Non-typhoidal salmonellosis is a severe, gastrointestinal disease caused by Salmonella enterica that places a large burden on global health. Salmonella encounter multiple host defenses during their associations with mammalian hosts, including the toxic radical nitric oxide (NO). My preliminary data indicate that NO induces the accumulation of the alarmone guanosine tetraphosphate (ppGpp). Synthesis of ppGpp is a hallmark of the stringent response of bacteria undergoing nutritional starvation. The stringent response-mediated up-regulation of transcription of amino acid biosynthesis could aid with the metabolic recovery of Salmonella undergoing nitrosative stress. I have noticed that the accumulation of ppGpp is dependent upon RelA which is ribosome associated and activated by halts in translation due to uncharged tRNAs. Oxidative damage of thiol groups or metal centers in enzymes involved in amino acid biosynthesis by NO could lead to drops in aminoacylated tRNAs and mediate the activation of the stringent response. I have developed a novel deep-sequencing technique to quantify the aminoacylation levels of all tRNA species within a single sample. My hypothesis predicts that the ppGpp- mediated transcriptional up-regulation of amino acid biosynthesis aids with the recovery of Salmonella undergoing nitrosative stress. Towards this goal, a library of bar-coded transposon Salmonella mutants has been challenged with NO. Antinitrosative resistant loci of Salmonella have been identified by deep-sequencing genomic analysis of input and output mutants in the library. My investigations will characterize metabolic events that help Salmonella adapt to nitrosative stress. In particular, the proposed research will help us understand the mechanisms by which the stringent response aids the recovery of bacteria from NO-induced cytotoxicity. State-of-the-art biochemical and genomic approaches will be used to answer these questions.

Public Health Relevance

Salmonella exposed to nitric oxide experience a drastic increase in the production of guanosine tetraphosphate (ppGpp) which I hypothesize contributes to the antinitrosative defenses of Salmonella by activating the transcription of amino acid biosynthetic genes. These data suggest intricate relationships between metabolism and stress resistance. The goals of this application are to identify the mechanism of how nitric oxide induces ppGpp and to characterize the role that ppGpp-mediated regulation of metabolism plays on resistance to nitrosative stress.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Alexander, William A
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University of Colorado Denver
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United States
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Tapscott, Timothy; Kim, Ju-Sim; Crawford, Matthew A et al. (2018) Guanosine tetraphosphate relieves the negative regulation of Salmonella pathogenicity island-2 gene transcription exerted by the AT-rich ssrA discriminator region. Sci Rep 8:9465
Kim, Ju-Sim; Liu, Lin; Fitzsimmons, Liam F et al. (2018) DksA-DnaJ redox interactions provide a signal for the activation of bacterial RNA polymerase. Proc Natl Acad Sci U S A 115:E11780-E11789
Fitzsimmons, Liam F; Liu, Lin; Kim, Ju-Sim et al. (2018) Salmonella Reprograms Nucleotide Metabolism in Its Adaptation to Nitrosative Stress. MBio 9:
Crawford, Matthew A; Tapscott, Timothy; Fitzsimmons, Liam F et al. (2016) Redox-Active Sensing by Bacterial DksA Transcription Factors Is Determined by Cysteine and Zinc Content. MBio 7:e02161-15