Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA

Most Listeria monocytogenes virulence genes are positively regulated by the PrfA protein, a transcription factor sharing sequence similarities with cy clic AMP (cAMP) receptor protein (CRP). Its coding gene, prfA, is regulated by PrfA itself via an autoregulatory loop mediated by the upstream PrfA-de pendent plcA promoter. We have recently characterized prfA* mutants from L. monocytogenes which, as a result of a single amino acid substitution in Pr fA, Gly145Ser, constitutively overexpress prfA and the genes of the PrfA vi rulence regulon. Here, we show that about 10 times more PrfA protein is pro duced in a prfA* strain than in the wild type. Thus, the phenotype of prfA* mutants is presumably due to the synthesis of a PrfA protein with higher p romoter-activating activity (PrfA*), which keeps its intracellular levels c onstantly elevated by positive feedback. We investigated the interaction of PrfA and PrfA* (Gly145Ser) with target DNA. Gel retardation assays perform ed with a DNA fragment carrying the PrfA binding site of the plcA promoter demonstrated that the PrfA* mutant form is much more efficient than wild-ty pe PrfA at forming specific DNA-protein complexes. In footprinting experime nts, the two purified PrfA forms interacted with the same nucleotides at th e target site, although the minimum amount required for protection was 6 to 7 times lower with PrfA*. These results shaw that the primary functional c onsequence of the Gly145Ser mutation is an increase in the affinity of PrfA for its target sequence. Interestingly, similar mutations at the equivalen t position in CRP result in a transcriptionally active, CRP* mutant form,wh ich binds with high affinity to target DNA in the absence of the activating cofactor, cAMP. Our observations suggest that the structural similarities between PrfA and CRP are also functionally relevant and support a model in which the PrfA protein, like CRP, shifts from transcriptionally inactive to active conformations by interaction with a cofactor.