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2004: Vega Yolanda; Rauch Markus; Banfield Mark J; Ermolaeva Svetlana; Scortti Mariela; Goebel Werner; Vázquez-Boland José A
New Listeria monocytogenes prfA* mutants, transcriptional properties of PrfA* proteins and structure-function of the virulence regulator PrfA.
Molecular microbiology 2004;52(6):1553-65.
PrfA, a transcription factor structurally related to Crp/Fnr, activates Listeria monocytogenes virulence genes during intracellular infection. We report two new PrfA* mutations causing the constitutive overexpression of the PrfA regulon. Leu-140Phe lies in alphaD adjacent to the DNA-binding motif in the C-terminal domain, like a previously characterized PrfA* mutation (Gly-145Ser). Ile-45Ser, in contrast, maps to the N-terminal beta-roll, a structure similar to that of the Crp cAMP binding site. The in vitro transcriptional properties of recombinant PrfA*(I45S) and PrfA*(G145S) were compared to those of PrfA(WT) at two differentially regulated PrfA-dependent promoters, PplcA and PactA. The two PrfA* mutations increased the affinity for the target DNA to a different extent, and the differences in DNA binding (PrfA*(G145S) > PrfA*(I45S) >>> PrfA(WT)) correlated with proportional differences in transcriptional activity. The use of the PrfA* proteins revealed that PplcA had a greater affinity for, and was more sensitive to, PrfA than PactA. RNA polymerase (RNAP) initiated transcription independently of PrfA at PplcA, but not at PactA, consistent with bandshift experiments suggesting that PplcA has a greater affinity for RNAP than PactA. Thus, differences in affinity for both PrfA and RNAP appear to determine the different expression pattern of PrfA-regulated promoters. Modelling of the PrfA* mutations in the crystal structure of PrfA and comparison with structure-function analyses of Crp, in which similar mutations lead to constitutively active (cAMP-independent) Crp* proteins, suggested that PrfA shares with Crp an analogous mechanism of cofactor-mediated allosteric shift. Our data support a regulatory model in which changes in PrfA-dependent gene expression are primarily accounted for by changes in PrfA activity.

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