Nitrate Assimilatory Genes and Their Transcriptional Regulation in a Unicellular Red Alga Cyanidioschyzon merolae: Genetic Evidence for Nitrite Reduction by a Sulfite Reductase-Like Enzyme
Nitrate Assimilatory Genes and Their Transcriptional Regulation in a Unicellular Red Alga Cyanidioschyzon merolae: Genetic Evidence for Nitrite Reduction by a Sulfite Reductase-Like Enzyme
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DOI：
10.1093/pcp/pcq043
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发表时间：
2010-05-01
影响因子：
4.9
通讯作者：
Tanaka, Kan
中科院分区：
文献类型：
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作者：
Imamura, Sousuke;Terashita, Masaru;Tanaka, Kan
Cyanidioschyzonmerolae是一种生活在酸性温泉中的单细胞红藻，能够以铵和硝酸盐为唯一氮源生长。基于全基因组序列，预测硝酸盐利用蛋白质硝酸盐转运蛋白（NRT）和硝酸盐还原酶（NR）分别由邻近的核基因CMG018C和CMG019C编码，但相似性搜索未发现典型的亚硝酸盐还原酶（NiR）基因。另一方面，发现了两个亚硫酸盐还原酶（SiR）的候选基因，其中一个（CMG021C）位于上述硝酸盐相关基因的旁边。考虑到CMG018 C、CMG019 C和CMG021 C的转录物在含硝酸盐的培养基中积累，但被铵抑制，并且SiR和NiR是结构相关的酶，我们假设CMG021 C基因产物在C中起NiR的作用。merolae。为了验证这一假设，我们开发了一种在C. merolae。为了支持我们的假设，我们发现，CMG021G无效突变体与亲本菌株相比，在含硝酸盐但不含铵的培养基中显示出细胞生长减少。此外，CMG021C在蓝细菌Leptolyngbya boryana（以前的Plectonema boryanum）的nirA突变体中的表达可以在遗传上弥补NiR缺陷。免疫荧光分析表明CMG021C定位在叶绿体中，因此，我们提出了一个整体方案，硝酸盐同化在C。merolae。
Cyanidioschyzon merolae is a unicellular red alga living in acid hot springs, which is able to grow on ammonium, as well as nitrate as sole nitrogen source. Based on the complete genome sequence, proteins for nitrate utilization, nitrate transporter (NRT) and nitrate reductase (NR), were predicted to be encoded by the neighboring nuclear genes CMG018C and CMG019C, respectively, but no typical nitrite reductase (NiR) gene was found by similarity searches. On the other hand, two candidate genes for sulfite reductase (SiR) were found, one of which (CMG021C) is located next to the above-noted nitrate-related genes. Given that transcripts of CMG018C, CMG019C and CMG021C accumulate in nitrate-containing media, but are repressed by ammonium, and that SiR and NiR are structurally related enzymes, we hypothesized that the CMG021C gene product functions as an NiR in C. merolae. To test this hypothesis, we developed a method for targeted gene disruption in C. merolae. In support of our hypothesis, we found that a CMG021G null mutant in comparison with the parental strain showed decreased cell growth in nitrate-containing but not in ammonium-containing media. Furthermore, expression of CMG021C in the nirA mutant of a cyanobacterium, Leptolyngbya boryana (formerly Plectonema boryanum), could genetically complement the NiR defect. Immunofluorescent analysis indicated the localization of CMG021C in chloroplasts, and hence we propose an overall scheme for nitrate assimilation in C. merolae.