Polizeli M del L, Noventa-Jordão M A, da Silva M M, Jorge J A, Terenzi H F
Departamento de Biologia, Faculdade de Filosofia, Universidade de Sö Paulo, Brazil.
Exp Mycol. 1995 Mar;19(1):35-47. doi: 10.1006/emyc.1995.1005.
The cell wall-less fz, sg, os-1 ("slime") triple mutant of Neurospora crassa lacks (1,3)-beta-D-glucan synthase activity. fz, sg, os-1 segregants from slime x 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)-beta-glucan, chitin, and other polysaccharides and possess (1,3)-beta-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 x 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)-beta-glucan synthase activity and cell wall.
粗糙脉孢菌的无细胞壁fz、sg、os-1（“黏液”）三重突变体缺乏（1,3）-β-D-葡聚糖合酶活性。来自黏液型与野生型杂交的fz、sg、os-1分离株最初以原质团（黏液样）形式萌发，但在数小时内发育出菌丝并获得稳定的菌丝体表型（菌丝体中间型）。通过在高渗透压培养基中进行过滤富集选择，可从菌丝体中间型中重新分离出无细胞壁表型（稳定黏液型）。对从单个黏液样分离株获得的成对菌丝体中间型和稳定黏液型进行了比较研究。菌丝体中间型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（1,3）-β-葡聚糖合酶活性，其性质显然正常（即与膜结合、稳定性、表观Km、Vmax、受GTP刺激）。用Tergitol NP-40和NaCl处理可使该酶解离为膜结合催化中心和一种在GTP存在下激活该酶的可溶性因子。异源重组试验表明，稳定黏液型原生质球具有正常的可溶性激活因子活性，但膜结合活性严重不足。稳定黏液型或菌丝体中间型与野生型杂交的可存活后代的遗传组成在黏液的两种极端表型之间未显示出差异。然而，对异核体的分析表明，稳定黏液型/野生型异核体的稳定黏液型同核后代不可存活。相比之下，菌丝体中间型/野生型异核体的行为正常。显然，稳定黏液型菌株与原始菌丝体中间型的不同之处在于，在对菌丝体中间型进行过滤富集选择以获得无细胞壁表型的过程中自发产生了一个或多个突变。这种新特性损害了分生孢子萌发，可能是（1,3）-β-葡聚糖合酶活性丧失和细胞壁缺失的实际原因。
