Phylogeny and functional significance of glutathionylspermidine in E. coli

我们目前对大肠杆菌的研究特别关注谷胱甘肽亚精胺的生理功能。谷胱甘肽亚精胺合成酶/氨基酶（Gss）和编码基因（Gss）仅在两个广泛分离的群体中报道；即大肠杆菌和几个动胞菌门的成员。在我们目前的工作中，我们研究了NCBI数据库中Gss的物种分布，发现Gss序列主要局限于某些细菌和动质体，而在所有无脊椎动物和脊椎动物物种、太古动物、植物和一些真细菌中都不存在。令人惊讶的是，几乎所有已测序的75种肠杆菌都含有与大肠杆菌Gss蛋白高度同源的序列。虽然谷胱甘肽亚精胺的最高积累是在固定相培养中发现的，其中大多数细胞内亚精胺转化为谷胱甘肽亚精胺，即使在对数相细胞中也有一些谷胱甘肽亚精胺的形成。同位素交换实验表明，细胞内谷胱甘肽亚精胺与亚精胺之间存在快速交换。通过对大肠杆菌gss+和gss-菌株的微阵列研究表明，gss基因缺失导致大量基因上调或下调。最显著的上调基因类别包括硫利用、谷氨酰胺和琥珀酸代谢、多胺和精氨酸代谢、嘌呤和嘧啶代谢。金属伴侣、钼、铜、锌和银离子转运基因与grxA （glutaredoxin 1，核糖核苷酸还原酶的氧化还原辅酶）和亚硝酸盐转运蛋白nirC一起下调。还进行了详细的蛋白质组学研究（与NIDDK质谱部门的Eric Anderson博士合作），使用SILAC技术比较了两种菌株的蛋白质组成。为此，我们构建了含有lysA和argA基因缺失的菌株。我们通过比较可溶性蛋白的质谱分析获得数据，发现突变体中有12个蛋白表达上调，8个蛋白表达下调。最近，我们正在通过进一步的SILAC分析比较整个蛋白质（可溶性和膜相关）。我们还开发了一种高通量分析技术，通过氟尿嘧啶耐药性分析gss+和gss-细胞突变频率的差异。我们目前正计划在无菌条件下进行小鼠实验，研究gss+和gss-细菌在小鼠肠道中的生存和定植差异。

Our current studies in E. coli have been particularly concerned with the physiologic function of glutathionylspermidine. Glutathionylspermidine synthetase/amidase (Gss) and the encoding gene (gss) have only been reported in two widely separated groups; namely Escherichia coli and several members of the Kinetoplastida phyla. In our current work we have studied the species distribution of Gss in the NCBI database, and have found that Gss sequences are largely limited to certain bacteria and Kinetoplastids, and are absent in all invertebrate and vertebrate species, Archea, plants and some Eubacteria. It is striking that almost all of the 75 Enterobacteria species that have been sequenced contain sequences with very high degree of homology to the E. coli Gss protein. Although highest accumulation of glutathionylspermidine is found in stationary phase cultures where most of the intracellular spermidine is converted to glutathionylspermidine, even in log phase cells there is some formation of glutathionylspermidine. Isotope exchange experiments show that there is a rapid exchange between intracellular glutathionylspermidine and spermidine. The microarray studies comparing gss+ and gss- strains of E. coli show that a large number of genes are either upregulated or downregulated by the loss of the gss gene. Most significant categories of up-regulated genes include sulfur utilization, glutamine and succinate metabolism, polyamine and arginine metabolism, and purine and pyrimidine metabolism.The genes for metalochaperone, molybdenum, copper, zinc and silver ion transport were down-regulated together with grxA (glutaredoxin 1, a redox coenzyme for ribonucleotide reductase), and nitrite transporter nirC. Detailed proteomic studies have also been carried out (in collaboration with Dr. Eric Anderson of the NIDDK Mass Spectrometry section) comparing the protein composition of the two strains using the SILAC technique. For this purpose we have constructed strains containing deletions in the lysA and argA genes. We have obtained data from this mass spectroscopy analysis by comparing the soluble proteins, and found 12 proteins are upregulated 8 proteins are downregulated in the mutant. Recently we are comparing the whole proteins (soluble and membrane associated) by further SILAC analyses. We also developed a technique to analyze differences in the mutational frequencies in the gss+ and gss- cells by fluorouracil resistance in a high throughput assay. We are currently planning mouse experiments in a germfree condition to study the differences in survival and colonization of gss+ and gss- bacteria in mouse intestine.