The regulation of genes of unknown function implicated in nitrosative stress tolerance in Escherichia coli K-12

Abstract

This study was designed to determine the regulatory network that controls expression from two \(Escherichia\) \(coli\) K-12 promoters, \(pyeaR\) and \(pogt\), during anaerobic growth. These promoters were identified from transcriptomic studies as being positively regulated by NarL independently of FNR, the master regulator of anaerobic respiration. Biochemical and genetic analyses presented in this study confirmed that expression from both the \(yeaR\) and \(ogt\) promoters is dependent upon NarL, which binds to a single site in the \(yeaR\) promoter and two sites in the \(ogt\) promoter. The nucleoid-associated protein, Fis, repressed transcription from both promoters, especially in rich medium, by binding to sites that overlap the NarL site, excluding the essential activator. Both promoters were more active in the absence of functional FNR. However, mutational analysis revealed that FNR does not bind to the \(yeaR\) promoter region, so this effect is indirect. How the absence of functional FNR might affect NarL-dependent nitrite signalling was investigated. The Ogt protein is known function as an O\(^6\)-alkyguanine methyltransferase. However, the functions of the gene products of \(yeaR-yoaG\) and another operon implicated in nitrosative stress management, \(hcp-hcr\), were unknown. Strains carrying a chromosomal \(yeaR-yoaG\) deletion were not more sensitive to nitric oxide or hydroxylamine compared with the parental strain, suggesting that the products of this operon are not essential for dealing with these toxic nitrogen species. Conversely, a strain deleted in \(hcp-hcr\) was shown to be slightly more sensitive to both nitric oxide and hydroxylamine, implicating Hcp and Hcr in nitrosative stress management.