Expression and Physiological Relevance of Agrobacterium tumefaciens Phosphatidylcholine Biosynthesis Genes

Microbial Biology, Ruhr-University Bochum, Bochum, Germany.
Journal of bacteriology (Impact Factor: 2.69). 11/2008; 191(1):365-74. DOI: 10.1128/JB.01183-08
Source: PubMed

ABSTRACT 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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Available from: Franz Narberhaus, Sep 01, 2015
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    • "The pcs gene of OV14 shares 92% and 85% protein sequence identity with 1021 and C58 respectively, while the pmtA gene of OV14 shares 83% and 67% protein sequence identity with 1021’s and C58’s respecitvely. The pcs pathway is dependent on the uptake of choline from the environment [55]. Screening OV14 for the choline ABC transporter genes choXWV (that have been identified in both C58 and 1021), revealed that the choX solute binding protein component (aproNOG00993) was represented by two orthologs in all three species (Table 3). "
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    ABSTRACT: Recently it has been shown that Ensifer adhaerens can be used as a plant transformation technology, transferring genes into several plant genomes when equipped with a Ti plasmid. For this study, we have sequenced the genome of Ensifer adhaerens OV14 (OV14) and compared it with those of Agrobacterium tumefaciens C58 (C58) and Sinorhizobium meliloti 1021 (1021); the latter of which has also demonstrated a capacity to genetically transform crop genomes, albeit at significantly reduced frequencies. The 7.7 Mb OV14 genome comprises two chromosomes and two plasmids. All protein coding regions in the OV14 genome were functionally grouped based on an eggNOG database. No genes homologous to the A. tumefaciens Ti plasmid vir genes appeared to be present in the OV14 genome. Unexpectedly, OV14 and 1021 were found to possess homologs to chromosomal based genes cited as essential to A. tumefaciens T-DNA transfer. Of significance, genes that are non-essential but exert a positive influence on virulence and the ability to genetically transform host genomes were identified in OV14 but were absent from the 1021 genome. This study reveals the presence of homologs to chromosomally based Agrobacterium genes that support T-DNA transfer within the genome of OV14 and other alphaproteobacteria. The sequencing and analysis of the OV14 genome increases our understanding of T-DNA transfer by non-Agrobacterium species and creates a platform for the continued improvement of Ensifer-mediated transformation (EMT).
    BMC Genomics 04/2014; 15(1):268. DOI:10.1186/1471-2164-15-268 · 4.04 Impact Factor
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    • "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. "
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    ABSTRACT: 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.
    Frontiers in Plant Science 03/2014; 5:109. DOI:10.3389/fpls.2014.00109 · 3.95 Impact Factor
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    • "PC-deficient mutants of Bradyrhizobium japonicum and Sinorhizobium meliloti exhibit drastically reduced symbiosis with their plant hosts [8], [9]. The plant pathogen Agrobacterium tumifaciens requires PC for assembly of the T4SS components which are critical for formation of crown-gall tumors on plants [10], [11]. In the human pathogen Legionella pneumophila, PC-deficient mutants were attenuated for virulence and had increased susceptibility to macrophage-mediated killing [12]. "
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    ABSTRACT: During growth in presence of choline, both laboratory and clinical Pseudomonas aeruginosa strains synthesize phosphatidylcholine (PC), and PC makes up ∼4% of the total membrane phospholipid content. In all the strains tested, PC synthesis occurred only when choline is provided exogenously. Mutants defective in synthesis of PC were generated in the strain backgrounds PAO1 and PA14. Minimum inhibitory concentration studies testing sensitivity of PC-deficient strains towards various antibiotics and cationic antimicrobial peptides revealed no differences as compared to wild-type strains. Mutants incapable of synthesizing PC were also found to be unaffected in motility and biofilm formation on abiotic surfaces, colonization of biotic surfaces and virulence in a mouse infection model. A global phenotypic microarray was further used to identify conditions wherein membrane PC may play a role of in P. aeruginosa. No culture conditions were identified wherein wild-type and PC-deficient mutants showed phenotypic differences. Membrane PC may serve a highly specific role during P. aeruginosa interactions with its eukaryotic hosts based on all the clinical strains tested retaining the ability to synthesize it during availability of choline.
    PLoS ONE 02/2012; 7(2):e30829. DOI:10.1371/journal.pone.0030829 · 3.23 Impact Factor
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