Nitrosative stress treatment of E. coli targets distinct set of thiol-containing proteins

Reactive nitrogen species (RNS) function as powerful antimicrobials in host defence, but so far little is known about their bacterial targets. In this study, we set out to identify Escherichia coli proteins with RNS-sensitive cysteines. We found that only a very select set of proteins contain cysteines that undergo reversible thiol modifications upon nitric oxide (NO) treatment in vivo . Of the 10 proteins that we identified, six (AtpA, AceF, FabB, GapA, IlvC, TufA) have been shown to harbour functionally important thiol groups and are encoded by genes that are considered essential under our growth conditions. Media supplementation studies suggested that inactivation of AceF and IlvC is, in part, responsible for the observed NO-induced growth inhibition, indicating that RNS-mediated modifications play important physiological roles. Interestingly, the majority of RNS-sensitive E. coli proteins differ from E. coli proteins that harbour H 2 O 2 -sensitive thiol groups, implying that reactive oxygen and nitrogen species affect distinct physiological processes in bacteria. We confirmed this specificity by analysing the activity of one of our target proteins, the small subunit of glutamate synthase. In vivo and in vitro activity studies confirmed that glutamate synthase rapidly inactivates upon NO treatment but is resistant towards other oxidative stressors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72397/1/j.1365-2958.2007.05964.x.pd

A study of a mutant elongation factor properties of E. coli HAK88 and its mutant elongation factor Tu

The E. coli chromosome contains two genes for elongation factor Tu, tufA (near the fusidic acid resistance marker) and tufB (near the rifampicin resistance marker). It has been discovered that the mutant E. coli K12 strain HAK88 bears a mutation in the tufB gene, which leads to the synthesis of a protein of increased acidity. To determine whether the mutation has altered the protein's function in peptide chain elongation, we have compared the reactivities of normal tufA EF-Tu and mutant tufB EF-Tu (purified together from HAK88) with the components of the AA-tRNA binding cycle. Normal tufA EF-Tu and mutant tufB EF-Tu are indistinguishable in their affinities for GDP, EF-Ts, and phe-tRNA, and differ only slightly in their affinities for ribosomes. Coupled with the results of a separate study showing the similarity of the normal tufA and tufB gene products, these experiments demonstrate that the mutation has not altered the function of tufB EF-Tu in peptide chain elongation. Contrary to the original report (Kuwano et al., 1974; J. Mol. Biol. 86 , 689–698) the HAK88 strains we have examined no longer possess a temperature-sensitive EF-Ts. The growth rates of HAK88 strains resemble the parent HAK8 strain in their lack of tRNA dependence but unlike HAK8 show varying degrees of temperature sensitivity. We conclude that HAK88 contains a physically altered but functionally intact tufB EF-Tu. The mutation in tufB should be valuable for studying in vivo the control of expression of the genes for EF-Tu.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47543/1/438_2004_Article_BF00401747.pd