Publications by authors named "Meriyem Aktas"

19 Publications

  • Page 1 of 1

Phospholipid -Methyltransferases Produce Various Methylated Phosphatidylethanolamine Derivatives in Thermophilic Bacteria.

Appl Environ Microbiol 2021 09 10;87(19):e0110521. Epub 2021 Sep 10.

Microbial Biology, Faculty of Biology, Ruhr University Bochumgrid.5570.7, Bochum, Germany.

One of the most common pathways for the biosynthesis of the phospholipid phosphatidylcholine (PC) in bacteria is the successive 3-fold -methylation of phosphatidylethanolamine (PE) catalyzed by phospholipid -methyltransferases (Pmts). Pmts with different activities have been described in a number of mesophilic bacteria. In the present study, we identified and characterized the substrate and product spectra of four Pmts from thermophilic bacteria. Three of these enzymes were purified in an active form. The Pmts from Melghirimyces thermohalophilus, Thermostaphylospora chromogena, and Thermobifida fusca produce monomethyl-PE (MMPE) and dimethyl-PE (DMPE). T. fusca encodes two Pmt candidates, one of which is inactivated by mutation and the other is responsible for the accumulation of large amounts of MMPE. The Pmt enzyme from Rubellimicrobium thermophilum catalyzes all three methylation reactions to synthesize PC. Moreover, we show that PE, previously reported to be absent in R. thermophilum, is in fact produced and serves as a precursor for the methylation pathway. In an alternative route, the strain is able to produce PC by the PC synthase pathway when choline is available. The activity of all purified thermophilic Pmt enzymes was stimulated by anionic lipids, suggesting membrane recruitment of these cytoplasmic proteins via electrostatic interactions. Our study provides novel insights into the functional characteristics of phospholipid -methyltransferases in a previously unexplored set of thermophilic environmental bacteria. In recent years, the presence of phosphatidylcholine (PC) in bacterial membranes has gained increasing attention, partly due to its critical role in the interaction with eukaryotic hosts. PC biosynthesis via a three-step methylation of phosphatidylethanolamine, catalyzed by phospholipid -methyltransferases (Pmts), has been described in a range of mesophilic bacteria. Here, we expand our knowledge on bacterial PC formation by the identification, purification, and characterization of Pmts from phylogenetically diverse thermophilic bacteria and thereby provide insights into the functional characteristics of Pmt enzymes in thermophilic actinomycetes and proteobacteria.
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http://dx.doi.org/10.1128/AEM.01105-21DOI Listing
September 2021

Promiscuous phospholipid biosynthesis enzymes in the plant pathogen Pseudomonas syringae.

Biochim Biophys Acta Mol Cell Biol Lipids 2021 07 22;1866(7):158926. Epub 2021 Mar 22.

Microbial Biology, Ruhr University Bochum, Bochum, Germany. Electronic address:

Bacterial membranes are primarily composed of phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). In the canonical PE biosynthesis pathway, phosphatidylserine (PS) is decarboxylated by the Psd enzyme. CL formation typically depends on CL synthases (Cls) using two PG molecules as substrates. Only few bacteria produce phosphatidylcholine (PC), the hallmark of eukaryotic membranes. Most of these bacteria use phospholipid N-methyltransferases to successively methylate PE to PC and/or a PC synthase (Pcs) to catalyze the condensation of choline and CDP-diacylglycerol (CDP-DAG) to PC. In this study, we show that membranes of Pseudomonas species able to interact with eukaryotes contain PE, PG, CL and PC. More specifically, we report on PC formation and a poorly characterized CL biosynthetic pathway in the plant pathogen P. syringae pv. tomato. It encodes a Pcs enzyme responsible for choline-dependent PC biosynthesis. CL formation is catalyzed by a promiscuous phospholipase D (PLD)-type enzyme (PSPTO_0095) that we characterized in vivo and in vitro. Like typical bacterial CL biosynthesis enzymes, it uses PE and PG for CL production. This enzyme is also able to convert PE and glycerol to PG, which is then combined with another PE molecule to synthesize CL. In addition, the enzyme is capable of converting ethanolamine or methylated derivatives into the corresponding phospholipids such as PE both in P. syringae and in E. coli. It can also hydrolyze CDP-DAG to yield phosphatidic acid (PA). Our study adds an example of a promiscuous Cls enzyme able to synthesize a suite of products according to the available substrates.
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http://dx.doi.org/10.1016/j.bbalip.2021.158926DOI Listing
July 2021

Small Lipoprotein Atu8019 Is Involved in Selective Outer Membrane Vesicle (OMV) Docking to Bacterial Cells.

Front Microbiol 2020 9;11:1228. Epub 2020 Jun 9.

Faculty of Biology and Biotechnology, Department of Microbial Biology, Ruhr University Bochum, Bochum, Germany.

Outer membrane vesicles (OMVs), released from Gram-negative bacteria, have been attributed to intra- and interspecies communication and pathogenicity in diverse bacteria. OMVs carry various components including genetic material, toxins, signaling molecules, or proteins. Although the molecular mechanism(s) of cargo delivery is not fully understood, recent studies showed that transfer of the OMV content to surrounding cells is mediated by selective interactions. Here, we show that the phytopathogen , the causative agent of crown gall disease, releases OMVs, which attach to the cell surface of various Gram-negative bacteria. The OMVs contain the conserved small lipoprotein Atu8019. An -deletion mutant produced wildtype-like amounts of OMVs with a subtle but reproducible reduction in cell-attachment. Otherwise, loss of did not alter growth, susceptibility against cations or antibiotics, attachment to plant cells, virulence, motility, or biofilm formation. In contrast, overproduction of Atu8019 in triggered cell aggregation and biofilm formation. Localization studies revealed that Atu8019 is surface exposed in cells and in OMVs supporting a role in cell adhesion. Purified Atu8019 protein reconstituted into liposomes interacted with model membranes and with the surface of several Gram-negative bacteria. Collectively, our data suggest that the small lipoprotein Atu8019 is involved in OMV docking to specific bacteria.
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http://dx.doi.org/10.3389/fmicb.2020.01228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296081PMC
June 2020

Virulence of Agrobacterium tumefaciens requires lipid homeostasis mediated by the lysyl-phosphatidylglycerol hydrolase AcvB.

Mol Microbiol 2019 01 14;111(1):269-286. Epub 2018 Nov 14.

Institute for Microbiology, Technische Universität Braunschweig, 38106, Braunschweig, Germany.

Agrobacterium tumefaciens transfers oncogenic T-DNA via the type IV secretion system (T4SS) into plants causing tumor formation. The acvB gene encodes a virulence factor of unknown function required for plant transformation. Here we specify AcvB as a periplasmic lysyl-phosphatidylglycerol (L-PG) hydrolase, which modulates L-PG homeostasis. Through functional characterization of recombinant AcvB variants, we showed that the C-terminal domain of AcvB (residues 232-456) is sufficient for full enzymatic activity and defined key residues for catalysis. Absence of the hydrolase resulted in ~10-fold increase in L-PG in Agrobacterium membranes and abolished T-DNA transfer and tumor formation. Overproduction of the L-PG synthase gene (lpiA) in wild-type A. tumefaciens resulted in a similar increase in the L-PG content (~7-fold) and a virulence defect even in the presence of intact AcvB. These results suggest that elevated L-PG amounts (either by overproduction of the synthase or absence of the hydrolase) are responsible for the virulence phenotype. Gradually increasing the L-PG content by complementation with different acvB variants revealed that cellular L-PG levels above 3% of total phospholipids interfere with T-DNA transfer. Cumulatively, this study identified AcvB as a novel virulence factor required for membrane lipid homeostasis and T-DNA transfer.
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http://dx.doi.org/10.1111/mmi.14154DOI Listing
January 2019

Dissection of membrane-binding and -remodeling regions in two classes of bacterial phospholipid N-methyltransferases.

Biochim Biophys Acta Biomembr 2017 Dec 11;1859(12):2279-2288. Epub 2017 Sep 11.

Microbial Biology, Faculty of Biology, Ruhr University Bochum, Bochum, Germany. Electronic address:

Bacterial phospholipid N-methyltransferases (Pmts) catalyze the formation of phosphatidylcholine (PC) via successive N-methylation of phosphatidylethanolamine (PE). They are classified into Sinorhizobium-type and Rhodobacter-type enzymes. The Sinorhizobium-type PmtA protein from the plant pathogen Agrobacterium tumefaciens is recruited to anionic lipids in the cytoplasmic membrane via two amphipathic helices called αA and αF. Besides its enzymatic activity, PmtA is able to remodel membranes mediated by the αA domain. According to the Heliquest program, αA- and αF-like amphipathic helices are also present in other Sinorhizobium- and Rhodobacter-type Pmt enzymes suggesting a conserved architecture of α-helical membrane-binding regions in these methyltransferases. As representatives of the two Pmt families, we investigated the membrane binding and remodeling capacity of Bradyrhizobium japonicum PmtA (Sinorhizobium-type) and PmtX1 (Rhodobacter-type), which act cooperatively to produce PC in consecutive methylation steps. We found that the αA regions in both enzymes bind anionic lipids similar to αA of A. tumefaciens PmtA. Membrane binding of PmtX1 αA is enhanced by its substrate monomethyl-PE indicating a substrate-controlled membrane association. The αA regions of all investigated enzymes remodel spherical liposomes into tubular filaments suggesting a conserved membrane-remodeling capacity of bacterial Pmts. Based on these results we propose that the molecular details of membrane-binding and remodeling are conserved among bacterial Pmts.
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http://dx.doi.org/10.1016/j.bbamem.2017.09.013DOI Listing
December 2017

Membrane Remodeling by a Bacterial Phospholipid-Methylating Enzyme.

mBio 2017 02 14;8(1). Epub 2017 Feb 14.

Microbial Biology, Faculty of Biology, Ruhr University Bochum, Bochum, Germany

Membrane deformation by proteins is a universal phenomenon that has been studied extensively in eukaryotes but much less in prokaryotes. In this study, we discovered a membrane-deforming activity of the phospholipid -methyltransferase PmtA from the plant-pathogenic bacterium PmtA catalyzes the successive three-step -methylation of phosphatidylethanolamine to phosphatidylcholine. Here, we defined the lipid and protein requirements for the membrane-remodeling activity of PmtA by a combination of transmission electron microscopy and liposome interaction studies. Dependent on the lipid composition, PmtA changes the shape of spherical liposomes either into filaments or small vesicles. Upon overproduction of PmtA in , vesicle-like structures occur in the cytoplasm, dependent on the presence of the anionic lipid cardiolipin. The N-terminal lipid-binding α-helix (αA) is involved in membrane deformation by PmtA. Two functionally distinct and spatially separated regions in αA can be distinguished. Anionic interactions by positively charged amino acids on one face of the helix are responsible for membrane recruitment of the enzyme. The opposite hydrophobic face of the helix is required for membrane remodeling, presumably by shallow insertion into the lipid bilayer. The ability to alter the morphology of biological membranes is known for a small number of some bacterial proteins. Our study adds the phospholipid -methyltransferase PmtA as a new member to the category of bacterial membrane-remodeling proteins. A combination of and methods reveals the molecular requirements for membrane deformation at the protein and phospholipid level. The dual functionality of PmtA suggests a contribution of membrane biosynthesis enzymes to the complex morphology of bacterial membranes.
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http://dx.doi.org/10.1128/mBio.02082-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5312082PMC
February 2017

Two Distinct Cardiolipin Synthases Operate in Agrobacterium tumefaciens.

PLoS One 2016 29;11(7):e0160373. Epub 2016 Jul 29.

Microbial Biology, Ruhr University Bochum, Bochum, Germany.

Cardiolipin (CL) is a universal component of energy generating membranes. In most bacteria, it is synthesized via the condensation of two molecules phosphatidylglycerol (PG) by phospholipase D-type cardiolipin synthases (PLD-type Cls). In the plant pathogen and natural genetic engineer Agrobacterium tumefaciens CL comprises up to 15% of all phospholipids in late stationary growth phase. A. tumefaciens harbors two genes, atu1630 (cls1) and atu2486 (cls2), coding for PLD-type Cls. Heterologous expression of either cls1 or cls2 in Escherichia coli resulted in accumulation of CL supporting involvement of their products in CL synthesis. Expression of cls1 and cls2 in A. tumefaciens is constitutive and irrespective of the growth phase. Membrane lipid profiling of A. tumefaciens mutants suggested that Cls2 is required for CL synthesis at early exponential growth whereas both Cls equally contribute to CL production at later growth stages. Contrary to many bacteria, which suffer from CL depletion, A. tumefaciens tolerates large changes in CL content since the CL-deficient cls1/cls2 double mutant showed no apparent defects in growth, stress tolerance, motility, biofilm formation, UV-stress and tumor formation on plants.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160373PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966929PMC
August 2017

Unconventional membrane lipid biosynthesis in Xanthomonas campestris.

Environ Microbiol 2015 Sep 11;17(9):3116-24. Epub 2015 Aug 11.

Microbial Biology, Ruhr University Bochum, Universitätsstrasse 150, NDEF 06/783, Bochum, D-44780, Germany.

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.
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http://dx.doi.org/10.1111/1462-2920.12956DOI Listing
September 2015

Membrane-binding mechanism of a bacterial phospholipid N-methyltransferase.

Mol Microbiol 2015 Jan 19;95(2):313-31. Epub 2014 Dec 19.

Microbial Biology, Ruhr University Bochum, Bochum, Germany.

The membrane lipid phosphatidylcholine (PC) is crucial for stress adaptation and virulence of the plant pathogen Agrobacterium tumefaciens. The phospholipid N-methyltransferase PmtA catalyzes three successive methylations of phosphatidylethanolamine to yield PC. Here, we asked how PmtA is recruited to its site of action, the inner leaflet of the membrane. We found that the enzyme attaches to the membrane via electrostatic interactions with anionic lipids, which do not serve as substrate for PmtA. Increasing PC concentrations trigger membrane dissociation suggesting that membrane binding of PmtA is negatively regulated by its end product PC. Two predicted alpha-helical regions (αA and αF) contribute to membrane binding of PmtA. The N-terminal helix αA binds anionic lipids in vitro with higher affinity than the central helix αF. The latter undergoes a structural transition from disordered to α-helical conformation in the presence of anionic lipids. The basic amino acids R8 and K12 and the hydrophobic amino acid F19 are critical for membrane binding by αA as well as for activity of full-length PmtA. We conclude that a combination of electrostatic and hydrophobic forces is responsible for membrane association of the phospholipid-modifying enzyme.
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http://dx.doi.org/10.1111/mmi.12870DOI Listing
January 2015

Enzymatic properties and substrate specificity of a bacterial phosphatidylcholine synthase.

FEBS J 2014 Aug 14;281(15):3523-41. Epub 2014 Jul 14.

Department of Microbial Biology, Ruhr University Bochum, Germany.

Unlabelled: Phosphatidylcholine (PC) is a rare membrane lipid in bacteria, but is crucial for virulence of the plant pathogen Agrobacterium tumefaciens and various other pathogens. Agrobacterium tumefaciens uses two independent PC biosynthesis pathways. One is dependent on the integral membrane protein PC synthase (Pcs), which catalyzes the conversion of cytidine diphosphate-diacylglycerol (CDP-DAG) and choline to PC, thereby releasing a cytidine monophosphate (CMP). Here, we show that Pcs consists of eight transmembrane segments with its N- and C-termini located in the cytoplasm. A cytoplasmic loop between the second and third membrane helix contains the majority of the conserved amino acids of a CDP-alcohol phosphotransferase motif (DGX2 ARX12 GX3 DX3 D). Using point mutagenesis, we provide evidence for a crucial role of this motif in choline binding and enzyme activity. To study the catalytic features of the enzyme, we established a purification protocol for recombinant Pcs. The enzyme forms stable oligomers and exhibits broad substrate specificity towards choline derivatives. The presence of CDP-DAG and manganese is a prerequisite for cooperative binding of choline. PC formation by Pcs is reversible and proceeds via two successive reactions. In a first choline- and manganese-independent reaction, CDP-DAG is hydrolyzed releasing a CMP molecule. The resulting phosphatidyl intermediate reacts with choline in a second manganese-dependent step to form PC.

Structured Digital Abstract: Pcs and Pcs bind by molecular sieving (1, 2, 3).
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http://dx.doi.org/10.1111/febs.12877DOI Listing
August 2014

Membrane lipids in Agrobacterium tumefaciens: biosynthetic pathways and importance for pathogenesis.

Front Plant Sci 2014 26;5:109. Epub 2014 Mar 26.

Microbial Biology, Department for Biology and Biotechnology, Ruhr University Bochum Bochum, Germany.

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, phosphatidylcholine (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 of A. 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.
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http://dx.doi.org/10.3389/fpls.2014.00109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3972451PMC
April 2014

Discovery of a bifunctional cardiolipin/phosphatidylethanolamine synthase in bacteria.

Mol Microbiol 2014 Jun 29;92(5):959-72. Epub 2014 Apr 29.

Microbial Biology, Ruhr University Bochum, Bochum, Germany.

Phosphatidylethanolamine (PE) and cardiolipin (CL) are major components of bacterial and eukaryotic membranes. In bacteria, synthesis of PE usually occurs via decarboxylation of phosphatidylserine (PS) by PS decarboxylases (Psd). CL is produced by various CL synthases (Cls). Membranes of the plant pathogen Xanthomonas campestris predominantly contain PE, phosphatidylglycerol (PG) and CL. The X. campestris genome encodes one Psd and six putative CLs. Deletion of psd resulted in loss of PE and accumulation of PS. The mutant was severely affected in growth and cell size. PE synthesis, growth and cell division were partially restored when cells were supplied with ethanolamine (EA) suggesting a previously unknown PE synthase activity. Via mutagenesis, we identified a Cls enzyme (Xc_0186) responsible for EA-dependent PE biosynthesis. Xanthomonas lacking xc_0186 not only lost its ability to utilize EA for PE synthesis but also produced less CL suggesting a bifunctional enzyme. Recombinant Xc_0186 in E. coli and in cell-free extracts uses cytidine diphosphate diacylglycerol (CDP-DAG) and PG for CL synthesis. It is also able to use CDP-DAG and EA for PE synthesis. Owing to its dual function in CL and PE production, we consider Xc_0186 the founding member of a new class of enzymes called CL/PE synthase (CL/PEs).
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http://dx.doi.org/10.1111/mmi.12603DOI Listing
June 2014

Phosphatidylcholine biosynthesis in Xanthomonas campestris via a yeast-like acylation pathway.

Mol Microbiol 2014 Feb 7;91(4):736-50. Epub 2014 Jan 7.

Microbial Biology, Ruhr University Bochum, Bochum, Germany.

Two principal phosphatidylcholine (PC) biosynthesis pathways are known in bacteria. S-adenosylmethionine (SAM)-dependent phospholipid N-methyltransferases (Pmt) catalyse the threefold N-methylation of phosphatidylethanolamine (PE) to PC. In an alternative pathway, the PC synthase (Pcs) condenses CDP-diacylglycerol and choline to produce PC. In this study, we investigated phospholipid biosynthesis in the plant pathogen Xanthomonas campestris that was found to contain significant amounts of monomethylated PE (MMPE) and small amounts of PC. We identified a Pmt enzyme that produces MMPE without methylating it further to PC. Surprisingly, PC production was independent of [(14) C]-SAM and [(14) C]-choline excluding canonical Pmt or Pcs pathways. Feeding experiments with various choline derivatives revealed a novel, yeast-like PC synthesis route in Xanthomonas, in which two acyl side-chains are added to a glycerophosphocholine (GPC) backbone. Two out of 12 tested acyltransferases from Xanthomonas were able to catalyse the second acylation step from lyso-PC to PC. This first description of GPC-dependent PC production in bacteria illustrates an unexpected diversity of PC biosynthesis pathways.
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http://dx.doi.org/10.1111/mmi.12492DOI Listing
February 2014

Choline uptake in Agrobacterium tumefaciens by the high-affinity ChoXWV transporter.

J Bacteriol 2011 Oct 29;193(19):5119-29. Epub 2011 Jul 29.

Microbial Biology, Universitätsstrasse 150, NDEF 06/786, Ruhr-Universität Bochum, D-44780 Bochum, Germany.

Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called "Venus flytrap mechanism" of substrate binding.
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http://dx.doi.org/10.1128/JB.05421-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187443PMC
October 2011

S-adenosylmethionine-binding properties of a bacterial phospholipid N-methyltransferase.

J Bacteriol 2011 Jul 20;193(14):3473-81. Epub 2011 May 20.

Ruhr-Universität Bochum, Bochum, Germany.

The presence of the membrane lipid phosphatidylcholine (PC) in the bacterial membrane is critically important for many host-microbe interactions. The phospholipid N-methyltransferase PmtA from the plant pathogen Agrobacterium tumefaciens catalyzes the formation of PC by a three-step methylation of phosphatidylethanolamine via monomethylphosphatidylethanolamine and dimethylphosphatidylethanolamine. The methyl group is provided by S-adenosylmethionine (SAM), which is converted to S-adenosylhomocysteine (SAH) during transmethylation. Despite the biological importance of bacterial phospholipid N-methyltransferases, little is known about amino acids critical for binding to SAM or phospholipids and catalysis. Alanine substitutions in the predicted SAM-binding residues E58, G60, G62, and E84 in A. tumefaciens PmtA dramatically reduced SAM-binding and enzyme activity. Homology modeling of PmtA satisfactorily explained the mutational results. The enzyme is predicted to exhibit a consensus topology of the SAM-binding fold consistent with cofactor interaction as seen with most structurally characterized SAM-methyltransferases. Nuclear magnetic resonance (NMR) titration experiments and (14)C-SAM-binding studies revealed binding constants for SAM and SAH in the low micromolar range. Our study provides first insights into structural features and SAM binding of a bacterial phospholipid N-methyltransferase.
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http://dx.doi.org/10.1128/JB.01539-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133305PMC
July 2011

Phosphatidylcholine biosynthesis and its significance in bacteria interacting with eukaryotic cells.

Eur J Cell Biol 2010 Dec 24;89(12):888-94. Epub 2010 Jul 24.

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

Phosphatidylcholine (PC), a typical eukaryotic membrane phospholipid, is present in only about 10% of all bacterial species, in particular in bacteria interacting with eukaryotes. A number of studies revealed that PC plays a fundamental role in symbiotic and pathogenic microbe-host interactions. Agrobacterium tumefaciens mutants lacking PC are unable to elicit plant tumors. The human pathogens Brucella abortus and Legionella pneumophila require PC for full virulence. The plant symbionts Bradyrhizobium japonicum 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 phosphatidylcholine synthase (Pcs) pathway. The methylation pathway involves a three-step methylation of phosphatidylethanolamine by at least one phospholipid N-methyltransferase to yield phosphatidylcholine. In the Pcs pathway, choline is condensed directly with CDP-diacylglycerol to form PC. This review focuses on the biosynthetic pathways and the significance of PC in bacteria with an emphasis on plant-microbe interactions.
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http://dx.doi.org/10.1016/j.ejcb.2010.06.013DOI Listing
December 2010

In vitro characterization of the enzyme properties of the phospholipid N-methyltransferase PmtA from Agrobacterium tumefaciens.

J Bacteriol 2009 Apr 30;191(7):2033-41. Epub 2009 Jan 30.

Ruhr-University, Bochum, Germany.

Agrobacterium tumefaciens requires phosphatidylcholine (PC) in its membranes for plant infection. The phospholipid N-methyltransferase PmtA catalyzes all three transmethylation reactions of phosphatidylethanolamine (PE) to PC via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE). The enzyme uses S-adenosylmethionine (SAM) as the methyl donor, converting it to S-adenosylhomocysteine (SAH). Little is known about the activity of bacterial Pmt enzymes, since PC biosynthesis in prokaryotes is rare. In this article, we present the purification and in vitro characterization of A. tumefaciens PmtA, which is a monomeric protein. It binds to PE, the intermediates MMPE and DMPE, the end product PC, and phosphatidylglycerol (PG) and phosphatidylinositol. Binding of the phospholipid substrates precedes binding of SAM. We used a coupled in vitro assay system to demonstrate the enzymatic activity of PmtA and to show that PmtA is inhibited by the end products PC and SAH and the antibiotic sinefungin. The presence of PG stimulates PmtA activity. Our study provides insights into the catalysis and control of a bacterial phospholipid N-methyltransferase.
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http://dx.doi.org/10.1128/JB.01591-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655499PMC
April 2009

Expression and physiological relevance of Agrobacterium tumefaciens phosphatidylcholine biosynthesis genes.

J Bacteriol 2009 Jan 31;191(1):365-74. Epub 2008 Oct 31.

Microbial Biology, Ruhr-University Bochum, Bochum, Germany.

Phosphatidylcholine (PC), or lecithin, is the major phospholipid in eukaryotic membranes, whereas only 10% of all bacteria are predicted to synthesize PC. In Rhizobiaceae, including the phytopathogenic bacterium Agrobacterium tumefaciens, PC is essential for the establishment of a successful host-microbe interaction. A. tumefaciens produces PC via two alternative pathways, the methylation pathway and the Pcs pathway. The responsible genes, pmtA (coding for a phospholipid N-methyltransferase) and pcs (coding for a PC synthase), are located on the circular chromosome of A. tumefaciens C58. Recombinant expression of pmtA and pcs in Escherichia coli revealed that the individual proteins carry out the annotated enzyme functions. Both genes and a putative ABC transporter operon downstream of PC are constitutively expressed in A. tumefaciens. The amount of PC in A. tumefaciens membranes reaches around 23% of total membrane lipids. We show that PC is distributed in both the inner and outer membranes. Loss of PC results in reduced motility and increased biofilm formation, two processes known to be involved in virulence. Our work documents the critical importance of membrane lipid homeostasis for diverse cellular processes in A. tumefaciens.
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http://dx.doi.org/10.1128/JB.01183-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2612428PMC
January 2009

Multiple phospholipid N-methyltransferases with distinct substrate specificities are encoded in Bradyrhizobium japonicum.

J Bacteriol 2008 Jan 9;190(2):571-80. Epub 2007 Nov 9.

Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum, NDEF 06/783, D-44780 Bochum, Germany.

Phosphatidylcholine (PC) is the major phospholipid in eukaryotic membranes. In contrast, it is found in only a few prokaryotes including members of the family Rhizobiaceae. In these bacteria, PC is required for pathogenic and symbiotic plant-microbe interactions, as shown for Agrobacterium tumefaciens and Bradyrhizobium japonicum. At least two different phospholipid N-methyltransferases (PmtA and PmtX) have been postulated to convert phosphatidylethanolamine (PE) to PC in B. japonicum by three consecutive methylation reactions. However, apart from the known PmtA enzyme, we identified and characterized three additional pmt genes (pmtX1, pmtX3, and pmtX4), which can be functionally expressed in Escherichia coli, showing different substrate specificities. B. japonicum expressed only two of these pmt genes (pmtA and pmtX1) under all conditions tested. PmtA predominantly converts PE to monomethyl PE, whereas PmtX1 carries out both subsequent methylation steps. B. japonicum is the first bacterium known to use two functionally different Pmts. It also expresses a PC synthase, which produces PC via condensation of CDP-diacylglycerol and choline. Our study shows that PC biosynthesis in bacteria can be much more complex than previously anticipated.
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http://dx.doi.org/10.1128/JB.01423-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2223695PMC
January 2008
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