Machado H B, Yates M G, Funayama S, Rigo L U, Steffens M B, Souza E M, Pedrosa F O
Department of Pharmacology, Universidade Federal do Paraná, Curitiba, PR, Brazil.
Can J Microbiol. 1995 Aug;41(8):674-84. doi: 10.1139/m95-093.
A cosmid able to complement the Nif- and nitrate-dependent growth phenotypes of the Azospirillum brasilense mutant FP9 was isolated from a genomic library of the wild-type strain FP2. A 6-kb DNA region was sequenced and showed two open reading frames (ORFs) identified as the ntrB and ntrC genes. An ORF1 located upstream from the ntrB gene and coding for a 36-kDa polypeptide showed similarity to the nifR3 gene of Rhodobacter capsulatus and the ORF1 of Rhizobium leguminosarum, both located upstream from the ntrB gene in a complex operon. Two other unidentified ORFs (ORF5 and partial ORF4) coding for hydrophobic polypeptides were also observed. delta ORF1-ntrBC, ORF1, ntrB, and ntrC mutants obtained by recombination of suicide plasmids containing an insertion of a promoterless lacZ kanamycin cassette showed decreased nitrogenase activities and were unable to grow on nitrate as the sole N source. These phenotypes were restored by complementation with plasmids containing the ntrC gene. Analysis of lacZ transcriptional fusions suggested that the ORF1-ntrBC operon in Azospirillum brasilense is expressed from a promoter located upstream from the ORF1 and that it is negatively regulated by the ntrC gene product.
从野生型菌株FP2的基因组文库中分离出一个能够互补巴西固氮螺菌突变体FP9的固氮和硝酸盐依赖生长表型的黏粒。对一个6 kb的DNA区域进行了测序，结果显示有两个开放阅读框（ORF），分别被鉴定为ntrB和ntrC基因。位于ntrB基因上游且编码一种36 kDa多肽的ORF1，与荚膜红杆菌的nifR3基因以及豌豆根瘤菌的ORF1相似，它们都位于一个复杂操纵子中ntrB基因的上游。还观察到另外两个编码疏水多肽的未鉴定ORF（ORF5和部分ORF4）。通过含有无启动子lacZ卡那霉素盒插入片段的自杀质粒重组获得的ΔORF1 - ntrBC、ORF1、ntrB和ntrC突变体，其固氮酶活性降低，并且不能以硝酸盐作为唯一氮源生长。用含有ntrC基因的质粒互补可恢复这些表型。对lacZ转录融合体的分析表明，巴西固氮螺菌中的ORF1 - ntrBC操纵子是从位于ORF1上游的一个启动子表达的，并且它受到ntrC基因产物的负调控。
