(2010) have reported slight increase in the sensitivity of a combined grxΔ and gssΔ double mutant to hydrogen peroxide, Doxorubicin supplier but no difference between gss+ and gssΔcells. They have also reported that glutathionyspermidine could form mixed disulfides with proteins, but their results do not exclude the possibility that comparable binding occurs with intracellular glutathione. In our C14-spermidine incorporation assays,

we found more than 98% of the counts are in the TCA supernatant, and only < 2% counts in TCA precipitate with the macromolecules. In this experiment, the gss+ cells showed twice more counts than gss− cells in the TCA precipitate (data not shown). Although we have not been able to define a specific function for the gss gene, we feel that the microarray results clearly show that this gene has a considerable effect on the physiology and gene expression of the bacteria. Comparison of the gss+ and gss− strains in the microarray studies showed marked differences in the regulation of different mRNAs. These differences have been listed in Tables 3, 4 and 5. Some of the gene expression changes in gss+ vs. gss− cells are in the polyamine metabolisms and arginine metabolisms pathways, as expected. We felt that it was important to show that glutathionylspermidine is not just an inactive end-product, but is metabolically active. Our isotope exchange experiments

show buy PD-0332991 that glutathionylspermidine is metabolically active in both logarithmically growing (data not shown) and stationary cultures (Tabor & Tabor, 1975). Thus, it could be possible that even in the log-phase cells, where glutathionylspermidine content is < 10%, there is always some change in spermidine and glutathione pools due to activities of both the synthetase and amidase domains of gss+ as compared to gss− cells. For further understanding of regulatory pathways involved in the gene PJ34 HCl expression pattern of up- or down-regulated genes in gss+ vs. gss− cells, we performed bioinformatics analyses.

The microarray results show an up-regulation of succinate metabolism (sdhD, sdhC, sdhA), which increases fumarate synthesis in the cells and on the other hand down-regulation of fumarate metabolism (frdC, frdD, and frdB), which could increase fumarate level in the gss− cells. The transcription of sdhCABD is enhanced during a switch from aerobic to anarerobic growth by ArcA transcriptional regulators (Maklashina et al., 1998). The carAB regulon is regulated by arginine, pyrimidine, and purine levels (Devroede et al., 2004). The genes for purine metabolisms (e.g. purM, purD, and purH) are regulated by PurRP (Meng et al., 1990). The precise mechanism of how these genes are regulated by gss gene deletion is not known. However, as shown in Table 5, fourteen transcriptional regulators are either up- or down-regulated in gss− culture.