Polyamine Biosynthesis And Physiological Functions


&#8232；多年来，我们一直在研究这些多胺是如何合成的，它们的生物合成和降解是如何被调节的，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零突变体，并制备了生物合成酶的过表达系统。以前，我们已经报道了大肠杆菌中所有多胺生物合成基因的完全缺失的构建。这些大肠杆菌细胞在空气中纯化的缺胺培养基中以正常生长速率的40-50%生长。然而，它们对氧化和厌氧应激高度敏感。我们的总体研究旨在利用这些突变体来阐明多胺的生理功能，并研究了多胺需求突变体的培养对添加多胺的反应。&#8232；在我们目前的研究中，我们已经开发了一种化学调节剂的使用，在这种情况下，生长受到多胺以外因素的限制。与我们自己和其他实验室之前报告的一些研究相反，我们认为很重要的一点是，不要让结果因添加多胺后生长速率的变化而复杂化，因为任何生长速率的变化都必然会产生多种影响，而不是由多胺添加引起的。我们最近的研究包括使用这种趋化技术进行微阵列分析，分析在我们的多胺缺乏突变体中添加多胺的效果。结果表明，添加多胺后，短时间内80个基因上调2倍以上，25个基因上调3倍以上。添加多胺也导致51个基因下调2倍以上。特别引人注目的是，大肠杆菌耐酸途径中许多基因（gadA、gadB、gadC、gadX、gadY、slp、ybaS、hdeD）的表达增加了2至5倍。这一发现特别有趣，因为大肠杆菌要在哺乳动物胃肠道中定植，必须通过胃的酸性环境才能存活下来。大肠杆菌AR系统中最有效的组分涉及两种谷氨酸脱羧酶（GAD）蛋白GadA、GadB和谷氨酸- gaba反转运蛋白GadC及其调节因子。&#8232；然后，我们直接测定了多胺突变体在酸胁迫下的谷氨酸脱羧酶活性，发现除非添加多胺，否则这些细胞没有谷氨酸脱羧酶活性。剂量反应研究表明，至少需要100微摩尔腐胺或10微摩尔亚精胺才能诱导大肠杆菌GAD活性；尸胺没有效果。为了揭示分子机制，我们寻找了这一途径的两个重要调控因子，rpoS和cAMP。这种需要多胺的突变体存在于标准的K12菌株中，该菌株在rpoS基因上也有琥珀色突变。因此，由于已知rpoS在GAD通路中是必需的，我们构建了一个新的多胺缺乏菌株，该菌株在rpoS基因中没有琥珀色突变。虽然有文献报道，抑制环AMP合成是多胺添加对gad基因表达的主要影响，但我们发现，与该报道相反，多胺添加到多胺缺陷突变体中会导致环AMP水平升高。为了进一步扩展我们的研究结果，我们用我们构建的新的多胺缺乏菌株（缺乏rpoS琥珀色突变）重复了上述研究，该菌株在环AMP合成或cAMP受体蛋白中也有缺失，并且发现这些菌株即使在酸胁迫下没有多胺也没有GAD活性，但在多胺存在时具有非常高的活性，这表明多胺效应可以在没有环AMP的情况下发生。我们计划在微阵列研究中对该酸岛的其他调控基因进行研究，并探讨多胺诱导大肠杆菌耐酸的分子机制。



For many years we have been studying how these 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. Previously, we have reported the construction of the complete deletion of all polyamine biosynthetic genes in E. coli. These E. coli cells grow at 40-50% of the normal growth rate in purified amine deficient medium in air. However, they are highly sensitive to oxidative and anaerobic stress. Our overall studies have aimed at the use of these mutants to elucidate the physiological functions of the polyamines and have involved studying the response of cultures of the polyamine-requiring mutants to the addition of polyamines. 
For our current studies we have developed the use of a chemostat where the growth is limited by factors other than the polyamines. As opposed to a number of previous studies reported from our own and other laboratories, we feel that it is very important not to have the results complicated by changes in the growth rates after polyamine addition since any changes in growth rate will necessarily have multiple effects other than those specifically resulting from the polyamine additions. Our most recent studies involve the use of this chemostat technique for microarray analyses on the effect of adding polyamines to our polyamine-deficient mutant. The results showed that 80 genes were up-regulated more than 2-fold and 25 genes were up-regulated more than 3-fold within a short time after the polyamine addition. Polyamine addition also caused more than 2-fold down-regulation of 51 genes. Particularly striking was the 2- fold to 5-fold increase in the expression of many genes in the E. coli acid resistance (AR) pathway (gadA, gadB, gadC, gadX, gadY, slp, ybaS, hdeD). This finding was of particular interest because to colonize the mammalian gastrointestinal tract, E. coli must survive passage through the acidic environment of the stomach. The most effective component of the E. coli AR system involves the two glutamate decarboxylase (GAD) proteins GadA, GadB and Glutamate-GABA antiporter GadC and their regulators. 
We then directly assayed the glutamic decarboxylase activity in response to acid stress in our polyamine mutant, and found that these cells had no glutamic decarboxylase activity unless polyamines were added. A dose response study showed that at least 100 micromolar putrescine or 10 microolar spermidine are needed to induce GAD activity in E. coli; cadavarine had no effect. To unravel the molecular mechanism, we looked for two important regulators of this pathway, rpoS and cAMP. This polyamine-requiring mutant was in the standard K12 strain that also has an amber mutation in the rpoS gene. Therefore since rpoS is known to be essential for the GAD pathway, we constructed a new polyamine-deficient strain that did not have the amber mutation in the rpoS gene. Although there is a report in the literature that inhibition of cyclic AMP synthesis is the primary effect of polyamine addition on gad gene expression, we have found, in contrast to this report, that polyamine addition to the polyamine deficient mutants causes an increase in the cyclic AMP levels. To extend our findings further, we repeated the above studies with the new polyamine-deficient strain that we constructed (lacking rpoS amber mutation) that also had deletions in either cyclic AMP synthesis or the cAMP receptor protein, and found that these strains even when acid stressed had no GAD activity in the absence of polyamines, but had very high activity in the presence of polyamines, indicating that the polyamine effect can occur in the absence of any cyclic AMP.. We plan to study the other regulatory genes in this acid island that are induced in our microarray study, and to investigate the molecular mechanisms of polyamine induced acid resistance in E. coli.