van Dillewijn P, Soto M J, Villadas P J, Toro N
Grupo de Ecología Genética, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain.
Appl Environ Microbiol. 2001 Sep;67(9):3860-5. doi: 10.1128/AEM.67.9.3860-3865.2001.
Highly efficient nitrogen-fixing strains selected in the laboratory often fail to increase legume production in agricultural soils containing indigenous rhizobial populations because they cannot compete against these populations for nodule formation. We have previously demonstrated, with a Sinorhizobium meliloti PutA- mutant strain, that proline dehydrogenase activity is required for colonization and therefore for the nodulation efficiency and competitiveness of S. meliloti on alfalfa roots (J. I. Jiménez-Zurdo, P. van Dillewijn, M. J. Soto, M. R. de Felipe, J. Olivares, and N. Toro, Mol. Plant-Microbe Interact. 8:492-498, 1995). In this work, we investigated whether the putA gene could be used as a means of increasing the competitiveness of S. meliloti strains. We produced a construct in which a constitutive promoter was placed 190 nucleotides upstream from the start codon of the putA gene. This resulted in an increase in the basal expression of this gene, with this increase being even greater in the presence of the substrate proline. We found that the presence of multicopy plasmids containing this putA gene construct increased the competitiveness of S. meliloti in microcosm experiments in nonsterile soil planted with alfalfa plants subjected to drought stress only during the first month. We investigated whether this construct also increased the competitiveness of S. meliloti strains under agricultural conditions by using it as the inoculum in a contained field experiment at León, Spain. We found that the frequency of nodule occupancy was higher with inoculum containing the modified putA gene for samples that were analyzed after 34 days but not for samples that were analyzed later.
在实验室中筛选出的高效固氮菌株，在含有本地根瘤菌种群的农业土壤中，往往无法提高豆科植物的产量，因为它们无法与这些种群竞争以形成根瘤。我们之前用苜蓿中华根瘤菌PutA突变株证明，脯氨酸脱氢酶活性对于定殖是必需的，因此对于苜蓿中华根瘤菌在苜蓿根上的结瘤效率和竞争力也是必需的（J. I. 希门尼斯 - 祖尔多、P. 范·迪勒维恩、M. J. 索托、M. R. 德·费利佩、J. 奥利瓦雷斯和N. 托罗，《分子植物 - 微生物相互作用》8:492 - 498，1995）。在这项工作中，我们研究了putA基因是否可以用作提高苜蓿中华根瘤菌菌株竞争力的一种手段。我们构建了一个载体，其中一个组成型启动子位于putA基因起始密码子上游190个核苷酸处。这导致该基因的基础表达增加，在有底物脯氨酸存在时这种增加更为明显。我们发现，含有这种putA基因构建体的多拷贝质粒的存在，在仅第一个月遭受干旱胁迫的种植苜蓿的非无菌土壤的微观实验中，提高了苜蓿中华根瘤菌的竞争力。我们通过在西班牙莱昂的一个封闭田间试验中使用该构建体作为接种物，研究了这种构建体在农业条件下是否也能提高苜蓿中华根瘤菌菌株的竞争力。我们发现，对于34天后分析的样品，含有修饰后的putA基因的接种物的根瘤占据频率更高，但对于之后分析的样品则不然。
