Regulation of Expression of the Nitrogen Fixation (nif) Genes of K. pneumoniae and Studies of the glnK-amtB operon

肺炎克雷伯菌固氮（nif）基因的表达调控及glnK-amtB操纵子的研究

• 批准号: 9874443
• 负责人: Sydney Kustu
• 金额: $ 30万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9874443&HistoricalAwards=false
• 关键词: Regulation; Expression; Nitrogen; Fixation; nif

Nitrogen must be obtained from the environment by all living things in order to synthesize compounds like proteins and nucleic acids. The NifA protein is required to activate transcription of the nitrogen fixation operons in a wide variety of proteobacteria. In all of them NifA activity is controlled by molecular oxygen (02) but the mechanism differs with the organism. In members of the y-subgroup, NifA activity is inhibited by a second protein, NifL. NifL can also inhibit NifA activity in the presence of combined nitrogen. Thus relief of NifL inhibition requires the absence of both 02 and combined nitrogen, and how such effects are coordinated is an important regulatory problem. NifL carries a FAD cofactor associated with its N-terminal domain. Reduction of the cofactor with the powerful reductant dithionite relieves NifL inhibition of NifA activity in a purified transcription system in vitro. The working hypothesis is that an unidentified iron-containing protein may be the physiological reductant for the FAD cofactor of NifL in Klebsiella pneumoniae. There is strong evidence that the nitrogen regulatory protein GlnK, a protein allosteric effector of a sort recently identified in all three domains of life, is also required for relief of NifL inhibition. It is postulated that GlnK enables reduction of the FAD co-factor of NifL under anaerobic conditions only when combined nitrogen is also limiting, e.g. by raising the mid-point potential for reduction into the physiological range. To test this hypothesis a combination of genetic, molecular biological and biochemical methods is being used to determine: 1) whether GlnK makes reduction of the FAD co-factor of NifL easier in vitro and thereby results in relief of NifL inhibition and 2) whether GlnK interacts directly with NifL. Although there was no previous report of a growth defect associated with disruption of the amtB (ammonium-methylammonium transport B) gene in bacteria, which lies downstream of glnK and in an operon with it, amtB mutant strains of enteric bacteria grow poorly at low concentrations of NH4+ at pH values below 7. Growth studies with a variety of mutant strains in combination with studies of transport of the NH4+ analogue l4Cmethylammonium are most parsimoniously interpreted to indicate that AmtB proteins, which are found in all three domains of life, transport the uncharged species NH3 rather than the charged species NH4, and that they facilitate the rate of diffusion of this species rather than concentrating it in an energy-dependent manner. Both interpretations are at odds with previous views from a number of other laboratories. To test them further, aim 3) is to study the role of the AmtB protein of enteric bacteria in acquisition of NH3 at pH 7.0, which must be done in continuous culture; aim 4)is to determine whether AmtB mediates loss of NH3 to the medium when strains are grown on poor nitrogen sources that generate it--i.e. whether diffusion is bi-directional; and aim 5) is to determine whether apparent concentrative uptake of 14Cmethylammonium by S. cerevisiae is due to "acid trapping" in yeast vacuoles. These studies are of interest with respect to understanding the coordination of transcriptional regulatory responses to different environmental signals, that is, in understanding how environmental signals are coordinated to control decoding of particular portions of an organism's DNA. They also bear on the functions of the enteric GlnK protein, part of a sophisticated "two-tiered" regulatory system for sensing nitrogen availability, and on those of AmtB, which is essential for acquisition of ammonia at low concentrations.

所有生物都必须从环境中获取氮，以合成蛋白质和核酸等化合物。 NifA蛋白是激活多种变形菌中固氮操纵子转录所必需的。在所有这些中，NifA活性由分子氧（02）控制，但机制因生物体而异。在y亚组的成员中，NifA活性被第二种蛋白NifL抑制。NifL还可以在结合氮的存在下抑制NifA活性。因此，NifL抑制的缓解需要不存在O2和结合氮两者，并且如何协调这些作用是重要的调节问题。NifL携带与其N-末端结构域相关的FAD辅因子。用强还原剂连二亚硫酸盐还原辅因子在体外纯化的转录系统中解除NifL对NifA活性的抑制。工作假设是，一个身份不明的含铁蛋白质可能是NifL的FAD辅因子在肺炎克雷伯氏菌的生理还原剂。有强有力的证据表明，氮调节蛋白GlnK，一种最近在所有三个生命领域中发现的蛋白质变构效应物，也是缓解NifL抑制所必需的。据推测，GlnK仅在结合氮也受限时才能在厌氧条件下还原NifL的FAD辅因子，例如通过将还原的中点电位提高到生理范围。为了检验这一假设，使用遗传、分子生物学和生物化学方法的组合来确定：1）GlnK是否使NifL的FAD辅因子在体外更容易减少，从而导致NifL抑制的缓解，和2）GlnK是否直接与NifL相互作用。尽管先前没有报道与细菌中amt B（铵-甲基铵转运B）基因（其位于glnK的下游并与其一起位于操纵子中）的破坏相关的生长缺陷，但肠道细菌的amt B突变菌株在pH值低于7的低浓度NH 4+下生长不良。生长研究与各种突变株结合的研究运输的NH 4+类似物14 C甲基铵是最吝啬的解释表明，AmtB蛋白，这是发现在所有三个领域的生活，运输不带电荷的物种NH3，而不是带电的物种NH 4，他们促进扩散的速度，而不是集中在一个能量依赖的方式。这两种解释都与许多其他实验室先前的观点不一致。为了进一步测试它们，目的3）是研究肠道细菌的AmtB蛋白在pH 7.0下获得NH3中的作用，这必须在连续培养中进行;目的4）是确定当菌株在产生NH3的贫氮源上生长时，AmtB是否介导NH3损失到培养基中-即扩散是否是双向的;目的5）确定S.酿酒酵母中的“酸捕获”是由于酵母液泡中的“酸捕获”。这些研究对于理解不同环境信号的转录调节反应的协调是有意义的，也就是说，理解环境信号如何协调以控制生物体DNA的特定部分的解码。它们还影响肠GlnK蛋白的功能，这是用于感测氮可用性的复杂的“双层”调节系统的一部分，并且影响AmtB的功能，这对于在低浓度下获得氨是必不可少的。