Lehneck Ronny, Elleuche Skander, Pöggeler Stefanie
Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August-University Göttingen, Göttingen, Germany.
Mol Microbiol. 2014 Jun;92(5):931-44. doi: 10.1111/mmi.12607. Epub 2014 May 2.
The rapid interconversion of carbon dioxide and bicarbonate (hydrogen carbonate) is catalysed by metalloenzymes termed carbonic anhydrases (CAs). CAs have been identified in all three domains of life and can be divided into five evolutionarily unrelated classes (α, β, γ, δ and ζ) that do not share significant sequence similarities. The function of the mammalian, prokaryotic and plant α-CAs has been intensively studied but the function of CAs in filamentous ascomycetes is mostly unknown. The filamentous ascomycete Sordaria macrospora codes for four CAs, three of the β-class and one of the α-class. Here, we present a functional analysis of CAS4, the S. macrospora α-class CA. The CAS4 protein was post-translationally glycosylated and secreted. The knockout strain Δcas4 had a significantly reduced rate of ascospore germination. To determine the cas genes required for S. macrospora growth under ambient air conditions, we constructed double and triple mutations of the four cas genes in all possible combinations and a quadruple mutant. Vegetative growth rate of the quadruple mutant lacking all cas genes was drastically reduced compared to the wild type and invaded the agar under normal air conditions. Likewise the fruiting bodies were embedded in the agar and completely devoid of mature ascospores.
二氧化碳和碳酸氢盐之间的快速相互转化由称为碳酸酐酶（CAs）的金属酶催化。碳酸酐酶在生命的所有三个域中都已被鉴定出来，并且可以分为五个进化上不相关的类别（α、β、γ、δ和ζ），它们没有显著的序列相似性。哺乳动物、原核生物和植物α-碳酸酐酶的功能已经得到深入研究，但丝状子囊菌中碳酸酐酶的功能大多未知。丝状子囊菌大孢粪壳菌编码四种碳酸酐酶，其中三种属于β类，一种属于α类。在这里，我们对大孢粪壳菌α类碳酸酐酶CAS4进行了功能分析。CAS4蛋白在翻译后进行了糖基化并被分泌。敲除菌株Δcas4的子囊孢子萌发率显著降低。为了确定在环境空气条件下大孢粪壳菌生长所需的cas基因，我们构建了四个cas基因所有可能组合的双突变体和三突变体以及一个四突变体。与野生型相比，缺乏所有cas基因的四突变体的营养生长速率大幅降低，并且在正常空气条件下侵入了琼脂。同样，子实体也嵌入琼脂中，完全没有成熟的子囊孢子。
