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Phosphatidic acid (PA) is the precursor of most phospholipids like phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. In bacteria, its biosynthesis begins with the acylation of glycerol‐3‐phosphate to lysophosphatidic acid (LPA), which is further acylated to PA by the PlsC enzyme. Some bacteria, like the plant pathogen Xanthomonas campestris, use a similar pathway to acylate lysophosphatidylcholine to phosphatidylcholine (PC). Previous studies assigned two acyltransferases to PC formation. Here, we set out to study their activity and found a second much more prominent function of these enzymes in LPA to PA conversion. This PlsC‐like activity was supported by the functional complementation of a temperature‐sensitive plsC‐deficient Escherichia coli strain. Biocomputational analysis revealed two further PlsC homologs in X. campestris. The cellular levels of the four PlsC‐like proteins varied with respect to growth phase and growth temperature. To address the question whether these enzymes have redundant or specific functions, we purified two recombinant, detergent‐solubilized enzymes in their active form, which enabled the first direct biochemical comparison of PlsC isoenzymes from the same organism. Overlapping but not identical acyl acceptor and acyl donor preferences suggest redundant and specialized functions of the X. campestris PlsC enzymes. The altered fatty acid composition in plsC mutant strains further supports the functional differentiation of these enzymes. [ABSTRACT FROM AUTHOR]

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.

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.

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 Bradyrhizobiumjaponicum. 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.

Knowledge on cortical development is based mainly on rodents besides primates and carnivores, all being altricial. Here, we analyzed a precocial animal, the pig, looking at dorsoparietal cortex from E45 to P90. At E45, most ionized calcium‐binding adapter molecule 1‐positive (Iba1+) cells had a macrophage‐like morphology and resided in meninges and choroid plexus. Only a few cells were scattered in the ventricular and subventricular zone (VZ and SVZ). At E60/E70, all laminar compartments displayed microglia cells at a low‐to‐moderate density, being highest in VZ and SVZ followed by intermediate zone/white matter (IZ/WM). The cortical plate and marginal zone displayed only a few Iba1+ cells. Cells were intensely labeled, but still had poorly arborized somata and many resembled ameboid, macrophage‐like microglia. Concurrent with a massive increase in cortical volume, microglia cell density increased until E85, and further until E100/E110 (birth at E114) to densities that resemble those seen postnatally. A fraction of microglia colabeled with Ki67 suggesting proliferation in all laminar compartments. Cell‐to‐cell distance decreased substantially during this time, and the fraction of microglia to all nuclei and to neurons increases in the laminar compartments. Eventually, of all cortical DAPI+ nuclei 7–12% were Iba1+ microglia. From E70 onwards, more and more cells with ramified processes were present in MZ down to IZ/WM, showing, for instance, a close association with NeuN+, NPY+, and GAD65/67+ somata and axon initial segments. These results suggested that the development of microglia cell density and morphology proceeds rapidly from mid‐gestation onwards reaching near‐adult status already before birth. [ABSTRACT FROM AUTHOR]

Summary: 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. In this study, we show that the virulence factor AcvB is a lysyl‐phosphatidylglycerol (L‐PG) hydrolase that controls proper L‐PG levels in A. tumefaciens membranes. Loss of AcvB results in increased L‐PG accumulation, which is accompanied with loss of virulence due to abolished T‐DNA transfer into plant cells. [ABSTRACT FROM AUTHOR]

Bacteria alter the biophysical properties of their membrane lipids in response to environmental cues, such as shifts in pH or temperature. In essence, lipid composition determines membrane structure, which in turn influences many basic functions, such as transport, secretion, and signaling. Like other members of the phylum Bacteroidetes, the oral anaerobe Porphyromonas gingivalis possesses the ability to synthesize a variety of novel membrane lipids, including species of dihydroceramides that are distinct, yet similar in structure to sphingolipids produced by the human host. The role of dihydroceramides in the physiology and pathogenic potential of the human microbiota is only beginning to be explored; yet there is increasing data indicating that these lipids play a role in human diseases, such as periodontitis and multiple sclerosis. Here, we report on the identification of a gene (PG1780) in the chromosome of P. gingivalis strain W83 encoding a putative serine palmitoyltransferase, the enzyme that catalyzes the first step in sphingolipid biosynthesis. While we were able to detect dihydroceramides in whole lipid extracts of P. gingivalis cells as well as crude preparations of outer membrane vesicles, sphingolipids were absent in the PG1780 mutant strain. Moreover, we show that the synthesis of sphingolipids plays an essential role in the long-term survival of the organism as well as its resistance to oxidative stress. Further, a PG1780 mutant displayed much lower activity of cell-associated arginine and lysine gingipains, yet slightly higher activity in the corresponding culture supernates, which we hypothesize is due to altered membrane properties and anchoring of these proteases to the cell surface. In addition, we determined that sphingolipid production is critical to the presentation of surface polysaccharides, with the mutant strain displaying less K-antigen capsule and more anionic polysaccharide (APS). Overall, we have discovered that, in addition to their role in pathogenicity, the synthesis of sphingolipids is critical to the cellular homeostasis and persistence of this important dental pathogen. [ABSTRACT FROM AUTHOR]

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 X anthomonas 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 X anthomonas, in which two acyl side-chains are added to a glycerophosphocholine ( GPC) backbone. Two out of 12 tested acyltransferases from X anthomonas 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. [ABSTRACT FROM AUTHOR]

Phosphatidylcholine (PC) is critical for the nitrogen-fixing symbiosis between rhizobia and legumes. We characterized three PC biosynthesis pathways in Rhizobium leguminosarum and evaluated their impact on nitrogen fixation in clover nodules. In the presence of choline, a PC synthase catalyzes the condensation of cytidine diphosphate-diacylglycerol with choline to produce PC. In the presence of lyso-PC, acyltransferases acylate this mono-acylated phospholipid to PC. The third pathway relies on phospholipid N-methyltransferases (Pmts), which sequentially methylate phosphatidylethanolamine (PE) through three rounds of methylation, yielding PC via the intermediates monomethyl-PE and dimethyl-PE. In R. leguminosarum, at least three Pmts participate in this methylation cascade. To elucidate the functions of these enzymes, we recombinantly produced and biochemically characterized them. We moved on to determine the phospholipid profiles of R. leguminosarum mutant strains harboring single and combinatorial deletions of PC biosynthesis genes. The cumulative results show that PC production occurs through the combined action of multiple enzymes, each with distinct substrate and product specificities. The methylation pathway emerges as the dominant PC biosynthesis route, and we pinpoint PmtS2, which catalyzes all three methylation steps, as the enzyme responsible for providing adequate PC amounts for a functional nitrogen-fixing symbiosis with clover. [ABSTRACT FROM AUTHOR]

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., (© 2018 John Wiley & Sons Ltd.)

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., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)