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The transcriptional activator Positive Regulatory Factor A (PrfA) regulates expression of genes essential for virulence in Listeria monocytogenes. To define the PrfA regulon, the 10403S wildtype (WT) strain, a constitutively active prfA* mutant, and an isogenic ∆prfA mutant were grown under PrfA-inducing conditions in a medium containing glucose-1-phosphate and pre-treated with 0.2% activated charcoal. RNA-seq-generated transcript levels were compared as follows (i) prfA* and WT; (ii) WT and ∆prfA and (iii) prfA* and ∆prfA. Significantly