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 H2O2-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.
Brandes
Nicolas
N
Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA.
Rinck
Andrea
A
Leichert
Lars Ingo
LI
Jakob
Ursula
U
D003470:Culture Media; D048168:Escherichia coli K12 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005970:Glutamate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018869:Heat-Shock Response; D006834:Hydrazines / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology*; D018384:Oxidative Stress; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*
D003470:Culture Media; D029968:Escherichia coli Proteins; D006834:Hydrazines; D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; C084012:1,1-diethyl-2-hydroxy-2-nitrosohydrazine; D005970:Glutamate Synthase