Migration of Polyphosphate Granules in Agrobacterium tumefaciens.

Agrobacterium tumefaciens has two polyphosphate (polyP) kinases, one of which (PPK1AT) is responsible for the formation of polyP granules, while the other (PPK2AT) is used for replenishing the NTP pools by using polyP as a phosphate donor to phosphorylate nucleoside diphosphates. Fusions of eYFP with PPK2AT or of the polyP granule-associated phosin PptA from Ralstonia eutropha always co-localized with polyP granules in A. tumefaciens and allowed the tracking of polyP granules in time-lapse microscopy experiments without the necessity to label the cells with the toxic dye DAPI. Fusions of PPK1AT with mCherry formed fluorescent signals often attached to, but not completely co-localizing with, polyP granules in wild-type cells. Time-lapse microscopy revealed that polyP granules in about one-third of a cell population migrated from the old pole to the new cell pole shortly before or during cell division. Many cells de novo formed a second (nonmigrating) polyP granule at the opposite cell pole before cell division was completed, resulting in two daughter cells each having a polyP granule at the old pole after septum formation. Migration of polyP granules was disordered in mitomycin C-treated or in PopZ-depleted cells, suggesting that polyP granules can associate with DNA or with other molecules that are segregated during the cell cycle.

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

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.

Two Distinct Cardiolipin Synthases Operate in Agrobacterium tumefaciens.

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.

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

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.

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

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.

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

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.

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

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.

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

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.