CAREER: Identification and Characterization of Phosphate- Starvation Inducible Genes in Rhizobium meliloti

[9420798]麦克德莫特研究计划：文献中有许多例子表明豆科植物与根瘤菌的共生关系对磷肥的施用有很好的反应，结瘤豆科植物比非结瘤豆科植物对磷的需求更高，以达到最佳生长。施磷显著增强了这一重要农艺学意义上的植物-微生物相互作用的各种共生特征。然而，我们对共生固氮增加磷需求的基础知之甚少，或者对磷反应的基础，特别是在这种反应中的类细菌的作用。在拟议的工作中，R. meliloti将用于开始对受磷有效性影响的根瘤菌基因进行定性和定量评估。R. meliloti，菌株104A14，突变了一个转座子，该转座子携带一个无启动子lacZ基因，当插入相对于活性启动子的正确方向时，该基因充当报告基因。经过广泛筛选，鉴定出19个突变体，当细胞受到磷胁迫时，它们的报告基因活性明显高于提供充足磷时的报告基因活性。报告突变体将被进一步表征，以确定它们在磷获取中起什么作用（如果有的话），野生型基因可能具有什么代谢作用，以及它们的表达是否受到其他环境刺激的影响。此外，它们的共生表型将与寄主基因型决定，这些基因型是磷利用效率高还是效率低，以及在寄主植物磷限制或过量的条件下。最后，选择的突变体将在序列水平上进行研究。受影响的基因将被克隆并测序，以帮助鉴定。教育计划：教育计划包括三个层次的教学：1)新课程的开发，2)传统的研究生和博士后科学家的培养，3)专业的本科生研究训练。正在开发的新课程包括一门本科课程和一门研究生课程，交替授课。一个专门的本科生研究培训计划也被描述。该计划的目的是发现有才华的本科生，并为他们提供一对一的培训。该计划旨在为那些计划攻读研究生和从事研究型职业的学生提供更多的培训机会。这些本科生所进行的研究将在PI的直接监督下进行，并将成为研究计划中描述的工作的一部分。

9420798 McDermott Research Plan: The literature contains numerous examples that shows the legume-Rhizobium symbiosis readily responds to the application of phosphorus (P) fertilizer, and that nodulated legumes have a higher P requirement for optimum growth than non-nodulated legumes. Various symbiotic features of this agronomically important plant-microbe interaction are significantly enhanced by P fertilization. However, we know little about the basis for increased P requirements of symbiotic nitrogen fixation, or the basis of the P respone, particularly the role of the bacterioids in this response. In the proposed work, R. meliloti will be used to begin a qualitative and quantitative assessment of Rhizobium genes that are affected by P availability. R. meliloti, strain 104A14, has been mutated with a transposon that carries a promoterless lacZ gene which behaves as a reporter gene when insertion is in the correct orientation relative to the active promoter. After extensive screening, 19 mutants were identified that exhibited significantly greater reporter gene activity when the cells were P-stressed relative to when the cells were provided adeuate P. The reporter mutants will be further characterized to determine what role, if any, they have in P acquisition, what metabolic role the wild-type gene may have, and if their expression is influenced by other environmental stimuli. Further, their symbiosis phenotype will be determined with host genotypes that are either P-use efficient or inefficient, and under conditions where P is either limiting or in excess for the host plant. Finally, selected mutants will be studied at the sequence level. The effected gene(s) will be cloned an sequenced to help in their identification. Education Plan: The Education Plan involves three levels of instruction: 1) development of new courses, 2) traditional graduate student and posdoctoral scientist training, and 3) specialized undergraduate research training. The n ew courses being developed include one undergraduate course and one graduate course, taught in alternate years. A specialized undergraudate research training program is also described. The purpose of this program is to identify talented undergraduate students and provide them with one-on- one training. The program is geared to enhance the training opportunities for those students who are planning on postgraduate school and a research-oriented career. The research conducted by these undergraduates will be under the direct supervision of the PI and will part of the work described in the Research Plan.