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European Journal of Cell Biology 89 (2010) 888–894

Contents lists available at ScienceDirect

European Journal of Cell Biology

journa l homepage: www.e lsev ier .de /e jcb

ini-review

hosphatidylcholine biosynthesis and its significance in bacterianteracting with eukaryotic cells

eriyem Aktas, Mirja Wessel, Stephanie Hacker, Sonja Klüsener,an Gleichenhagen, Franz Narberhaus ∗

uhr-Universität Bochum, Lehrstuhl für Biologie der Mikroorganismen, Universitätsstrasse 150, NDEF 06/783, 44780 Bochum, Germany

r t i c l e i n f o

eywords:embrane lipids

hospholipid biosynthesishosphatidylethanolaminehosphatidylcholinehospholipid N-methyltransferase

a b s t r a c t

Phosphatidylcholine (PC), a typical eukaryotic membrane phospholipid, is present in only about 10% ofall bacterial species, in particular in bacteria interacting with eukaryotes. A number of studies revealedthat PC plays a fundamental role in symbiotic and pathogenic microbe–host interactions. Agrobacteriumtumefaciens mutants lacking PC are unable to elicit plant tumors. The human pathogens Brucella abortusand Legionella pneumophila require PC for full virulence. The plant symbionts Bradyrhizobium japonicum

hosphatidylcholine synthaseymbioticathogeniclant microbe–interactionholine-Adenosylmethionine-Adenosylhomocysteine

and Sinorhizobium meliloti depend on wild-type levels of PC to establish an efficient root nodule symbiosis.Two pathways for PC biosynthesis are known in bacteria, the methylation pathway and the phos-

phatidylcholine synthase (Pcs) pathway. The methylation pathway involves a three-step methylationof phosphatidylethanolamine by at least one phospholipid N-methyltransferase to yield phosphatidyl-choline. In the Pcs pathway, choline is condensed directly with CDP-diacylglycerol to form PC. Thisreview focuses on the biosynthetic pathways and the significance of PC in bacteria with an emphasison plant–microbe interactions.

hosphatidylcholine – a membrane lipid more widespreadn bacteria than previously thought

PC is a zwitterionic glycerophospholipid with choline as headroup (Fig. 1A). As the major membrane lipid in non-photosyntheticukaryotic cells it accounts for approximately half of all membraneipids. Besides its structural role in membranes PC serves as a mod-lator of a wide variety of cellular functions (Exton, 1994; Kent,990; LeBlanc et al., 1998; Zeisel and Blusztajn, 1994).

In contrast to eukaryotes, much less is known about the rolef PC in prokaryotes. Bacteria are surrounded by one (Gram-ositive) or two membranes (Gram-negative). The outer membranef Gram-negative bacteria is asymmetric exposing lipopolysaccha-ides towards the surface. The major membrane lipids in mostacteria are phosphatidylethanolamine (PE), phosphatidylglycerolPG), and cardiolipin (CL) (Fig. 1B). However, in some bacteriather membrane lipids like phosphatidylserine, phosphatidylinos-

tol or PC are found (Cronan, 2003; Geiger et al., 2010; Goldfine,984; Zhang and Rock, 2008). The occurrence of PC in prokary-tes has been underestimated for a long time since neither of thewo most frequently used bacterial model organisms, Escherichia

∗ Corresponding author. Tel.: +49 234 32 23100; fax: +49 234 32 14620.E-mail address: franz.narberhaus@rub.de (F. Narberhaus).

171-9335/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.oi:10.1016/j.ejcb.2010.06.013

© 2010 Elsevier GmbH. All rights reserved.

coli and Bacillus subtilis, contains PC. However, already more than25 years ago it was reported that phototrophic bacteria containingextensive internal membrane structures and some bacteria livingin association with eukaryotes in fact possess PC as membranephospholipid (Goldfine, 1984). A recent systematic inspection ofbacterial genome sequences suggested that PC is present in about10% of all bacterial species (Sohlenkamp et al., 2003). As PC isfrequently found in bacteria interacting with eukaryotic hosts,research focused on its function in infection processes (Comerci etal., 2006; Conde-Alvarez et al., 2006; Conover et al., 2008; Minderet al., 2001; Sohlenkamp et al., 2003; Wessel et al., 2006). MostPC-containing bacteria belong to the alpha- and gamma-subgroupof Proteobacteria (e.g. Acetobacter aceti, Agrobacterium tumefaciens,Bradyrhizobium japonicum, Brucella abortus, Legionella pneumophilaand Pseudomonas aeruginosa). However, PC-containing specieswere also found in the beta-subgroup of the Proteobacteria andin distantly related groups like Gram-positives, the Bacteroides-Flavobacterium group and the Spirochetes (López-Lara and Geiger,2001; Sohlenkamp et al., 2003).

The amount of PC detected in different species ranges from a few

percent of total membrane lipids in P. aeruginosa (Wilderman et al.,2002) to up to 73% PC in A. aceti (Hanada et al., 2001). Membranefractionation experiments suggest that PC is present in both theinner and outer membranes (Fig. 1) (Hindahl and Iglewski, 1984;Klüsener et al., 2009; Wilderman et al., 2002).

M. Aktas et al. / European Journal of C

Fig. 1. Structure of phosphatidylcholine (A) and occurrence in a Gram-negative bac-tLC

Pe

aaab(2

Cdim(S

(dNBmtWmP2eP1(Pm

w

to rRNA methylases, whereas Rhodobacter-like Pmts are similar to

erial cell envelope (B). Abbreviations: OM, outer membrane; IM, inner membrane;PS, lipopolysaccharide; PE, phosphatidylethanolamine; PG, phosphatidylglycerol;L, cardiolipin.

hosphatidylcholine synthase (Pcs) – a unique bacterialnzyme

Bacteria synthesize PC via two distinct routes, the methylationnd the Pcs pathway (Fig. 2). Many bacteria, especially plant-ssociated Rhizobiaceae, produce PC by both pathways (Hacker etl., 2008b; Sohlenkamp et al., 2000; Wessel et al., 2006). Otheracteria use either the methylation pathway or the Pcs pathwayArondel et al., 1993; Sohlenkamp et al., 2003; Wilderman et al.,002).

The bacterial Pcs pathway is distinct from the eukaryoticDP-choline pathway, in which activated CDP-choline is con-ensed with diacylglycerol (DAG). In the bacterial pathway, choline

s condensed directly with CDP-DAG to form PC and cytidineonophosphate (CMP) in a reaction catalyzed by Pcs (Fig. 2)

de Rudder et al., 1999, 2000; Martínez-Morales et al., 2003;ohlenkamp et al., 2000).

Since the discovery of the Sinorhizobium meliloti Pcs enzymede Rudder et al., 1997, 1999) numerous homologues have beenescribed, but only a few have been studied experimentally.otably, some important pathogens (P. aeruginosa, B. abortus andorrelia burgdorferi) exclusively use the Pcs pathway for PC for-ation and are thought to depend on choline supplied from

heir hosts (Comerci et al., 2006; Martínez-Morales et al., 2003;ilderman et al., 2002). In contrast, A. tumefaciens, B. japonicum, S.eliloti, Rhizobium leguminosarum and Mesorhizobium loti use both

C biosynthesis pathways (de Rudder et al., 1999; Hacker et al.,008b; Martínez-Morales et al., 2003; Minder et al., 2001; Wesselt al., 2006). In S. meliloti, choline required for PC synthesis via thecs pathway is supplied by its legume host plants (de Rudder et al.,999) and transported via a high-affinity choline ABC-transporterDupont et al., 2004). In case choline is supplied by the host, the

cs pathway is energetically more favourable than the threefoldethylation of PE.Pcs enzymes are predicted to be highly hydrophobic proteins

ith 6–8 transmembrane helices. BLAST searches revealed signifi-

ell Biology 89 (2010) 888–894 889

cant similarities between Pcs enzymes and other CDP-alcohol phos-phatidyltransferases (López-Lara and Geiger, 2001; Sohlenkamp etal., 2000, 2003). The motif D-G-X2-A-R-X8-G-X3-D-X3-D which ischaracteristic for CDP-alcohol phosphatidyltransferases (Williamsand McMaster, 1998) is conserved in Pcs enzymes (D-G-X2-A-R-X8-P-X3-G-X3-D-X3-D, differences between both sequencesare underlined) (Sohlenkamp et al., 2003). Owing to the similarreaction catalyzed by these enzymes, Pcs is related to phos-phatidylserine synthases (Pss) catalysing the condensation ofserine with CDP-DAG to yield phosphatidylserine and CMP (López-Lara and Geiger, 2001; Sohlenkamp et al., 2003).

Initial biochemical characterization of the Pcs enzyme wasperformed using S. meliloti cell free extracts (de Rudder etal., 1999). The 241 amino acid protein showed optimal activ-ity at pH 8.0. Pcs activity strictly depended on bivalent cationswith Mn2+ being more effective than Mg2+. In addition, Pcsactivity required Triton X-100 with maximum activity at 0.2%(w/v) Triton X-100. Higher Triton concentrations inhibited Pcsactivity. At choline concentrations >50 mM, the enzyme activ-ity reached typical saturation kinetics, when the molar ratioof CDP-DAG varied. At choline concentrations <100 mM kineticcurves were sigmoidal suggesting a positive cooperative effectof CDP-DAG or substrate inhibition by high concentrations ofcholine.

PE methylation in bacteria – simple and complex pathways

Both eukaryotes and prokaryotes use methylation pathwaysto produce PC by three successive methylations of PE via theintermediates monomethyl-PE (MMPE) and dimethyl-PE (DMPE).The reactions are catalyzed by one or several phospholipid N-methyltransferase(s) (Pmt enzymes) using S-adenosylmethionine(SAM) as methyl donor (López-Lara and Geiger, 2001; Sohlenkampet al., 2003). During transmethylation SAM is converted to S-adenosylhomocysteine (SAH) (Fig. 2).

Despite similar activities, sequences of eukaryotic and prokary-otic Pmt enzymes differ substantially, and the number of Pmtenzymes participating in the methylation pathway varies amongorganisms. In mammals, all three methylation reactions dependon a single gene coding for two protein isoforms of Pmt enzymes(PEMT1 and PEMT2) (Cui et al., 1993; Vance, 1990; Vance et al.,2007). In contrast, two different Pmts with distinct substrate speci-ficities are required for the methylation pathway in yeast andNeurospora (Crocken and Nyc, 1964; Kodaki and Yamashita, 1987).Depending on the species one or more Pmt enzymes catalyze theformation of PC from PE in bacteria (de Rudder et al., 1997, 2000;Hacker et al., 2008b; Klüsener et al., 2009; Minder et al., 2001;Wessel et al., 2006).

While Pmt enzymes from mammals and yeast are biochemicallywell characterized (Gaynor and Carman, 1990; Shields et al., 2003;Vance et al., 1997), the activity of their bacterial counterparts islargely unexplored. The majority of prokaryotic Pmt enzymes arecytosolic in contrast to the membrane-bound eukaryotic enzymes(Sohlenkamp et al., 2003; Vance and Schneider, 1981; Vance et al.,1997).

Two Pmt families can be distinguished in bacteria, theSinorhizobium- and the Rhodobacter-type. Members of the two fam-ilies are only distantly related, and they are often more similarto methyltransferases with different substrate specificities thanto each other. Sinorhizobium-type Pmt enzymes show homology

ubiquinone/menaquinone biosynthesis methyltransferases. Simi-larities between Rhodobacter PmtA, Sinorhizobium PmtA and otherSAM-dependent methyltransferases from prokaryotes and eukary-otes are restricted to the motif V-L-E/D-X-G-X-G-X-G (SAM motif

890 M. Aktas et al. / European Journal of Cell Biology 89 (2010) 888–894

F s tranp yltrana .

I1

seo

FbD

ig. 2. Principle PC biosynthesis pathways in prokaryotes. The three methylgrouprotein with 6 transmembrane helices. Abbreviations: Pmt, phospholipid N-methdenosylhomocysteine; CMP, cytidine monophosphate; CDP, cytidine diphosphate

) which is discussed to bind the methyl donor SAM (Haydock et al.,

991; Ingrosso et al., 1989).

The first bacterial pmt gene isolated from Rhodobacterphaeroides (pmtA) encodes a 22.9 kDa soluble protein (Arondelt al., 1993). Rhodobacter PmtA catalyzes the sequential transferf three methyl groups onto PE (Arondel et al., 1993). Expression

ig. 3. PC biosynthesis in A. tumefaciens and B. japonicum. Solid arrows and boldface lettrackets are not expressed in B. japonicum wild type but are functional when expressed in EMPE, dimethyl-PE; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; CDP, cy

sferred by the Pmt enzyme are indicated by circles. Pcs is shown as membranesferase; Pcs, phosphatidylcholine synthase; SAM, S-adenosylmethionine; SAH, S-

of R. sphaeroides pmtA in E. coli results in PC formation with-

out detectable amounts of methylated intermediates, MMPE andDMPE. Pmt enzymes from A. aceti, L. pneumophila and P. aerugi-nosa belong to the Rhodobacter-family of Pmts (Conover et al., 2008;Hanada et al., 2001; Martínez-Morales et al., 2003; Wilderman etal., 2002).

ers indicate the predominant reaction(s) performed by each enzyme. Enzymes in. coli (Hacker et al., 2008b). PE, phosphatidylethanolamine; MMPE, monomethyl-PE;tidine diphosphate; DAG, diacylglycerol.

M. Aktas et al. / European Journal of Cell Biology 89 (2010) 888–894 891

Table 1PC biosynthesis and associated phenotypes of selected bacteria.

Proteobacteria PC level Pmt type Pcs PreferredPC-pathway

PC-deficientphenotypes

References

�-groupA. aceti 73.4% rPmtAa – – Reduced growth in

medium containingacetic acid

Hanada et al.(2001)

A. tumefaciens ∼23% sPmtAa Yesa Both Heat- andSDS-sensitive; lossof TIVSS; no tumorformation; motilitydefect and reducedflagellinproduction inminimal medium

Wessel et al. (2006)and Klüsener et al.(2009, 2010)

B. abortus 26.8% sPmtAa Yesa Pcs Altered cellenvelope;virulence defect inmice

Comerci et al.(2006) andConde-Alvarez etal. (2006)

B. japonicum 52% sPmtAa; rPmtX1a; rPmtX2a;sPmtX3a; sPmtX4a

Yesa Pmt: PmtA; PmtX1 Reduced noduleoccupancy,leghemoglobincontent andnitrogen fixation;reduced survivalrates upon freezing

Minder et al.(2001), Hacker etal. (2008a,b), andHacker(unpublished)

Bradyrhizobium sp. SEMIA6144

∼48% sPmtAa; sPmtX1; sPmtX2 Yesa Pmt Reduced cell size,motility andcompetitiveness insymbiosis

Medeot et al.(2009)

R. sphaeroides 27.1% rPmtAa Yes Pmt Extended lag phaseupon transitionfrom aerobic tophoto-heterotrophicconditions;decreasedB800-850 complex

Arondel et al.(1993), López-Laraet al. (2003), andKim et al. (2007)

S. meliloti 36.5% sPmtAa Yesa Both Growth defect; nonodule formationon alfalfa

de Rudder et al.(2000), andSohlenkamp et al.(2000, 2003)

Z. mobilis 5.3% PmtAa No – – Tahara et al. (1986,1987a,b, 1994) andSeo et al. (2005)

�-groupL. pneumophila ∼32% rPmtAa Yesa Pcs Less flagellin

production;compromisedmacrophagebinding; nonfunctional Dot/IcmTIVSS

Conover et al.(2008)

P. aeruginosa n.q. rPmtA Yesa Pcs Reduced survival Wilderman et al.

n

2ebaPtSKW

fimh1

.q.: not quantified; s = Sinorhizobium-type Pmt; r = Rhodobacter-type Pmt.a Activity was demonstrated experimentally.

The Sinorhizobium pmtA gene encodes a soluble protein of2 kDa, and, like R. sphaeroides pmtA, it can be functionallyxpressed in E. coli. In contrast to R. sphaeroides PmtA, sinorhizo-ial PmtA production in E. coli results in formation of significantmounts of the intermediates MMPE and DMPE in addition toC (Arondel et al., 1993; de Rudder et al., 2000). Sinorhizobium-ype pmt genes were cloned from B. japonicum, Bradyrhizobium sp.EMIA 6144, B. abortus and A. tumefaciens (Comerci et al., 2006;lüsener et al., 2009; Medeot et al., 2009; Minder et al., 2001;essel et al., 2006).

A Pmt enzyme catalyzing all three methylation steps was puri-

ed to homogeneity from the membrane fraction of Zymomonasobilis. Z. mobilis Pmt is a membrane protein, which formsomotrimers consisting of 42 kDa subunits (Tahara et al., 1986,987a,b, 1994). In this respect, the enzyme differs from other bac-

rates upon freezing (2002)

terial Pmts, and therefore, might represent a third type of bacterialPmt enzymes (Sohlenkamp et al., 2003). Z. mobilis pmt mutants lackPC indicating that the methylation pathway is the only route for PCformation in this organism (Seo et al., 2005; Tahara et al., 1994).

Methylation pathway in A. tumefaciens – PC is formed by asingle Pmt enzyme

More than 40 years ago, two distinct phospholipid N-methyltransferase activities in A. tumefaciens cell extracts were

proposed, a soluble methyltransferase capable of only the first reac-tion and a particulate enzyme catalyzing all three methylationsconverting PE to PC (Kaneshiro and Law, 1964; Sherr and Law,1965). According to the genome sequence (Goodner et al., 2001;Wood et al., 2001), however, A. tumefaciens possesses only a sin-

8 al of C

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pRlsiPsRTjtMwaup2Bti(p2fs

Fi

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nhtpm

92 M. Aktas et al. / European Journ

le pmtA gene, which is constitutively expressed (Klüsener et al.,009). The pmtA gene encodes a soluble 21.9 kDa enzyme of theinorhizobium family catalyzing all three methylation steps (Aktasnd Narberhaus, 2009; Klüsener et al., 2009). Functional expressionf pmtA in E. coli results in significant amounts of the methylatedntermediates MMPE and DMPE and the end product PC confirminghat PmtA catalyzes all three methylation steps (Fig. 3) (Klüsenert al., 2009). Biochemical characterization of recombinant PmtAevealed that it binds SAM only in the presence of one of the sub-trate lipids (PE, MMPE or DMPE) or the end product PC. In vitroctivity of PmtA is inhibited by both end products, PC and SAH.n the other hand, PmtA activity is stimulated by PG, one of theain phospholipids found in A. tumefaciens membranes (Aktas andarberhaus, 2009).

ethylation pathway in B. japonicum – PC is formed by theoncerted action of two distinct Pmt enzymes

Based on genome sequences, some bacteria are predicted toossess two or more Pmt enzymes (López-Lara et al., 2003).hodopseudomonas palustris for example encodes three Pmt homo-

ogues, two related to PmtA from R. sphaeroides and one with highimilarity to S. meliloti PmtA (López-Lara et al., 2003). B. japon-cum even encodes five pmt genes. PmtA, PmtX3 (∼21 kDa) andmtX4 (∼24 kDa) are similar to S. meliloti PmtA (∼53 and 50%imilarity), whereas PmtX1 (∼23 kDa) and PmtX2 (∼25 kDa) arehodobacter-type enzymes (∼56% similarity) (Hacker et al., 2008b).he methylation pathway is the main route for PC synthesis in B.aponicum, and is catalyzed by coordinated activities of two dis-inct Pmt enzymes (PmtA and PmtX1; Fig. 3) (Hacker et al., 2008b;

inder et al., 2001). PmtA predominantly converts PE to MMPE,hich is then methylated by PmtX1 via DMPE to PC. The three

dditional pmt genes (pmtX2, pmtX3 and pmtX4) are not expressednder free living conditions in the wild-type strain. However, allmt genes can be functionally expressed in E. coli (Hacker et al.,008b). PmtX4 exhibits a PmtA-like activity and PC depletion in a. japonicum pmtA mutant is partially compensated via the induc-ion of pmtX4 expression. The pmtA mutant is greatly compromisedn colonization of soybean roots and symbiotic nitrogen fixationMinder et al., 2001). A pmtX1 mutant could not be generated. ThemtX2-4 genes do not seem to play a role in symbiosis (Hacker et al.,008b). B. japonicum is the first bacterium known to use two dif-erent phospholipid N-methyltransferases with distinct substratepecificities in a yeast-like manner (Gaynor and Carman, 1990).

unctional importance of PC in bacteria – in microbe–hostnteraction and beyond

PC is an essential membrane component of all eukaryotes.n contrast, PC biosynthesis genes may be deleted in some PC-ynthesizing bacteria without loss of viability. An overview ofC-associated phenotypes in bacteria is given in Table 1. A Pmt-eficient Z. mobilis mutant lacking PC did not exhibit any noticeableefect (Tahara et al., 1994). This is contrasted by an A. aceti pmtAutant, which grew slowly and reached lower maximum cell den-

ities when grown in medium containing acetic acid. There is someorrelation between PC biosynthesis and acetic acid resistanceecause PC accumulated in wild-type cells with increasing acidityHanada et al., 2001).

A R. sphaeroides pmtA mutant unable to synthesize PC did

ot show any growth defect (Arondel et al., 1993). However, itad an extended lag phase upon transition from aerobic to pho-oheterotrophic conditions. Furthermore, PC is required for theroper formation of the B800-850 complex in the intracytoplasmicembrane of R. sphaeroides (Kim et al., 2007).

ell Biology 89 (2010) 888–894

A PC-deficient P. aeruginosa pcs mutant showed drasticallyreduced survival rates upon freezing (Wilderman et al., 2002).A similar defect was observed in the B. japonicum pmtA mutant(Hacker and Narberhaus, unpublished). A critical role of PCin plant–microbe interaction was first demonstrated with thismutant. It produced only low amounts of PC, namely 6% of totallipids as compared to more than 50% in the wild type (Minder etal., 2001). The generation time of the B. japonicum pmtA mutantwas slightly delayed under aerobic conditions. This growth defectwas more severe when cells grew under micro-aerobic condi-tions. Soybeans inoculated with B. japonicum pmtA mutants formedinefficient nodules with a greatly reduced number of normallyshaped bacteroids, lower amounts of leghemoglobin and increasedamounts of starch granules. As a consequence, symbiotic nitro-gen fixation activity of nodules harboring the pmtA mutant wasdecreased to 18% as compared to soybean plants infected with B.japonicum wild type.

A genome-wide survey for differentially expressed genes in aB. japonicum pmtA mutant revealed that PC reduction affects tran-scription of a strictly confined set of genes (Hacker et al., 2008a). 11genes were upregulated, among them the pmtA isogenes pmtX3 andpmtX4. Genes of two typical two-component systems, two proteinsof a RND-type (resistance nodulation cell division) efflux systemand a MarR-like regulator were differentially expressed in the pmtAmutant. Despite the pronounced defect in plant interaction, reduc-tion of the PC content in B. japonicum membranes induced a rathersubtle and specific transcriptional response under normal growthconditions.

A Bradyrhizobium sp. SEMIA 6144 pmtA mutant with 50%reduced PC content was recently reported to be severely affectedin motility and cell size. The mutant formed wild-type like noduleson its host plant but was less competitive in co-inoculation experi-ments than the wild type (Medeot et al., 2009). PC-deficiency in a S.meliloti pmtA,pcs double mutant resulted in severe growth defects(de Rudder et al., 2000) and prevented formation of nitrogen fixingroot nodules on their legume host alfalfa (Sohlenkamp et al., 2003).

A number of interesting PC-associated phenotypic changes wereobserved in A. tumefaciens, a plant pathogen inducing crown galltumors on dicotylic plants. Mutants lacking the methylation andPcs pathway were non-virulent based on the impaired expressionof genes encoding the type IV secretion machinery responsiblefor transfer of the oncogenic T-DNA and effector proteins intoplant cells (Wessel et al., 2006). Apart from the virulence defecta PC-deficient A. tumefaciens mutant was hyper-sensitive towardsthe detergent SDS, showed a growth defect at elevated temper-ature and was characterized by reduced motility and enhancedbiofilm formation when cells were propagated in minimal medium(Klüsener et al., 2009; Wessel et al., 2006). The motility defect canbe explained by reduced levels of the flagellar proteins FlaA andFlaB as revealed by a comparative proteomic approach with wildtype and PC-deficient A. tumefaciens cells. At the whole-genomelevel, the loss of PC was correlated with altered expression of upto 13% of all genes. Most of these genes encode membrane ormembrane-associated proteins and proteins with functions in theextracytoplasmic stress response (Klüsener et al., 2010).

Most interestingly, the requirement for bacterial PC is notrestricted to plant associations. At least two important PC-forminghuman pathogens are compromised in virulence when depleted forPC. PC synthesis in the human pathogen B. abortus occurs via the Pcspathway. A pcs mutant displayed a clear virulence defect in miceindicating that PC is necessary to sustain chronic infection (Comerci

et al., 2006). PC biosynthesis mutants of B. abortus have altered cellenvelopes and can hardly escape fusion with lysosomes (Conde-Alvarez et al., 2006). PC synthesis in the respiratory pathogen L.pneumophila occurs either via the PmtA or the Pcs pathway, withthe latter pathway being of predominant importance. A strain lack-

al of C

imscq(

C

wpmSSloTsm

sTsdsboct

iiraenAptpewl1sfb

tps

A

Rlt

R

A

A

M. Aktas et al. / European Journ

ng PC produced less flagellin and was compromised in binding toacrophages. Furthermore, the Dot/Icm apparatus delivering sub-

trates for intracellular growth of L. pneumophila within infectedells was strongly affected in a PC-deficient mutant. As a conse-uence, this mutant showed lowered infection of macrophagesConover et al., 2008).

onclusion and perspectives

The typical eukaryotic membrane phospholipid PC is much moreidespread in bacteria and biosynthetic pathways are more com-lex than previously anticipated. Diverse classes of Pmt enzymesight derive from different origins (López-Lara and Geiger, 2001;

ohlenkamp et al., 2003). Apart from the Rhodobacter-type andinorhizobium-type Pmt enzymes, the protein size and membraneocalization of Z. mobilis Pmt suggests the existence of a third classf Pmts (López-Lara and Geiger, 2001; Sohlenkamp et al., 2003;ahara et al., 1987a). Given the fact that PC biosynthesis has beentudied in only a few bacterial species, additional Pmt familiesight be anticipated.Most PC-containing organisms characterized so far use a

ingle Pmt enzyme for the three-step methylation of PE.he involvement of two Pmt enzymes with distinct sub-trate specificities in the methylation pathway of B. japonicumisclosed a new eukaryotic-like principle of PC biosynthe-is in bacteria. One interesting aspect for future studies wille to study the mechanistic differences between the vari-us Pmt enzymes biochemically, especially in cases in whichlosely related sequences produce different product spec-ra.

The most fascinating aspect of PC formation in bacteria is themportance of this membrane lipid for symbiotic and pathogenicnteraction. Absence of PC also interferes with motility, bacte-ial growth and stress response in some organisms (Conover etl., 2008; Hanada et al., 2001; Medeot et al., 2009; Sohlenkampt al., 2003; Wessel et al., 2006). The specific molecular mecha-isms underlying these diverse phenotypes are largely unexplored.part from their general function in maintaining cellular integrity,hospholipids are known to stabilize or activate membrane pro-eins. Protein function is influenced by the bulk physico-chemicalroperties of the membrane, which is strictly determined by thexact lipid composition of the bilayer. Several membrane proteinshose topological organization and function is affected by their

ipid environment have been described (Bogdanov and Dowhan,999; Bogdanov et al., 2008a,b; Zhang et al., 2005). Recent studieshow, that replacing PE with PC causes concomitant structural andunctional changes of the ABC multidrug exporter of Lactococcusrevis (Gustot et al., 2010).

It certainly is worth pursuing the physiological role of PC in bac-eria. The importance for host–microbe interactions might open upromising avenues for novel antimicrobial strategies targeting thetrictly bacteria-specific Pmt and Pcs enzymes.

cknowledgements

The work was in part supported by grants from the Germanesearch Foundation (DFG; SFB 480 and NA 240/7-1) to FN and a fel-

owship from the Promotionskolleg of the Ruhr-University Bochumo MA.

eferences

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Conde-Alvarez, R., Grilló, M.J., Salcedo, S.P., de Miguel, M.J., Fugier, E., Gorvel, J.P.,Moriyón, I., Iriarte, M., 2006. Synthesis of phosphatidylcholine, a typical eukary-otic phospholipid, is necessary for full virulence of the intracellular bacterialparasite Brucella abortus. Cell. Microbiol. 8, 1322–1335.

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