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New Listeria monocytogenes prfA * mutants, transcriptional properties of PrfA* proteins and structure–function of the virulence regulator PrfA
Author(s) -
Vega Yolanda,
Rauch Markus,
Banfield Mark J.,
Ermolaeva Svetlana,
Scortti Mariela,
Goebel Werner,
VázquezBoland José A.
Publication year - 2004
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2004.04052.x
Subject(s) - biology , promoter , allosteric regulation , regulon , transcription (linguistics) , virulence , rna polymerase , mutant , microbiology and biotechnology , biochemistry , gene , rna , gene expression , enzyme , linguistics , philosophy
Summary 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 αD 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 β‐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, P plcA and P actA . 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 P plcA had a greater affinity for, and was more sensitive to, PrfA than P actA . RNA polymerase (RNAP) initiated transcription independently of PrfA at P plcA , but not at P actA , consistent with bandshift experiments suggesting that P plcA has a greater affinity for RNAP than P actA . 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.