Phosphate starvation in fungi induces the replacement of phosphatidylcholine with the phosphorus-free betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine

Abstract

Diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) is a phosphorus-free betaine-lipid analog of phosphatidylcholine (PtdCho) synthesized by many soil bacteria, algae, and nonvascular plants. Synthesis of DGTS and other phosphorus-free lipids in bacteria occurs in response to phosphorus (P) deprivation and results in the replacement of phospholipids by nonphosphorous lipids. The genes encoding DGTS biosynthetic enzymes have previously been identified and characterized in bacteria and the alga Chlamydomonas reinhardtii. We now report that many fungal genomes, including those of plant and animal pathogens, encode the enzymatic machinery for DGTS biosynthesis, and that fungi synthesize DGTS during P limitation. This finding demonstrates that replacement of phospholipids by nonphosphorous lipids is a strategy used in divergent eukaryotic lineages for the conservation of P under P-limiting conditions. Mutants of Neurospora crassa were used to show that DGTS synthase encoded by the BTA1 gene is solely responsible for DGTS biosynthesis and is under the control of the fungal phosphorus deprivation regulon, mediated by the NUC-1/Pho4p transcription factor. Furthermore, we describe the rational reengineering of lipid metabolism in the yeast Saccharomyces cerevisiae, such that PtdCho is completely replaced by DGTS, and demonstrate that essential processes of membrane biogenesis and organelle assembly are functional and support growth in the engineered strain.

FIG 1

Schematic of proposed P_i starvation-responsive synthesis of DGTS in fungi. (A) Synthesis of DGTS in bacteria is carried out by the proteins BtaA and BtaB, which both use S-adenosylmethionine as a cosubstrate. The structure of PtdCho is shown as a comparison to DGTS, highlighting structural similarities. DAG, diacylglycerol; DGHS, diacylglycerylhomoserine; DGTS, diacylglyceryl-N,N,N-trimethylhomoserine; PtdCho, phosphatidylcholine. (B) Proposed scheme for P_i starvation-induced activation of DGTS synthesis in fungi. P_i starvation induces the nuclear translocation of the Pho4 transcription factor, which binds to and activates genes of the PHO regulon via a common upstream activation sequence (UAS). Fungal BTA1 gene promoters contain multiple P_i starvation response elements; thus, P_i starvation is expected to induce expression of the BTA1 protein. (C) The fungal BTA1 protein is homologous to algal BTA1 proteins and consists of two domains, corresponding to the bacterial BtaA and BtaB proteins, with corresponding enzymatic functions in the DGTS biosynthetic pathway, as indicated in panel A.

FIG 2

P_i starvation induces DGTS accumulation in fungi. (A and B) N. crassa was grown in minimal medium containing the indicated amounts of P_i and then harvested for lipid analysis. Two-dimensional TLC of P_i-replete (A) and P_i-limited (B) N. crassa was performed as described in the text, and lipids were visualized with I₂ vapor. Lipid spots are numbered as follows, in reference to authentic standards: 1, PtdIns; 2, PtdSer; 3, PtdCho; 4, PtdEtn; 5, cardiolipin; 6, DGTS. (C) K. lactis cultures were grown to saturation in minimal medium with the indicated initial concentration of P_i, and lipids were extracted, separated by standard TLC, and visualized as described in the text. Authentic standard compounds were purchased from Avanti Polar Lipids (Alabaster, AL, USA) or, in the case of DGTS, purified from Chlamydomonas reinhardtii lipid extracts.

FIG 3

DGTS accumulation in N. crassa is dependent on the BTA-1 structural gene and P starvation transcription factor NUC-1. P_i-replete (A) and -limited (B) liquid cultures of strains of the indicated genotype were prepared as described in the text, and lipids were prepared and analyzed by TLC and I₂ vapor staining.

FIG 4

DGTS accumulation rescues choline auxotrophy in pem1Δ pem2Δ S. cerevisiae. (A) A choline (Cho) auxotrophic pem1Δ pem2Δ yeast strain bearing a plasmid containing either the KlBTA1 gene (pKlBTA1) or an empty vector control (pYES2.1) was grown with or without choline (Cho) for 40 h, and the cultures were photographed to demonstrate that the pKlBTA1 plasmid causes reversion of Cho auxotrophy in the pem1Δ pem2Δ strain background. (B) Cells were harvested and lipids extracted from these cultures and examined by TLC and staining with I₂ vapor as described in the text, and representative results from a single experiment are represented as individual lanes under the culture from which they originate. (C) Iodine-stained bands were quantified by analysis with ImageJ software and are presented as the fraction of total pixel counts for the lane. Data presented in panel C represent the means ± standard errors of the means from 3 independent cultures.

FIG 5

Growth phenotypes of DGTS-accumulating S. cerevisiae strains. Cultures of the indicated genotype were prepared as described for Fig. 4A and inoculated at an initial OD₆₀₀ of 0.05 in SC galactose (A) or SC lactate (B), and growth was monitored by measurement of the OD₆₀₀ at regular intervals. Results shown are representative of 4 individual independent experiments.

FIG 6

Lipid droplet morphology of DGTS-accumulating S. cerevisiae strains. Cultures of the indicated genotype were grown to late log phase, stained with Nile red, and subjected to fluorescence microscopy as described in Materials and Methods, using an Evos Fl GFP light cube and 100× oil immersion objective. Fields of cells are representative of at least three independent cultures.

FIG 7

Endocytosis and vacuolar morphology of DGTS-accumulating S. cerevisiae strains. Cultures of the indicated genotype were grown to mid-log phase, stained with FM4-64 as described in Materials and Methods, and subjected to fluorescence microscopy using an Evos Fl RFP light cube and 100× oil immersion objective. Fields of cells are representative of at least three independent cultures and the corresponding uptake experiments.