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

所有生物必须从环境中获取氮，才能合成蛋白质和核酸等化合物。NIFA蛋白是激活多种变形杆菌中固氮操纵子转录所必需的。在所有这些化合物中，NIFA的活性都是由分子氧(02)控制的，但其作用机制因生物体而异。在y-亚群的成员中，NIFA活性被第二种蛋白质NIFL抑制。在结合态氮存在时，NIFL也能抑制NIFA的活性。因此，解除NIFL抑制需要同时缺乏02和结合氮，如何协调这些影响是一个重要的调控问题。NIFL携带与其N-末端结构域相关的FAD辅因子。在体外纯化的转录系统中，用强大的还原剂二亚硫酸盐还原辅因子可以解除NIFL对NIFA活性的抑制。工作假说是，一种未知的含铁蛋白可能是肺炎克雷伯菌中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也是解除NIFL抑制所必需的。GlnK是最近在所有三个生命领域中发现的一种蛋白质变构效应器。据推测，只有当结合氮也是有限的时，GlnK才能在厌氧条件下降低NIFL的FAD辅助因子，例如通过提高还原到生理范围的中点电位。为了验证这一假说，我们采用了遗传学、分子生物学和生物化学相结合的方法来确定：1)GlnK是否使NIFL的FAD辅助因子在体外更容易降低，从而导致NIFL抑制的解除；2)GlnK是否直接与NIFL相互作用。尽管以前还没有关于细菌amtB(氨甲基铵运输B)基因的生长缺陷的报道，amtB基因位于glnK下游和带有它的操纵子中，但肠道细菌amtB突变株在pH值低于7的低浓度NH4+下生长不良。对各种突变菌株的生长研究以及对NH4+类似物14C甲基铵运输的研究被最简明地解释为表明，在所有三个生命领域都发现的amtB蛋白运输的是未带电的物种NH3，而不是带电的物种NH4。它们促进了这种物种的扩散速度，而不是以一种依赖能量的方式将其集中。这两种解释都与其他一些实验室之前的观点不符。为了进一步测试它们，目的3)是研究肠道细菌的AmtB蛋白在获得pH为7.0的NH3中的作用，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的贫乏氮源--即AmtB--时，AmtB是否介导了NH3向培养基的流失。扩散是否是双向的；目标5)是确定酿酒酵母对~(14)C甲基铵的表观集中吸收是否是由于酵母液泡中的“酸捕获”。这些研究对于理解转录调控反应对不同环境信号的协调，即理解环境信号是如何协调以控制生物体DNA特定部分的解码是有意义的。它们还与肠道GlnK蛋白和AmtB的功能有关。GlnK蛋白是一个复杂的“两级”调节系统的一部分，用于感知氮素的有效性；AmtB是获取低浓度氨的关键。

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.