Stigter J, Schneider M, de Bruijn F J
MSU-DOE Plant Research Laboratory, East Lansing 48824.
Mol Plant Microbe Interact. 1993 Mar-Apr;6(2):238-52. doi: 10.1094/mpmi-6-238.
Using site-directed mutagenesis, mutations were introduced in the -24/-12 promoter element of the Azorhizobium caulinodans nifA gene, and chimeric nifA-lacZ reporter gene fusions were constructed. Single base-pair mutations in the conserved -25 or -13 G residues were found to reduce or abolish nifA promoter activity, respectively, demonstrating that the -24/-12 promoter element is important for nifA gene expression and suggesting the involvement of a sigma 54 (NtrA/RpoN)-type transcription factor in nifA gene regulation. A 2-bp mutation at positions -25 and -16 was found to create a relatively nitrogen-control-independent, highly expressed nifA promoter. Using a heterologous ntrA(rpoN) gene probe, an A. caulinodans ntrA(rpoN)-like gene was cloned and the DNA sequence of this gene and flanking regions was determined. The presence of three open reading frames (ORF1-3) was demonstrated. ORF2 was found to contain regions sharing a high degree of homology with all characterized bacterial ntrA(rpoN) genes. ORF1 was found to share homology with ORFs found upstream of other bacterial ntrA(rpoN) genes, which have been postulated to encode members of a superfamily of ATP-binding proteins. Transposon Tn5 insertion mutations were introduced into the cloned ntrA(rpoN) gene, and chromosomal ntrA(rpoN)::Tn5 A. caulinodans mutants were created. The resulting mutants were found to be unable to fix nitrogen in the free-living state (Nif- in culture) or in stem or root nodules induced on Sesbania rostrata (Fix- in planta), and to be unable to grow aerobically in the presence of nitrate as sole nitrogen source (Ntr-). A nifH-lacZ gene fusion was found to be silent in ntrA(rpoN)::Tn5 mutant strains, but a nifA-lacZ gene fusion was found to be expressed at a wild-type level, suggesting that the ntrA(rpoN) gene identified here controls the expression of some of the A. caulinodans nif genes, but not the central nif regulatory gene nifA. Based on these results, a new model for the regulation of nif/fix gene expression in A. caulinodans is proposed.
利用定点诱变技术，在茎瘤固氮根瘤菌nifA基因的-24/-12启动子元件中引入突变，并构建了嵌合的nifA-lacZ报告基因融合体。发现在保守的-25或-13 G残基处的单碱基对突变分别降低或消除了nifA启动子活性，这表明-24/-12启动子元件对nifA基因表达很重要，并提示一种σ54（NtrA/RpoN）型转录因子参与nifA基因调控。发现在-25和-16位的一个2碱基对突变产生了一个相对不依赖氮控制、高表达的nifA启动子。使用异源ntrA（rpoN）基因探针，克隆了茎瘤固氮根瘤菌一个类似ntrA（rpoN）的基因，并测定了该基因及其侧翼区域的DNA序列。证实存在三个开放阅读框（ORF1-3）。发现ORF2包含与所有已鉴定的细菌ntrA（rpoN）基因具有高度同源性的区域。发现ORF1与在其他细菌ntrA（rpoN）基因上游发现的ORF具有同源性，这些ORF被推测编码一个ATP结合蛋白超家族的成员。将转座子Tn5插入突变引入克隆的ntrA（rpoN）基因，并构建了染色体ntrA（rpoN）::Tn5茎瘤固氮根瘤菌突变体。结果发现，所得突变体在自由生活状态下（培养物中固氮缺陷）或在大托叶田菁诱导的茎或根瘤中（植物体内固氮缺陷）不能固氮，并且在以硝酸盐作为唯一氮源的情况下不能有氧生长（氮调节缺陷）。发现nifH-lacZ基因融合体在ntrA（rpoN）::Tn5突变体菌株中不表达，但nifA-lacZ基因融合体以野生型水平表达，这表明这里鉴定的ntrA（rpoN）基因控制茎瘤固氮根瘤菌一些nif基因的表达，但不控制nif中央调控基因nifA的表达。基于这些结果，提出了一个茎瘤固氮根瘤菌中nif/fix基因表达调控的新模型。
