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

9420798麦克德莫特研究计划：文献中包含了大量的例子，表明豆科植物-根瘤菌的共生很容易对磷肥的施用做出反应，而且节状豆科植物比非节状豆类植物对磷的需求更高，以实现最佳生长。施磷肥显著增强了这一农学上重要的植物-微生物相互作用的各种共生特征。然而，我们对共生固氮需磷增加的基础或磷反应的基础，特别是类细菌在这一反应中的作用知之甚少。在这项拟议的工作中，将利用苜蓿根瘤菌开始对受磷供应影响的根瘤菌基因进行定性和定量评估。苜蓿根瘤菌104A14菌株已被突变为转座子，该转座子携带无启动子的lacZ基因，当插入相对于活性启动子的正确方向时，该基因起报告基因的作用。经过广泛的筛选，发现了19个突变体，它们在磷胁迫下表现出比细胞在去磷时明显更高的报告基因活性。报告突变体将被进一步鉴定，以确定它们在磷的获取中起什么作用，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，它们的共生表型将根据P利用效率或低效的寄主基因型以及在P对寄主植物限制或过剩的条件下确定。最后，将在序列水平上对选定的突变体进行研究。受影响的基因(S)将被克隆和测序，以帮助鉴定他们。教育计划：教育计划包括三个层次的教学：1)开发新课程，2)传统研究生和博士后科学家培训，3)专业本科生研究培训。正在开发的新课程包括一门本科课程和一门研究生课程，每隔一年教授一次。还描述了一个专门的本科研究培训计划。该项目旨在发掘有才华的本科生，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.