Polyamine Biosynthesis And Physiological Functions



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调节的，它们的生理功能以及它们在体内的作用。为了这个目的，我们已经构建了空突变体在大肠杆菌和酿酒酵母的生物合成步骤中的每一个，并已准备过表达系统的生物合成酶。在此之前，我们已经报道了在大肠杆菌中构建了所有多胺生物合成基因的完全缺失体。杆菌这些E.大肠杆菌细胞在空气中的纯化的胺缺乏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，并涉及研究多胺需要突变体的培养物对添加多胺的反应。
对于我们目前的研究，我们已经开发了恒化器的使用，其中生长受到多胺以外的因素的限制。与我们自己和其他实验室以前报道的许多研究相反，我们认为，在加入多胺后，不要使生长速率的变化使结果复杂化，这是非常重要的，因为生长速率的任何变化都必然会产生多种效应，而不是多胺加入所引起的效应。我们最近的研究涉及使用这种恒化器技术的微阵列分析的效果，添加多胺到我们的多胺缺陷突变体。结果表明，在短时间内，80个基因的表达上调了2倍以上，25个基因的表达上调了3倍以上。多胺的加入也导致了51个基因的2倍以上的下调。特别引人注目的是，在大肠杆菌中许多基因的表达增加了2倍至5倍。大肠杆菌耐酸性（AR）途径（gadA、gadB、gadC、gadX、gadY、slp、ybaS、hdeD）。这一发现特别令人感兴趣，因为E.大肠杆菌必须在胃的酸性环境中存活。E.大肠杆菌AR系统涉及两个谷氨酸脱羧酶（GAD）蛋白GadA、GadB和谷氨酸-GABA反向转运蛋白GadC及其调控因子。
然后，我们直接测定了谷氨酸脱羧酶活性，在我们的多胺突变体酸胁迫的反应，并发现，这些细胞没有谷氨酸脱羧酶活性，除非多胺添加。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E. cadavarine没有影响。为了阐明分子机制，我们寻找了该途径的两个重要调节因子，rpoS和cAMP。这种需要多胺的突变体存在于标准K12菌株中，该菌株在rpoS基因中也具有琥珀突变。因此，由于已知rpoS是GAD途径所必需的，我们构建了一种新的多胺缺陷型菌株，其在rpoS基因中不具有琥珀突变。尽管文献中有报道称，多胺对gad基因表达的主要作用是抑制环AMP合成，但我们发现，与此报道相反，多胺对多胺缺陷型突变体的作用是增加环AMP水平。为了进一步扩展我们的发现，我们用我们构建的新的多胺缺陷菌株重复了上述研究（缺乏rpoS琥珀突变），其在环AMP合成或cAMP受体蛋白中也具有缺失，并且发现这些菌株即使在酸应激时在不存在多胺的情况下也没有GAD活性，但是在多胺存在下具有非常高的活性，表明多胺效应可以在不存在任何环AMP的情况下发生。我们计划研究在我们的微阵列研究中诱导的这个酸岛上的其他调控基因，并探讨多胺诱导大肠杆菌耐酸性的分子机制。杆菌



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.