Abstract
Bacterial small RNAs (sRNAs) function as vital regulators in response to various environmental stresses by base pairing with target mRNAs. The sRNA DsrA, an important posttranscriptional regulator, has been reported to play a crucial role in defense against oxidative stress in Salmonella enterica serovar Typhimurium, but its regulatory mechanism remains unclear. The transcriptome sequencing (RNA-seq) results in this study showed that the genes involved in glycolysis, pyruvate metabolism, the tricarboxylic acid (TCA) cycle, and NADH-dependent respiration exhibited significantly different expression patterns between S. Typhimurium wild type (WT) and the dsrA deletion mutant (ΔdsrA strain) before and after H2O2 treatment. This indicated the importance 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 candidate genes (acnA, srlE, tdcB, nuoH, katG, and pflB) with green fluorescent protein (GFP) were constructed, and the fluorescence analysis showed that the expression of pflB encoding pyruvate-formate lyase was repressed by DsrA. Furthermore, site-directed mutagenesis and RNase E-dependent experiments showed that the direct base pairing of DsrA with pflB mRNA could recruit RNase E to degrade pflB mRNA and reduce the stability of pflB mRNA. In addition, the NAD+/NADH ratio in WT-ppflB-pdsrA was significantly lower than that in WT-ppflB, suggesting that the repression of pflB by DsrA could contribute greatly to the redox balance in S. Typhimurium. Taken together, a novel target of DsrA was identified, and its regulatory role was clarified, which demonstrated that DsrA could modulate CCM and redox balance by directly repressing pflB expression in S. Typhimurium. IMPORTANCE Small RNA DsrA plays an important role in defending against oxidative stress in bacteria. In this study, we identified a novel target (pflB, encoding pyruvate-formate lyase) of DsrA and demonstrated its potential regulatory mechanism in S. Typhimurium by transcriptome analysis. In silico prediction revealed a direct base pairing between DsrA and pflB mRNA, which was confirmed in site-directed mutagenesis experiments. The interaction of DsrA-pflB mRNA could greatly contribute to the regulation of central carbon metabolism and intracellular redox balance in S. Typhimurium. These findings provided a better understanding of the critical roles of small RNA in central metabolism and stress responses in foodborne pathogens.