Phylogeny and functional significance of glutathionylspermidine in E. coli

项目#3 目的和目标： 多胺，腐胺，亚精胺和精胺，是所有活细胞中的主要多元化合物。这些胺对于与生长和分化相关的许多系统是重要的。我们的研究关注这些多胺是如何合成和代谢的，以及它们的生理功能，它们在体内是如何起作用的。在本项目中，我们特别感兴趣的是阐明多胺结合谷胱甘肽（谷胱甘肽亚精胺）在大肠杆菌中的功能。大肠杆菌的基因芯片、蛋白质组学和生物信息学技术。 摘要： 我们继续对谷胱甘肽亚精胺的生理功能感兴趣。 我们已经对编码谷胱甘肽培脒合酶/酰胺酶的序列进行了分类学分析，发现同源序列几乎完全限于 肠杆菌属和动质体门的几个成员（包括锥虫），并且在所有无脊椎动物和脊椎动物物种、古细菌、植物和大多数真细菌中明显缺失（只有少数例外）。特别引人注目的是不同肠杆菌物种之间的非常高的同源性（>60%）。虽然谷胱甘肽亚精胺的最高积累被发现在静止期的文化，其中大部分的细胞内亚精胺被转化为谷胱甘肽亚精胺，我们的同位素交换实验表明，细胞内亚精胺和谷胱甘肽亚精胺之间的快速交换，即使在对数期细胞。我们早期的基因芯片研究比较了大肠杆菌gss+和gss-菌株。大肠杆菌中的表达表明，大量的基因由于gss基因的缺失而上调或下调。最重要的上调基因类别包括硫利用、谷氨酰胺和琥珀酸代谢、多胺和精氨酸代谢以及嘌呤和嘧啶代谢。随后，我们进行了蛋白质组学研究（与Eric安德森博士合作 NIDDK质谱部分）使用SILAC技术比较两种菌株的蛋白质组成。 为此，我们构建了在gss+和gss背景中含有lysA和argA基因缺失的菌株。 从SILAC测定中，我们发现，与gss+细胞相比，在gss-细胞中有12种蛋白质上调，8种蛋白质下调。一些上调的蛋白质是ZapA、YghU、MgtA、AroH、Ecfk、p53。下调的肽包括Flu、GlpX、FldA、YcbB、DscB、YghD。最引人注目的是流感蛋白的变化。 然而，在进一步的研究中，我们发现流感蛋白的变化与gss缺失无关，而是流感基因的可转移性的结果。这是一个非常有趣的蛋白质，是相位可变外膜蛋白Ag 43。在进一步的研究中，我们发现用作构建gss的亲本的背景菌株具有特别高的flu+转化为flu-的频率。 为了进一步研究，我们正在分离具有相同流感蛋白表达的gss+/gss菌株，其将用于SILAC和其他比较分析，以找出谷胱甘肽亚精胺在大肠杆菌中的生理意义。杆菌

Project #3 Goals and Objectives: The polyamines, putrescine, spermidine, and spermine, are major polybasic compounds in all living cells. These amines are important for many systems related to growth and differentiation. Our studies are concerned with how these polyamines are synthesized and metabolized and their physiologic functions, how they act in vivo. In this project we are particularly interested in elucidating the functions of polyamine conjugated glutathione (glutathionylspermidine) in E. coli by the use of microarrays, proteomics and bioinformatics techniques. Summary: We continued to be interested in the physiologic function of glutathionylspermidine. We have carried out taxonomic analysis of the sequences that encode glutathionlyspermidine synthase/amidase, and have found that homologous sequences are almost completely restricted to Enterobacter and several members of the Kinetoplastida phyla (including trypanosomes), and are strikingly absent in all invertebrate and vertebrate species, Archea, plants and most Eubacteria (with only a few exceptions). Particularly striking is the very high homology (>60%) among different Enterobacterial species. Although highest accumulation of glutathionylspermidine is found in stationary phase cultures where most of the intracellular spermidine is converted to glutathionylspermidine, our isotope exchange experiments show that there is a rapid exchange between intracellular spermidine and glutathionylspermidine even in log phase cells. Our earlier 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. Subsequently we performed proteomic studies (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 in the gss+ and gssΔ background. From the SILAC assay we found that 12 proteins were up-regulated and 8 proteins were down-regulated in gss- cells as compared to gss+ cells. Some of the up-regulated proteins are ZapA, YghU, MgtA, AroH, Ecfk, Pal. The down-regulated peptides include Flu, GlpX, FldA, YcbB, DscB, YghD. Most striking was the change in the Flu protein. However, on further investigation, we found that the change in the Flu protein was not related to the gss deletion, but rather was the result of the matastable nature of the flu gene. This is a very interesting protein and is the phase-variable outer membrane protein Ag43. On further studies we found that the background strain used as the parent for the construction of the gssΔ had a particularly high frequency of conversion of flu+ to flu-. For further studies we are isolating gss+/gssΔ strains with identical expression of flu proteins, which will be used for SILAC and other comparative analyses to find out the physiological significance of glutathionylspermidine in E. coli.