmtA belong to dif-
ferent transcription units (Fig. 3B).
The second PC biosynthesis gene, pcs, is located 212 bp
downstream of ubiH, encoding a predicted 2-octaprenyl-6-
methoxyphenol hydroxylase which is transcribed in the oppo-
site direction (Fig. 3C). Two hundred one base pairs further
downstream of pcs are four genes oriented in the same direc-
tion. The gene products are annotated as an ABC transporter
with unknown substrate. We tested whether the genes abc1 to
abc4 might form an operon with pcs. The primer pair P3 and
P4, covering an internal DNA fragment of pcs, yielded the
expected product of 586 bp (Fig. 3D), demonstrating that the
pcs gene is expressed under the tested conditions. In contrast,
the primer set N3 and N4, designed to amplify a DNA frag-
ment overlapping the borders of ubiH and pcs, did not produce
a PCR product, as expected since the two genes are oriented in
opposite directions. The primer pair C and D, designed to
amplify a DNA fragment overlapping the gene borders of pcs
and abc1, resulted in a PCR product of 342 bp (Fig. 3D).
Hence, it appears that pcs and the abc1-4 genes form an
Constitutive expression of A. tumefaciens PC biosynthesis
genes. To further analyze the expression of pmtA and pcs,
transcriptional lacZ fusions were constructed (Fig. 3A and C).
In addition, an abc1-lacZ fusion was created to test whether
abc expression depends exclusively on the pcs promoter (Fig.
3C). The resulting reporter gene plasmids, pBO380 (pmtA-
lacZ), pBO377 (pcs-lacZ), and pBO1264 (abc1-lacZ), were
electroporated into wild-type A. tumefaciens. To analyze if the
presence or absence of PC influences the expression of PC
biosynthesis genes and/or abc1, the lacZ fusions were also
introduced into existing PC biosynthesis mutants. It is known
that the two single deletion ?pmtA and ?pcs mutants are able
to synthesize PC through the remaining pathway, whereas the
double deletion ?pmtA ?pcs mutant is entirely PC deficient
(45). All reporter strains were grown in YEB complex medium
and in AB minimal medium at 30°C. The PC biosynthesis
genes pmtA and pcs, as well as abc1, were clearly expressed
under all conditions tested. The expression levels were three-
to fourfold higher in minimal medium (data not shown). Ex-
FIG. 3. RT-PCR of the pmtA and pcs gene regions. The PCR strategy is outlined in panels A and C. The positions of primers used for the
reverse transcription and PCRs are given below the corresponding gene regions. Putative promoters and constructed lacZ fusions are indicated.
dnaN encodes ?-chain of DNA polymerase III; pmtA encodes phospholipid N-methyltransferase; pyrF encodes orotidine 5?-monophosphate
decarboxylase; atu0299, atu0297, and atu0296 are hypothetical open reading frames; ubiH encodes 2-octaprenyl-6-methoxyphenol hydroxylase; pcs
encodes PC synthase; abc1 encodes a nucleotide binding ABC transporter; abc2 and abc3 encode membrane-spanning proteins forming an ABC
transporter; and abc4 encodes a lipoprotein ABC transporter. In panels B and D, the results of RT-PCRs with RNA from wild-type A. tumefaciens
C58 cells are presented. The primer pairs used and lengths of PCR products are indicated. c, PCR products using chromosomal DNA as template; ?,
standard RT-PCR; ?, negative control in which no RT had been added to the reaction mixture.
VOL. 191, 2009 PC BIOSYNTHESIS IN AGROBACTERIUM TUMEFACIENS 369
pression was not altered in the PC biosynthesis mutants (Fig.
4), indicating that pmtA, pcs, and abc expression is indepen-
dent of the presence of PC, MMPE, and DMPE. The signifi-
cant ?-galactosidase activity of the abc1-lacZ fusion demon-
strates that the abc1-4 genes possess their own promoter in
addition to the one upstream of pcs (Fig. 3D). In all cases, the
level of expression of pmtA was higher than that of pcs (Fig. 4).
This might be taken as an additional line of support for our
previous assumption that the methylation pathway is more
relevant than the Pcs pathway in A. tumefaciens. Stronger ev-
idence for this conclusion derives from the following findings.
Only traces of PC were detectable after growth in AB minimal
medium in the presence of choline (45), indicating that the
still-existing Pcs pathway cannot fully compensate for the
methylation pathway. In addition, Kalanchoe ¨ leaves infected
with the ?pmtA mutant showed decreased tumor formation
(45). The significance of the alternative PC biosynthesis path-
ways in prokaryotes differs. In contrast to A. tumefaciens, the
Pcs pathway is of predominant importance in L. pneumophila
(7) and Pseudomonas aeruginosa (46).
In S. meliloti, choline is taken up by an ABC transport
system (14). Given the genetic organization of the abc1-4 genes
immediately downstream of the pcs gene (Fig. 3C), we specu-
lated that choline might be a substrate of this putative ABC
transporter. In bacteria, the expression and activity of ABC
transporters is often regulated via their substrates. The expres-
sion of the fructose uptake ABC transporter in S. meliloti is
induced in the presence of this sugar (25). Likewise, the
rhamnose uptake system in Rhizobium leguminosarum is con-
trolled by rhamnose (36). To check if the expression of the abc
operon is choline dependent, choline was added to the AB
minimal medium at a final concentration of 0.1 mM. The
pmtA-lacZ fusion was considered a negative control since the
expression of the methylation pathway should not be depen-
dent on choline. The expression of all three genes (abc1, pcs,
and pmtA) was not altered in the presence of 0.1 mM choline
(Fig. 4) or 0.5 mM or 1 mM choline (data not shown), indi-
cating that the expression of the Pcs pathway and the putative
ABC transporter is choline independent.
We have previously shown that the deletion of pmtA and pcs
causes a drastic virulence defect (45). Genes involved in cho-
line uptake and metabolism in S. meliloti are highly expressed
under symbiotic conditions (14, 28). To further inspect the link
between virulence and PC biosynthesis in A. tumefaciens, we
tested if virulence conditions change the expression of the PC
biosynthesis genes. The addition of 0.1 mM acetosyringone, an
artificial virulence gene inducer, to the AB minimal medium
did not affect the expression of pmtA, pcs, and abc1 (data not
shown). We conclude that all three genes are constitutively
expressed and not regulated by (i) the PC content, (ii) the
availability of choline, and (iii) the presence of acetosyringone.
The abc1-4 genes do not encode a critical choline uptake
system. PC formation in an A. tumefaciens pmtA mutant de-
pends on the Pcs pathway and the presence of choline in the
growth medium (45). To address whether the predicted ABC
transporter encoded downstream of the pcs gene is responsible
for choline uptake, we monitored PC biosynthesis in a pmtA
abc1-4 double mutant grown in YEB complex medium by TLC
analysis. As expected, wild-type A. tumefaciens membranes
consist of PE, MMPE, DMPE, and PC, whereas a ?pmtA
mutant produces only PC but not the intermediates MMPE
and DMPE (Fig. 5). The ?pmtA ?pcs mutant is no longer able
to synthesize PC (45). Wild-type-like amounts of PC in the
pmtA abc1-4 deletion strain (Fig. 5) indicate that the Pcs path-
way is efficiently supplied with its substrate choline. We con-
clude that the abc1-4 genes are not responsible for choline
uptake or that additional transporters compensate for the lack
of the ABC transporter. Residual choline uptake activity has
also been reported in the absence of the high-affinity ChoX
system in S. meliloti (14). Bacillus subtilis contains two closely
related ABC transport systems for the uptake of choline as
precursor for the osmoprotectant glycine betaine (22). All
these findings suggest that bacteria might be equipped with
alternative routes for choline uptake. Based on its genome
sequence, A. tumefaciens is predicted to contain 667 compo-
nents of ABC transporters (16, 48). There clearly is potential
for alternative choline transport systems.
Membrane localization of PC. The results shown in Fig. 5
and previous TLC analyses (23, 43) suggested that the relative
PC content in A. tumefaciens membranes amounts to approx-
FIG. 4. ?-Galactosidase activities of plasmid-encoded transcrip-
tional lacZ fusions in wild-type A. tumefaciens C58 and PC biosynthesis
mutants. Cells were grown in AB minimal medium in the absence or
presence of 0.1 mM choline at 30°C. The error bars indicate standard
deviations of the results from three independent assays. The plasmid
pAC01 containing the promoterless lacZ gene was used as negative
control, and the background activity was below 3 Miller units (MU). 1,
wild-type; 2, ?pmtA; 3, ?pcs; 4, ?pmtA ?pcs.
FIG. 5. PC formation in the A. tumefaciens abc1-4 mutant. A. tu-
mefaciens wild-type, ?pmtA, ?pmtA ?pcs, and ?pmtA ?abc cells were
grown in YEB complex medium at 30°C. Lipids were extracted, sep-
arated by one-dimensional TLC, and visualized by Cu2SO4staining.
370KLU ¨SENER ET AL. J. BACTERIOL.
imately 25%. The quantification of radiolabeled phospholipids
revealed that PC levels indeed reach 23% of total phospholip-
ids (data not shown). Since it was unknown whether PC is
localized in the inner, the outer, or in both membranes of A.
tumefaciens, we isolated membranes of wild-type A. tumefa-
ciens and subsequently separated the inner and outer mem-
branes by sucrose density gradient ultracentrifugation (Fig.
6A). As reported for R. leguminosarum (12), two major lipid
fractions were visible as a diffuse cream-colored upper band
and a distinct white lower band. Measuring the protein con-
centration of the individual fractions revealed two major peaks
with maxima in fractions 3 to 7 and 19 to 21 (Fig. 6B). Both
peaks contain a clearly distinguishable collection of proteins
spanning the entire molecular-mass range (Fig. 6C). Fractions
from the inner and outer membranes were clearly distinguish-
able. To assign the peaks to a membrane compartment,
NADH oxidase activity, which is a marker enzyme for the inner
membrane, was determined. As expected, the NADH oxidase
activity was predominantly found in fractions 3 to 7 (data not
To determine the distribution of PC, membrane lipids from
pooled fractions 3 to 7 and 19 to 21 were analyzed by TLC. The
pooled inner membrane fraction mainly consists of PE and PC,
with little MMPE and only traces of DMPE (Fig. 6D). The
outer membrane is also composed of PE, MMPE, and PC.
DMPE was not visible, probably because the amount was be-
low the detection limit. We conclude that A. tumefaciens con-
tains PC in both the inner and outer membrane.
PC and PE are also the most-abundant phospholipids in the
pathogens L. pneumophila (20) and P. aeruginosa (46), and it
has been shown that they are localized in both membrane
compartments. The presence of PC is critical for adaptation to
different environmental conditions in these organisms. The
loss of PC in L. pneumophila led to reduced bacterial binding
to macrophages during the infection process (7), and PC seems
to be important for P. aeruginosa in specific responses to stress
(46). An A. tumefaciens PC-deficient mutant showed sensitivity
toward elevated temperature and SDS (45). The presence of
PC in the outer membrane, which is in direct contact with the
environment, might be important for coping with stressful con-
PC-deficient mutants are impaired in motility. Since the
lipid composition impacts bacterial fitness, we hypothesized
that PC might be of particular importance for biological pro-
cesses mediated through the membranes. The presence of PC
is required for the induction of the type IV secretion system in
A. tumefaciens (45). Another process that relies on a complex
membrane-spanning system is bacterial motility. Swimming
motility in A. tumefaciens is mediated by flagella which are
typically localized as a small tuft at or around a single cell pole
(3). To investigate the motility phenotype, the wild-type strain
and the single deletion ?pmtA and ?pcs mutants, as well as the
PC-deficient mutant (?pmtA ?pcs), were plated on soft agar
medium. Consistent with previous reports (29), A. tumefaciens
is highly motile, as indicated by the formation of large concen-
tric swim rings (Fig. 7A). Both single mutants showed swim-
ming behavior comparable to that of the wild-type (data not
shown). In contrast, the PC-deficient mutant was severely im-
paired in motility. The motility defect was partially restored
when the ?pmtA ?pcs mutant was complemented with either
pmtA or pcs expressed from low-copy-number vector pVS_P-
mtA or pVS_Pcs. It is known that many members of the Rhi-
zobiaceae are nonmotile in medium lacking divalent cations
but retain good motility in medium containing calcium, mag-
nesium, barium, or strontium (37). Therefore, we checked
whether calcium and/or magnesium could rescue the motility
defect of the double mutant and analyzed its motility in the
absence or presence of different concentrations of both ions.
Neither the addition of MgSo4nor of CaCl2restored the mo-
tility defect (data not shown). Motility was also inspected mi-
croscopically. The wild type showed normal motility, whereas
FIG. 6. Localization of PC in membranes of wild-type A. tumefa-
ciens C58. (A) Schematic representation of bands observed after cen-
trifugation. (B) Separation of agrobacterial inner and outer membrane
by discontinuous sucrose density gradient centrifugation. Fractions
were collected as 500-?l aliquots from the top of the sucrose gradient.
Protein concentrations (F) and sugar densities (Œ) of the gradient
fractions are shown. (C) Fractions 5 and 6 (IM), fraction 12, and
fractions 20 and 21 (OM) were analyzed by SDS-PAGE and visualized
with Coomassie blue staining. (D) Lipids were extracted, and fractions
3 to 7 and 19 to 21 were pooled, followed by one-dimensional TLC
analysis and molybdenum blue staining. Barely detectable lipids are
marked with arrows. PE, MMPE, DMPE, and PC were used as stan-
dards. M, phospholipid standard; IM, inner membrane; OM, outer
VOL. 191, 2009 PC BIOSYNTHESIS IN AGROBACTERIUM TUMEFACIENS371
the ?pmtA ?pcs mutant showed uncoordinated circular move-
ment (data not shown).
A L. pneumophila PC-deficient mutant has also been shown
to be impaired in motility (7). This phenotype was due to the
lack of flagellum proteins. Hence, we examined whether these
proteins are present in the agrobacterial PC-deficient mutant.
Total protein extracts of the wild type and the ?pmtA ?pcs
mutant were analyzed by Western blotting using a flagellum
protein-specific antibody raised against flagellar proteins of A.
tumefaciens (B. Scharf, personal communication). In contrast
to the L. pneumophila PC mutant, the flagellar proteins FlaA
(modified by glycosylation) (11), FlaB, FlaC, and FlaD were
detected in the A. tumefaciens PC-deficient mutant (Fig. 7B),
indicating that reduced motility is not caused by the absence of
the flagellar proteins. The presence of flagellum filaments was
confirmed by silver nitrate staining of intact cells (data not
PC influences A. tumefaciens biofilm formation. A. tumefa-
ciens forms complex biofilms on abiotic surfaces and plant
tissues, with an equilibrium between the sessile and motile
lifestyle (10). When microbes directly encounter a surface, the
outer membrane represents the interface between cell and
substratum. It has been proposed that systems presumably
involved in the cell envelope stress response, such as the Cpx or
Rcs systems of E. coli, are participating in the transition from
the planktonic to the attached life style (15, 34). In addition, it
has been shown that motility often correlates with the coloni-
zation of surfaces. An aflagellate (Fla?) E. coli mutant is de-
ficient in biofilm formation (35), and several pseudomonads
show biofilm formation and architecture dependent on flagella,
as well as type IV pili (24, 33). Therefore, we hypothesized that
changes in the composition of the outer membrane might also
influence the ability of A. tumefaciens to form microbial com-
munities. To investigate biofilm formation in the absence of
PC, we compared the wild-type and PC-deficient mutant
(?pmtA ?pcs) strains in a hydrodynamic flow chamber system
in which the attached cells were constantly provided with fresh
AB minimal medium. After 24 h, the PC-deficient mutant
formed thicker and denser communities than the wild type,
with numerous large towering structures (Fig. 8A). The differ-
ence became even more obvious after 48 h. In addition to the
enhanced structure formation, the mutant strains displayed
almost-complete surface coverage, while the wild-type commu-
nities exhibited more-unsettled surface areas (Fig. 8B). Except
for the increase in height and biomass, no alterations occurred
in the basic architecture of the structures formed by the two
strains. Cells harvested during the exponential and stationary
growth phases showed similar behavior in biofilm formation. In
summary, it appears that in the PC-deficient mutant, the equi-
librium between the sessile and motile cells is shifted more
toward the side of attachment, favoring the accumulation of
larger amounts of biomass. Consistent with our results, a sim-
ilar phenotype was described by Merritt et al. (29) for a non-
motile agrobacterial mutant (?flgE). The biofilms formed by
the ?flgE mutant showed increases in biomass, surface cover-
age, and average structure height compared to those of the
wild type. Interestingly, the ?flgE mutant biofilms grown in
static culture were highly reduced relative to those of the wild
type (29). In preliminary experiments, we detected a similar
effect for the PC-deficient mutant when grown in static culture
(data not shown). Several reasons might account for the bio-
FIG. 7. Motility assay of an agrobacterial PC-deficient mutant and
complemented strains. (A) Motility of the wild type, the PC-deficient
mutant (?pmtA ?pcs), and the mutant complemented with either
pVS_pmtA or pVS_pcs was assayed on AB minimal medium plate
containing 0.3% (wt/vol) agar. (B) Cell lysates of wild type and PC-
deficient mutant were analyzed by SDS-PAGE and Western blotting
using a flagellin antibody (1:30,000).
FIG. 8. Biofilm formation of a PC-deficient mutant of A. tumefa-
ciens C58. Flow cell biofilms of wild-type and mutant (?pmtA ?pcs)
expressing GFP were grown in AB minimal medium at 30°C. Micro-
scopic visualization of biofilms was carried out using an inverted Zeiss
LSM510 confocal laser-scanning microscope equipped with 20?/0.5 W
Achroplan objective. Image data were obtained after 24 h (A) and 48 h
(B) and further processed using the IMARIS software package (Bit-
plane AG, Zu ¨rich, Switzerland) and Adobe Photoshop.
372 KLU ¨SENER ET AL. J. BACTERIOL.
film phenotype of the ?pmtA ?pcs mutant. First, it might be
attributed to a motility defect similar to that of the ?flgE
mutant. Second, changes in the envelope stress response elic-
ited by alterations in the membrane composition might play a
role. Third, it is possible that the altered membrane composi-
tion affects biofilm matrix composition. Exopolysaccharides
and cellulose are known to influence the attachment and bio-
film formation of A. tumefaciens (9).
Although we are now beginning to appreciate the biological
significance of bacterial PC biosynthesis, we are far from un-
derstanding the molecular details of how PC influences bacte-
rial physiology and host-microbe interactions. We revealed
that PC in the membrane of A. tumefaciens plays a critical role
in seemingly diverse processes, such as stress response, motil-
ity, social behavior, and virulence. Further studies will be di-
rected toward revealing the underlying mechanisms of PC ac-
tion in bacterial membranes.
We are grateful to Ehr-Min Lai for plasmid pAC01, Yun-long Tsai
for advice on membrane separation, and Birgit Scharf for antiflagella
sera. We thank Christiane Fritz for excellent technical assistance,
Stephanie Hacker for quantitative phospholipid analysis, Robbin
Stantscheff for mutant construction, and Bernd Masepohl for helpful
comments on the manuscript.
The work was in part supported by a grant from the German Re-
search Foundation (DFG; SFB 480) to F.N. and a fellowship from the
Promotionskolleg of the Ruhr-University Bochum to M.A.
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374KLU ¨SENER ET AL. J. BACTERIOL.