Muro-Pastor M I, Reyes J C, Florencio F J
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-Consejo Superior de Investigaciones Científicas, Seville, Spain.
J Bacteriol. 1996 Jul;178(14):4070-6. doi: 10.1128/jb.178.14.4070-4076.1996.
NADP+-isocitrate dehydrogenase (NADP+-IDH) activity and protein levels in crude extracts from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 and the filamentous, dinitrogen-fixing Anabaena sp. strain PCC 7120 were determined under different nitrogen conditions. The highest NADP+-IDH activity and protein accumulation were found under dinitrogen-fixing conditions for the Anabaena strain and under nitrogen starvation for Synechocystis sp. PCC 6803. The icd gene that encodes the NADP+-IDH from Synechocystis sp. strain PCC 6803 was cloned by heterologous hybridization with the previously isolated icd gene from Anabaena sp. strain PCC 7120. The two cyanobacterial icd genes show 81% sequence identity and share a typical 44-amino-acid region different from all the other icd genes sequenced so far. The icd gene seems to be essential for Synechocystis growth since attempts to generate a completely segregated icd mutant were unsuccessful. Transcripts of 2.0 and 1.6 kb were detected by Northern (RNA) blot analysis, for the Anabaena and Synecho-cystis icd genes, respectively. Maximal icd mRNA accumulation was reached after 5 It of nitrogen starvation in Synechocystis cells and under dinitrogen-fixing conditions in Anabaena cells. Primer extension analysis showed that the structure of the Synechocystis icd gene promoter resembles those of the NtcA-regulated promoters. In addition, mobility shift assays demonstrated that purified Synechocystis NtcA protein binds to the promoter of the icd gene. All these data suggest that the expression of the icd gene from Synechocystis sp. strain PCC 6803 may be subjected to nitrogen control mediated by the positively acting regulatory protein NtcA.
在不同氮条件下，测定了单细胞蓝藻集胞藻PCC 6803菌株和丝状固氮鱼腥藻PCC 7120菌株粗提物中的NADP⁺ -异柠檬酸脱氢酶（NADP⁺ -IDH）活性及蛋白质水平。对于鱼腥藻菌株，在固氮条件下NADP⁺ -IDH活性和蛋白质积累最高；对于集胞藻PCC 6803菌株，在氮饥饿条件下最高。通过与先前从鱼腥藻PCC 7120菌株中分离的icd基因进行异源杂交，克隆了集胞藻PCC 6803菌株中编码NADP⁺ -IDH的icd基因。这两个蓝藻icd基因显示出81%的序列同一性，并且共享一个与迄今测序的所有其他icd基因不同的典型44个氨基酸区域。icd基因似乎对集胞藻的生长至关重要，因为尝试构建完全分离的icd突变体未成功。通过Northern（RNA）印迹分析分别检测到鱼腥藻和集胞藻icd基因的2.0 kb和1.6 kb转录本。集胞藻细胞在氮饥饿5小时后以及鱼腥藻细胞在固氮条件下达到icd mRNA的最大积累。引物延伸分析表明，集胞藻icd基因启动子的结构类似于NtcA调控的启动子。此外，迁移率变动分析表明，纯化的集胞藻NtcA蛋白与icd基因的启动子结合。所有这些数据表明，集胞藻PCC 6803菌株icd基因的表达可能受到正调控蛋白NtcA介导的氮控制。
