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REVIEW ARTICLE
published: 26 March 2014
doi: 10.3389/fpls.2014.00109
Membrane lipids in Agrobacterium tumefaciens:
biosynthetic pathways and importance for pathogenesis
Meriyem Aktas, Linna Danne, Philip Möller and Franz Narberhaus*
Microbial Biology, Department for Biology and Biotechnology, Ruhr University Bochum, Bochum, Germany
Edited by:
Erh-Min Lai, Academia Sinica,Taiwan
Reviewed by:
Christian Sohlenkamp, Universidad
Nacional Autónoma de México,
Mexico
Georg Hölzl, University Bonn,
Germany
*Correspondence:
Franz Narberhaus, Microbial Biology,
Department for Biology and
Biotechnology, Ruhr University
Bochum, Universitätsstrasse 150,
NDEF 06/783, 44780 Bochum,
Germany
e-mail: franz.narberhaus@rub.de
Many cellular processes critically depend on the membrane composition. In this review, we
focus on the biosynthesis and physiological roles of membrane lipids in the plant pathogen
Agrobacterium tumefaciens.The major components of A. tumefaciens membranes are the
phospholipids (PLs), phosphatidylethanolamine (PE), phosphatidylglycerol, phosphatidyl-
choline (PC) and cardiolipin, and ornithine lipids (OLs). Under phosphate-limited conditions,
the membrane composition shifts to phosphate-free lipids like glycolipids, OLs and a
betaine lipid. Remarkably, PC and OLs have opposing effects on virulence ofA. tumefaciens.
OL-lacking A. tumefaciens mutants form tumors on the host plant earlier than the wild
type suggesting a reduced host defense response in the absence of OLs. In contrast,
A. tumefaciens is compromised in tumor formation in the absence of PC. In general, PC is a
rare component of bacterial membranes but amount to ∼22% of all PLs in A. tumefaciens.
PC biosynthesis occurs via two pathways. The phospholipid N-methyltransferase PmtA
methylates PE via the intermediates monomethyl-PE and dimethyl-PE to PC. In the second
pathway, the membrane-integral enzyme PC synthase (Pcs) condenses choline with CDP-
diacylglycerol to PC. Apart from the virulence defect, PC-deficient A. tumefaciens pmtA
and pcs double mutants show reduced motility, enhanced biofilm formation and increased
sensitivity towards detergent and thermal stress. In summary, there is cumulative
evidence that the membrane lipid composition of A. tumefaciens is critical for agrobacterial
physiology and tumor formation.
Keywords: membrane lipids, phospholipid biosynthesis, phosphatidylcholine, phosphorus-free lipids, ornithine
lipids, glycolipids, betaine lipids, Agrobacterium tumefaciens
INTRODUCTION
The structure of biological membranes is mainly defined by
heterogeneous amphipathic phospholipids (PLs) forming the
phospholipid bilayer. PLs contain a diacylglycerol (DAG) as
hydrophobic component with saturated or unsaturated fatty acyl
chains of variable length and a polar head group attached to
the phosphate group (Korn, 1966;van Meer et al., 2008;Wolf
and Quinn, 2008). The general structure of PLs and common
head groups are shown in Figure 1. Phosphatidylethanolamine
(PE) and phosphatidylcholine (PC) are zwitterionic lipids whereas
phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin
(CL), phosphatidylserine (PS), and phosphatidylinositol (PI)
represent the anionic lipid class. Contrary to previous assump-
tions based on the fluid mosaic model (Singer and Nicolson,
1972), the lipid distribution in pro- and eukaryotic mem-
branes is dynamic and asymmetric (Fadeel and Xue, 2009;
Clifton et al., 2013). Specialized lipid micro domains (in eukary-
otes referred to as lipid rafts) serve as platform for various
cellular processes such as signal transduction and transport
(Edidin, 2003;Zhang et al., 2005;Pike, 2006;Donovan and
Bramkamp, 2009;Lingwood and Simons, 2010;LaRocca et al.,
2013).
All biological membranes share the same basic membrane
structure but the lipid composition differs tremendously between
the domains of life and even within a domain. The lipid repertoire
of eukaryotic cells is very complex. Combination of different head
groups and variations in fatty acid tails results in more than a
thousand different lipids. The major lipids in eukaryotes are PLs
with PC as the most abundant, followed by PE, PS, PI, and PA
(van Meer et al., 2008). PG is also present in eukaryotes and is
used as precursor for CL synthesis, exclusively found in mitochon-
dria (Bligny and Douce, 1980). Further important constituents of
eukaryotic membranes are sphingolipids (SLs) and cholesterol,
which are enriched in lipid rafts (Lingwood and Simons, 2010;
Sonnino and Prinetti, 2013).
Bacterial membrane lipids are more diverse than previously
thought (Parsons and Rock, 2013). Most bacteria, like the Gram-
negative model organism Escherichia coli have a simple membrane
lipid composition with the major PLs PE, PG, and CL (Ames, 1968;
Cronan, 2003;López-Lara et al., 2003;Dowhan, 2009). However,
many other bacteria are known to produce additional and uncom-
mon lipids. PS is abundant in eukaryotic membranes but most
prokaryotes contain only minor PS amounts as it serves as precur-
sor for PE biosynthesis (Bunn and Elkan, 1971;López-Lara et al.,
2003). Although PI is a rare component of bacterial membranes
it is a major lipid in Mycobacterium tuberculosis where it is essen-
tial for viability (Jackson et al., 2000). SLs have been described in
Sphingobacterium, Sphingomonas, and Bacteroides species (Heung
et al., 2006). Sphingomonas paucomobilis contains two glyco-SLs
in its outer membrane important for pathogenesis (Kinjo et al.,
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Aktas et al. Membrane lipids in Agrobacterium tumefaciens
FIGURE 1 |General structure of phospholipids and common head
groups. PLs contain two fatty acids ester-linked to glycerol at C-1 and C-2,
and a polar head group attached at C-3 via a phosphodiester bond.The fatty
acids in PLs can vary in carbon group length and saturation degree. The
different common polar head groups and charges are indicated. PA,
phosphatidic acid; PE, phosphatidylethanolamine; PC, phosphatidylcholine;
PS, phosphatidylserine; PG, phosphatidylglycerol; CL, cardiolipin; PI,
phosphatidylinositol.
2005;Mattner et al., 2005). Some bacteria such as Methylococcus
capsulatus or Rhodopseudomonas palustris TIE-1 can also syn-
thesize steroid lipids and/or sterol homologues (hopanoid lipids;
Tippelt et al., 1998;Bode et al., 2003;Doughty et al., 2011). The
membrane of the Gram-positive model organism Bacillus sub-
tilis comprises lysyl-PG (LPG) and up to 40% neutral glycolipids
(GLs; Salzberg and Helmann, 2008). In some bacteria such as
Agrobacterium tumefaciens,Sinorhizobium meliloti, and Rhodobac-
ter spaeroides phosphate limitation stimulates the production of
phosphate-free lipids including ornithine lipids (OLs), sulfolipids,
betaine lipids, and GLs (López-Lara et al., 2003,2005;Vences-
Guzmán et al., 2012;Geske et al., 2013;Parsons and Rock, 2013).
The major eukaryotic membrane lipid PC is not widespread in
bacteria. It has been estimated that ∼15% of all bacterial species
produce PC (Sohlenkamp et al., 2003;Aktas et al., 2010;Geiger
et al., 2013). It is frequently found in symbionts or pathogens and
in bacteria with extensive intracytoplasmic membranes (Hagen
et al., 1966;Goldfine, 1984;Geiger et al., 2013). Often, PC is critical
for bacteria–host interactions.
COMMON METABOLIC PATHWAYS FOR PHOSPHOLIPIDS IN
BACTERIA
All major PLs in bacteria are formed from a common precursor,
namely cytidine diphosphate diacylglycerol (CDP-DAG) gener-
ated by a CDP-DAG synthase (CdsA) using PA and cytidine
triphosphate (CTP; Figure 2;Zhang and Rock, 2008;Parsons
FIGURE 2 |Phospholipid pathways and enzymes in bacteria. CDP-DAG
is the central precursor for synthesis of the PLs. Thick arrows and boldface
letters indicate the most common pathways and enzymes in bacteria. For
details see text. CMP, cytidine monophosphate; CTP, cytidine triphosphate;
EA, ethanolamine; cho, choline; G3P, glycerol 3-phosphate; gly, glycerol; lys,
lysine; L-ser, L-serine; myo-I-P, myo-inositol 1-phosphate; SAM,
S-adenosylmethionine; SAH, S-adenosylhomocysteine.
and Rock, 2013). CDP-DAG can be directly converted to PS, PG
phosphate (PGP) or in some bacteria to PI phosphate (PIP) and
PC. These reactions are catalyzed by specific CDP-alcohol phos-
phatidyltransferases releasing a CMP molecule from CDP-DAG
and transferring the phosphatidyl moiety to different polar head
groups (Sohlenkamp et al., 2003;Parsons and Rock, 2013). PS syn-
thases (Pss) use L-serine as the phosphatidyl acceptor to generate
the anionic lipid PS, which serves as precursor for PE synthesis via
PS decarboxylases (Psd). In mycobacteria, a PIP synthase (Pips)
converts CDP-DAG and myo-inositol 1-phosphate to PIP which is
dephosphorylated via a PIP phosphatase (Pipp) to PI (Morii et al.,
2010;Morii et al., 2014). PG synthases (Pgs) transfer the phos-
phatidyl group from CDP-DAG to a glycerol-3-phosphate (G3P)
resulting in PGP, which serves as precursor for PG synthesis by
PGP phosphatases (Pgp). Two PG molecules are condensed via a
cardiolipin synthase (Cls) to CL. Most bacteria possess more than
one Cls. E. coli encodes three Cls with distinct specificities. ClsA
uses two PG molecules for CL formation whereas ClsC condenses
a PE and PG molecule to CL. Like the other Cls enzymes, ClsB
utilizes PG but the second substrate is unknown (Pluschke et al.,
1978;Nishijima et al., 1988;Guo and Tropp, 2000;Tan et al., 2012).
In Streptomyces coelicolor a eukaryotic-type Cls using CDP-DAG
and PG for CL synthesis was identified. This enzyme belongs to
the CDP-alcohol phosphatidyltransferase family and seems to be
common in actinobacteria (Sandoval-Calderón et al., 2009).
In several Gram-positive bacteria such as Staphylococcus aureus
and Bacillus subtilis, PG is converted to the positively charged lipid
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Aktas et al. Membrane lipids in Agrobacterium tumefaciens
LPG by aminoacylation using lysyl-tRNA as the lysine donor by
the Mprf (multiple peptide resistant factor) enzyme (Ernst and
Peschel, 2011). In Staphylococcus aureus, LPG confers resistance
towards cationic antimicrobial peptides (CAMPs) by perturbation
of the electrostatic attraction of CAMPs (Kilelee et al., 2010;Andrä
et al., 2011). MprF homologs namely LpiA (low pH inducible A)
are also present in some Gram-negative bacteria such as Rhi-
zobium tropici and Sinorhizobium medicae and confer tolerance
to acid stress and selected cationic peptides (Reeve et al., 2006;
Sohlenkamp et al., 2007).
Two common PC synthesis pathways operate in bacteria: the
PE-methylation pathway and the PC synthase (Pcs) route. Several
bacteria contain both PC synthesis pathways such as A. tume-
faciens and S. meliloti. However, some species like Rhodobacter
sphaeroides or Zymomonas mobilis only have the methylation
pathway for PC synthesis. Some important pathogens including
Borrelia burgdorferi,Brucella abortus, or Pseudomonas aerugi-
nosa only possess the Pcs pathway (Martínez-Morales et al., 2003;
Sohlenkamp et al., 2003;Aktas et al., 2010;Geiger et al., 2013).
In the methylation pathway, one or several phospholipid N-
methyltransferase (Pmt) enzymes transfer a methyl group from
S-adenosylmethionine (SAM) to the amino group of PE generat-
ing the intermediates monomethyl-PE (MMPE) and dimethyl-PE
(DMPE) and finally PC (Figure 2). The methyldonor SAM is con-
verted to S-adenosylhomocysteine (SAH) during this reaction. In
the bacteria-specific Pcs pathway, choline is condensed with CDP-
DAG to PC releasing a CMP molecule (Sohlenkamp et al., 2000;
Aktas et al., 2010;Solís-Oviedo et al., 2012;Geiger et al., 2013).
A eukaryotic-like CDP-choline pathway has been postulated in
Treponema denticola (Kent et al., 2004) and might be also present
in other Treponema species. This pathway involves a choline kinase
(LicA) generating choline phosphate which serves as substrate
for a CTP: phosphocholine cytidylyl transferase (LicC) to pro-
duce CDP-choline. In the final step, PC is formed by transferring
the phosphocholine moiety to DAG by a CDP-choline transferase
(CPT; Kent et al., 2004;Geiger et al., 2013).
Recently, a new PC biosynthesis route was discovered in Xan-
thomonas campestris, which produces PC via a yeast-like two-step
acylation of the precursor glycerophosphocholine (Moser et al.,
2014) demonstrating that quite different strategies acting on the
head or tail group have evolved for PC synthesis in bacteria.
Following this general information, the remainder of this
review will present an overview of biosynthetic pathways and
enzymes for membrane lipids in the plant pathogen A.tumefa-
ciens and discuss the physiological relevance of those lipids in this
organism.
MEMBRANE LIPID REPERTOIRE AND PHOSPHOLIPID
BIOSYNTHESIS ENZYMES IN A. tumefaciens
Agrobacterium membranes contain a rich setup of polar lipids
(Randle et al., 1969;Das et al., 1979;Thompson et al., 1983;
Vences-Guzmán et al., 2013;Moser et al., 2014). The lipid reper-
toire of several Agrobacterium strains has been quantified. Under
full nutrition, A. tumefaciens membranes are mainly composed
of the PLs PE and PG (account together ∼45%), PC (∼22%),
CL (∼15%), MMPE (∼15%) and traces of DMPE (∼4%; Moser
et al., 2014). Two-dimensional thin layer chromatography and
mass spectrometry analysis revealed that A. tumefaciens mem-
branes also contain two OLs (Geske et al., 2013;Vences-Guzmán
et al., 2013). A broad variety of membrane lipids in this organism
is reflected by a lysine-containing lipid with a backbone structure
similar to OLs (Tahara et al., 1976). Most of the PL synthesis path-
ways and enzymes in Agrobacterium, except for PC synthesis, are
still uncharacterised. However, with the exception of a pgp gene,
homologs for all common PL biosynthesis genes described above
are encoded in the A. tumefaciens genome (Figure 3;Wood et al.,
2001).
The putative A. tumefaciens pss (atu1062) gene is homol-
ogous to the pss gene from the non-pathogenic, high beta-
1,3-glucan (curdlan) producing Agrobacterium sp. ATCC31749
(Karnezis et al., 2002;Ruffing et al., 2011). Functional analy-
sis of its recombinant Pss protein in E. coli demonstrated a
Mn2+-dependent [3H]serine incorporation into a chloroform-
soluble product, most likely PS. Localisation studies in E. coli and
topology predictions suggest that Pss is an integral membrane
protein of ∼30 kDa with eight transmembrane domains (TM).
A cytosolic loop connecting the second and third TM contains a
conserved motif (DX2DGX2ARX5S/TX2GX3DSX2D) characteris-
tic for amino alcohol phosphatidyltransferases and thought to be
involved in catalysis. A pss mutant is unable to produce PE sug-
gesting that PE synthesis exclusively occurs via decarboxylation
of PS. Loss of PE seems to be compensated by increased PG and
CL levels in the pss mutant. Interestingly, the PE-deficient mutant
is dramatically reduced in curdlan production and grows poorly
in minimal medium. This growth defect can be compensated by
Mg2+ions, which presumably stabilize the membrane. However,
curdlan production of the mutant strain cannot be cured by Mg2+.
FIGURE 3 |Phospholipid synthesis in A. tumefaciens. Characterized
pathways are indicated by thick arrows and enzymes in boldface letters.
Dashed arrows indicate the putative pathways. For details see text.
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Aktas et al. Membrane lipids in Agrobacterium tumefaciens
PE seems to be required for proper assembly and function of the
integral inner membrane protein curdlan synthase as shown for
several other membrane proteins (Karnezis et al., 2002,2003;Raja,
2011;Bogdanov and Dowhan, 2012).
A. tumefaciens codes for a putative LPG synthase (Atu2521,
LpiA) but LPG has not yet been identified in this organism. LPG
is a major lipid in some Gram-positive bacteria but only low lev-
els are formed in Gram-negatives. Transcription of the related
lpiA gene in S. medicae is activated at low pH and is required for
survival during acid stress. However, LPG was not detected even
under acidic conditions in this organism suggesting production
of very small amounts or rapid turnover of LPG (Reeve et al.,
2006). Small amounts of LpiA-produced LPG were detected in
R. tropici CIAT899 (∼1% of the total lipids) in low pH minimal
medium. Here, LPG confers resistance against the cationic peptide
polymyxin B under acidic growth conditions (Sohlenkamp et al.,
2007). Interestingly, lpiA/mprF homologs are present in many bac-
teria interacting with eukaryotes such as symbionts, pathogens and
commensals suggesting that LPG might be important for bacteria–
host interactions (Vinuesa et al., 2003;Sohlenkamp et al., 2007).
Since low pH is one of the signals inducing virulence factors in A.
tumefaciens, it will be of great interest to determine whether lpiA
contributes to Agrobacterium pathogenesis.
THE METHYLATION PATHWAY IN A. tumefaciens
The two PC biosynthesis pathways and corresponding enzymes
(Pcs and PmtA) in A. tumefaciens have been well characterized
(Figure 3). Initial work on PC synthesis in Agrobacterium demon-
strated incorporation of the 14 C-methyl moiety of SAM into
MMPE, DMPE, and PC and 14 C-choline uptake and incorpora-
tion into PC (Kaneshiro and Law, 1964;Sherr and Law, 1965;
Kaneshiro, 1968). In earlier studies, two distinct Pmts were postu-
lated in A. tumefaciens. A soluble Pmt catalyzing MMPE formation
only and a Pmt associated with the particulate cell fraction pro-
ducing all methylated PE-derivatives (Kaneshiro and Law, 1964).
The A. tumefaciens genome, however, contains only a single con-
stitutively expressed pmt gene (pmtA,atu0300) on the circular
chromosome (Wessel et al., 2006;Klüsener et al., 2009). The lack
of MMPE, DMPE, and PC in a pmtA mutant grown without
choline demonstrated that PmtA is the only enzyme responsible
for MMPE, DMPE, and PC synthesis via the methylation pathway
(Wessel et al., 2006). Purification of recombinant PmtA from the
soluble cell fraction suggests that it is a peripheral membrane pro-
tein reversibly attaching to its site of action, the membrane (Aktas
and Narberhaus, 2009;Aktas et al., 2011a). PmtA is a monomeric
small enzyme (∼22 kDa) catalyzing the methylation of PE to
MMPE, DMPE, and PC. In vitro lipid binding experiments with
PmtA revealed strong binding to the anionic lipids PI and PG.
Interestingly, overall PmtA activity is stimulated by PG. Associ-
ation of peripheral proteins with membranes is often mediated
via electrostatic interactions with negatively charged PLs such as
PG and a similar mechanism is proposed for the A. tumefaciens
PmtA enzyme (Figure 4). SAM binding by PmtA occurs only in
the presence of its substrates PE, MMPE, DMPE or the end prod-
uct PC. PG alone does not influence SAM binding suggesting that
two distinct binding sites for its substrates or products and for PG
exist (Aktas and Narberhaus, 2009).
FIGURE 4 |Phosphatidylcholine biosynthesis in A. tumefaciens. In the
PC synthase pathway, the integral membrane protein Pcs condenses
CDP-DAG and choline to PC. Choline is taken up via the ChoXWV
transporter. In the PmtA pathway, a single peripheral phospholipid
N-methyltransferase (PmtA) converts PE via three successive methylations
to PC. PmtA is stimulated by the anionic lipid PG and inhibited by PC and
SAH. CM, cytoplasmic membrane.
In vitro PmtA activity is negatively regulated by the end prod-
ucts SAH (via interfering with SAM binding) and by PC. End
product-mediated inhibition might also be relevant in vivo to bal-
ance proper lipid composition (Aktas and Narberhaus, 2009). Like
all Pmt enzymes, PmtA contains a highly conserved N-terminal
SAM binding motif [VL(E/D)XGXGXG] (Sohlenkamp et al.,
2003). Within this motif, the amino acids E58, G60, G62, and E84
were found to be essential for activity and SAM binding (Aktas
et al., 2011a). A. tumefaciens PmtA seems to follow an ordered Bi–
Bi reaction mechanism with initial substrate binding followed by a
conformational change allowing SAM binding. Subsequently, the
methyl group might be transferred to the lipid substrate releasing
the first product SAH followed by the release of the methylated
lipid product (Aktas et al., 2010).
Bacterial Pmts are classified into Sinorhizobium and Rhodobac-
ter type enzymes. Enzymes belonging to the Sinorhizobium family
including A. tumefaciens PmtA, are homologous to rRNA methy-
lases, whereas Rhodobacter-like Pmt enzymes are similar to UbiE,
ubiquinone/menaquinone biosynthesis methyltransferases. Sim-
ilarities between these two Pmt families are restricted to the
conserved SAM-binding motif (Sohlenkamp et al., 2003;Aktas
et al., 2010;Geiger et al., 2013). The product spectrum of Pmt
enzymes varies in different organisms. While A. tumefaciens and
S. meliloti pmtA release small amounts of the intermediates MMPE
and DMPE, expression of R. sphaeroides pmtA in E. coli exclusively
resulted in PC formation (Arondel et al., 1993;de Rudder et al.,
2000;Klüsener et al., 2009). The Sinorhizobium type PmtA from
X. campestris produces MMPE exclusively and is unable to further
methylate it to DMPE and PC (Moser et al., 2014).
Most bacteria contain one Pmt enzyme for all three methylation
steps but in some cases several Pmts with different specifici-
ties are required (Sohlenkamp et al., 2003). In the soybean
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Aktas et al. Membrane lipids in Agrobacterium tumefaciens
symbiont Bradyrhizobium japonicum, PmtA methylates PE to
MMPE, which serves as substrate for PmtX1-catalyzed methy-
lation to DMPE and PC. B. japonicum encodes three further
Pmt enzymes with distinct specificities (PmtX2-4), which are
not expressed under standard laboratory conditions. PmtX1 and
PmtX2 are similar to R. sphaeroides PmtA, whereas PmtA, PmtX3
and PmtX4 are homologous to S. meliloti PmtA (Minder et al.,
2001;Hacker et al., 2008a,b). Like B. japonicum,Rhizobium legu-
minosarum,Rhodopseudomonas palustris, and Rhizobium etli seem
to encode more than one pmt homolog (López-Lara et al., 2003;
Martínez-Morales et al., 2003).
THE PC SYNTHASE PATHWAY: A MEMBRANE-INTEGRATED ENZYME
USES EXOGENOUS CHOLINE FOR PC SYNTHESIS
The second PC synthesis pathway in A. tumefaciens is catalyzed by
the Pcs enzyme (Figure 4). Like pmtA, the pcs gene (atu1793)is
located on the circular chromosome and is constitutively expressed
(Wessel et al., 2006;Klüsener et al., 2009;Wilms et al., 2012).
Pcs uses exogenous choline, which is transported via the high-
affinity choline transport system ChoXWV. A choXWV-deficient
strain is largely impaired in choline transport but can still pro-
duce PC when choline is present suggesting alternative choline
uptake systems in A. tumefaciens (Aktas et al., 2011b). Similar to
A. tumefaciens, the Pcs pathway in S. meliloti and B. abortus rely
on exogenous choline delivered by a homologous Cho transport
system (de Rudder et al., 1999;Dupont et al., 2004;Herrmann
et al., 2013). Choline is a major component of eukaryotic mem-
branes liberated by phospholipases from PC. Large amounts of free
choline is found in homogenized plant tissues (Zeisel et al., 2003)
and a recent study showed that considerable choline pools are
also present on leaf surfaces. Pseudomonas syringae produces PC
exclusively via the Pcs pathway and contains three choline trans-
port systems with different specificities (Chen and Beattie, 2008).
P. syringae exhibits chemotaxis towards choline and other quater-
nary amines. Extracellular choline is scavenged by P. syringae and
enhances fitness during leaf colonization (Chen et al., 2013).
An A. tumefaciens pcs mutant produces PC via the remaining
PmtA pathway and conversely PC production in a pmtA mutant
depends on extracellular choline which might be delivered by the
host plant. Only a pmtA/pcs double mutant lacks PC exclud-
ing alternative PC synthesis pathways in this organism (Wessel
et al., 2006). Both A. tumefaciens PC biosynthesis pathways can be
functionally reconstituted in E. coli demonstrating that PmtA and
Pcs do not require A. tumefaciens specific cofactors or substrates
(Klüsener et al., 2009).
The best-characterized Pcs enzyme derives from S. meliloti
(de Rudder et al., 1999;Sohlenkamp et al., 2000;Solís-Oviedo
et al., 2012). It catalyses the transfer of a phosphatidyl group
from CDP-DAG to choline releasing a CMP molecule and PC.
Enzyme activity depends on divalent cations like Mn2+or
Mg2+and on detergents such as triton X100 (de Rudder et al.,
1999). A topological study suggested that sinorhizobial Pcs is
an integral membrane protein containing eight TM with N-
and C- termini located in the cytosol. Pcs is a member of the
CDP-alcohol phosphotransferase (CDP-OH-PT) protein super-
family containing a modified version of a conserved CDP-OH-PT
motif (DX2DGX2ARX12GX3GX3D) characteristic for this enzyme
family. Most of the conserved amino acids are located within a
cytosolic loop connecting the TM domains II and III and are crit-
ical for enzyme activity as shown via mutagenesis (Solís-Oviedo
et al., 2012;Geiger et al., 2013). Since the membrane-bound nature
of Pcs enzymes has precluded their purification and biochemical
characterisation, the precise reaction mechanism of Pcs enzymes
is presently unknown but most likely proceeds via a sequential
Bi–Bi reaction as in other CDP-OH-PT enzymes (Geiger et al.,
2013).
It is not clear why two PC biosynthesis pathways operate simul-
taneously in Agrobacterium and some other bacteria. Although
the Pcs pathway is energetically more favorable than the PE-
methylation route, under conditions of choline limitation during
competition with other bacteria, the Pmt pathway might be ben-
eficial. In Agrobacterium both pathways seem to be constitutively
present. PmtA activity is detected even in the presence of choline,
when Pcs is active (Wessel et al., 2006;Klüsener et al., 2009). When
two alternative PC synthesis pathways are present in eukaryotes,
PC production via PE-methylation is repressed in the presence
of choline used by the CDP-choline dependent pathway (Vance
and Ridgway, 1988;Vance et al., 1997). It remains to be examined
whether PmtA and Pcs pathways produce distinct PC pools with
different fatty acyl chains as it is the case in eukaryotes (DeLong
et al., 1999). Clearly, PC biosynthesis in Agrobacterium deserves
further studies.
NON-PHOSPHORUS LIPIDS AND BIOSYNTHETIC PATHWAYS
Since inorganic phosphate is limiting in most soils, bacteria have
evolved exquisite strategies to deal with phosphate deficiency. One
strategy is to partially replace membrane PLs by phosphorus-
free lipids as shown for S. meliloti,Pseudomonas fluorescens,
R. sphaeroides, and A. tumefaciens. Various phosphorus-free lipids
appear in these organisms upon phosphate limitation such as
sulfolipids, GLs, betaine lipids, or OLs (Benning et al., 1995;
López-Lara et al., 2003;Geiger et al., 2010;Zavaleta-Pastor et al.,
2010;Geske et al., 2013).
The A. tumefaciens-related α-proteobacterium S. meliloti has
served as model system in this context. Its membranes are
composed of the PLs PG, PE, MMPE, and PC when grown
under phosphate-rich conditions. Phosphate limitation triggers
the degradation of PE, MMPE, and PC and accumulation of the
phosphate-lacking lipids DGTS-(N,N,N,-trimethyl)homoserine
(DGTS), sulfoquinovosyl-DAG (SQD), and OLs. Phosphate-
dependent membrane remodeling is regulated by the PhoR-PhoB
system: under phosphate-limitation, the response regulator PhoB
activates expression of genes responsible for OL and DGTS syn-
thesis and for synthesis of an intracellular phospholipase C (PlcP).
PlcP degrades the PLs PE, MMPE, and PC to the correspond-
ing phosphoalcohols and DAG. Inorganic phosphate is released
from the phosphoalcohols by yet unknown phosphatases and is
used as a source for essential phosphate-dependent biological pro-
cesses. The released DAG serves as substrate for the formation of
the non-phosphorus lipids DGTS and SQD (Geiger et al., 1999;
Zavaleta-Pastor et al., 2010).
Ornithine lipids are fatty-acylated amino acids free of phos-
phate and glycerol. The non-proteinogenic amino acid ornithine
is connected via its α-amino group to a 3-hydroxy fatty acid and
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Aktas et al. Membrane lipids in Agrobacterium tumefaciens
FIGURE 5 |Structure of the ornithine lipids OLS1/OLS2 and
diacylglycerol trimethylhomoserine (DGTS) in A. tumefaciens.The OLs
contain C16 3OH and C19:0 cyclo fatty acids. OLS2 is hydroxylated within
the ornithine moiety. DGTS contains 18:1 and 19:0 cyclo fatty acids (Geske
et al., 2013;Vences-Guzmán et al., 2013).
a second fatty acid chain is esterified to the 3-hydroxy group of
the first fatty acid (Figure 5). OLs are widely distributed among
eubacteria but absent from archaea and eukaryotes. Biosynthesis
of OLs occurs via an acyl-ACP dependent two-step acylation of
ornithine by two different acyltransferases. The first OL acyltrans-
ferases were discovered in S. meliloti (Weissenmayer et al., 2002;
Gao et al., 2004). Acylation of ornithine occurs here via OlsB at
the α-amino group to form lyso-ornithine (LOL), which in turn
is acylated by OlsA at the 3-hydroxyl group to form OL (Gao
et al., 2004;Geiger et al., 2013). Some bacteria modify their OLs by
hydroxylationof the ornithine moiety or the ester- or amide-linked
fatty acid. Three different OL hydroxylases are known in bacteria
so far. OlsE homologs hydroxylate the ornithine moiety and the
fatty acid portion is hydroxylated by OlsD (amide-linked) or OlsC
(ester-linked) hydroxylases (Geiger et al., 2010;González-Silva
et al., 2011;Vences-Guzmán et al., 2012). Several studies showed
a contribution of hydroxylated OLs in microbe–host interactions
and pH or thermal stress resistance (Rojas-Jiménez et al., 2005;
González-Silva et al., 2011;Vences-Guzmán et al., 2011,2012). It
has been suggested that the additional hydroxyl groups increase the
interaction between lipids via hydrogen bonds and thus, decrease
the membrane fluidity and permeability, which might be advan-
tageous under different stress conditions (Geiger et al., 2013). A
recent study revealed a modification of OLs via methylation of the
ornithine head group to mono-, di- and trimethyl-OL in planc-
tomycetes isolated from an acidic and nutrient-poor ecosystem
(Moore et al., 2013). Methylation of OLs increases their polarity
and confers a more cylindrical shape, which possibly increases
membrane stability similar to the bilayer forming lipid PC. There-
fore, producing methyl-OLs might be an adaptation strategy to
cope with acidity and nutrient scarcity in these organisms (Moore
et al., 2013).
FIGURE 6 |Phosphorus-free lipid synthesis pathways in A.
tumefaciens. (A) Characterized pathways are indicated by thick arrows and
enzymes in boldface letters. Putative biosynthetic pathways/enzymes are
indicated with dashed arrows (Geske etal., 2013;Vences-Guzmán et al.,
2013;Semeniuk et al., 2014). For details see text. (B) Relative proportion of
phosphorus-free lipids in A. tumefaciens C58C1 under phosphate-replete
(+P) and phosphate depleted (−P) conditions (Geske et al., 2013). Gal,
galactose; Glu, glucose; Glca, glucoronic acid.
TWO ORNITHINE LIPIDS ARE SYNTHESIZED IN A. tumefaciens
In contrast to S. meliloti and R. sphaeroides, which produce only
minor amounts of OLs under phosphate-replete conditions, A.
tumefaciens accumulates significant amounts of two different OLs
namely OLS1 and OLS2 even under full nutrient supply (Geske
et al., 2013;Vences-Guzmán et al., 2013). In A. tumefaciens the
OLs are composed of the fatty acids C16 3OH and C19:0 cyclo
as shown by mass spectrometry analyses. OLS2 is the hydrox-
ylated form of OLS1 containing the hydroxyl group within the
ornithine moiety (Geske et al., 2013;Vences-Guzmán et al., 2013;
Figure 5). Agrobacterium encodes the three ols homologs olsA
(atu0355), olsB (atu0344), and olsE (atu0318) on the circular chro-
mosome (Vences-Guzmán et al., 2012). olsE and olsB mutants in
the A. tumefaciens A208 strain revealed that olsB is essential for
formation of both OLS1 and OLS2 whereas olsE is only required
for OLS2 synthesis (Figure 6). Heterologous expression of olsE
resulted in OLS2 formation providing further evidence that OlsE
is the hydroxylase responsible for OLS2 formation. Thus, the first
step in OL synthesis in Agrobacterium is mediated by the acyl-
transferase OlsB forming ornithine to the lyso-ornithine lipid
(LOL). Subsequently, LOL might be acylated via the putative
OlsA to form OLS1. OLS2 formation is completed by hydrox-
ylation of the ornithine moiety by OlsE (Vences-Guzmán et al.,
2013).
Under low phosphate conditions, both OLs accumulate to a
total amount of 45–50% whereas the total PL content decreases in
A. tumefaciens A208. A putative Pho box is located in the promoter
region of olsB suggesting PhoB-induced expression under phos-
phate starvation (Geske et al., 2013;Vences-Guzmán et al., 2013).
In the olsB mutant, lack of OLs seems to be compensated by an
Frontiers in Plant Science |Plant-Microbe Interaction March 2014 |Volume 5 |Article 109 |6
Aktas et al. Membrane lipids in Agrobacterium tumefaciens
increase in DGTS and GL accumulation under phosphate reduced
conditions (Vences-Guzmán et al., 2013).
Ornithine lipids production under phosphate starvation condi-
tions seems to vary in different Agrobacterium strains. In contrast
to A. tumefaciens A208, the total amount of OLs does not change
under phosphate-limiting conditions in A. tumefaciens C58C1 but
the degree of hydroxylation is ninefold increased (Geske et al.,
2013;Vences-Guzmán et al., 2013). Deviations in the experimen-
tal setups such as the growth media and the precise phosphate
concentrations might account for these differences.
A. tumefaciens PRODUCES FOUR DIFFERENT GLYCOLIPIDS AND A
BETAINE LIPID UNDER PHOSPHATE DEPRIVATION
Glycolipids contain carbohydrateresidues, which are glycosidically
bound to the 3-position of a sn-1,2-DAG (Shaw, 1970). Different
GLs are produced in bacteria under phosphate starvation. The
photosynthetic bacterium R. sphaeroides produces the unique GL
glucosylgalactosyl-DAG (GGD) with α-glucose (1−→ 4)-linked
to β-galactose (Benning et al., 1995). A series of GLs found in
the nitrogen-fixing symbiont Mesorhizobium loti differs from the
rhodobacterial GL. M. loti produces the GLs GGD, digalactosyl-
DAG (DGD), and different molecular species of triglycosyl-DAG
with various combinations of galactose and glucose in the head.
All of the sugars are in β-configuration and (1−→ 6)-linked to
each other. Additionally, M. loti contains two further GLs with yet
unknown head groups (Devers et al., 2011).
A. tumefaciens produces under phosphate deprivation four dif-
ferent GLs and DGTS accounting to 35% of the total lipids (Geske
et al., 2013). The GLs have been identified recently as GGD and
DGD with a β-configuration and monoglucosyl-DAG (MGlcD)
and glucuronosyl-DAG (GlcAD) with a α-configuration (Geske
et al., 2013;Semeniuk et al., 2014). The relative amount of these
lipids in A. tumefaciens C58C1 is given in Figure 6B. Similar to
M. loti, GGD and DGD are synthesized in A. tumefaciens viaapro-
cessive glycosyltransferase namely Pgt (Figure 6) by a successive
transfer of glucosyl and/or galactosyl residues to DAG. Functional
characterisation of Pgt in E. coli and Pichia pastoris and overex-
pression in Agrobacterium revealed a broad substrate specificity
concerning the glycosyl acceptor (DAG or ceramides) and sugar
residues (uridine diphospho, UDP-galactose or UDP-glucose).
However, Pgt favors DAG over ceramide and UDP-galactose over
UDP-glucose (Hölzl et al., 2005). The promoter region of pgt con-
tains a predicted Pho box suggesting an induced Pgt synthesis upon
phosphate limitation mediated via the PhoR-PhoB system. A pgt
mutant lacks GGD and DGD but the remaining lipids accumulate
wild type-like (Geske et al., 2013).
Synthesis of MGlcD and the acidic GlcAD in A. tumefaciens is
catalyzed by a single promiscuous glycosyltransferase namely Agt
encoded by atu2297 (Figure 6). Enzyme assays with recombinant
Agt in E. coli protein extracts provided evidence that Agt uses UDP-
glucose and UDP-glucuronic acid as sugar donors for MGlcD and
GlcAD synthesis, repectively (Figure 6;Semeniuk et al., 2014).
A. tumefaciens Agt is the first described glycosyltransferase using
sugars with different chemistry. An A. tumefaciens agt mutant is
deficient in MGlcD and GlcAD formation and loss of these GLs is
compensated by a twofold increase in GGD and DGD. Remarkably,
while DGTS and all other PLs are not influenced in the agt mutant,
PC amount is strongly reduced. Deletion of both pgt and agt genes
results in the loss of all GLs, which is compensated by a strong
DGTS accumulation. Similar to the single agt mutant, the PC
content of the double mutant is strongly reduced. One reason
might be that PC is degraded to provide DAG for GGD/DGD
synthesis in case of the agt mutant or for DGTS synthesis in the
double mutant. It is unclear, however, why specifically PC and
no other PL is turned over to supply DAG for the synthesis of
phosphate-free lipids. Another reason might be that reduction of
the bilayer-stabilizing PC in membranes missing the acidic GlcAD
is necessary to sustain membrane structure and fluidity (Semeniuk
et al., 2014). In S. meliloti, loss of the acidic glycolipid SQD is
compensated by an increase of the anionic and bilayer-forming
lipid PG (Weissenmayer et al., 2000).
Since loss of all GLs has no impact on growth and virulence even
under phosphate-limited conditions, A. tumefaciens seemstocom-
pensate the lack of all GLs by DGTS (Geske et al., 2013;Semeniuk
et al., 2014). A S. meliloti mutant deficient in all phosphate-free
lipids shows decreased growth under phosphate starvation but is
not influenced in nodule formation on its host alfalfa (López-Lara
et al., 2005) suggesting that these lipids function as bulk membrane
lipids. Whether lack of all GLs and DGTS impacts A. tumefaciens
physiology and virulence remains to be seen.
The acidic GlcAD in A. tumefaciens might be the counter-
part of the glycolipid SQD which is absent in Agrobacterium but
widespread in photosynthetic organisms and present in a few non-
photosynthetic bacteria such as some rhizobia (López-Lara et al.,
2003). The role of SQD in these organisms is still unclear. It has
been speculated that SQD might have a special role in photosyn-
thesis or is required for nodule formation and nitrogen fixation.
However, SQD-free mutants of the photosynthetic purple bac-
terium R. sphaeroides and the nitrogen fixing S. meliloti are not
compromised in photosynthesis and symbiosis, respectively sug-
gesting no general function of bacterial SQD in these processes
(Benning et al., 1993;Weissenmayer et al., 2002;López-Lara et al.,
2003).
DGTS-(N,N,N,-trimethyl)homoserine is a betaine-ether linked
glycerolipid abundant in membranes of plants, algae, and fungi
and is found in a few bacteria (Dembitsky, 1996;López-Lara
et al., 2003). In Agrobacterium membranes DGTS is a major
non-phosphorus lipid (∼20 mol%) during phosphate starvation
(Figure 6B). Similar to PC, DGTS is a zwitterionic lipid con-
taining a quaternary amino head group (Figure 5). It has been
observed that the content of PC and DGTS within a cell is recip-
rocal. Organisms containing major amounts of PC produce only
traces of DGTS and vice versa (Geiger et al., 2010). The struc-
tural similarity and the inverse relationship between DGTS and
PC concentrations led to the speculation that these two lipids are
functionally interchangeable (López-Lara et al., 2003;Geiger et al.,
2010;Devers et al., 2011).
DGTS-(N,N,N,-trimethyl)homoserine synthesis in R. sphae-
roides and S. meliloti occurs via the BtaA/B system (Klug
and Benning, 2001;López-Lara et al., 2005). BtaA is a
SAM/DAG 3-amino-3-carboxypropyl transferase that converts
DAG to DAG-homoserine (DGHS) using SAM as homoseryl
donor. Subsequently, DGHS is threefold methylated via
BtaB, a SAM:DAG-homoserine-N-methyltransferase, to DGTS.
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Aktas et al. Membrane lipids in Agrobacterium tumefaciens
Expression of the sinorhizobial btaA and btaB genes is PhoB
regulated. BtaA (atu2119) and BtaB (atu2120) homologs which
have not been characterized yet are encoded in the A. tumefaciens
genome suggesting a similar DGTS biosynthesis and regulation
(Yuan et al., 2006;Figure 6). In A. tumefac iens, DGTS and GL accu-
mulation under phosphate limitation also seems to be controlled
not only on transcriptional level of the responsible biosynthesis
genes but also via DAG substrate availability. A PlcP homolog,
encoded by atu1649 in the A. tumefaciens genome (Geske et al.,
2013) suggests a similar membrane remodeling mechanism as
described in S. meliloti (Zavaleta-Pastor et al., 2010;Geiger et al.,
2013). Interestingly, phosphate starvation results not only in the
replacement of PLs by non-phosphorus lipids in A. tumefaciens
but also in changes in fatty acid composition of DAG and PLs
with a shift from 18:1 to 19:0 cyclo fatty acids (Geske et al., 2013).
Whereas under full nutrition PLs are mainly composed of 18:1
(50–60%) fatty acids and contain low proportions of 19:0 cyclo
(20 and 40% in PC) fatty acid, phosphate limitation results in a
decrease in 18:1 (10%) and a strong increase in 19:0 cyclo (60%)
fatty acids (Geske et al., 2013). A. tumefaciens codes for a putative
cyclopropane fatty acid (CFA) synthase presumably responsible
for this modification (Geske et al., 2013). Cyclopropanation of
pre-existing unsaturated fatty acids is widespread in bacteria and
maximal activity is observed during stationary phase. The biologi-
cal role of CFA containing lipids in bacteria is not fully understood.
Accumulation of CFAs in E. coli is correlated with acid tolerance
and seems to be important for pathogenic bacteria–host interac-
tions as shown for Mycobacterium tuberculosis (Chang and Cronan,
1999;Glickman et al., 2000;Zhang and Rock, 2009). A twofold
increase in CFA content under phosphate starvation and acid con-
ditions is also observed in S. meliloti. Here, two CFA synthases have
been described, with Cfa1 essential for cyclopropanation of fatty
acids under tested conditions. Both cfa genes are not required
for symbiotic nitrogen fixation in S. meliloti (Saborido Bascon-
cillo et al., 2009). Whether cyclopropanated lipids are required for
A. tumefaciens virulence remains to be determined.
IMPORTANCE OF MEMBRANE LIPIDS FOR A. tumefaciens
PHYSIOLOGY AND PATHOGENESIS
PHOSPHATIDYLCHOLINE IS CRUCIAL FOR AGROBACTERIUM
VIRULENCE
Although the typical eukaryotic membrane lipid PC is rarely found
in bacteria it is a main constituent of A. tumefaciens inner and
outer membranes suggesting an important role for this organism
(Klüsener et al., 2009). Indeed, loss of PC causes different phys-
iological defects. A PC-deficient mutant is impaired in growth
on solid medium at elevated temperatures and is unable to grow
in the presence of the anionic detergent SDS. Furthermore, it
is less motile and produces larger amounts of surface-attached
biomass (Klüsener et al., 2009). The motility defect is explained
by reduced flagellar proteins (FlaA and FlaB) in minimal medium
(Klüsener et al., 2009,2010). The most striking phenotype of a
PC-deficient mutant is its defect in tumor formation due to loss
of the VirB/D4 Type 4 SS (T4SS) essential for T-DNA transfer
(Wessel et al., 2006). In response to plant stimuli the two compo-
nent system VirA/G controls the expression of 11 transcriptional
units, among them the virB and virD operons encoding the T4SS.
The homodimeric histidine kinase VirA is anchored in the inner
membrane. Plant-released signals, e.g., phenolic compounds are
recognized by a cytoplasmic linker domain whereas acidic pH and
monosaccharides are perceived by the periplasmic domain (Nair
et al., 2011). The global response to PC-deficiency in A. tumefaciens
as determined by proteomics and transcriptomics shows that the
VirA/G-controlled vir gene expression under virulence-induced
conditions is drastically reduced thus explaining the absence of
the T4SS (Klüsener et al., 2010). Only a limited set of other
genes coding for membrane-related proteins were changed in the
absence of PC. Expression of virG in the PC-deficient mutant
was also dramatically reduced suggesting that lack of virulence
gene induction is due to low virG expression. Since the loss of
vir gene expression in a PC-deficient mutant cannot be comple-
mented by expression of a plasmid-encoded wild type virG but
by a constitutively active VirG, it seems that a non-functional
VirA sensor kinase is responsible for the loss of virulence gene
expression in the PC-lacking Agrobacterium mutant. These obser-
vations suggest that signal transduction between VirA and VirG
is impaired in the absence of PC, possibly due to limitations in
membrane insertion or folding of VirA (Figure 7). It remains
to be seen whether the observed phenotypic defects in the PC-
deficient mutant are PC-specific or a consequence of altered bulk
physico-chemical properties of the membrane in the absence of
PC. The structural organization of membranes is defined by the
physical properties and shape of membrane lipids. Cylindrical-
shaped lipids such as PG or PC are bilayer-forming lipids whereas
cone-shaped lipids such as PE are considered non-bilayer forming
lipids (van Meer et al., 2008). However, non-bilayer lipids can form
bilayer-structures depending on solvent conditions, alkyl chain
composition, and temperature.
We do not know yet whether loss of other PLs such as PE,
PG, or CL in A. tumefaciens causes similar effects on physiology
and VirA/G-mediated signal transduction. It has been shown that
PE can act as molecular chaperone for proper folding and func-
tion of membrane proteins such as the lactose permease LacY
in E. coli (Bogdanov et al., 1999;Bogdanov and Dowhan, 2012).
Replacement of PE by PC during reconstitution of the ABC mul-
tidrug exporter HorA from Lactobacillus brevis into membrane
vesicles altered the orientation of TM helices and abolished trans-
port function (Gustot et al., 2010).TheeffectofPCdepletionon
membrane proteins (Klüsener et al., 2010) suggests that PC and
probably other PLs play a role in membrane protein homeostasis
in A. tumefaciens.
It is important to note that the requirement of PC for produc-
tive host–microbe interactions is not restricted to A. tumefaciens.
PC-deficient S. meliloti mutants are unable to establish nitrogen-
fixing symbiosis with their host plant alfalfa (Sohlenkamp et al.,
2003).ReducedPClevelsinB. japonicum, the symbiont of the
soybean Glycine max cause formation of nodules with impaired
nitrogen fixation activity (Minder et al., 2001). A PC-deficient
mutant of the intracellular human pathogen Legionella pneu-
mophila shows lowered cytotoxicity and adhesion to the host cell.
Loss of PC affects the Dot/Icm T4SS, system which delivers viru-
lence factors into the cytosol of infected cells and is required for
intracellular growth (Conover et al., 2008). In P. syringae PC is
essential for secretion of the HrpZ harpin effector protein possibly
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Aktas et al. Membrane lipids in Agrobacterium tumefaciens
FIGURE 7 |Model of PC- or OL-dependent effects onA. tumefaciens
infection efficiency. OL-lacking A. tumefaciens induces accelerated tumor
formation on potato disks compared to wild type (WT) probably due to
reduced plant defense (Vences-Guzmán etal., 2013). A PC-deficient mutant is
unable to elicit tumors on Kalanchoë leaves since VirA/G controlled vir gene
expression is impaired. As a consequence, the type4 secretion system (T4SS)
is not produced which is essential for tumor formation (Wessel et al., 2006;
Klüsener et al., 2010).
due to a non-functional T3SS (Xiong et al., 2014). B. abortus, the
causative agent of brucellosis produces PC via the Pcs pathway.
Apcs mutant is defective in PC formation and attenuated in vir-
ulence when assayed in the mouse model (Comerci et al., 2006).
PC is not generally critical for physiology or microbe–host inter-
actions. Loss of PC in the opportunistic pathogen P.aeruginosa
did not affect physiology and virulence (Malek et al., 2012). It is
important to note here that PC is only a minor (∼4%) component
of P. aeruginosa membranes (Geiger et al., 2013).
LACK OF THE HYDROXYLATED ORNITHINE LIPID OLS2 IN A. tumefaciens
CAUSES ACCELERATED TUMOR FORMATION
Although various bacteria deficient in OL biosynthesis have been
characterized, the function of OLs still is largely unclear. OLs have
been implicated in high-temperature tolerance in Burkholderia
cepacia (Taylor et al., 1998). In Bordetella pertussis and Flavobac-
terium meningosepticum OLs are involved in hemagglutination
and stimulation of macrophages (Kawai and Yano, 1983;Kawai
and Akagawa, 1989;Kawai et al., 1999). In Rhodobacter capsulatus
OL is critical for optimal yields of cytochrome c (Aygun-Sunar
et al., 2006). In Gram-negative bacteria OLs are enriched in the
outer membrane. It has been postulated that the zwitterionic OLs
increase outer membrane stability via stabilization of the nega-
tive charges of LPS. Hydroxylation of OLs often correlates with
bacterial stress response (Vences-Guzmán et al., 2012). It is spec-
ulated that the additional OH group increases hydrogen bonding
between the lipid molecules as shown for the 2-hydroxylated lipid
AinSalmonella typhimurium. This would decrease the mem-
brane fluidity and make it less permeable (Gibbons et al., 2000;
Vences-Guzmán et al., 2012).
In A. tumefaciens A208 grown at low temperatures (15◦C)
unmodified OLS1 is completely hydroxylated to OLS2 suggest-
ing a role of this modified OL in temperature stress. However, lack
of both OLs has no impact on growth even under high osmo-
larity or at low temperature. Interestingly, A. tumefaciens A208
mutants devoid of OLS2 induce about 1 week earlier tumors and
consequently, the tumor size is increased compared to wild type
induced tumors (Vences-Guzmán et al., 2013). OLs share a 3-acyl-
oxyacylamide structure with lipid A of Gram-negative bacteria,
which is an elicitor in plant–microbe interactions (Scheidle et al.,
2005;Silipo et al., 2008;Madala et al., 2011). It has been specu-
lated that hydroxylated OLs cause a plant defense response, which
might be lowered in the absence of OLs thus explaining accelerated
tumor formation (Figure 7).
The role of OLs in plant interaction cannot be generalized and
it seems that OLs have different functions in different bacteria. In
contrast to A. tumefaciens, the two modified OLs P1 and P2 are
necessary for a successful symbiotic interaction in the nitrogen-
fixing symbiont R. tropici CIAT899, which is highly tolerant to
different environmental stresses (Rojas-Jiménez et al., 2005). The
OL in S. meliloti is required for normal growth under phosphate-
limiting conditions but not necessary for symbiotic performance
(López-Lara et al., 2005).
CONCLUSION
Recent progress in lipid analysis technologies has revealed a sur-
prising diversity in bacterial membrane lipid biosynthesis. The
membrane composition is very dynamic and substantially remod-
eled in response to environmental changes. A future challenge will
be to define the physiological role of specific lipids at the molecular
www.frontiersin.org March 2014 |Volume 5 |Article 109 |9
Aktas et al. Membrane lipids in Agrobacterium tumefaciens
level. The phenotypic characterisation of lipid biosynthesis
mutants has already provided interesting insights into the in vivo
function of various lipids but has considerable limitations. Some-
times it is difficult to interpret whether the observed phenotypes
are direct or indirect because most bacteria are able to compensate
the loss of one lipid by changing the overall lipid composition.
One interesting model organism in this context is A. tumefaciens,
the natural genetic engineer of plants. Two specific membrane
lipids, the PL PC and a phosphate-free lipid OL affect virulence
with opposing outcomes. PC-deficiency causes a loss of viru-
lence gene expression and tumor formation whereas lack of OLS2
accelerates tumorigenesis. Biophysical and biochemical studies
combined with genetic manipulation are needed to understand
the precise molecular mechanisms, by which these lipids influ-
ence membrane properties and Agrobacterium-mediated tumor
formation.
ACKNOWLEDGMENTS
Funding for this project is provided by the German Research
Foundation (DFG NA240/9-1). We thank Roman Moser for crit-
ical reading of the manuscript and Simon Czolkoss for drawing
chemical structures.
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