Environmental Microbiology (2015) 17(9), 3116–3124


Minireview Unconventional membrane lipid biosynthesis in Xanthomonas campestris Meriyem Aktas and Franz Narberhaus* Microbial Biology, Ruhr University Bochum, Universitätsstrasse 150, NDEF 06/783, Bochum D-44780, Germany. Summary All bacteria are surrounded by at least one bilayer membrane mainly composed of phospholipids (PLs). Biosynthesis of the most abundant PLs phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL) is well understood in model bacteria such as Escherichia coli. It recently emerged, however, that the diversity of bacterial membrane lipids is huge and that not yet explored biosynthesis pathways exist, even for the common PLs. A good example is the plant pathogen Xanthomonas campestris pv. campestris. It contains PE, PG and CL as major lipids and small amounts of the N-methylated PE derivatives monomethyl PE and phosphatidylcholine (PC = trimethylated PE). Xanthomonas campestris uses a repertoire of canonical and non-canonical enzymes for the synthesis of its membrane lipids. In this minireview, we briefly recapitulate standard pathways and integrate three recently discovered pathways into the overall picture of bacterial membrane biosynthesis. Introduction The bacterial membrane is the first barrier to external factors such as temperature, changes in pH or osmolarity and antimicrobial or toxic compounds. This makes membrane biosynthesis and membrane homeostasis critically important for fitness and survival of the cell (Hazel and Williams, 1990; Cronan, 2003; Zhang and Rock, 2008b). Glycerophospholipids [from now on referred to as phospholipids (PLs)] are the fundamental building blocks of eukaryotic and bacterial membranes (van Meer et al., 2008). They determine the physicochemical properties of Received 27 March, 2015; revised 3 June, 2015; accepted 14 June, 2015. *For correspondence: E-mail: [email protected]; Tel. 49 (234) 322 3100; Fax 49 (234) 321 4620.

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the membrane including fluidity, surface charge, thickness and intrinsic curvature. In response to environmental challenges, bacteria remodel their membrane lipid composition by modifying the lipid head group or fatty acid composition to maintain optimal membrane function (Yano et al., 1998; Koga, 2012; Murinova and Dercova, 2014). In response to phosphate limitation, some bacteria degrade their PLs to provide phosphate for important physiological processes. Phospholipids are then replaced by phosphate-free lipids such as ornithine lipids and glycolipids (Vences-Guzmán et al., 2012; Geske et al., 2013). Work over the last decade uncovered a huge diversity of bacterial membrane lipids (Geiger et al., 2010; 2013; Parsons and Rock, 2013; Yao and Rock, 2015). The primary PLs found in bacteria are phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). In addition, many bacteria are able to produce various unusual lipids such as sphingolipids, sterol-like lipids (hopanoids), alanylPG, lysyl-PG or methylated PE derivatives like phosphatidylcholine (PC) (Sohlenkamp et al., 2003; 2007; Hannich et al., 2011; Arendt et al., 2012; 2013; Geiger et al., 2013). The relative amount of these membrane lipids varies from one organism to another and even within an organism under different environmental conditions. A proper membrane composition is required for many physiological processes. Escherichia coli cells lacking PE are defective in cell division (Mileykovskaya et al., 1998). Phosphatidylethanolamine, PG and CL have been implicated in protein translocation and folding (Dowhan and Bogdanov, 2009; Renner and Weibel, 2012). Cardiolipin is required for osmo adaptation and oxidative phosphorylation in bacteria (Haines and Dencher, 2002; Catucci et al., 2004; Romantsov et al., 2008; 2009; Arias-Cartin et al., 2012). Due to its physical properties based on four fatty acid chains and a cylindrical shape, CL preferentially accumulates at regions of negative curvature in rod-shaped bacteria (Mileykovskaya and Dowhan, 2009; Renner and Weibel, 2011; Barak and Muchova, 2013) where they might function as landmarks to recruit specific proteins to the poles (Romantsov et al., 2010; Renner and Weibel, 2011). A few bacteria are able

Lipid biosynthesis in Xanthomonas


Fig. 1. Overview of phospholipid synthesis pathways in bacteria. Details on the individual enzyme reactions are given in the text. The unconventional pathways identified in X. campestris pv. campestris are highlighted in red. G3P, glycerol 3-phosphate; CTP, cytidine triphosphate; CMP, cytidine monophosphate; L-ser, L-serine; cho, choline; gly; glycerol.

to produce PC, the major phospholipid in eukaryotes (Goldfine and Ellis, 1964; Goldfine, 1984). In some of these bacteria, PC is important for a productive pathogenic or symbiotic host interaction, for example in Agrobacterium tumefaciens, Legionella pneumophila and Sinorhizobium melioti (Sohlenkamp et al., 2003; Wessel et al., 2006; Conover et al., 2008; Aktas et al., 2014a). Metabolic pathways and enzymes for the synthesis of the major phospholipids (PE, PG and CL) are well conserved in bacteria and have been studied extensively in E. coli (Fig. 1) (Cronan, 2003; Parsons and Rock, 2013). In all organisms, the phospholipid biosynthesis starts with a two-step acylation of sn-glycerol-3-phosphate (G3P) via lyso phosphatidic acid (LPA) to phosphatidic acid (PA). These reactions are catalysed by the combined activity of G3P acyltransferase enzymes. Two acyltransferase systems have been described in bacteria. Escherichia coli utilizes the PlsB/PlsC pathway using either acyl-ACP or acyl-coenzyme A as acyl chain donor. In the most widely distributed PlsX/PlsY/PlsC pathway, acyl-ACP is converted by PlsX to acyl-phosphate

which in turn is acylated by PlsY at 1-position to LPA. In a final acylation step, PlsC converts LPA to PA (Zhang and Rock, 2008a; Yao and Rock, 2013). Phosphatidic acid is then converted to the central precursor of all phospholipids, cytidine diphosphate-diacylglycerol (CDP-DAG), by a CDP-DAG synthase (CdsA in E. coli). For synthesis of the most abundant zwitterionic membrane lipid PE, a phosphatidylserine synthase (PssA) converts CDP-DAG to phosphatidylserine (PS), which is decarboxylated by a PS decarboxylase (Psd) to finally produce PE (Fig. 1). A recently discovered novel PE synthesis route in Xanthomonas campestris pv. campestris bypasses the serine-dependent pathway and uses CDP-DAG and ethanolamine (EA) for PE synthesis (see below) (Moser et al., 2014b). Formation of the anionic lipid PG follows a condensation of CDP-DAG with G3P to form the intermediate PG phosphate (PGP), which is then dephosphorylated by a PGP phosphatase (Pgp) to PG. In E. coli, the latter reaction is catalysed by three different Pgp enzymes (PgpA/ B/C) (Lu et al., 2011; Dowhan, 2013).

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M. Aktas and F. Narberhaus

Cardiolipin is another abundant anionic lipid in bacteria, and in eukaryotes it is exclusively present in the inner mitochondrial membrane (Schlame, 2008). Various CL synthesis pathways have been described, and many bacteria encode multiple CL synthases (Cls) (Lopez et al., 2006; Koprivnjak et al., 2011; Tan et al., 2012). In most bacteria, CL is formed by a reversible transesterification reaction of two PG molecules catalysed by phospholipase D (PLD)-type Cls enzymes (ClsA/B in E. coli, Fig. 1) (Schlame, 2008). Cardiolipin synthases of this type were considered typical bacterial enzymes. However, recent research on the protozoan parasite Trypanosoma brucei provided evidence that PLD-type Cls are not restricted to bacteria (Serricchio and Bütikofer, 2012). A different PLDtype enzyme (ClsC) has recently been discovered in E. coli. It uses a PG and a PE molecule instead of two PG molecules for CL formation (Tan et al., 2012), demonstrating the substrate diversity of these enzymes. Like in eukaryotes, actinobacteria and some proteobacteria seem to use CDP-DAG and PG for CL synthesis (Tian et al., 2012) via a Cls-II enzyme belonging to the CDPalcohol phosphatidyltransferase family as demonstrated in Streptomyces coelicolor (Fig. 1) (Sandoval-Calderón et al., 2009). As will be discussed later, similar to Cls-II, the CL/PE synthase of X. campestris utilizes CDP-DAG and PG to produce CL (Moser et al., 2014b). Two well characterized PC pathways operate in bacteria (Sohlenkamp et al., 2003; Geiger et al., 2013). N-methylation of pre-existing PE occurs via the intermediates monomethyl PE (MMPE) and dimethyl PE (DMPE) by a single or by multiple phospholipid N-methyltransferase (Pmt) enzyme(s). The methyl group is provided by S-adenosylmethionine (SAM), which is converted to S-adenosylhomocysteine (SAH) (Fig. 1). Some organisms such as A. tumefaciens or S. melioti use a single Pmt enzyme to catalyse all three methylation steps (Sohlenkamp et al., 2003; Aktas and Narberhaus, 2009; Aktas et al., 2011; 2014a). The soybean symbiont Bradyrhizobium japonicum produces PC by the combined action of two Pmt enzymes (PmtA and PmtX1) (Minder et al., 2001; Hacker et al., 2008). In an alternative pathway, a PC synthase (Pcs) enzyme produces PC by condensation of choline and CDP-DAG (Sohlenkamp et al., 2000; Aktas et al., 2014b). Some bacteria encode only one of the two pathways, whereas other species use both pathways simultaneously (Geiger et al., 2013). An entirely different strategy for PC production operating in X. campestris will be described in the following section (Moser et al., 2014a). Xanthomonas has unique solutions for membrane lipid biosynthesis Bacteria of the Xanthomonas genus (Xanthomonads) comprise a ubiquitous group of Gram-negative plant-

pathogenic bacteria causing a variety of diseases in economically important crop plants. Xanthomonas campestris pv. campestris (from now on called X. campestris) is the causal agent of black rot in crucifers. It invades the xylem and colonizes the mesophyll. With the aim to develop antimicrobial strategies against the phytopathagen, Xanthomonads are well studied model organisms. The most important factors contributing to virulence are the Type3 secretion system and effector proteins delivered by this system such as the transcription activator-like effectors (TALEs) unique to Xanthomonads (Boch et al., 2009; Boch and Bonas, 2010; Büttner and Bonas, 2010). Recent research on membrane lipid biosynthesis in X. campestris suggests that Xanthomonads are not only unique in pathogenicity concepts but also have established non-conventional pathways for membrane lipid synthesis (Moser et al., 2014a,b). Xanthonomas campestris membranes contain the typical bacterial phospholipids PE (∼50% of the total PL composition), CL (∼33%) and PG (∼13%) and small amounts of MMPE (∼4%). When grown in complex medium, Xanthomonas is able to produce small amounts of PC (∼6%) (Moser et al., 2014a,b). Xanthonomas campestris contains the complete gene repertoire required for conventional biosynthesis of the common phospholipid precursor CDP-DAG and the major PLs PE, PG and CL (Fig. 1, highlighted in red). Most of these PL synthesis genes are still uncharacterized. Among bacteria, Xanthomonadales are unique since PA synthesis is catalysed exclusively by the PlsB/PlsC system encoded by xc_4220 and xc_4099 respectively (Lu et al., 2006; Zhang and Rock, 2008a; Röttig and Steinbüchel, 2013). Many bacteria, in particular the Firmicutes, lack a PlsB homologue and use exclusively the PlsX/PlsY system. Like Xanthomonadales, archaea and eukarya have no PlsX/PlsY homologues and use exclusively a PlsB/PlsC homologous system (Lu et al., 2006; Zhang and Rock, 2008a; Röttig and Steinbüchel, 2013). The reasons for the PlsB/PslC preference are unclear. The system utilizes not only acyl-ACP originating from the type II fatty acid pathway, but also acyl-CoA derived from exogenous fatty acids as acyl donor and thus allows the incorporation of exogenous fatty acids into membrane phospholipids. In the alternative system acyl-phosphate produced by PlsX is the sole acyl donor. Phosphatidic acid might be converted in Xanthomonas by the CdsA homologue (XC_2870) to the universal bacterial PL precursor CDP-DAG. Xanthomonas encodes at least one PgsA homologue (XC_0511 as most likely candidate) for PGP synthesis, but a Pgp homologue responsible for dephosphorylation of PGP to PG has not been annotated yet. Overall, it seemed initially that Xanthomonas uses the same paradigmatic pathways for

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 3116–3124

Lipid biosynthesis in Xanthomonas


Fig. 2. Recently described phospholipid biosynthesis pathways and enzymes in X. campestris. A detailed description is given in the text. LPC, lyso-PC; PS, phosphatidylserine; CDP, cytidine diphosphate; ???, unknown proteins.

PL biosynthesis as other bacteria. Although it does so, we recently learned that membrane biosynthesis in this phytopathogen is surprisingly diverse. PE synthesis – conventional and non-conventional strategies Phosphatidylethanolamine synthesis in X. campestris occurs to a large extent via the canonical PS decarboxylation reaction carried out by the Psd enzyme XC_1563 (Moser et al., 2014b). Deletion of the psd gene resulted in a dramatic decrease of PE and accumulation of the precursor PS, which is presumably produced by the annotated PssA homologue XC_3594 in Xanthomonas via condensation of CDP-DAG and L-serine (Fig. 1). The psd mutant exhibited severe defects in growth and cell size suggesting a crucial role of PE in this organism. Phosphatidylethanolamine synthesis and the defects in growth and cell size could be partially restored when cells were grown in the presence of EA, suggesting a novel bacterial pathway that uses exogenous EA for PE production. The responsible activity was found to depend on XC_0186, annotated as PLD-type CL synthase. In vivo and in vitro activity assays suggested that this enzyme uses CDP-DAG as phosphatidyldonor and externally provided EA to synthesize PE (Fig. 2). In addition to EA, this enzyme accepts methylated EA or propanolamine derivatives to generate the corresponding phospholipid products. The substrate specificity of XC_0186 is even broader. It can convert CDP-DAG and PG to CL. Thus, we designated it as CL/PE synthase (CL/PEs).

Although unprecedented in bacteria, EA as a source for PE synthesis is well known in eukaryotes, which can produce PE via the PS decarboxylation pathway and an EA-dependent route. In the Kennedy pathway, EA is activated to CDP-EA, which in turn is converted together with DAG to PE and CMP by a choline/ethanolamine phosphotransferase (Fig. 3, top panel) (Bleijerveld et al., 2007; Gibellini and Smith, 2010; Vance and Tasseva, 2013). This enzyme can also use CDP-choline for PC synthesis. In addition, a CDP-choline-specific choline phosphotransferase (CPT1) is available for PC biosynthesis in eukaryotes (Fig. 3, lower panel) (Bleijerveld et al., 2007). In bacteria, EA usually serves as carbon/nitrogen source (Garsin, 2010). Beyond its role as nutrient, EA is involved as a signalling molecule in bacterial pathogenesis (Garsin, 2010; Kendall et al., 2012; Khatri et al., 2012). Following transport into the cytoplasm by specific transporter or passive diffusion, EA is degraded in specific micro compartments to ammonia and then to acetaldehyde by EA ammonia lyases (encoded by eutB and eutC). Xanthomonas encodes a putative EA transporter (XC_1856) and ammonia lyase subunits (XC_1854/ 1855), suggesting its capacity to catabolize EA. Since an xc_1856 mutant still produced PE from exogenous EA, alternative EA transporters or passive diffusion might be responsible for uptake (Moser et al., 2014b). The use of EA as an alternative source for PE synthesis does not seem to be a common strategy in bacteria. The plant pathogenic bacterium A. tumefaciens for instance is not able to incorporate EA into PE (Moser et al., 2014b).

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3120 M. Aktas and F. Narberhaus

Fig. 3. Comparison of selected phospholipid biosynthesis pathways between Xanthomonas, other bacteria and eukaryotes. Phosphatidylethanolamine, CL and PC pathways in Xanthomonas resemble the situation in eukaryotes (here shown for yeast). A detailed description of the different pathways is found in the text. ALE1, lyso-PE/PC acyltransferase; CPT1, CDP-choline:1,2-DAG choline phosphotransferase; DAG, diacylglycerol; GPC, glycerophosphocholine; HNM1, choline/ethanolamine transporter; cho, choline; CDP, cytidine diphosphate; CMP, cytidine monophosphate; Cho2, class II methyltransferase; NTE1, neuropathy target esterase 1; OPI3, class I methyltransferase, Pmt, phospholipid N-methyltransferase; ABC, choline ABC-transporter; ???, unknown proteins.

Why Xanthomonas has established two routes for PE synthesis remains an open question. Ethanolamine is not essential for Xanthomonas demonstrating that the CL/PE synthase-dependent pathway is dispensable for PE synthesis. Most likely, conventional decarboxylation of PS represents the primary route for PE production. The EA-specific pathway (that we discovered in the rather sick psd mutant) might be a backup system under conditions where the Psd enzyme is not able to function properly, for example when serine is limiting. Serine is not only required for PS synthesis but can also serve a precursor for glycine and cysteine in Xanthomonas (Schatschneider et al., 2011). Thus, it is conceivable that under certain conditions, serine might become a limiting factor. The relative contribution of the EA- and serine-dependent pathways to overall PE biosynthesis might vary depending on the availability of the substrates.

Cardiolipin synthesis – an unusual CL synthase with extra talents The relative content of CL in bacteria depends on the growth phase and culture conditions and can vary from a few to 30%. As in most bacteria, CL is a predominant lipid in early stationary phase in X. campestris (Moser et al., 2014a,b). Many bacteria including E. coli, Staphylococcus aureus and Bacillus subtilis contain multiple CL synthases (Lopez et al., 2006; Tan et al., 2012; Kuhn et al., 2015). It is notable that the genome of X campestris harbours six annotated cls genes belonging to different Cls families (Fig. 1). Four of the predicted gene products are similar to the PLD-Type Cls (ClsA/ClsB) from E. coli. In addition, XC_1408 shares similarities with the E. coli ClsC protein indicative of the ability to use PG and PE for CL formation. Experimental characterization of these five predicted Cls

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Lipid biosynthesis in Xanthomonas will provide insights into the complexity of CL biosynthesis in Xanthomonas. The only characterized Cls enzyme in Xanthomonas so far is the bifunctional CL/PEs (XC_0186) introduced in the previous section. It is an exceptional enzyme that utilizes CDP-DAG and PG for CL formation like eukaryotic CL synthases and the Cls-II enzyme from S. coelicolor (Sandoval-Calderón et al., 2009). Quite surprisingly, in contrast to these CDP-alcohol phosphotransferase-type enzymes, Xanthomonas CL/PEs belongs to the PLD-family CL synthases. A Xanthomonas CL/PEs-deficient mutant is characterized by decreased CL content and growth defect under osmotic stress conditions (Fig. 2). Apparently, the remaining putative Cls enzymes cannot compensate these defects (Moser et al., 2014b). Heterologous expression of the Xanthomonas CL/PEs gene in E. coli resulted in CL accumulation and EA-dependent PE production providing conclusive evidence for the bifunctional activity. The CL/PEs contains two HKD motifs typical for PLD-type enzymes which are both essential for enzyme activity (Moser et al., 2014b). Xanthomonas campestris might not be the only bacterium having the bifunctional enzyme. Related sequences were found in the genomes of Xanthomonadales and Pseudomonadales (Moser et al., 2014b). Compared with other PLD-type Cls enzymes with a molecular mass ranging from 38–53 kDa, CL/PEs homologues are larger proteins (∼70 kDa) and might represent a new family of PLD enzymes (Moser et al., 2014b). Xanthomonas and Pseudomonas species frequently interact with plants producing high amounts of EA, which is an important metabolite in higher plants for the synthesis of choline and membrane lipids, such as PE and PC. In contrast to animals, plants can de novo synthesize EA from serine via direct decarboxylation using a unique soluble serine decarboxylase. Even methylated EA derivatives, which also serve as substrates for the CL/PE synthase, are produced in different plants and might be available in planta for Xanthomonas (Mudd and Datko, 1989a,b; Rontein et al., 2001; 2003; Lykidis, 2007). Thus, it is conceivable that in nature, EA might be provided by the host plant. The acquisition of PE synthase activity by the original CL synthase during evolution might provide these bacteria with a competitive advantage in the presence of other microbes. PC is obtained via a eukaryotic-like acylation pathway In contrast to typical N-methylation pathways known from bacteria and eukaryotes, the methylation route in X. campestris produces solely MMPE without methylating it further to DMPE and PC. Xanthomonas encodes a single pmt homologue (XC_0035, designated as XC_PmtA) related to the Sinorhizobium Pmt enzyme


family. A Xanthomonas pmtA mutant lacks MMPE, and heterologous expression of xc_pmtA in E. coli leads to MMPE formation confirming that PmtA is responsible for MMPE production. Similar to Xanthomonas, PmtA of B. japonicum and Cho2 of yeast produce predominantly MMPE. However, these organisms have a second enzyme that converts MMPE to DMPE and PC (PmtX1 in B. japonicum and OPI3 in yeast) (Kodaki and Yamashita, 1987; McGraw and Henry, 1989; Hacker et al., 2008). Such an activity is missing in Xanthomonas (Fig. 3). Monomethyl PE appears not to be a critical component in Xanthomonas membranes since a pmtA mutant was not affected in growth, stress sensitivity, virulence, protein secretion, xanthan production or biofilm formation (Moser et al., 2014a). However, the MMPE-deficient mutant exhibited reduced motility despite the presence of a flagellum. This suggests a role of MMPE in motility as shown for PC in other bacteria such as A. tumefaciens and L. pneumophila (Wessel et al., 2006; Conover et al., 2008). In search for the PC-forming activity in Xanthomonas, in vivo feeding experiments with radiolabeled SAM and choline were conducted. Surprisingly, PC production turned out to be independent of these precursors for the two known PC biosynthesis pathways in bacteria (Moser et al., 2014a). Instead of those pathways operating at the lipid head group, Xanthomonas uses an acylation mechanism starting from glycerophosphocholine (GPC), which is esterified with two fatty acids via the intermediate lyso-PC to PC. While the enzyme(s) responsible for GPC acylation remain(s) elusive, two redundant membrane-bound acyltransferases (XC_0188 and XC_0238) were found to catalyse acylation of lyso-PC to PC in an acyl-CoAdependent manner (Fig. 2). At present, X. campestris is the only prokaryote known to use GPC for PC synthesis. Neither A. tumefaciens nor E. coli was able to use GPC for PC production. A GPCacylation pathway, however, has been described in yeast and plants (Stålberg et al., 2008; Lager et al., 2015). Phosphatidylcholine turnover generates GPC and fatty acids by the action of GPC acyltransferases (NTE1 in yeast). Glycerophosphocholine can be re-acylated via an unknown acyltransferase to lyso-PC, which is further acylated via a CoA-dependent lysophospholipid acyltransferase with broad substrate specificity (ALE1 in yeast) to PC (Fig. 3). This pathway can not only recycle internally generated GPC but can also utilize externally provided GPC, which is transported via the glycerophosphoinositol transporter GIT1 (Stålberg et al., 2008). De-acylation of PC to GPC in yeast is strongly enhanced at elevated temperature. This is different in Xanthomonas where GPC acylation to PC is increased at elevated temperature (Moser et al., 2014a). Furthermore, in contrast to eukaryotes, PC formation in Xanthomonas

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 3116–3124

3122 M. Aktas and F. Narberhaus is strictly dependent on exogenous GPC supply, which might derive from the host in the natural environment. Glycerophosphocholine accumulates in different plants during membrane turnover under various abiotic stresses (Van Der Rest et al., 2004). Like in most plants, in Arabidopsis (a host plant for Xanthomonas), PC is the major membrane lipid and its synthesis is increased under osmotic and cold stress (Tasseva et al., 2004). At high salt concentrations or phosphate starvation, PC is degraded releasing GPC and free fatty acids (Tasseva et al., 2004; Lager et al., 2015). Glycerophosphocholine is then hydrolysed via GPC hydrolases to glycerol-3-phosphate and choline (Van Der Rest et al., 2004; Cheng et al., 2011). In many plants, choline is converted to glycine betaine, a compatible solute providing resistance for different environmental stresses such as salt and cold stress (Sakamoto and Murata, 2002). Whether PC plays a crucial role for Xanthomonas virulence, stress tolerance, motility and biofilm formation like in other pathogenic bacteria needs to be determined. Concluding remarks and perspectives Recent studies on Xanthomonas membranes have revealed an unprecedented diversity of lipid biosynthesis pathways. The phytopathogen uses several pathways entirely different from canonical bacterial lipid biosynthesis. The common PlsX/Y pathway for PA synthesis seems to be missing. Moreover, Xanthomonas employs two eukaryotic-like strategies to synthesize PE, CL and PC. The EA-dependent PE synthesis route is unique to X. campestris but the EA dependence resembles the eukaryotic Kennedy pathway. The same is true for the GPC-dependent PC synthesis that has previously been found only in yeast and plants (Fig. 3). These findings demonstrate that the strict classification into eukaryotic and bacterial lipid biosynthesis pathways needs to be revisited. In the future, it will be of interest to find out whether these novel enzymatic activities are restricted to Xanthomonas species or more widely distributed in the bacterial kingdom. Another important goal will be to understand the enzymology and regulation of these biosynthetic pathways. It is well known that PLs play essential roles in various physiological processes such as cell signalling and virulence. The first glimpse of the complexity of membrane lipid homeostasis in this plant pathogen has already opened a number of fascinating avenues for future research. Acknowledgements This study was financially supported by the German Research Foundation (DFG; NA 240/9-1).

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Unconventional membrane lipid biosynthesis in Xanthomonas campestris.

All bacteria are surrounded by at least one bilayer membrane mainly composed of phospholipids (PLs). Biosynthesis of the most abundant PLs phosphatidy...
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