RHO1 (YlRHO1) is a non-essential gene in Yarrowia lipolytica and complements rho1Delta lethality in Saccharomyces cerevisiae

The synthesis of beta-1,3-glucan, the structural component of the yeast cell wall that gives shape to the cell, occurs at the plasma membrane and is the result of the activity of at least a two-component complex. Fks1p is the catalytic subunit directly responsible for the synthesis of beta-1,3-glucan, whilst the second subunit, Rho1p, has a GTP-dependent regulatory role (Yamochi et al., 1994). RHO1 has been characterized in Saccharomyces cerevisiae (Yamochi et al., 1994), and in several other fungal species. In this work, we have used degenerate oligonucleotides derived from the conserved regions of Rho1ps to isolate the RHO1 gene of Yarrowia lipolytica. The gene isolated in this way, which we have named YlRHO1, encodes a 204 amino acid protein that shows a high degree of homology with other Rho1ps. However, unlike S. cerevisiae, the ylrho1Delta disruptant strain in Y. lipolytica is viable, although it exhibits an increased sensitivity to Calcofluor white and Congo red. Also, YlRHO1 complements rho1 lethality in S. cerevisiae at both 28 degrees C and 37 degrees C. The complete sequence of YlRHO1 can be obtained from GenBank under Accession No. AF279915.     

Yeast Lrg1p acts as a specialized RhoGAP regulating 1,3-beta-glucan synthesis.

Selection of an extragenic suppressor of fks1-1154 Deltafks2, mutations in the catalytic subunits of yeast 1,3-beta-glucan synthase (GS) conferring temperature-sensitivity, led to the LRG1 gene, which was originally identified as a LIM-RhoGAP homologous gene. Mutations in the LRG1 gene restore impaired 1,3-beta-glucan synthesis in the fks1-1154 Deltafks2 mutant as well as that in rho1-2, a temperature-sensitive mutant of Rho-type GTPase that functions as a regulatory subunit of GS. Two-hybrid analyses of Lrg1p, which contains a sequence conserved among Rho GTPase-activating proteins (GAPs), revealed its specific interactions with the active form of Rho1p. Among eight potential yeast RhoGAPs, Lrg1p is the only member that negatively regulates GS activity: mutations in the rest of GAPs, including bem2, Deltabem3, Deltasac7, Deltabag7, Deltarga1, Deltarga2 and Deltargd1, do not suppress impairment of 1,3-beta-glucan synthesis. Analyses of Mpk1p phosphorylation revealed the inability of Lrg1p to regulate the Pkc1p-MAP kinase cascade, a distinct Rho1p-regulating signalling pathway known to be affected by the GAPs, Bem2p and Sac7p. Thus, different groups of Rho1p GAPs control the activity of different Rho1p-effector proteins.     

Mutations in Fks1p affect the cell wall content of beta-1,3- and beta-1,6-glucan in Saccharomyces cerevisiae

Fks1p and Fks2p are related proteins thought to be catalytic subunits of the beta-1,3-glucan synthase. Analysis of fks1 delta mutants showed a partial K1 killer toxin-resistant phenotype and a 30% reduction in alkali-soluble beta-1,3-glucan that was accompanied by a modest reduction in beta-1,6-glucan. The gas1 delta mutant lacking a 1,3-beta-glucanosyltransferase displayed a similar reduction in alkali-soluble beta-1,3-glucan but did not share the beta-1,6-glucan defect, indicating that beta-1,6-glucan reduction is not a general phenotype among beta-1,3-glucan biosynthetic mutants. Overexpression of FKS2 suppressed the killer toxin phenotype of fks1 delta mutants, implicating Fks2p in the biosynthesis of the residual beta-1,6-glucan present in fks1 delta cells. In addition, eight out of 12 fks1ts fks2 delta mutants had altered beta-glucan levels at the permissive temperature: the partial killer resistant FKS1F1258Y N1520D allele was severely affected in both polymers and displayed a 55% reduction in beta-1,6-glucan, while the in vitro hyperactive allele FKS1T605I M761T increased both beta-glucan levels. These beta-1,6-glucan phenotypes may be due to altered availability of, and structural changes in, the beta-1,3-glucan polymer, which might serve as a beta-1,6-glucan acceptor at the cell surface. Alternatively, Fks1p and Fks2p could actively participate in the biosynthesis of both polymers as beta-glucan transporters. We analysed Fks1p and Fks2p in beta-1,6-glucan deficient mutants and found that they were mislocalized and that the mutants had reduced in vitro glucan synthase activity, possibly contributing to the observed beta-1,6-glucan defects.     

Importance of cell wall mannoproteins for septum formation in Saccharomyces cerevisiae

The mannosyltransferase mutants mnn9 and mnn10 were isolated in a genetic screen for septation defects in Saccharomyces cerevisiae. Ultrastructural examination of mutant cell walls revealed markedly thin septal structures and occasional failure to construct trilaminar septa, which then led to the formation of bulky default septa at the bud neck. In the absence of a functional septation apparatus, mnn10 mutants are unable to complete cytokinesis and die as cell chains with incompletely separated cytoplasms, indicating that mannosylation defects impair the ability to form remedial septa. We could not detect N-linked glycosylation of the beta(1,3)glucan synthase Fks1p and mnn10 defects do not change the molecular weight or abundance of the protein. We discuss a model explaining the pleiotropic effects of impaired N-linked protein glycosylation on septation in S. cerevisiae.     

Molecular cloning and characterization of a glucan synthase gene from the human pathogenic fungus Paracoccidioides brasiliensis

1,3-beta-D-glucan is a fungal cell wall polymer synthesized by the multi-subunit enzyme 1,3-beta-D-glucan synthase. A subunit of this integral membrane protein was first described as the product of the FKS1 gene from Saccharomyces cerevisiae using echinocandin mutants. Other FKS1 genes were also reported for Candida albicans, Aspergillus nidulans and Cryptococcus neoformans. Here, we report the nucleotide sequence of the first homologous FKS gene cloned from the pathogenic fungus Paracoccidioides brasiliensis. An open reading frame of 5942 bp was identified in the complete sequence, interrupted by two putative introns, the first close to the 5' end and the second close to the 3' end of the gene. A promoter region is also described containing consensus sequences such as canonical TATA and CAAT boxes and, possibly, multiple sites for glucose regulation by creA protein. The deduced sequence of 1926 amino acid show more than 85% similarity to FksAp from A. nidulans, and 71% to Fks1p and Fks2p from S. cerevisiae. Computational analysis of P. brasiliensis Fks1p suggests a similar structure to transmembrane proteins, such as FksAp, with the presence of two domains composed by hydrophobic helices that limit the putative highly hydrophilic catalytic domain within the cytoplasm.     

β‐1,6‐glucan synthesis‐associated genes are required for proper spore wall formation in Saccharomyces cerevisiae

The yeast spore wall is an excellent model to study the assembly of an extracellular macromolecule structure. In the present study, mutants defective in β ‐1,6‐glucan synthesis, including kre1? , kre6? , kre9? and big1? , were sporulated to analyse the effect of β ‐1,6‐glucan defects on the spore wall. Except for kre6? , these mutant spores were sensitive to treatment with ether, suggesting that the mutations perturb the integrity of the spore wall. Morphologically, the mutant spores were indistinguishable from wild‐type spores. They lacked significant sporulation defects partly because the chitosan layer, which covers the glucan layer, compensated for the damage. The proof for this model was obtained from the effect of the additional deletion of CHS3 that resulted in the absence of the chitosan layer. Among the double mutants, the most severe spore wall deficiency was observed in big1? spores. The majority of the big1?chs3? mutants failed to form visible spores at a higher temperature. Given that the big1? mutation caused a failure to attach a GPI‐anchored reporter, Cwp2‐GFP, to the spore wall, β ‐1,6‐glucan is involved in tethering of GPI‐anchored proteins in the spore wall as well as in the vegetative cell wall. Thus, β ‐1,6‐glucan is required for proper organization of the spore wall. Copyright © 2017 John Wiley & Sons, Ltd.     

Soluble forms of YlCrh1p and YlCrh2p, cell wall proteins of Yarrowia lipolytica, have beta-1,3-glycosidase activity

Crh1p and Crh2p of Saccharomyces cerevisiae are cell wall proteins covalently attached to cell wall glucan and are thought to be putative glycosidases involved in cell wall remodelling. We investigated whether YlCrh1p and YlCrh2p, the Yarrowia lipolytica proteins homologous to ScCrh1p and ScCrh2p, had the required glycosidase activity for cell wall biosynthesis and maintenance. Ylcrh1Delta and Ylcrh2Delta mutants showed sensitivity to compounds that interfere with cell wall construction. Soluble forms of YlCrh1p and YlCrh2p that lacked the C-terminal consensus sequence for GPI anchoring showed glycosidase activity on laminarin, a substrate carrying beta-1,3-glycosidic linkage. Our study suggests that the YlCrh1p and YlCrh2p may participate in cell wall biosynthesis and remodelling through their beta-1,3-glycosidase activity.     

The trehalose pathway and intracellular glucose phosphates as modulators of potassium transport and general cation homeostasis in yeast

Trk, encoded by the partially redundant genes TRK1 and TRK2, is the major potassium transporter of Saccharomyces cerevisiae. This system is specific for potassium and rubidium but, by reducing the electrical membrane potential of the plasma membrane, Trk decreases the uptake of toxic cations such as lithium, calcium, aminoglycosides and polyamines, which are transported by other systems. Gain- and loss-of-function studies indicate that TPS1, a gene encoding trehalose-6-phosphate synthase and known to modulate glucose metabolism, activates Trk and reduces the sensitivity of yeast cells to many toxic cations. This effect is independent of known regulators of Trk, such as the Hal4 and Hal5 protein kinases and the protein phosphatase calcineurin. Mutants defective in isoform 2 of phosphoglucomutase (pgm2) and mutants defective in isoform 2 of hexokinase (hxk2) exhibit similar phenotypes of reduced Trk activity and increased sensitivity to toxic cations compared with tps1 mutants. In all cases Trk activity was positively correlated with levels of glucose phosphates (glc-1-P and glc-6-P). These results indicate that Tps1, like Pgm2 and Hxk2, increases the levels of glucose phosphates and suggest that these metabolites, directly or indirectly, activate Trk.     

vga Mutants of Kluyveromyces lactis show cell integrity defects

We studied the cell wall alterations that occur in mutants of Kluyveromyces lactis impaired in glycosylation. The mutants belong to four complementation groups named vga1 to vga4 (vanadate glycosylation affected), characterized by sodium orthovanadate resistance and alteration of the glycosylation profile of native invertase. A drastic reduction of the alkali-soluble fraction of the beta-D-glucan was observed in vga1, vga2 and vga3 cells, accompanied by an increase in the chitin content of the cell wall. In vga4 cells, both beta-D-glucan fractions (alkali-soluble and alkali-insoluble) were reduced to about half of the corresponding wild-type value but the chitin content was normal. A protein related to Fks1p, the catalytic subunit of the major 1,3-beta-D-glucan synthase of S. cerevisiae, was detected in K. lactis. The amount of this Fks1p-like protein increased 7-10 times in vga1, vga2 and vga3 mutants as compared to wild-type cells; the same strains released significant amounts of beta-D-glucan in the culture supernatant. These mutations also resulted in abnormally thick cell walls with conspicuous irregularities in the structure, as revealed by electron microscopy and by an altered resistance to Zymolyase. The observed high responsiveness of cell wall phenotypes to alterations of glycosylation make K. lactis an attractive system for studying the interconnections between these processes.     

Dissecting the role of dolichol in cell wall assembly in the yeast mutants impaired in early glycosylation reactions

Evidence is presented that temperature-sensitive Saccharomyces cerevisiae mutants, impaired in dolichol kinase (Sec59p) or dolichyl phosphate mannose synthase (Dpm1p) activity have an aberrant cell wall composition and ultrastructure. The mutants were oversensitive to Calcofluor white, an agent interacting with the cell wall chitin. In accordance with this, chemical analysis of the cell wall alkali-insoluble fraction indicated an increased amount of chitin and changes in the quantity of beta1,6- and beta1,3-glucan in sec59-1 and dpm1-6 mutants. In order to unravel the link between the formation of dolichyl phosphate and dolichyl phosphate mannose and the cell wall assembly, we screened a yeast genomic library for a multicopy suppressors of the thermosensitive phenotype. The RER2 and SRT1 genes, encoding cis-prenyltransferases, were isolated. In addition, the ROT1 gene, encoding protein involved in beta1,6-glucan synthesis (Machi et al., 2004) and protein folding (Takeuchi et al., 2006) acted as a multicopy suppressor of the temperature-sensitive phenotype of the sec59-1 mutant. The cell wall of the mutants and of mutants bearing the multicopy suppressors was analysed for carbohydrate and mannoprotein content. We also examined the glycosylation status of the plasma membrane protein Gas1p, a beta1,3-glucan elongase, and the degree of phosphorylation of the Mpk1/Slt2 protein, involved in the cell wall integrity pathway.     

KlROM2 encodes an essential GEF homologue in Kluyveromyces lactis

Cellular integrity in yeasts is ensured by a rigid cell wall whose synthesis is controlled by a MAP kinase signal transduction cascade. In Saccharomyces cerevisiae upstream regulatory components of this MAP kinase pathway involve a single protein kinase C, which is regulated in part by interaction with the small GTPase Rho1p. This small G protein is in turn rendered inactive (GDP-bound) or is activated (GTP-bound) by the influence of GTPase activating proteins (GAPs) and the GDP/GTP exchange factors (GEFs), respectively. We report here on the isolation of a gene from Kluyveromyces lactis, KlROM2, which encodes a member of the latter protein family. The nucleotide sequence contains an open reading frame of 1227 amino acids, with an overall identity of 57% to the Rom2 protein of S. cerevisiae. Four conserved sequence motifs could be identified: a RhoGEF domain, a DEP sequence, a CNH domain and a less conserved pleckstrin homology (PH) sequence. Klrom2 null mutants show a lethal phenotype, which indicates that the gene may encode the only functional GEF regulating the cellular integrity pathway in K. lactis. Conditional genomic expression of KlROM2 resulted in sensitivity towards caffeine and Calcofluor white as typical phenotypes of mutants defective in this pathway. Overexpression of KIROM2 from multicopy plasmids under the control of the ScGAL1 promoter severely impaired growth in both S. cerevisiae and in K. lactis. The fact that the lethal phenotype was not prevented in mpk1 deletion mutants indicates that growth inhibition is not simply caused by hyperactivation of the Pkc1p signal transduction pathway.     

A Study of Saccharomyces cerevisiae Cell Wall Glucans

Much research has been carried out over the years examining cell wall glucans from Saccharomyces cerevisiae and this study further examines aspects of the binding of (1r?4)‐α‐D‐glucan in the yeast cell wall, using a number of isolation techniques as well as monoclonal antibodies able to recognize a mixed (1r?4)‐α‐D‐glucan/(1r?6)‐β‐D‐glucan. Extraction of purified glucan, from S. cerevisiae cell wall, with 0.1N HCl, at 80°C for 6 h, released into the solution (1r?4)‐α‐D‐glucan and (1r?6)‐β‐D‐glucan as the major polysaccharides, along with an insoluble pellet highly enriched in (1r?3)‐β‐D‐glucan. The released (1r?4)‐α‐D‐glucan was composed of a high molecular size >100 kDa fraction (7.2% w/w) and a medium 5–50 kDa polysaccharide (10.2% w/w), with the (1r?4)‐α‐D‐glucan covalently bound to the (1r?6)‐β‐D‐glucan. The average molar ratio of the α:β glucan was 47: 53 in this mixed polysaccharide. The structure of this polysaccharide was different from the structure of plant starch or animal glycogen as monoclonal antibodies specific to yeast (1r?4)‐α‐D‐glucan/(1r?6)‐β‐D‐glucan did not recognize the plant starch or animal glycogen standards.     

Mapping and characterizing a new DNA replication mutant in Saccharomyces cerevisiae

A detailed characterization of the mak1-3 mutation of Saccharomyces cerevisiae has been made possible by modifying its genetic background. The mak1-3 mutation, which confers temperature sensitivity for growth, was originally identified as one of four mak1 mutations (Wickner and Leibowitz, 1976). Mak1-1, 1-2 and 1-4 mutants are deficient in DNA topoisomerase I activity and thus have been renamed 'top1' (Thrash et al., 1984). Studies presented here show that the map position of MAK1-3 on chromosome XVI distinguishes it from TOP1 which maps on chromosome XV (Wickner and Leibowitz, 1976). An investigation of in vivo macromolecular synthesis in the mak1-3 mutant shows that it is deficient in DNA replication at the restrictive temperature. Experiments in which DNA synthesis was measured in synchronized cell populations indicate that the mak1-3 mutant is deficient in the initiation step of DNA synthesis. Furthermore, crude extracts from the mak1-3 mutant cells support temperature-sensitive in vitro DNA synthesis on yeast chromosomal DNA replication origin containing plasmid pARS1, suggesting that the MAK1 gene product is directly required for in vitro DNA replication. The conclusion that mak1-3 is a newly identified DNA replication mutation is based on the observations that it (1) complements all DNA synthesis mutants examined, (2) maps to a previously undetected chromosomal location and (3) has a distinct terminal morphology. In light of these distinctions and of the role mak1-3 plays in DNA replication, it has been renamed 'dna1'.     

Disruption of LRG1 inhibits mother-daughter separation in Saccharomyces cerevisiae

LRG1, previously characterized as a Rho-GAP that regulates beta-1,3-glucan synthesis, was identified using a filtration screen designed to isolate genes that promote cell aggregation in Sigma1278b Saccharomyces cerevisiae. Disruption of LRG1 in haploid cells resulted in enhanced invasive growth and a strain-specific 'clustered' phenotype that is a consequence of failed separation of mother and daughter cells. Genetic analysis revealed that clustering required functional ROM2, FKS1 and STE12 but not FLO8 or FLO11. Additionally, this phenotype required sufficient nitrogen. beta-1,3-Glucan content was elevated in lrg1 haploids and these cells possessed a thicker cell wall and septum than wild-type strains. Additional glucan at the bud site inhibited separation of lrg1 mutants as the clustered phenotype was repressed by expression of the glucanases ENG1 or EGT2.     

Ynl038wp (Gpi15p) is the Saccharomyces cerevisiae homologue of human Pig-Hp and participates in the first step in glycosylphosphatidylinositol assembly.

Glycosylphosphatidylinositols (GPIs) are found in all eukaryotes and are synthesized in a pathway that starts with the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol (PI). This reaction is carried out by a protein complex, three of whose subunits in humans, hGpi1p, Pig-Cp and Pig-Ap, have sequence and functional homologues in the Saccharomyces cerevisiae Gpi1, Gpi2 and Gpi3 proteins, respectively. Human GlcNAc-PI synthase contains two further subunits, Pig-Hp and PigPp. We report that the essential YNL038w gene encodes the S. cerevisiae homologue of Pig-Hp. Haploid YNL038w-deletion strains were created, in which Ynl038wp could be depleted by repressing YNL038w expression using the GAL10 promoter. Depletion of Ynl038wp from membranes virtually abolished in vitro GlcNAc-PI synthetic activity, indicating that Ynl038wp is necessary for GlcNAc-PI synthesis in vitro. Further, depletion of Ynl038wp in an smp3 mutant background prevented the formation of the trimannosylated GPI intermediates that normally accumulate in this late-stage GPI assembly mutant. Ynl038wp is therefore required for GPI synthesis in vivo. Because YNL038w encodes a protein involved in GPI biosynthesis, we designate the gene GPI15. Potential Pig-Hp/Gpi15p counterparts are also encoded in the genomes of Schizosacchomyces pombe and Candida albicans.     

Increase in gene expression by respiratory-deficient mutation

Respiratory-deficient mutants (rho- cells) of Saccharomyces cerevisiae produced about 10 times as much human (h-) lysozyme as did wild-type strains (rho+ cells) when the GAL10 promoter was used in an expression plasmid with the h-lysozyme gene. Introduction of intact mitochondria into the rho- cells resulted in a significant decrease in the production of h-lysozyme, indicating that the rho- mutation increased the expression of the h-lysozyme gene. The copy number of the expression plasmid was not responsible for the increased expression. The level of h-lysozyme mRNA in the rho- cells was also much higher than that in the rho+ cells especially at the stationary phase. The increased expression of the h-lysozyme gene was also observed when a glyceraldehyde-3-phosphate dehydrogenase gene promoter and the PHO5 promoter were used in the expression plasmid. The rho- mutation also increased the expression of the PHO5 gene under the control of the HIS5 promoter in a plasmid and the ACT1 gene in the yeast chromosome, but did not increase the expression of the ribosomal RNA gene. In contrast to the rho- mutants, pet mutants did not show higher gene expression compared with wild-type strains.     

酿酒酵母表达侧耳源单基因生物合成麦角硫因

运用生物信息学方法对侧耳属蘑菇（平菇、杏鲍菇和白灵菇）的麦角硫因合成酶基因Egt 1进行挖掘,对其蛋白结构和功能进行预测,并构建酿酒酵母表达载体,在酿酒酵母EC 1118中进行表达研究。结果表明：克隆得到侧耳属平菇、杏鲍菇和白灵菇麦角硫因合成基因PoEgt 1、PeEgt 1和PtEgt 1,三者核苷酸序列同源性为97.03%,氨基酸序列同源性为97.93%,联合菌体破碎法并通过体外酶促反应后检测到麦角硫因,产量为（2.5±0.08）mg/L,证明了该基因具有单基因合成麦角硫因的活性。     

Quantification of uncertainty using Bayesian and bootstrap models to simulate the impact of nitrogen fertilisation on β‐glucan levels in barley

BACKGROUND: β‐Glucans have enjoyed renewed interest as a functional food ingredient, with current attention focused on optimising β‐glucan levels in finished products without compromising final product quality. In order to measure the uncertainty about the level of β‐glucans in barley, two different statistical methods (Bayesian inference and Bootstrap technique) were applied to measured levels of β‐glucan in three different varieties of barley grain (n = 83). RESULTS: The resulting probability density distributions were similar for the full data set and also when applied to smaller sample sizes, highlighting the potential for either method in quantifying the total uncertainty in β‐glucan levels. Bayesian inference was used to model the effect of nitrogen treatment on β‐glucan and protein contents in barley. The model found that a low level of fertilisation (50 kg N ha^?1) did not have a significant effect on β‐glucan or protein content. However, fertilisation above this level did result in an increase in β‐glucan and protein levels, the effect seeming to plateau at 100 kg N ha^?1. In addition, the uncertainty distributions were significantly different for two consecutive years of data, highlighting the potential environmental influence on β‐glucan content. CONCLUSION: The model developed in this study could be a useful tool for processors to quantify the uncertainty about the initial level of β‐glucan in barley and to evaluate the influence of environmental factors, thus enabling them to formulate their ingredient base to optimise levels of β‐glucan without compromising final product quality. Copyright © 2009 Society of Chemical Industry     

十味中草药提取液的体外抗菌活性研究

本文选用黄连、黄柏、黄芩、连翘、厚朴、甘草、艾叶、川芎、金银花及五味子十味中草药水提液,对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、啤酒酵母、黄曲霉及绿色木霉这六种菌进行了体外抗菌试验。目的是通过对抗菌效果的比较,筛选出抗菌活性好的中草药及有效的提取方法。实验结果表明这十味中草药提取液对六种供试菌有不同程度的抗菌效果,其抗细菌活性比抗真菌活性强,其中黄连、黄柏和黄芩提取液的总抗菌活性最强;结果还表明70%乙醇水溶液比水提取药液的抗菌活性要强。