title: A naturally occurring mutation K220T in the pleiotropic activator PrfA of Listeria monocytogenes results in a loss of virulence due to decreasing DNA-binding affinity.
abstract: The sequencing of prfA, encoding the transcriptional regulator of virulence genes, in 26 low-virulence field Listeria monocytogenes strains showed that eight strains exhibited the same single amino-acid substitution: PrfAK220T. These strains exhibited no expression of PrfA-regulated proteins and thus no virulence. This substitution inactivated PrfA, since expression of the PrfAK220T mutant gene in an EGDDeltaprfA strain did not restore the haemolytic and phosphatidylcholine phospholipase C activities, in contrast to the wild-type prfA gene. The substitution of the lysine at position 220 occurred in the helix alphaH. However, the data showed that the PrfAK220T protein is dimerized just as well as its wild-type counterpart, but does not bind to PrfA-boxes. PrfAK220T did not form a PrfA-DNA complex in electrophoretic mobility shift assays, but low concentrations of CI complexes (PrfAK220T-RNA polymerase-DNA complex) were formed by adding RNA polymerase, suggesting that PrfA interacted with RNA polymerase in solution in the absence of DNA. Formation of some transcriptionally active complexes was confirmed by in vitro runoff transcription assays and quantitative RT-PCR. Crystallographic analyses described the structure of native PrfA and highlighted the key role of allosteric changes in the activity of PrfA and especially the role of the Lys220 in the conformation of the helix-turn-helix (HTH) motif.
authors: Institut National de la Recherche Agronomique, UR1282 Infectiologie animale et santé publique, 37380 Nouzilly, France.
authors: P
authors: P
authors: Velge
authors: Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften der Universität Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors: M
authors: M
authors: Herler
authors: Institut Pasteur, Unité des Interactions Bactéries-Cellules, 28 rue du Docteur Roux, 75015 Paris, France.
authors: J
authors: J
authors: Johansson
authors: Institut National de la Recherche Agronomique, UR1282 Infectiologie animale et santé publique, 37380 Nouzilly, France.
authors: S M
authors: SM
authors: Roche
authors: Institut National de la Recherche Agronomique, UR1282 Infectiologie animale et santé publique, 37380 Nouzilly, France.
authors: S
authors: S
authors: Témoin
authors: Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors: A A
authors: AA
authors: Fedorov
authors: Institut National de la Recherche Agronomique, UR1282 Infectiologie animale et santé publique, 37380 Nouzilly, France.
authors: P
authors: P
authors: Gracieux
authors: Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors: S C
authors: SC
authors: Almo
authors: Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften der Universität Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors: W
authors: W
authors: Goebel
authors: Institut Pasteur, Unité des Interactions Bactéries-Cellules, 28 rue du Docteur Roux, 75015 Paris, France.
authors: P
authors: P
authors: Cossart
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000378:metabolism*; D019281:Dimerization; D019077:Helix-Turn-Helix Motifs; D006459:Hemolysin Factors / Q000235:genetics; D006801:Humans; D008089:Listeria monocytogenes / Q000737:chemistry / Q000378:metabolism / Q000472:pathogenicity*; D008958:Models, Molecular; D009154:Mutation; D010454:Peptide Termination Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014774:Virulence
chemical_list: D004269:DNA, Bacterial; D010454:Peptide Termination Factors; D015534:Trans-Activators