DsrA modulates central carbon metabolism and redox balance by directly repressing pflB expression in Salmonella Typhimurium

Authors: DONG, RUI - Shanghai Jiaotong University; LIANG, YUAN - Shanghai Jiaotong University; HE, SHOUKUI - Shanghai Jiaotong University; CUI, YAN - Shanghai Jiaotong University; SHI, CHUNLEI - Shanghai Jiaotong University; He, Yiping; SHI, XIANMING - Shanghai Jiaotong University

Submitted to: Microbiology Spectrum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/3/2022
Publication Date: 2/2/2022
Citation: Dong, R., Liang, Y., He, S., Cui, Y., Shi, C., He, Y., Shi, X. 2022. DsrA modulates central carbon metabolism and redox balance by directly repressing pflB expression in Salmonella Typhimurium. Microbiology Spectrum. 10(1):1. Article e01522-21. https://doi.org/10.1128%2Fspectrum.01522-21.
DOI: https://doi.org/10.1128%2Fspectrum.01522-21

Interpretive Summary: Small RNA DsrA plays important roles in defending oxidative stress in bacteria. This manuscript identified a novel target (pflB, encoding pyruvate-formate lyase) of DsrA and its potential regulatory mechanism in Salmonella Typhimurium by using several advanced molecular techniques. In silico prediction revealed a direct base-pairing between dsrA and pflB mRNA sequences, which was confirmed in site-directed mutagenesis experiments. Transcriptome and phenotype studies showed that the interaction of DsrA-pflB mRNA could greatly contribute to the regulation of central carbon metabolism and intracellular redox balance in S. Typhimurium. The findings of this research provided a better understanding of the critical roles of small RNA in central metabolism and stress responses in foodborne pathogens.

Technical Abstract: Bacterial sRNA functions as a vital regulator in response to various environmental stresses by base-paring with target mRNA. The sRNA DsrA, an important post-transcriptional regulator, has been found to play an important role in defense against oxidative stress in S. Typhimurium, but its regulatory mechanism remains unclear. To clarify the mechanism of DsrA in oxidative stress resistance of S. Typhimurium, transcriptome sequencing was performed on S. Typhimurium wild type (WT) and the dsrA deletion mutant ('dsrA) before and after H2O2 treatment in this study. RNA-seq data showed that the genes involved in glycolysis, pyruvate metabolism, tricarboxylic acid (TCA) cycle, and NADH-dependent respiration exhibited significantly different expression patterns, indicating the critical role of DsrA in regulating central carbon metabolism (CCM) and NAD(H) homeostasis of S. Typhimurium. To reveal the direct target of DsrA action, fusion proteins of six candidates (acnA, srlE, tdcB, nuoH, katG, and pflB) with GFP were constructed and analyzed, showing that the expression of pflB (encoding pyruvate-formate lyase) was repressed by DsrA. Furthermore, in silico predicted base-pairing between dsrA and pflB mRNA sequences was confirmed by site-directed mutagenesis. Lastly, the NAD+/NADH ratio in WT-ppflB-pdsrA was significantly lower than in WT-ppflB, suggesting the direct interaction of DsrA-pflB mRNA could contribute greatly to the redox balance in S. Typhimurium. Taken together, this study identified a new target of DsrA and its regulatory mechanism in oxidative stress resistance in S. typhimurium, which demonstrated that a key gene (pflB) in central metabolism was functionally associated with sRNA-mediated stress resistance.