Release of nitric oxide by the Escherichia coli YtfE (RIC) protein and its reduction by the hybrid cluster protein in an integrated pathway to minimize cytoplasmic nitrosative stress.

Synthesis of the Escherichia coli YtfE protein, also known as RIC, for the repair of damaged iron centres, is highly induced during anaerobic growth under conditions of nitrosative stress. How YtfE repairs nitrosative damage remains unclear. Contrary to previous reports, we show that strains defective in YtfE that lack the high-affinity NO reductase activity of the hybrid cluster protein (Hcp) are less sensitive to nitrosative stress than isogenic ytfE+ strains, which are extremely sensitive. Evidence that this sensitivity is due to YtfE-dependent release of NO into the cytoplasm includes: relief of growth inhibition by PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide), which degrades NO; relief of nitrosative stress by deletion of narG encoding the nitrate reductase that is the major source of NO from nitrite; partial suppression of nitrosative stress due to loss of Hcp function by a further mutation in ytfE; YtfE-dependent loss of aconitase and fumarase activities in the absence of Hcp; and YtfE-dependent relief of NsrR repression of the hcp promoter in response to cytoplasmic NO. We suggest that a major role for YtfE is to reverse nitrosative damage by releasing, directly or indirectly, NO from nitrosylated proteins into the cytoplasm where the high-affinity NO reductase activity of Hcp ensures its reduction to N2O. If so, the concerted action of YtfE and Hcp would not only maintain the cytoplasmic concentration of NO in the low nM range, but also provide a rationalization for the coordinate regulation of Hcp and YtfE synthesis by NsrR.

Anaerobic Bacterial Response to Nitrosative Stress.

This chapter provides an overview of current knowledge of how anaerobic bacteria protect themselves against nitrosative stress. Nitric oxide (NO) is the primary source of this stress. Aerobically its removal is an oxidative process, whereas reduction is required anaerobically. Mechanisms required to protect aerobic and anaerobic bacteria are therefore different. Several themes recur in the review. First, how gene expression is regulated often provides clues to the physiological function of the gene products. Second, the physiological significance of reports based upon experiments under extreme conditions that bacteria do not encounter in their natural environment requires reassessment. Third, responses to the primary source of stress need to be distinguished from secondary consequences of chemical damage due to failure of repair mechanisms to cope with extreme conditions. NO is generated by many mechanisms, some of which remain undefined. An example is the recent demonstration that the hybrid cluster protein combines with YtfE (or RIC protein, for repair of iron centres damaged by nitrosative stress) in a new pathway to repair key iron-sulphur proteins damaged by nitrosative stress. The functions of many genes expressed in response to nitrosative stress remain either controversial or are completely unknown. The concentration of NO that accumulates in the bacterial cytoplasm is essentially unknown, so dogmatic statements cannot be made that damage to transcription factors (Fur, FNR, SoxRS, MelR, OxyR) occurs naturally as part of a physiologically relevant signalling mechanism. Such doubts can be resolved by simple experiments to meet six proposed criteria.