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

By Derrick J.P. Squire

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

Topics: QH301 Biology, QR Microbiology
Year: 2009
OAI identifier: oai:etheses.bham.ac.uk:476

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Citations

  1. (1995). As observed previously, the absolute activity of the promoter in the absence of functional
  2. (2007). at the start of this project, no promoter had been shown to be activated by NarL independently of FNR. Data generated during this study and in a parallel independent study
  3. (2006). In a reassessment of the extent of the FNR regulon, a group of genes were identified as being upregulated by NarL in response to nitrate, even in an FNR null strain (Constantinidou
  4. (2007). In the case of NsrR, a putative binding site in the yeaR promoter has been identified in this study and previously (Filenko et al.,
  5. (2006). In vivo transcription activation of pyeaR is repressed by FNR and NsrR Due to the apparent repression of pyeaR by FNR and NsrR, revealed by micro-array analysis (Constantinidou
  6. (2009). pCP20 Confers AmpR and CmR resitance and a temperature sensitive origin of replication. Expresses the FLP recombinase. (Cherepanov and Wackernagel,
  7. (1991). Purified Fis protein, which was purified by the method described previously
  8. (1998). The global transcription activator, FNR, can function as both a class I and a class II activator (Li et al.,
  9. (2007). The regulation of phcp has been studied in detail and is known to be FNR-dependent, and enhanced by the presence of nitrite via de-repression of NsrR (Filenko

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