Glutathionylspermidine synthetase/amidase (Gss) and the encoding gene (gss) have only been studied in Escherichia coli and several members of the Kinetoplastida phyla. In the present article, we have studied the phylogenetic distribution of Gss and have found that Gss sequences are largely limited to certain bacteria and Kinetoplastids and are absent in a variety of 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. To find out the physiological significance of glutathionylspermidine in E. coli, we have performed global transcriptome analyses. The microarray studies comparing gss+ and Δgss strains of E. coli show that a large number of genes are either up-regulated (76 genes more than threefold) or down-regulated (35 genes more than threefold) 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.