During infection or denitrification, bacteria encounter reactive nitrogen species. Although the molecular targets of and defensive response against nitric oxide (NO) in Escherichia coli are well studied, the response elements specific to S-nitrosothiols are less clear. Previously, we employed an integrated systems biology approach to unravel the E. coli NO-response network. Here we use a similar approach to confirm that S-nitrosoglutathione (GSNO) primarily impacts the metabolic and regulatory programs of E. coli in minimal medium by reaction with homocysteine and cysteine and subsequent disruption of the methionine biosynthesis pathway. Targeting of homocysteine and cysteine results in altered regulatory activity of MetJ, MetR, and CysB, activation of the stringent response and growth inhibition. Deletion of metJ or supplementation with methionine strongly attenuated the effect of GSNO on growth and gene expression. Furthermore, GSNO inhibited the ArcAB two-component system. Consistent with the underlying nitrosative and thiol-oxidative chemistry, growth inhibition and the majority of the regulatory perturbations were dependent upon GSNO internalization by the Dpp dipeptide transporter. Contrastingly, perturbation of NsrR appeared to be a result of the submicromolar levels of NO released from GSNO and did not require GSNO internalization.