Moshiri F, Smith E G, Taormino J P, Maier R J
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
J Biol Chem. 1991 Dec 5;266(34):23169-74.
Cytochrome d has been postulated to be the "respiratory protection" oxidase of Azotobacter vinelandii, allowing this organism to fix nitrogen under aerobic growth conditions. We have previously cloned and characterized the structural genes for the A. vinelandii cytochrome d (cydA and cydB). The cyd genes are co-transcribed, yielding an mRNA of approximately 3.6 kilobase pairs. The level of the cyd message was 2-3-fold higher in cells that were fixing nitrogen, as compared with non-nitrogen-fixing cells. RNase protection analysis was used to determine the transcriptional start site at 275 bases upstream of the initiator ATG of cydA, and this start site was the same for nitrogen-fixing and non-nitrogen-fixing cells. The cyd promoter has sequence similarities to the canonical Escherichia coli promoters, which are transcribed by the major sigma 70 form of RNA polymerase. Plasmid-borne lacZ transcriptional fusions were constructed, using approximately 650 base pairs of 5'-upstream sequences of the cyd structural genes. This region had a strong promoter activity which was further up-regulated 1.5-2.5-fold upon the induction of nitrogen fixation. The cyd-lacZ fusions were characterized in a nifA- as well as an ntrA- background. Mutations in neither of these nif regulatory genes affected the constitutive expression of cyd under non-nitrogen-fixing conditions. However, the up-regulation of this promoter during the induction of nitrogen fixation was abolished only in the ntrA- background. Based on these results, the cytochrome d promoter of A. vinelandii belongs to a new class of nitrogen-regulated promoters which, unlike the authentic nif genes, does not require the ntrA gene product for its expression. The up-regulation of this promoter during nitrogen fixation, however, requires the ntrA gene product.
细胞色素d被推测为维涅兰德固氮菌的“呼吸保护”氧化酶，使该生物体能够在有氧生长条件下固氮。我们之前已经克隆并表征了维涅兰德固氮菌细胞色素d的结构基因（cydA和cydB）。cyd基因共同转录，产生约3.6千碱基对的mRNA。与非固氮细胞相比，固氮细胞中cyd信息的水平高2 - 3倍。核糖核酸酶保护分析用于确定cydA起始ATG上游275个碱基处的转录起始位点，固氮细胞和非固氮细胞的这个起始位点相同。cyd启动子与典型的大肠杆菌启动子具有序列相似性，后者由RNA聚合酶的主要σ70形式转录。使用cyd结构基因约650个碱基对的5'上游序列构建了质粒携带的lacZ转录融合体。该区域具有很强的启动子活性，在固氮诱导时进一步上调1.5 - 2.5倍。在nifA以及ntrA背景下对cyd - lacZ融合体进行了表征。这两个nif调节基因中的任何一个发生突变都不会影响非固氮条件下cyd的组成型表达。然而，只有在ntrA背景下，固氮诱导期间该启动子的上调才被消除。基于这些结果，维涅兰德固氮菌的细胞色素d启动子属于一类新的氮调节启动子，与真正的nif基因不同，其表达不需要ntrA基因产物。然而，固氮期间该启动子的上调需要ntrA基因产物。
