MUTANTS IN BDELLOVIBRIO DEVELOPMENT

描述：蜈蚣弧菌属细菌的成员是专性的 其他革兰氏阴性细菌的捕食者。他们过着两相的生活 具有自由游泳攻击期和细胞内生长的周期 相位。目前还没有关于基因调控的任何知识。 实际上，人们对它们的一般遗传学知之甚少。 然而，最近有一种将dna导入蛭弧菌的系统。 已经有报道，使这种有机体中的一些遗传方法成为可能。 调查员提议分离和描述条件 已经失去在草坪上产生斑块的能力的突变体 敏感的猎物细胞。总体目标是隔离 温度敏感(Ts)突变体的发育周期 来鉴定这些突变体，并分离和 确定突变DNA序列的特征。蜈蚣弧菌将被化学处理 致突变，突变株在34摄氏度下不能产生斑块，但 能够在20摄氏度下形成斑块的人将被隔离。每个突变体都被分离出来 将根据发育的哪个阶段来表征 循环受阻。无法穿透猎物的突变体 将进一步检查周质间隙是否有改变的酶 渗透专一性酶的活性。来自每个类别的突变体 将与携带野生型蜈蚣弧菌基因组的大肠杆菌交配 可传播载体中的文库。已经夺回了 在34摄氏度下产生斑块的能力将被分离，质粒DNA 提取的突变序列进行限制性内切酶分析和 DNA测序分析与野生型序列的比较。数列 获得的序列将与Genbank中的已知序列进行筛选 同源。与同源序列的鉴定可能暗示着 涉及的基因产物的功能。这项工作提供了一个初步的 进一步了解被捕食的蜈蚣弧菌细胞的性质 互动。

DESCRIPTION: Members of the bacterial genus Bdellovibrio are obligate predators of other Gram negative bacteria. They follow a biphasic life cycle with a free swimming attack phase and an intracellular growth phase. Nothing is currently known regarding gene regulation in bdellovibrios, indeed little is known about their genetics in general. However, a system for introducing DNA into bdellovibrios has recently been reported, making some genetic approaches possible in this organism. The investigator proposes to isolate and characterize conditional mutants that have lost the ability to produce plaques on lawns of susceptible prey cells. The overall objectives are to isolate temperature-sensitive (ts) mutants of the developmental cycle of the bdellovibrios, to characterize those mutants, and to isolate and characterize mutant DNA sequences. Bdellovibrios will be chemically mutagenized, and mutants unable to produce plaques at 34 degrees C but able to plaque at 20 degrees C will be isolated. Each mutant isolated will be characterized according to which phase in the developmental cycle is blocked. Mutants unable to penetrate into the prey's periplasmic space will be further examined for altered enzymatic activity of penetration-specific enzymes. Mutants from each category will be mated with E. coli carrying wild-type Bdellovibrio genomic libraries in transmissible vectors. Conjugants that have regained the ability to produce plaques at 34 degrees C will be isolated, plasmid DNA extracted and mutant sequences characterized by restriction analysis and DNA sequencing analysis in comparison to wild-type sequences. Sequences obtained will be screened against known sequences in Genbank for homology. Identification with homologous sequences may suggest possible functions of the gene products involved. This work provides an initial step towards understanding the nature of the Bdellovibrio-prey cell interaction.