A Homologue of the Tryptophan-Rich Sensory Protein TspO and FixL Regulate a Novel Nutrient Deprivation-Induced Sinorhizobium meliloti Locus

A nutrient deprivation-induced locus in Sinorhizobium meliloti strain 1021 was identified by use of a Tn5-luxAB reporter gene transposon. The tagged locus is comprised of two open reading frames (ORFs) designated ndiA and ndiB for nutrient deprivation-induced genes A and B. Comparison of the deduced amino acid sequences of both ndiA and ndiB to the protein databases failed to reveal similarity to any known genes. The expression of the ndi locus was found to be induced by carbon and nitrogen deprivation, osmotic stress, and oxygen limitation and during entry into stationary phase. To identify regulatory components involved in the control of ndi gene expression, a second round of mutagenesis was performed on the primary ndiB::Tn5-luxAB-tagged strain (C22) with transposon Tn1721. A double-mutant strain was obtained that lacked ndi locus transcriptional activity under all of the inducing conditions tested. The Tn1721-tagged gene showed a high degree of similarity to tryptophan-rich sensory protein TspO from Rhodobacter sphaeroides, as well as to mitochondrial benzodiazepine receptor pK18 from mammals. Induction of the ndi::Tn5-luxAB reporter gene fusion was restored under all inducing conditions by introducing the tspO coding region, from either S. meliloti or R. sphaeroides, in trans. Furthermore, it was found that, in addition to tspO, fixL, which encodes the sensor protein of an oxygen-sensing two-component system, is required for full expression of the ndi locus, but only under low oxygen tension.

研究人员借助Tn5-luxAB报告基因转座子（Tn5-luxAB reporter gene transposon），在苜蓿中华根瘤菌（Sinorhizobium meliloti）1021菌株中鉴定出一处营养匮乏诱导型基因座。该被标记的基因座包含两个开放阅读框（open reading frame, ORF），分别命名为ndiA和ndiB，即营养匮乏诱导基因A和B。将ndiA与ndiB推导得到的氨基酸序列与蛋白质数据库进行比对，未发现其与任何已知基因存在序列相似性。研究发现，ndi基因座的表达可被碳源匮乏、氮源匮乏、渗透胁迫以及氧限制所诱导，且在菌株进入稳定生长期时表达上调。为鉴定参与调控ndi基因表达的调控元件，研究人员以携带ndiB::Tn5-luxAB标记的初始菌株C22为对象，使用转座子Tn1721开展第二轮诱变实验。最终获得一株双突变菌株，其在所有测试诱导条件下均无法检测到ndi基因座的转录活性。被Tn1721标记的基因与球形红杆菌（Rhodobacter sphaeroides）的富色氨酸传感蛋白TspO（tryptophan-rich sensory protein TspO），以及哺乳动物线粒体苯二氮䓬受体pK18（mitochondrial benzodiazepine receptor pK18）均具有高度序列相似性。通过反式导入苜蓿中华根瘤菌或球形红杆菌的tspO编码区，可在所有诱导条件下恢复ndi::Tn5-luxAB报告基因融合的表达诱导活性。此外，研究还发现，除tspO外，编码氧感应双组分系统（two-component system）传感蛋白的fixL基因，仅在低氧张力条件下才对ndi基因座的完整表达不可或缺。