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.

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