Polyamine Biosynthesis And Physiological Functions

多胺生物合成和生理功能

• 批准号: 9549810
• 负责人: Herbert Tabor
• 金额: $ 29.53万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9549810
• 关键词: Acids; Affect; Amination; Amines; Anabolism; Attention; Bacteria; Bacteria sigma factor KatF protein; Biological Assay; Cells; Code; DNA Damage; DNA-Directed RNA Polymerase; Data; Deletion Mutation; Development; Differentiation and Growth; Employee Strikes; Environment; Enzymes; Escherichia coli; Gastrointestinal tract structure; Genes; Glutamate Decarboxylase; Glutamates; Glutamic Acid; Growth; Hydrogen Peroxide; Island; Laboratories; Measurement; Measures; Methods; Modeling; Molecular Profiling; Mutation; Optics; Oxygen; Pathway interactions; Phase; Physiological; Plasmids; Polyamines; Positioning Attribute; Proteins; Proteomics; Putrescine; RNA; Regulation; Regulator Genes; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Saccharomyces cerevisiae; Series; Sigma Factor; Spermidine; Spermine; Stomach; Stress; System; Techniques; Testing; Time; Transfer RNA; Uncertainty; acid stress; aminobutyrate; antiporter; density; experimental study; fitness; genome-wide; in vivo; mutant; overexpression; promoter; protective effect; response

For many years we have been studying how polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions and how they act in vivo. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared over-expression systems for the biosynthetic enzymes. Despite many studies for years from our own and other laboratories there has been uncertainty of the specific mechanisms involved in the pleotropic effects of polyamines. Our current studies using microarray and proteomic techniques have characterized the specific mechanism for many of the polyamine effects. In our earlier microarray studies we found that within 60 minutes after polyamine addition 54 genes were up-regulated more than 2-fold and 15 genes were up-regulated more than 3-fold; 94 genes were down-regulated more than 2-fold. It was striking that most affected system involved the genes involved in the development of acid-resistance in E. coli. Not only were the two genes for glutamate decarboxylases and the gene for glutamate gamma-aminobutyrate antiporter (gadC) induced by the polyamine addition, but also the various genes involved in the regulation of this system were induced. This acid-resistance system is of importance because to colonize the gastrointestinal tract, E. coli must survive passage through the acidic environment of the stomach. We confirmed the importance of polyamines for the induction of the proteins of the acid-resistance system by direct measurement of glutamate decarboxylase protein and activity and by measuring acid-survival at pH 2.5. In the absence of added polyamines there was no induction of glutamate decarboxylase and no protection against acid stress. The protection from acid stress required polyamines, glutamic acid, and glutamate decarboxylase. The effect of deletions of the regulatory genes on this system and the effect of overproduction of three of these genes were also studied. Deletion of either rpoS (coding for the alternate Sigma factor of RNA polymerase) or gadE or rcsB resulted in complete loss of glutamate decarboxylase activity and acid resistance even in cultures containing polyamines. In conclusion, these data show that a major function of polyamines in E. coli is protection against acid stress by increasing the synthesis of glutamate decarboxylase, presumably by increasing the levels of the rpoS and gadE regulators. In our more recent studies we focused our attention on the regulatory network responsible for this polyamine induced glutamate decarboxylase activation, and also carried out preliminary studies on some other systems in which polyamines protect E. coli from other conditions of stress. For these studies we constructed a new polyamine-deficient strain containing well-defined deletion mutations in all of the genes involved in polyamine biosynthesis. Further confirmation of the regulatory roles of rpoS and gadE is shown by a comparison of genome-wide expression profiling data from a series of microarrays comparing the genes induced by polyamine addition to polyamine-free rpoS+/gadE+ cells with genes induced by polyamine addition to polyamine-free delta rpoS/gadE+ and rpoS+/delta gadE cells. The results indicate that a large percentage of the genes induced by polyamine addition (including the GDAR system) require an intact rpoS gene. Polyamine addition resulted in greater than 3-fold increased expression of 85 genes, and it also suppressed 70 genes at least three-fold. Among the pathways most affected by the addition of polyamines were the genes of the E. coli acid response pathway and genes in the acid fitness island including gadA, gadB, gadC, gadE, hdeD hdeA, slp, dctR, and yhiD; Most striking were the very large increases in gadA and gadB expression (20-fold and 26-fold), confirming our previous results. Many oof these inductions were markedly decreased if the cells contained a deletion in rpoS or gadE. To supplement the microarray data, we also carried out quantitative-real-time PCR analyses on the effect of rpoS or gadE deletions on polyamine induced expression of six of these mostly affected genes. These results confirm the microarray data that gadE and rpoS as well as polyamines are critical for the induced expression of most of the genes in the E. coli acid response system. Polyamine addition increased glutamate decarboxylase activity 50-70-fold in a stationary phase culture of rpoS+/gadE+ cells; in contrast the rpoS gadE+ and the rpoS+ gadE strains had no activity even if the culture contained polyamines. We also constructed a polyamine mutant that lacked both the rpoS and gadE genes, and repeated the above experiments in presence of prpoS, or pgadE or both. Overexpression of the RpoS protein from a plasmid had no effect in the absence of the gadE gene. In contrast overexpression of a gadE plasmid was able to partially replace an rpoS deletion. Addition of plasmids overexpressing both RpoS and GadE proteins from both plasmids plus polyamine addition resulted in a very large increase in glutamate decarboxylase activity. Hence, gadE is most directly involved in the synthesis of and activity of glutamate decarboxylase via the activation of gadA and gadB, and rpoS acts by stimulating gadE synthesis. The above conclusion on the direct involvement of gadE for the GDAR system is further supported by survival assays at pH 2.5. From the above experiments it is clear that gadE expression is directly correlated with polyamine induced expression of glutamate decarboxylase activity. Using a RT-PCR method we have used the RNA from our polyamine-free mutant strain (HT873- rpoS+/gadE+) to study the effect of polyamine addition on each of the three promoters. All the three promoters showed enhanced activity after polyamine treatment, particularly P2. We next tested the effect of an rpoS deletion (HT874-rpoS/gadE+) on the polyamine induction of gadE promoters by quantitative real time PCR. In the absence of rpoS there was no induction by polyamines of any of the three promoters of gadE. Most strikingly we found that polyamine addition markedly increased the RpoS protein level within twenty minutes after addition of polyamines to the amine-deprived culture. In the early growth phase in the absence of polyamines, there was only a trace amount of RpoS protein. However addition of polyamines for a very short time (20 min) resulted in a large increase in RpoS protein level at all optical densities. We postulate a cascade model in which the primary action of the polyamine addition to the polyamine-deficient cells is the very rapid, increase in the RpoS level with subsequent induction of gadE expression, that in turn increases the GDAR system. This observation that polyamines increase the amount of RpoS protein is a major breakthrough in understanding the physiological role of polyamines in bacteria. This finding is of importance not only for understanding the requirement of polyamines for the glutamate-dependent-acid-response system but also for the pleotropic effects of polyamines in regulating the large number of systems already known to involve RpoS. We have already confirmed this mechanism for the protective effect of polyamines against oxygen stress (H2O2) and DNA damaging agents, and for long time survival. We have also found that some effects of polyamines are not dependent on the RpoS system. As part of these studies we found two mutants (mnmE and mnmG) that modify the U34 position in tRNA,and which we show have an absolute requirement of polyamines for growth. When we add either of these mutations to the strain that we had constructed that lacked the genes required for polyamine bioynthesis, the resultant strain does not grow unless polyamines are added to the me

多年来，我们一直在研究多胺是如何合成的、它们的生物合成和降解是如何调节的、它们的生理功能以及它们在体内的作用。为此，我们在大肠杆菌和酿酒酵母的每个生物合成步骤中构建了无效突变体，并制备了生物合成酶的过表达系统。 尽管我们自己和其他实验室多年来进行了许多研究，但多胺多效性的具体机制仍存在不确定性。我们目前使用微阵列和蛋白质组技术的研究已经表征了许多多胺效应的具体机制。 在我们早期的微阵列研究中，我们发现添加多胺后 60 分钟内，54 个基因上调超过 2 倍，15 个基因上调超过 3 倍； 94 个基因下调超过 2 倍。令人惊讶的是，大多数受影响的系统涉及大肠杆菌中与耐酸性发展相关的基因。多胺的添加不仅诱导了谷氨酸脱羧酶的两个基因和谷氨酸γ-氨基丁酸逆向转运蛋白（gadC）基因，而且还诱导了参与该系统调节的各种基因。 这种耐酸系统非常重要，因为为了在胃肠道定殖，大肠杆菌必须在胃的酸性环境中存活下来。我们通过直接测量谷氨酸脱羧酶蛋白和活性以及测量 pH 2.5 下的酸存活率，证实了多胺对于诱导耐酸系统蛋白的重要性。在不添加多胺的情况下，没有谷氨酸脱羧酶的诱导，也没有针对酸应激的保护作用。防止酸应激需要多胺、谷氨酸和谷氨酸脱羧酶。还研究了调控基因的缺失对该系统的影响以及其中三个基因的过量产生的影响。 rpoS（编码 RNA 聚合酶的替代 Sigma 因子）或 gadE 或 rcsB 的缺失会导致谷氨酸脱羧酶活性和耐酸性完全丧失，即使在含有多胺的培养物中也是如此。总之，这些数据表明，大肠杆菌中多胺的主要功能是通过增加谷氨酸脱羧酶的合成（可能是通过增加 rpoS 和 gadE 调节剂的水平）来防止酸应激。 在我们最近的研究中，我们将注意力集中在负责这种多胺诱导的谷氨酸脱羧酶激活的调节网络上，并且还对一些其他系统进行了初步研究，在这些系统中多胺保护大肠杆菌免受其他应激条件的影响。在这些研究中，我们构建了一种新的多胺缺陷菌株，其中所有参与多胺生物合成的基因均含有明确的缺失突变。 通过比较一系列微阵列的全基因组表达谱数据，进一步证实了 rpoS 和 gadE 的调节作用，比较了无聚胺 rpoS+/gadE+ 细胞中添加多胺诱导的基因与无聚胺 delta rpoS/gadE+ 和 rpoS+/delta gadE 细胞中添加多胺诱导的基因。结果表明，大部分由多胺添加诱导的基因（包括 GDAR 系统）需要完整的 rpoS 基因。添加多胺导致 85 个基因的表达增加了 3 倍以上，并且还抑制了 70 个基因至少三倍。受添加多胺影响最大的途径是大肠杆菌酸反应途径的基因和酸性适应岛中的基因，包括 gadA、gadB、gadC、gadE、hdeD hdeA、slp、dctR 和 yhiD；最引人注目的是 gadA 和 gadB 表达的大幅增加（20 倍和 26 倍），证实了我们之前的结果。如果细胞含有 rpoS 或 gadE 缺失，则许多诱导作用都会显着减少。 为了补充微阵列数据，我们还对 rpoS 或 gadE 缺失对多胺诱导的其中六个最受影响的基因的表达的影响进行了定量实时 PCR 分析。这些结果证实了微阵列数据，即gadE和rpoS以及多胺对于大肠杆菌酸反应系统中大多数基因的诱导表达至关重要。 在 rpoS+/gadE+ 细胞的稳定期培养物中，添加多胺可使谷氨酸脱羧酶活性提高 50-70 倍；相反，即使培养物含有多胺，rpoS gadE+和rpoS+ gadE菌株也没有活性。我们还构建了缺乏 rpoS 和 gadE 基因的多胺突变体，并在 prpoS 或 pgadE 或两者都存在的情况下重复上述实验。在没有 gadE 基因的情况下，质粒中 RpoS 蛋白的过表达没有效果。 相反，gadE 质粒的过表达能够部分替代 rpoS 缺失。添加过表达来自两种质粒的 RpoS 和 GadE 蛋白的质粒，加上多胺的添加，导致谷氨酸脱羧酶活性大幅增加。因此，gadE通过激活gadA和gadB最直接地参与谷氨酸脱羧酶的合成和活性，而rpoS通过刺激gadE合成起作用。上述关于 gadE 直接参与 GDAR 系统的结论得到了 pH 2.5 下存活测定的进一步支持。从以上实验可以清楚地看出，gadE表达与多胺诱导的谷氨酸脱羧酶活性表达直接相关。 通过 RT-PCR 方法，我们使用来自无多胺突变株 (HT873-rpoS+/gadE+) 的 RNA 来研究多胺添加对三个启动子中每一个启动子的影响。所有三个启动子在多胺处理后均显示出增强的活性，尤其是 P2。接下来，我们通过定量实时 PCR 测试了 rpoS 缺失 (HT874-rpoS/gadE+) 对 gadE 启动子多胺诱导的影响。在不存在 rpoS 的情况下，gadE 的三个启动子中的任何一个的多胺都不会诱导。 最引人注目的是，我们发现，在将多胺添加到缺乏胺的培养物中后二十分钟内，添加多胺显着增加了RpoS蛋白水平。在缺乏多胺的生长早期，仅存在微量的RpoS蛋白。然而，在很短的时间内（20 分钟）添加多胺会导致所有光密度下的 RpoS 蛋白水平大幅增加。 我们假设一个级联模型，其中多胺添加到缺乏多胺的细胞中的主要作用是非常快速地增加 RpoS 水平，随后诱导 gadE 表达，从而增加 GDAR 系统。多胺增加 RpoS 蛋白量的这一观察结果是理解多胺在细菌中的生理作用的重大突破。这一发现不仅对于理解谷氨酸依赖性酸反应系统对多胺的需求很重要，而且对于多胺在调节已知涉及 RpoS 的大量系统中的多效作用也很重要。我们已经证实了多胺对氧应激 (H2O2) 和 DNA 损伤剂的保护作用以及长期存活的这种机制。 我们还发现多胺的一些作用并不依赖于 RpoS 系统。作为这些研究的一部分，我们发现了两个突变体（mnmE 和 mnmG），它们修改了 tRNA 中的 U34 位置，并且我们证明它们的生长绝对需要多胺。当我们将这些突变中的任何一个添加到我们构建的缺乏多胺生物合成所需基因的菌株时，除非将多胺添加到菌株中，否则所得菌株不会生长。