Functional, transcriptomic, and lipidomic studies of the choC gene encoding a phospholipid methyltransferase in Aspergillus fumigatus.

IMPORTANCE: This study explored the phospholipid metabolic pathway in A. fumigatus and its relationship with fungal growth, metabolism, and pathogenicity. ChoC, based on its critical roles in many aspects of the fungus and relatively conserved characteristics in filamentous fungi with low similarity with mammalian ones, can be a novel target of new antifungal drugs.

The importance of Arabidopsis PHOSPHOLIPID N-METHYLTRANSFERASE in glycerolipid metabolism and plant growth.

Phosphatidylcholine (PC) is a major class of phospholipids that are essential for post-embryonic growth in plants. In Arabidopsis, three copies of the phospho-base N-methyltransferase, PMT1, PMT2, and PMT3, are known to account for PC biosynthesis because the triple-knockout mutant is devoid of biosynthesis and shows lethality in post-embryonic but not embryonic growth. Arabidopsis also contains a distinct phospholipid N-methyltransferase (PLMT) that is homologous with yeast and animal PLMT that methylates phospholipids to produce PC. However, the knockout mutant of PLMT does not show morphological phenotypes or decreased PC content, so the role of PLMT remains unclear. Here, we show that PLMT is ubiquitously expressed in different organs and localized at the endoplasmic reticulum, where PC is produced. Overexpression of PLMT in planta increased the content of phospholipids including PC and affected vegetative but not reproductive growth. Although silique lengths were shorter, pollen remained viable and mature seeds were produced. Intriguingly, seed triacylglycerol content was increased with altered fatty acid composition. We conclude that PLMT might be a functional enzyme in PC biosynthesis and play an organ-specific role in developing seeds, where rapid accumulation of triacylglycerol dominates the entire glycerolipid metabolic flux.

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

One of the most common pathways for the biosynthesis of the phospholipid phosphatidylcholine (PC) in bacteria is the successive 3-fold N-methylation of phosphatidylethanolamine (PE) catalyzed by phospholipid N-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 N-methyltransferases in a previously unexplored set of thermophilic environmental bacteria. IMPORTANCE 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 N-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.

The topology of the ER-resident phospholipid methyltransferase Opi3 of Saccharomyces cerevisiae is consistent with in trans catalysis.

Phospholipid N-methyltransferases (PLMTs) synthesize phosphatidylcholine by methylating phosphatidylethanolamine using S-adenosylmethionine as a methyl donor. Eukaryotic PLMTs are integral membrane enzymes located in the endoplasmic reticulum (ER). Recently Opi3, a PLMT of the yeast Saccharomyces cerevisiae was proposed to perform in trans catalysis, i.e. while localized in the ER, Opi3 would methylate lipid substrates located in the plasma membrane at membrane contact sites. Here, we tested whether the Opi3 active site is located at the cytosolic side of the ER membrane, which is a prerequisite for in trans catalysis. The membrane topology of Opi3 (and its human counterpart, phosphatidylethanolamine N-methyltransferase, expressed in yeast) was addressed by topology prediction algorithms and by the substituted cysteine accessibility method. The results of these analyses indicated that Opi3 (as well as phosphatidylethanolamine N-methyltransferase) has an N-out C-in topology and contains four transmembrane domains, with the fourth forming a re-entrant loop. On the basis of the sequence conservation between the C-terminal half of Opi3 and isoprenyl cysteine carboxyl methyltransferases with a solved crystal structure, we identified amino acids critical for Opi3 activity by site-directed mutagenesis. Modeling of the structure of the C-terminal part of Opi3 was consistent with the topology obtained by the substituted cysteine accessibility method and revealed that the active site faces the cytosol. In conclusion, the location of the Opi3 active site identified here is consistent with the proposed mechanism of in trans catalysis, as well as with conventional catalysis in cis.

SMRT sequencing reveals differential patterns of methylation in two O111:H- STEC isolates from a hemolytic uremic syndrome outbreak in Australia.

In 1995 a severe haemolytic-uremic syndrome (HUS) outbreak in Adelaide occurred. A recent genomic analysis of Shiga toxigenic Escherichia coli (STEC) O111:H- strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes encoded within prophage regions. The structure of the Stx2-converting prophages could not be fully resolved using short-read sequence data alone and it was not clear if there were other genomic differences between 95JB1 and 95NR1. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) sequencing to characterise the genome and methylome of 95JB1 and 95NR1. We completely resolved the structure of all prophages including two, tandemly inserted, Stx2-converting prophages in 95NR1 that were absent from 95JB1. Furthermore we defined all insertion sequences and found an additional IS1203 element in the chromosome of 95JB1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5'-CTGCm6AG-3') in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethylated in the 95JB1 genome, and included at least 177 potential promoter regions that could contribute to regulatory differences between the strains. IS1203 mediated deactivation of a novel type IIG methyltransferase in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the capability of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

Roles of phospholipid methyltransferases in pycnidia development, stress tolerance and secondary metabolism in the taxol-producing fungus Pestalotiopsis microspore.

Phosphatidylcholine (PC) is an important membrane component of the eukaryotic cell. In yeast fungi, two phospholipid methyltransferases catalyze consecutive steps of methylation in the formation of phosphatidylcholine from phosphatidylethanolamine. However, roles of phospholipid methyltransferases in filamentous fungi remains less investigated. We report here the characterization of two genes, choA and choC, that putatively encoded phospholipid methyltransferases in the taxol-producing fungus Pestalotiopsis microspora. Deletion of choC resulted in defects in PC production, vegetative growth and development of asexual structure. The mutant strains exhibited multiple morphological abnormalities, e.g. swollen hyphal tips and enhanced hyphal branching, and even mycelial autolysis. Some novel roles for the genes were also revealed, for instance, the deletion of either choC or choA impaired the development of pycnidia and conidia, the cell wall integrity. The mutant strains displayed a hypersensitivity to stress conditions, e.g. osmotic stress, cold and metal ions. The osmotic hypersensitivity indicates a crosstalk of PC pathways to other signaling pathways, such as the HOG pathway. Still more, choA, but not choC, was required for the production of secondary metabolites, e.g. pestalotiollide B, suggesting distinct roles of the two genes. This work would contribute to better understanding the function of phospholipid methyltransferases in fungi.

Petit-High Pressure Carbon Dioxide stress increases synthesis of S-Adenosylmethionine and phosphatidylcholine in yeast Saccharomyces cerevisiae.

Petit-High Pressure Carbon Dioxide (p-HPCD) is a promising nonthermal technology for foods pasteurization. Cluster analysis of gene expression profiles of Saccharomyces cerevisiae exposed to various stresses exhibited that gene expression profile for p-HPCD stress (0.5MPa, 25°C) was grouped into a cluster including profiles for Sodium Dodecyl Sulfate and Roundup herbicide. Both are detergents that can disorder membrane structurally and functionally, which suggests that cell membrane may be a target of p-HPCD stress to cause cell growth inhibition. Through metabolomic analysis, amount of S-Adenosylmethionine (AdoMet) that is used as methyl donor to participate in phosphatidylcholine synthesis via phosphatidylethanolamine (PE) methylation pathway, was increased after p-HPCD treatment for 2h. The key gene OPI3 encoding phospholipid methyltransferase that catalyzes the last two steps in PE methylation pathway was confirmed significantly induced by RT-PCR. Transcriptional expression of genes (MET13, MET16, MET10, MET17, MET6 and SAM2) related to AdoMet biosynthesis was also significantly induced. Choline as the PC precursor and ethanolamine as PE precursor in Kennedy pathway were also found increased under p-HPCD condition. We also found that amounts of most of amino acids involving protein synthesis were found decreased after p-HPCD treatment for 2h. Moreover, morphological changes on cell surface were observed by scanning electron microscope. In conclusion, the effects of p-HPCD stress on cell membrane appear to be a very likely cause of yeast growth inhibition and the enhancement of PC synthesis could contribute to maintain optimum structure and functions of cell membrane and improve cell resistance to inactivation.

The choC gene encoding a putative phospholipid methyltransferase is essential for growth and development in Aspergillus nidulans.

Phosphatidylcholines (PCs) are a class of major cell membrane phospholipids that participate in many physiological processes. Three genes, choA, choB and choC, have been proposed to function in the endogenous biosynthesis of PC in Aspergillus nidulans. In this study, we characterize the choC gene encoding a putative highly conserved phospholipid methyltransferase. The previously reported choC3 mutant allele results from a mutation leading to the E177K amino acid substitution. The transcript of choC accumulates at high levels during vegetative growth and early asexual developmental phases. The deletion of choC causes severe impairment of vegetative growth, swelling of hyphal tips and the lack of both asexual and sexual development, suggesting the requirement of ChoC and PC in growth and development. Noticeably, supplementation of the mutant with the penultimate precursor of PC N, N-dimethylaminoethanol leads to full recovery of vegetative growth, but incomplete progression of asexual and sexual development, implying differential roles of PC and its intermediates in fungal growth and development. Importantly, while the choC deletion mutant shows reduced vegetative growth and precocious cell death until day 4, it regains hyphal proliferation and cell viability from day 5, indicating the presence of an alternative route for cellular membrane function in A. nidulans.

Expression and physiological relevance of Agrobacterium tumefaciens phosphatidylcholine biosynthesis genes.

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 synthesis is required for optimal function of Legionella pneumophila virulence determinants.

The function of phosphatidylcholine (PC) in the bacterial cell envelope remains cryptic. We show here that productive interaction of the respiratory pathogen Legionella pneumophila with host cells requires bacterial PC. Synthesis of the lipid in L. pneumophila was shown to occur via either phospholipid N-methyltransferase (PmtA) or phosphatidylcholine synthase (PcsA), but the latter pathway was demonstrated to be of predominant importance. Loss of PC from the cell envelope caused lowered yields of L. pneumophila within macrophages as well as loss of high multiplicity cytotoxicity, while mutants defective in PC synthesis could be complemented either by reintroduction of PcsA or by overproduction of PmtA. The lowered yields and reduced cytotoxicity in mutants with defective PC biosynthesis were due to three related defects. First, there was a poorly functioning Dot/Icm apparatus, which delivers substrates required for intracellular growth into the cytosol of infected cells. Second, there was reduced bacterial binding to macrophages, possibly due to loss of PC or a PC derivative on the bacterium that is recognized by the host cell. Finally, strains lacking PC had low steady-state levels of flagellin protein, a deficit that had been previously associated with the phenotypes of lowered cytotoxicity and poor cellular adhesion.