RHIZOBIUM NODULATION GENE EXPRESSION AND GENE PRODUCTS

我们研究细菌细胞与特定真核宿主的相互作用， 在一个既与多细胞发育事件有关的系统中 生物体，以及细菌的致病机理。紫花苜蓿根瘤菌入侵和 在包括紫花苜蓿在内的特定寄主植物中诱导结瘤 (紫花苜蓿)。当细菌入侵时，它们会改变宿主的细胞壁 合成，诱导正常不分裂的细胞恢复细胞分裂，以及 刺激新的宿主基因表达。这种细菌还能分化 在结构上和表达新基因。我们已经鉴定并克隆了 用于入侵和诱导结节的细菌基因。自由生活 细菌很少或没有与nod基因片段同源的RNA， 这表明它们可能不会在自由生活的细胞中转录。不含蛋白质 观察到野生型细菌和转座子Tn5之间的差异 诱导结节突变体。NODC和NODC的框内翻译基因融合 LacZ已被构建，并用于nod基因的分析 在不同的生长条件和感染期间表达。DNA 这个区域的大部分序列已经确定，在体外， 实现了开放阅读的小细胞和大细胞表达 Nod基因片段中的框架。正在产生对野田佳彦的抗体 已经在大肠杆菌中过度表达，并预测了两个合成肽 从NODC的DNA序列中构建并连接到 承运人。我们将继续研究nod基因的表达和nod基因。 通过构建进一步的操纵子和NOD基因之间的基因融合而产生的产品 以及指示基因Lacz、cat和lux。体外融合将基于 这些共生基因的完整DNA序列。随机操纵子融合 将利用放置LacZ或CAT指示器的TN5衍生品 插入部位启动子控制下的基因。我们将使用基因 融合作为定位和突变结瘤调控基因的检测方法。 Nod基因蛋白将在大肠杆菌中生产并提纯用于 产生抗体，该抗体将用于确认 基因，在自由生活的生长和过程中跟随基因的表达 感染，并确定基因产物的细胞定位 在活体内。这些基因的表达时机和细胞定位 产品将提供对其在入侵期间可能的功能的洞察 并在寄主中诱导结瘤。

We study the interaction of bacterial cells with specific eukaryotic hosts, in a system which relates both to developmental events in multicellular organisms, and to bacterial pathogenesis. Rhizobium meliloti invades and induces nodule formation in specific host plants including alfalfa (Medicago sativa). As they invade, the bacteria alter the host's cell wall synthesis, induce normally non-dividing cells to resume cell division, and stimulate new host gene expression. The bacteria also differentiate structurally and express new genes. We have identified and cloned the bacterial genes used in invasion and nodule induction. Free living bacteria have little or no RNA homologous to the nod gene fragments, suggesting they may not be transcribed in free-living cells. No protein differences were observed between wild type bacteria and transposon Tn5 induced Nod- mutants. An in-frame translational gene fusion of nodC and lacZ has been constructed and this is being used to analyze nod gene expression in various growth conditions and during infection. The DNA sequence has been determined for most of this region, and in vitro, mini-cell and maxi-cell expression has been achieved for open reading frames in the nod gene segment. Antibody is being generated to nodA which has been overexpressed in E. coli, and two synthetic peptides predicted from the DNA sequence of nodC have been constructed and coupled to carrier. We will continue our studies of nod gene expression and nod gene products by constructing further operon and gene fusions between nod genes and indicator genes lacZ, CAT, and lux. In vitro fusions will be based on the complete DNA sequence for these symbiotic genes. Random operon fusions will take advantage of Tn5 derivatives which place lacZ or CAT indicator genes under control of promoters at the insertion site. We will use gene fusions as assays for locating and mutating regulatory nodulation genes. The nod gene proteins will be produced in E. coli and purified for generating antibody, which will be used to confirm the identity of the genes, to follow gene expression in free-living growth and during infection, and to determine the cellular localization of the gene products in vivo. The timing of expression and cellular location of these gene products will provide insight into their possible function during invasion of and nodule induction in their host.