A Two-Component Regulator Mediates Population-Density-Dependent Expression of the Bradyrhizobium japonicum Nodulation Genes

Bradyrhizobium japonicum nod gene expression was previously shown to be population density dependent. Induction of the nod genes is highest at low culture density and repressed at high population densities. This repression involves both NolA and NodD2 and is mediated by an extracellular factor found in B. japonicum conditioned medium. NolA and NodD2 expression is maximal at high population densities. We demonstrate here that a response regulator, encoded by nwsB, is required for the full expression of the B. japonicum nodYABC operon. In addition, NwsB is also required for the population-density-dependent expression of both nolA and nodD2. Expression of nolA and nodD2 in the nwsB mutant remained at a basal level, even at high culture densities. The nwsB defect could be complemented by overexpression of a second response regulator, NodW. Consistent with the fact that NolA and NodD2 repress nod gene expression, the expression of a nodY-lacZ fusion in the nwsB mutant was unaffected by culture density. In plant assays with GUS fusions, nodules infected with the wild type showed no nodY-GUS expression. In contrast, nodY-GUS expression was not repressed in nodules infected with the nwsB mutant. Nodule competition assays between the wild type and the nwsB mutant revealed that the addition of conditioned medium resulted in a competitive advantage for the nwsB mutant.

此前已有研究证实，大豆慢生根瘤菌（Bradyrhizobium japonicum）的结瘤基因（nod gene）表达具有群体密度依赖性。结瘤基因的诱导活性在低培养物密度时达最高水平，而在高种群密度下会被阻遏。该阻遏过程同时涉及NolA与NodD2，并由大豆慢生根瘤菌条件培养基中检出的胞外因子所介导。NolA与NodD2的表达在高种群密度时达到峰值。本研究表明，由nwsB编码的应答调控因子（response regulator）是大豆慢生根瘤菌nodYABC操纵子完整表达所必需的。此外，NwsB同样参与nolA与nodD2的群体密度依赖性表达调控。即便在高培养物密度条件下，nwsB突变株中nolA与nodD2的表达仍维持在基础水平。通过过量表达另一应答调控因子NodW，可互补nwsB基因的功能缺陷。鉴于NolA与NodD2可阻遏结瘤基因表达，nwsB突变株中nodY-lacZ融合基因的表达不受培养物密度的影响。在携带GUS融合基因的植物试验中，野生型菌株侵染形成的根瘤未检测到nodY-GUS表达；与之形成鲜明对比的是，nwsB突变株侵染形成的根瘤中，nodY-GUS表达并未被阻遏。野生型菌株与nwsB突变株的根瘤竞争试验显示，添加条件培养基可使nwsB突变株获得竞争优势。