title: New Listeria monocytogenes prfA* mutants, transcriptional properties of PrfA* proteins and structure-function of the virulence regulator PrfA.
abstract: 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.
authors.affiliation: Grupo de Patogénesis Molecular Bacteriana, Unidad de Microbiología e Inmunología, Facultad de Veterinaria, Universidad Complutense de Madrid and Universidad de León, Spain.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Vega
authors.forename: Markus
authors.initials: M
authors.lastname: Rauch
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Banfield
authors.forename: Svetlana
authors.initials: S
authors.lastname: Ermolaeva
authors.forename: Mariela
authors.initials: M
authors.lastname: Scortti
authors.forename: Werner
authors.initials: W
authors.lastname: Goebel
authors.forename: José A
authors.initials: JA
authors.lastname: Vázquez-Boland
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008089:Listeria monocytogenes / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D008565:Membrane Proteins / Q000235:genetics; D008958:Models, Molecular; D020125:Mutation, Missense; D010454:Peptide Termination Factors; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics; D018085:Regulon; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics; D014158:Transcription, Genetic; D014774:Virulence / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010454:Peptide Termination Factors; D011956:Receptors, Cell Surface; D015534:Trans-Activators; D014157:Transcription Factors; C481662:crp protein, E coli; C072980:actA protein, Listeria monocytogenes; D012321:DNA-Directed RNA Polymerases