eTheses Repository

# Stress, damage and repair during anaerobic bacterial growth

Bacteria are exposed to nitrosative stress generated by at least three mechanisms, all of which result in the accumulation of nitric oxide. The presence of NO results in nitrosation of iron atoms in iron-sulphur clusters. In enzymes such as aconitase and fumarase this causes inactivation. Many transcription factors containing metal cofactors, such as NsrR, sense and bind NO. NsrR is a key player in the response to nitrosative stress. When these transcription regulators bind NO, their functions are inactivated and gene expression is altered. Genes repressed by NsrR include those that code for the flavohemoglobin, Hmp, the hybrid cluster protein, Hcp, and other genes of unknown function, including ytfE. The ytfE gene encodes the di-iron protein YtfE and is reported to be involved in repair of iron-sulphur clusters damaged by nitrosative stress. We propose that Hcp and YtfE play critical roles in the response to nitrosative stress. An isogenic set of strains that lack all known (or proposed) $$E.$$ $$coli$$ NO reductases is used to investigate their roles. Strains deficient in Hcp show more sensitivity to NO under anaerobic conditions. Both hcp and another gene of unknown function, $$ydbC$$, are ruled out as encoding the missing NO reductase. The kinetics of the rate of NO reduction by this missing NO reductase is used to estimate a value for the Km suggesting it has a very high affinity for NO. The toxic product produced by YtfE is shown to be NO. NsrR appears to be inactivated in an Hcp mutant even in the absence of nitrosative stress. The combined data suggest that there are multiple responses to nitrosative stress in $$E.$$ $$coli.$$