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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]

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: The bacterial membrane is constantly remodelled in response to environmental conditions and the external supply of precursor molecules. Some bacteria are able to acquire exogenous lyso‐phospholipids and convert them to the corresponding phospholipids. Here, we report that some soil‐dwelling bacteria have alternative options to metabolize lyso‐phosphatidylglycerol (L‐PG). We find that the plant‐pathogen Agrobacterium tumefaciens takes up this mono‐acylated phospholipid and converts it to two distinct isoforms of the non‐canonical lipid bis(monoacylglycero)phosphate (BMP). Chromatographic separation and quadrupole‐time‐of‐flight MS/MS analysis revealed the presence of two possible BMP stereo configurations acylated at either of the free hydroxyl groups of the glycerol head group. BMP accumulated in the inner membrane and did not visibly alter cell morphology and growth behaviour. The plant‐associated bacterium Sinorhizobium meliloti was also able to convert externally provided L‐PG to BMP. Other bacteria like Pseudomonas fluorescens and Escherichia coli metabolized L‐PG after cell disruption, suggesting that BMP production in the natural habitat relies both on dedicated uptake systems and on head‐group acylation enzymes. Overall, our study adds two previously overlooked phospholipids to the repertoire of bacterial membrane lipids and provides evidence for the remarkable condition‐responsive adaptation of bacterial membranes. [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]

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 E scherichia 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 X anthomonas 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). X anthomonas 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. [ABSTRACT FROM AUTHOR]

The membrane lipid phosphatidylcholine ( PC) is crucial for stress adaptation and virulence of the plant pathogen A grobacterium 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 (α A and α F) contribute to membrane binding of PmtA. The N-terminal helix α A binds anionic lipids in vitro with higher affinity than the central helix α F. 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 α A 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. [ABSTRACT FROM AUTHOR]

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 DX3D). 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 and by (, , ) [ABSTRACT FROM AUTHOR]

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 X anthomonas 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. X anthomonas 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). [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]