Maria de Lourdes T.M. Polizeli, Maria A. Noventa-Jordāo, Maira Marques da Silva, Joāo A. Jorge, Héctor F. Terenzi
{"title":"(1,3)-β- d -葡聚糖合成酶在粗神经孢子菌fz, sg, os-1(“黏液”)突变株菌丝和细胞壁无表型中的活性","authors":"Maria de Lourdes T.M. Polizeli, Maria A. Noventa-Jordāo, Maira Marques da Silva, Joāo A. Jorge, Héctor F. Terenzi","doi":"10.1006/emyc.1995.1005","DOIUrl":null,"url":null,"abstract":"<div><p>Polizeli, M. L. T. M., Noventa-Jordāo, M. A., Marques da Silva, M., Jorge, J. A., and Terenzi, H. F. 1995. (1,3)-β-D-Glucan synthase activity in mycelial and cell wall-less phenotypes of the <em>fz, sg, os-1</em> (\"slime\") mutant strain of <em>Neurospora crassa. Experimental Mycology</em> 19, 35-47. The cell wall-less <em>fz, sg, os-1</em> (\"slime\") triple mutant of <em>Neurospora crassa</em> lacks (1,3)-βD-glucan synthase activity. <em>fz, sg, os-1</em> segregants from slime × wild-type crosses initially germinate as a plasmodium (slime-like), but develop hyphae in a few hours and acquire a stable mycelial phenotype (mycelial intermediate). The cell wall-less phenotype (stable slime) can be reisolated from mycelial intermediates by filtration-enrichment selection in medium of high osmolarity. Pairs of mycelial intermediate and stable slime obtained from a single slime-like segregant were comparatively studied. Mycelial intermediate strains synthesize a cell wall with normal amounts of (1,3)-β-glucan, chitin, and other polysaccharides and possess (1,3)-β-glucan synthase activity with apparently normal properties (i.e., association with membranes, stability, <em>K</em><sub>m</sub> app, <em>V</em><sub>max</sub>, stimulation by GTP). The enzyme was dissociated by treatment with Tergitol NP-40 and NaCl into a membrane-bound catalytic center and a soluble factor which activates the enzyme in the presence of GTP. Heterologous reconstitution assays demonstrated that stable slime spheroplasts had normal activity of the soluble activating factor, but were severely deficient in membrane-bound activity. The genetic composition of the viable progeny of stable slime or mycelial intermediate × wild-type crosses failed to show differences between the two extreme phenotypes of slime. However, the analysis of heterokaryons demonstrated that the stable slime homokaryotic progeny of stable slime/wild-type heterokaryons were not viable. In contrast, the behavior of mycelial intermediate/wild-type heterokaryons was normal. Apparently, stable slime strains differed from the original mycelial intermediate in a mutation(s) which arose spontaneously during the filtration-enrichment selection applied to mycelial intermediates in order to obtain the cell wall-less phenotype. This new trait impaired conidial germination and might be the actual cause of the loss of (1,3)-β-glucan synthase activity and cell wall.</p></div>","PeriodicalId":12110,"journal":{"name":"Experimental Mycology","volume":"19 1","pages":"Pages 35-47"},"PeriodicalIF":0.0000,"publicationDate":"1995-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/emyc.1995.1005","citationCount":"1","resultStr":"{\"title\":\"(1,3)-β-D-Glucan Synthase Activity in Mycelial and Cell Wall-less Phenotypes of the fz, sg, os-1 (\\\"Slime\\\") Mutant Strain of Neurospora crassa\",\"authors\":\"Maria de Lourdes T.M. Polizeli, Maria A. Noventa-Jordāo, Maira Marques da Silva, Joāo A. Jorge, Héctor F. Terenzi\",\"doi\":\"10.1006/emyc.1995.1005\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Polizeli, M. L. T. M., Noventa-Jordāo, M. A., Marques da Silva, M., Jorge, J. A., and Terenzi, H. F. 1995. (1,3)-β-D-Glucan synthase activity in mycelial and cell wall-less phenotypes of the <em>fz, sg, os-1</em> (\\\"slime\\\") mutant strain of <em>Neurospora crassa. Experimental Mycology</em> 19, 35-47. The cell wall-less <em>fz, sg, os-1</em> (\\\"slime\\\") triple mutant of <em>Neurospora crassa</em> lacks (1,3)-βD-glucan synthase activity. <em>fz, sg, os-1</em> segregants from slime × wild-type crosses initially germinate as a plasmodium (slime-like), but develop hyphae in a few hours and acquire a stable mycelial phenotype (mycelial intermediate). The cell wall-less phenotype (stable slime) can be reisolated from mycelial intermediates by filtration-enrichment selection in medium of high osmolarity. Pairs of mycelial intermediate and stable slime obtained from a single slime-like segregant were comparatively studied. Mycelial intermediate strains synthesize a cell wall with normal amounts of (1,3)-β-glucan, chitin, and other polysaccharides and possess (1,3)-β-glucan synthase activity with apparently normal properties (i.e., association with membranes, stability, <em>K</em><sub>m</sub> app, <em>V</em><sub>max</sub>, stimulation by GTP). The enzyme was dissociated by treatment with Tergitol NP-40 and NaCl into a membrane-bound catalytic center and a soluble factor which activates the enzyme in the presence of GTP. Heterologous reconstitution assays demonstrated that stable slime spheroplasts had normal activity of the soluble activating factor, but were severely deficient in membrane-bound activity. The genetic composition of the viable progeny of stable slime or mycelial intermediate × wild-type crosses failed to show differences between the two extreme phenotypes of slime. However, the analysis of heterokaryons demonstrated that the stable slime homokaryotic progeny of stable slime/wild-type heterokaryons were not viable. In contrast, the behavior of mycelial intermediate/wild-type heterokaryons was normal. Apparently, stable slime strains differed from the original mycelial intermediate in a mutation(s) which arose spontaneously during the filtration-enrichment selection applied to mycelial intermediates in order to obtain the cell wall-less phenotype. This new trait impaired conidial germination and might be the actual cause of the loss of (1,3)-β-glucan synthase activity and cell wall.</p></div>\",\"PeriodicalId\":12110,\"journal\":{\"name\":\"Experimental Mycology\",\"volume\":\"19 1\",\"pages\":\"Pages 35-47\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"1995-03-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1006/emyc.1995.1005\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Experimental Mycology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0147597585710055\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Experimental Mycology","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0147597585710055","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 1
摘要
Polizeli, m.l.t.m., Noventa-Jordāo, m.a., Marques da Silva, m.a., Jorge, j.a.,和Terenzi, h.f. 1995。(1,3)-β- d -葡聚糖合成酶在粗神经孢子菌fz, sg, os-1(“粘液”)突变株菌丝和细胞壁无表型中的活性。实验真菌学19,35-47。无细胞壁的粗神经孢子虫fz, sg, os-1(“粘液”)三重突变体缺乏(1,3)-β d -葡聚糖合成酶活性。黏液x野生型杂交的Fz, sg, os-1分离体最初作为疟原虫(黏液样)萌发,但在几小时内发育出菌丝并获得稳定的菌丝表型(菌丝中间体)。在高渗透压培养基中,通过过滤富集选择,可以从菌丝中间体中分离出无壁表型(稳定黏液)。对从单一黏液样分离得到的菌丝中间黏液和稳定黏液对进行了比较研究。菌丝中间菌株合成的细胞壁含有正常量的(1,3)-β-葡聚糖、几丁质和其他多糖,并具有明显正常性质的(1,3)-β-葡聚糖合成酶活性(即与膜的关联、稳定性、Km app、Vmax、GTP刺激)。用Tergitol NP-40和NaCl将酶解离成一个膜结合的催化中心和一个在GTP存在下激活酶的可溶性因子。异源重组实验表明,稳定的黏液球质体具有正常的可溶性激活因子活性,但严重缺乏膜结合活性。稳定黏液或菌丝中间型×野生型杂交的活代遗传组成在黏液的两种极端表型之间没有差异。然而,对异核体的分析表明,稳定黏液/野生型异核体的稳定黏液同核后代是不可活的。相比之下,菌丝中间型/野生型异核体的行为正常。显然,稳定的黏液菌株与原始的菌丝中间体不同,其突变是在对菌丝中间体进行过滤富集选择以获得无壁表型时自发产生的。这一新性状损害了分生孢子的萌发,可能是(1,3)-β-葡聚糖合成酶活性和细胞壁丧失的实际原因。
(1,3)-β-D-Glucan Synthase Activity in Mycelial and Cell Wall-less Phenotypes of the fz, sg, os-1 ("Slime") Mutant Strain of Neurospora crassa
Polizeli, M. L. T. M., Noventa-Jordāo, M. A., Marques da Silva, M., Jorge, J. A., and Terenzi, H. F. 1995. (1,3)-β-D-Glucan synthase activity in mycelial and cell wall-less phenotypes of the fz, sg, os-1 ("slime") mutant strain of Neurospora crassa. Experimental Mycology 19, 35-47. The cell wall-less fz, sg, os-1 ("slime") triple mutant of Neurospora crassa lacks (1,3)-βD-glucan synthase activity. fz, sg, os-1 segregants from slime × wild-type crosses initially germinate as a plasmodium (slime-like), but develop hyphae in a few hours and acquire a stable mycelial phenotype (mycelial intermediate). The cell wall-less phenotype (stable slime) can be reisolated from mycelial intermediates by filtration-enrichment selection in medium of high osmolarity. Pairs of mycelial intermediate and stable slime obtained from a single slime-like segregant were comparatively studied. Mycelial intermediate strains synthesize a cell wall with normal amounts of (1,3)-β-glucan, chitin, and other polysaccharides and possess (1,3)-β-glucan synthase activity with apparently normal properties (i.e., association with membranes, stability, Km app, Vmax, stimulation by GTP). The enzyme was dissociated by treatment with Tergitol NP-40 and NaCl into a membrane-bound catalytic center and a soluble factor which activates the enzyme in the presence of GTP. Heterologous reconstitution assays demonstrated that stable slime spheroplasts had normal activity of the soluble activating factor, but were severely deficient in membrane-bound activity. The genetic composition of the viable progeny of stable slime or mycelial intermediate × wild-type crosses failed to show differences between the two extreme phenotypes of slime. However, the analysis of heterokaryons demonstrated that the stable slime homokaryotic progeny of stable slime/wild-type heterokaryons were not viable. In contrast, the behavior of mycelial intermediate/wild-type heterokaryons was normal. Apparently, stable slime strains differed from the original mycelial intermediate in a mutation(s) which arose spontaneously during the filtration-enrichment selection applied to mycelial intermediates in order to obtain the cell wall-less phenotype. This new trait impaired conidial germination and might be the actual cause of the loss of (1,3)-β-glucan synthase activity and cell wall.