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

为了合成蛋白质和核酸等化合物，所有生物都必须从环境中获取氮。在各种各样的变形菌中，NifA蛋白是激活固氮操纵子转录所必需的。在所有这些NifA活性都受分子氧（02）的控制，但其机制因生物体而异。在y亚群成员中，NifA活性被第二种蛋白NifL抑制。在复合氮存在下，NifL也能抑制NifA的活性。因此，NifL抑制的解除需要缺乏02和组合氮，而如何协调这些作用是一个重要的调控问题。NifL携带与其n端结构域相关的FAD辅因子。在体外纯化的转录系统中，用强大的还原剂二硫代酸还原辅助因子减轻NifL对NifA活性的抑制。工作假设是一种未知的含铁蛋白可能是肺炎克雷伯菌NifL的FAD辅因子的生理还原剂。有强有力的证据表明，氮调节蛋白GlnK，一种最近在生命的所有三个领域中发现的蛋白质变构效应，也是缓解NifL抑制所必需的。据推测，在厌氧条件下，只有当结合氮也有限制时，GlnK才能使NifL的FAD辅助因子还原，例如，通过将还原的中点电位提高到生理范围。为了验证这一假设，我们使用了遗传学、分子生物学和生化方法的结合来确定：1)GlnK是否使NifL的FAD辅助因子在体外更容易降低，从而减轻NifL的抑制作用；2)GlnK是否直接与NifL相互作用。虽然之前没有关于细菌中amtB（氨甲基铵转运B）基因破坏相关生长缺陷的报道，但amtB突变菌株在pH值低于7的低浓度NH4+下生长不良。对多种突变菌株的生长研究，结合对NH4+类似物l4Cmethylammonium运输的研究，最简洁地解释了AmtB蛋白，它在生命的所有三个领域中都发现，运输未带电的NH3而不是带电的NH4，并且它们促进了这种物种的扩散速度，而不是以能量依赖的方式集中它。这两种解释都与之前许多其他实验室的观点不一致。为了进一步验证，目的3)是研究肠道细菌AmtB蛋白在pH 7.0下获取NH3的作用，这必须在连续培养中完成；目的4)是确定当菌株生长在产生NH3的贫氮源上时，AmtB是否介导NH3向培养基的损失。扩散是否双向；目的5)是确定酿酒酵母对14cmethy铵的明显富集是否由于酵母液泡中的“酸捕获”。这些研究对理解对不同环境信号的转录调节反应的协调很有兴趣，也就是说，理解环境信号如何协调来控制生物体DNA特定部分的解码。它们还影响肠道GlnK蛋白的功能，GlnK蛋白是一个复杂的“双层”调节系统的一部分，用于感知氮的有效性，以及AmtB的功能，AmtB对于低浓度氨的获取至关重要。

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.