State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Fungal Genet Biol. 2013 Jan;50:11-20. doi: 10.1016/j.fgb.2012.11.002. Epub 2012 Nov 29.
T-DNA inserted mutants of Acremonium chrysogenum were constructed by Agrobacterium tumefaciens-mediated transformation (ATMT). One mutant 1223 which grew slowly was selected. TAIL-PCR and sequence analysis indicated that a putative septation protein encoding gene AcsepH was partially deleted in this mutant. AcsepH contains nine introns, and its deduced protein AcSEPH has a conserved serine/threonine protein kinase catalytic (S_TKc) domain at its N-terminal region. AcSEPH shows high similarity with septation H proteins from other filamentous fungi based on the phylogenetic analysis of S_TKc domains. In sporulation (LPE) medium, the conidia of AcsepH mutant was only about one-seventh of the wild-type, and more than 20% of conidia produced by the mutant contain multiple nuclei which were rare in the wild-type. During fermentation, the AcsepH disruption mutant grew slowly and its cephalosporin production was only about one quarter of the wild-type, and the transcription analysis showed that pcbC expression was delayed and the expressions of cefEF, cefD1 and cefD2 were significantly decreased. The vegetative hyphae of AcsepH mutant swelled abnormally and hardly formed the typical yeast-like cells. The amount of yeast-like cells was about one-tenth of the wild-type after fermentation for 5days. Comparison of hyphal viabilities revealed that the cells of AcsepH mutant died easily than the wild-type at the late stage of fermentation. Fluorescent stains revealed that the absence of AcsepH in A. chrysogenum led to reduction of septation and formation of multinucleate cells. These data indicates that AcsepH is required for the normal cellular septation and differentiation of A. chrysogenum, and its absence may change the cellular physiological status and causes the decline in cephalosporin production.
棘孢木霉的 T-DNA 插入突变体是通过根癌农杆菌介导的转化(ATMT)构建的。选择了一个生长缓慢的突变体 1223。TAIL-PCR 和序列分析表明,该突变体中一个假定的隔膜蛋白编码基因 AcsepH 部分缺失。AcsepH 包含 9 个内含子,其推导的蛋白 AcSEPH 在其 N 端区域具有保守的丝氨酸/苏氨酸蛋白激酶催化(S_TKc)结构域。基于 S_TKc 结构域的系统发育分析,AcSEPH 与其他丝状真菌的隔膜 H 蛋白具有高度相似性。在产孢(LPE)培养基中,AcsepH 突变体的分生孢子仅约为野生型的七分之一,并且突变体产生的超过 20%的分生孢子含有多个核,这在野生型中很少见。在发酵过程中,AcsepH 敲除突变体生长缓慢,其头孢菌素产量仅为野生型的四分之一左右,转录分析表明 pcbC 的表达被延迟,并且 cefEF、cefD1 和 cefD2 的表达显著降低。AcsepH 突变体的营养菌丝异常肿胀,几乎不能形成典型的酵母样细胞。发酵 5 天后,酵母样细胞的数量约为野生型的十分之一。菌丝活力比较表明,在发酵后期,AcsepH 突变体细胞比野生型细胞更容易死亡。荧光染色表明,棘孢木霉中 AcsepH 的缺失导致隔膜减少和多核细胞的形成。这些数据表明 AcsepH 是棘孢木霉正常细胞隔膜和分化所必需的,其缺失可能改变细胞的生理状态并导致头孢菌素产量下降。