Manipulation of major membrane lipid synthesis and its effects on sporulation in Saccharomyces cerevisiae

During the sporulation process of Saccharomyces cerevisiae, meiotic progression is accompanied by de novo formation of the prospore membrane inside the cell. However, it remains to be determined whether certain species of lipids are required for spore formation in yeast. In this study, we analyzed the requirement of the synthesis of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and ergosterol for spore formation using strains in which the synthesis of these lipids can be controlled. When synthesis of PE and PC was repressed, sporulation efficiency decreased. This suggests that synthesis of these phospholipids is vital to proper sporulation. In addition, sporulation was also impaired in cells with a lowered sterol content, raising the possibility that sterol content is also important for spore formation.     

Effects of modification of membrane lipid composition on Bacillus subtilis sporulation and spore properties

Aims: To determine effects of inner membrane lipid composition on Bacillus subtilis sporulation and spore properties. Methods and Results: The absence of genes encoding lipid biosynthetic enzymes had no effect on B. subtilis sporulation, although the expected lipids were absent from spores’ inner membrane. The rate of spore germination with nutrients was decreased c. 50% with mutants that lacked the major cardiolipin (CL) synthase and another enzyme for synthesis of a major phospholipid. Spores lacking the minor CL synthase or an enzyme essential for glycolipid synthesis exhibited 50–150% increases in rates of dodecylamine germination, while spores lacking enzymes for phosphatidylethanolamine (PE), phosphatidylserine (PS) and lysylphosphatidylglycerol (l‐PG) synthesis exhibited a 30–50% decrease. Spore sensitivity to H₂O₂ and tert‐butylhydroperoxide was increased 30–60% in the absence of the major CL synthase, but these spores’ sensitivity to NaOCl or Oxone^? was unaffected. Spores of lipid synthesis mutants were less resistant to wet heat, with spores lacking enzymes for PE, PS or l‐PG synthesis exhibiting a two to threefold decrease and spores of other strains exhibiting a four to 10‐fold decrease. The decrease in spore wet heat resistance correlated with an increase in core water content. Conclusions: Changing the lipid composition of the B. subtilis inner membrane did not affect sporulation, although modest effects on spore germination and wet heat and oxidizing agent sensitivity were observed, especially when multiple lipids were absent. The increases in rates of dodecylamine germination were likely due to increased ability of this compound to interact with the spore’s inner membrane in the absence of some CL and glycolipids. The effects on spore wet heat sensitivity are likely indirect, because they were correlated with changes in core water content. Significance and Impact of the Study: The results of this study provide insight into roles of inner membrane lipids in spore properties.     

Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.     

脱落酸对丛枝菌根真菌侵染和产孢的调控效应研究

【目的】揭示脱落酸(ABA)对丛枝菌根(AM)真菌侵染和产孢的影响,建立利用外源ABA促进孢子产量的高效菌剂扩繁方法。【方法】利用番茄毛状根和AM真菌Rhizophagus irregularis DAOM 197198建立双重培养体系,通过外源施用ABA、赤霉素(GA)或者使用ABA、GA的缺陷突变体,染色观察菌根侵染,荧光定量PCR测定丛枝发育和脂质合成运输相关基因的表达,统计丛枝和孢子的数量,从而揭示ABA对AM真菌侵染和产孢的影响。【结果】ABA缺陷突变体not中的F%(侵染频率)、a%(丛枝丰度)、丛枝数量,以及丛枝发育特异性相关基因EXO70A1-like (LOC101253481)、脂质合成运输相关基因RAM2和STR2的表达均显著低于其野生型MT;外源施用ABA显著促进了F%、M%(侵染强度)、丛枝数量、孢子产量,以及脂质合成运输相关基因RAM2和STR2的表达,外源添加ABA处理的孢子产量约为不添加处理的4.5倍;外源GA处理极显著抑制了菌根侵染的所有指标和孢子产量;GA缺陷突变体gib3与其野生型MM的AM真菌侵染之间没有显著差异,但gib3的孢子产量显著高于MM...     

Unconventional membrane lipid biosynthesis in Xanthomonas campestris

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.     

Lipid composition of plasma membranes from barley leaves and roots, spinach leaves and cauliflower inflorescences

Highly purified plasma membranes (PM) were obtained from barley (Hordeum vulgare L. cv. Kristina) leaves and roots, spinach (Spinacia oleracea L. cv. Viking II) leaves, and cauliflower (Brassica oleracea) inflorescences by partitioning in an aqueous polymer two-phase system. The sterol and polar lipid composition of the PM, including the fatty acid composition of the glycerolipids, was determined. Dominating lipids were free sterols, glucocerebroside, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), although large variations in content were observed between the PM of the different species and organs. Thus, the spinach leaf PM contained only 7% (mol %) free sterol compared to over 30% free sterol in the other PM analysed, with the barley root PM as the other extreme (57% free sterol). On the other hand, sterol derivatives were more abundant in the spinach leaf PM, containing 13% acylated sterol glycosides. Cerebroside constituted 16% of the lipids in the barley leaf PM but only 3% in cauliflower. The phospholipids PC and PE ranged from 25 and 24%, respectively, in the spinach leaf PM to 8 and 7%, respectively, in the barley root PM. As a result of the large variations in sterol and phospholipid content, the ratio of free sterol to phospholipid varied from 2.2 in the barley root PM to only 0.1 in the spinach leaf PM. Sitosterol, campesterol and stigmasterol were the completely dominating sterols in the barley and cauliflower PM, whereas the unique sterol composition of spinach was dominated by spinasterol. Palmitic (16:0), linoleic (18:2) and linolenic (18:3) acid were the major glycerolipid fatty acids. The fatty acid composition of the barley root PM was the most saturated (44% 16:0, 13% 18:3), whereas that of the cauliflower PM was the most unsaturated (21% 16:0,42% 18:3). Thus, very large variations were observed in both total lipid and fatty acid composition of the PM investigated, which represent both mono— and dicotyledons, as well as both photosynthetic and non-photosynthetic tissue. The consequences of this large diversity in composition of the lipid bilayer for the function of integral PM proteins are discussed.     

The program of protein synthesis during sporulation in Bacillus subtilis

The program of protein synthesis was examined during sporulation in Bacillus subtilis as an index of the control of gene expression. At various stages of growth and spore formation, cells of B. subtilis were pulse-labeled with ³⁵S-methionine. Protein was extracted from the radioactively labeled bacteria and then subjected to high resolution one-dimensional and two-dimensional slab gel electrophoresis. We report that sporulating cells restricted or “turned off” the synthesis of certain polypeptides characteristic of the vegetative phase of growth. In certain cases, this “turn off” was prevented in a mutant (SpoOa-5NA) blocked at the first stage of spore formation. Sporulating bacteria also elaborated new polypeptide species that could not be detected in vegetatively growing cells or in cells of the asporogenous mutant SpoOa-5NA in sporulation medium. The synthesis of these sporulation-specific proteins was “turned on” in a temporally defined sequence throughout the period of spore formation. Spore coat protein, for example, was first synthesized at 4 hr after the onset of sporulation, the time at which refractile prespores appeared. Certain sporulation-specific polypeptides including the coat protein were among the most actively produced polypeptides in sporulating cells.     

Effect of nitrogen limitation and sporulation on sterol and lipid formation inSaccharomyces cerevisiae

The content of sterols and lipids was compared in the cells ofSaccharomyces cerevisiae cultivated in sporulation and the sterol-induction nitrogen-limited media. After 24 h the measured values in the two cultivations did not significantly differ. However, after subsequent 24 h, further formation of lipid globules and a corresponding increase of lipid and sterol content was detected only in the sterol-induction medium. To demonstrate the similarity of physiological state during the first day of the two cultivations, the combined cultivations were performed. Maximum sporulation, suggesting maximum similarity, of the two processes was achieved when the cells were grown in the sterol-induction medium for 15 h and then transferred to a sporulation medium.     

Chemical and Morphological Studies of Bacterial Spore Formation : III. The Effect of 8-Azaguanine on Spore and Parasporal Protein Formation in Bacillus cereus var. Alesti

The purine analogue, 8-azaguanine, was added to cultures of the parasporal crystal-forming organism Bacillus cereus var. alesti at different times during growth and synchronous sporulation. The effect of its incorporation has been studied with particular reference to cell growth, nucleic acid composition, cytology, and the synthesis of the spore and crystal protein. Additions of the analogue during any stage of growth prevented further cell proliferation and all spore and crystal formation. Since both nucleic acids continued to be formed, cells of an increased size developed, containing large masses of chromatin in the form of condensed balls or axial cords. Lipid-containing inclusions also appeared following these additions and were usually aggregated at the centre or poles of the cells. The analogue could be isolated as the ribonucleotide from both the acid soluble and RNA fractions of these inhibited cells. Additions of the analogue following commencement of sporulation did not prevent either spore or crystal formation or affect the nucleic acid content of the sporulating cells. However, as before, the 8-azaguanine was incorporated into both the acid soluble and RNA of the cells, but not into these fractions of the spores ultimately formed. The implications of these findings are discussed in relation to crystal protein synthesis.     

Bichemical genetics of bacterial sporulation

Summary Pleitropic interactions among genes controlling the formation of bacterial spores and of sporulation-associated products are studied. In order to obtain sporulation mutants, spores have been germinated in the presence of chloramphenicol and then treated with nitrosoguanidine. In the most favorable conditions 25% of sporulation mutants have been found among the 40% surviving bacteria. This number is at least four times higher than the number of auxotrophic mutants, therefore a rough estimate of the number of genes involved in sporulation is 800.Rapid plate-tests have been developed for the oxidation of terrazolium salts, the formation of various proteolytic enzymes and the production of antibiotics. Although the exact biochemical nature of the products is not yet known, the results suggest that distinct factors, probably various enzymes (including several proteases) are detected by these tests. All of them are associated with spore formation and absent from a large number of sporulation mutants. Using these tests, the phenotypes of 500 randomly selected sporulation mutants were determined. No important differences were found between asporogenous and oligosporogenous mutants. The number of mutants deficient for several sporulation-associated characters is large, pleiotropic interactions following a defined pattern are observed. Statistical analysis indicates the existence of a unidirectional pleiotropic system. All the results agree with the hypothesis of sequential gene activation. Consequently, the sporulation-associated characters can be ordered into a linear sequence, presumably reflecting the consecutive steps in spore formation. The order obtained is the following: gelatinase, proteases acting on casein and on denatured albumin, oxidation of tetrazolium No 7, digestion of protamine, production of antibiotics (against a Staphylococcus and a Bacillus), hydrolysis of hemoglobin, oxidation of tetrazolium No 2, digestion of native albumin, synthesis of elastase. Another category of mutants, blocked in a late step of sporulation and apparently derepressed for the formation of elastase, is also described.In conclusion, arguments are put forward in favor of sequential gene activation. Sporulation genes, related by unidirectional pleiotropic interactions, form a sporulon. Generalization of this concept to other differentiating systems (a differon), its predictions and possible experimental confirmation are considered.The author was a Gosney Research Fellow in 1966/67, on leave of absence from the Centre National de la Recherche Scientifique, Paris. Present adress: see end of paper.     

Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation‐specific genes

Three classes of low‐G+C Gram‐positive bacteria (Firmicutes), Bacilli, Clostridia and Negativicutes, include numerous members that are capable of producing heat‐resistant endospores. Spore‐forming firmicutes include many environmentally important organisms, such as insect pathogens and cellulose‐degrading industrial strains, as well as human pathogens responsible for such diseases as anthrax, botulism, gas gangrene and tetanus. In the best‐studied model organism Bacillus subtilis, sporulation involves over 500 genes, many of which are conserved among other bacilli and clostridia. This work aimed to define the genomic requirements for sporulation through an analysis of the presence of sporulation genes in various firmicutes, including those with smaller genomes than B. subtilis. Cultivable spore‐formers were found to have genomes larger than 2300 kb and encompass over 2150 protein‐coding genes of which 60 are orthologues of genes that are apparently essential for sporulation in B. subtilis. Clostridial spore‐formers lack, among others, spoIIB, sda, spoVID and safA genes and have non‐orthologous displacements of spoIIQ and spoIVFA, suggesting substantial differences between bacilli and clostridia in the engulfment and spore coat formation steps. Many B. subtilis sporulation genes, particularly those encoding small acid‐soluble spore proteins and spore coat proteins, were found only in the family Bacillaceae, or even in a subset of Bacillus spp. Phylogenetic profiles of sporulation genes, compiled in this work, confirm the presence of a common sporulation gene core, but also illuminate the diversity of the sporulation processes within various lineages. These profiles should help further experimental studies of uncharacterized widespread sporulation genes, which would ultimately allow delineation of the minimal set(s) of sporulation‐specific genes in Bacilli and Clostridia.     

Poly(3‐hydroxybutyrate) fuels the tricarboxylic acid cycle and de novo lipid biosynthesis during Bacillus anthracis sporulation

Numerous bacteria accumulate poly(3‐hydroxybutyrate) (PHB) as an intracellular reservoir of carbon and energy in response to imbalanced nutritional conditions. In Bacillus spp., where PHB biosynthesis precedes the formation of the dormant cell type called the spore (sporulation), the direct link between PHB accumulation and efficiency of sporulation was observed in multiple studies. Although the idea of PHB as an intracellular carbon and energy source fueling sporulation was proposed several decades ago, the mechanisms underlying PHB contribution to sporulation have not been defined. Here, we demonstrate that PHB deficiency impairs Bacillus anthracis sporulation through diminishing the energy status of the cells and by reducing carbon flux into the tricarboxylic acid (TCA) cycle and de novo lipid biosynthesis. Consequently, this metabolic imbalance decreased biosynthesis of the critical components required for spore integrity and resistance, such as dipicolinic acid (DPA) and the spore's inner membrane. Supplementation of the PHB deficient mutant with exogenous fatty acids overcame these sporulation defects, highlighting the importance of the TCA cycle and lipid biosynthesis during sporulation. Combined, the results of this work reveal the molecular mechanisms of PHB contribution to B. anthracis sporulation and provide valuable insight into the metabolic requirements for this developmental process in Bacillus species.     

Lipid‐dependent pore formation by antimicrobial peptides arenicin‐2 and melittin demonstrated by their proton transfer activity

This work presents a comparative study of proton transfer activity (PTA) of two cationic (+6) antimicrobial peptides, β‐structural arenicin‐2 and α‐helical melittin. A new approach was proposed for the detection of passive proton transfer by using proteoliposomes containing bacteriorhodopsin, which creates a small light‐induced electrochemical proton gradient ?ΔpH. Addition of several nanomoles of the peptides lowers ?ΔpH that is proximately indicative of the pore formation. The quantitative analysis of sigmoidal dependences of ?pH on the peptides concentration was carried out using liposomes prepared from PC, PC/PE, PC/PE/PI and PC/PG. Substitution of PC‐containing liposomes with PE‐containing ones, having negative spontaneous curvature, reduced the PTA of α‐helical melittin and increased that of β‐structural arenicin‐2. This result indicates an essential difference in the pore formation by these peptides. Further increase of PTA in response to arenicin‐2 (in contrast to melittin) was observed in the liposomes prepared from PC/PE/PI. The data analysis leads to the conclusion that PTA is influenced by (i) efficiency of the pore assemblage, which depends on the structure of pore‐forming peptides, and the spontaneous curvature of lipids and (ii) the presence of mobile protons in the polar head groups of phospholipids. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Sensitivity ofBacillus subtilis sporulation to methylene blue inhibition

The dye methylene blue was found to inhibit sporulation inBacillus subtilis 168. The compound blocked spore formation at concentrations subinhibitory to vegetative growth while allowing synthesis of serine protease, antibiotic, and certain catabolite-repressed enzymes. The sporulation process was sensitive to the inhibitor through T₆, but germination and outgrowth were not affected by the presence of the compound. The inhibition of sporulation may be related to the ability of the compound to inhibit oxidative phosphorylation.     

Spatiotemporally regulated proteolysis to dissect the role of vegetative proteins during Bacillus subtilis sporulation: cell‐specific requirement of σH and σA

Sporulation in Bacillus subtilis is a paradigm of bacterial development, which involves the interaction between a larger mother cell and a smaller forespore. The mother cell and the forespore activate different genetic programs, leading to the production of sporulation‐specific proteins. A critical gap in our understanding of sporulation is how vegetative proteins, made before sporulation initiation, contribute to spore formation. Here we present a system, spatiotemporally regulated proteolysis (STRP), which enables the rapid, developmentally regulated degradation of target proteins, thereby providing a suitable method to dissect the cell‐ and developmental stage‐specific role of vegetative proteins. STRP has been used to dissect the role of two major vegetative sigma factors, σ^H and σ^A, during sporulation. The results suggest that σ^H is only required in predivisional cells, where it is essential for sporulation initiation, but that it is dispensable during subsequent steps of spore formation. However, evidence has been provided that σ^A plays different roles in the mother cell, where it replenishes housekeeping functions, and in the forespore, where it plays an unexpected role in promoting spore germination and outgrowth. Altogether, the results demonstrate that STRP has the potential to provide a comprehensive molecular dissection of every stage of sporulation, germination and outgrowth.     

Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts

Triacylglycerols (TAG) and steryl esters (SE) are the principal storage lipids in all eukaryotic cells. In yeasts, these storage lipids accumulate within special organelles known as lipid bodies (LB). In the lipid accumulation-oriented metabolism of the oleaginous yeast Yarrowia lipolytica, storage lipids are mostly found in the form of TAG, and only small amounts of SE accumulate. We report here the identification of a new DAG acyltransferase gene, DGA2, homologous to the ARE genes of Saccharomyces cerevisiae. This gene encodes a member of the type 1 acyl-CoA:diacylglycerol acyltransferase family (DGAT1), which has not previously been identified in yeasts, but is commonly found in mammals and plants. Unlike the Are proteins in S. cerevisiae, Dga2p makes a major contribution to TAG synthesis via an acyl-CoA-dependent mechanism and is not involved in SE synthesis. This enzyme appears to affect the size and morphology of LB, suggesting a direct role of storage lipid proteins in LB formation. We report that the Are1p of Y. lipolytica was essential for sterol esterification, as deletion of the encoding gene (ARE1) completely abolished SE synthesis. Unlike its homologs in yeasts, YlARE1 has no DAG acyltransferase activity. We also reconsider the role and function of all four acyltransferase enzymes involved in the final step of neutral lipid synthesis in this oleaginous yeast.