A naturally occuring mutation K220T in the pleiotropic activator PrfA of Listeria monocytogenes results in a loss of virulence due to decreasing DNA-binding affinity

Philippe Velge, M. Herler, J. Johansson, S. M. Roche, S. Témoin, A. A. Fedorov, P. Gracieux, Steven C. Almo, W. Goebel, P. Cossart

Research output: Contribution to journalArticle

22 Citations (Scopus)

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 EGDΔprfA 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 αH. 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 Cl 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.

Original languageEnglish (US)
Pages (from-to)995-1005
Number of pages11
JournalMicrobiology
Volume153
Issue number4
DOIs
StatePublished - Apr 2007

Fingerprint

Listeria monocytogenes
DNA-Directed RNA Polymerases
Virulence
Mutation
DNA
Helix-Turn-Helix Motifs
Type C Phospholipases
Electrophoretic Mobility Shift Assay
Amino Acid Substitution
Regulator Genes
Phosphatidylcholines
Genes
Lysine
Proteins
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Microbiology

Cite this

A naturally occuring mutation K220T in the pleiotropic activator PrfA of Listeria monocytogenes results in a loss of virulence due to decreasing DNA-binding affinity. / Velge, Philippe; Herler, M.; Johansson, J.; Roche, S. M.; Témoin, S.; Fedorov, A. A.; Gracieux, P.; Almo, Steven C.; Goebel, W.; Cossart, P.

In: Microbiology, Vol. 153, No. 4, 04.2007, p. 995-1005.

Research output: Contribution to journalArticle

Velge, Philippe ; Herler, M. ; Johansson, J. ; Roche, S. M. ; Témoin, S. ; Fedorov, A. A. ; Gracieux, P. ; Almo, Steven C. ; Goebel, W. ; Cossart, P. / A naturally occuring mutation K220T in the pleiotropic activator PrfA of Listeria monocytogenes results in a loss of virulence due to decreasing DNA-binding affinity. In: Microbiology. 2007 ; Vol. 153, No. 4. pp. 995-1005.
@article{c8a2683b296445c59d07aec1b5e9b74f,
title = "A naturally occuring 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 EGDΔprfA 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 αH. 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 Cl 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.",
author = "Philippe Velge and M. Herler and J. Johansson and Roche, {S. M.} and S. T{\'e}moin and Fedorov, {A. A.} and P. Gracieux and Almo, {Steven C.} and W. Goebel and P. Cossart",
year = "2007",
month = "4",
doi = "10.1099/mic.0.2006/002238-0",
language = "English (US)",
volume = "153",
pages = "995--1005",
journal = "Microbiology (United Kingdom)",
issn = "1350-0872",
publisher = "Society for General Microbiology",
number = "4",

}

TY - JOUR

T1 - A naturally occuring mutation K220T in the pleiotropic activator PrfA of Listeria monocytogenes results in a loss of virulence due to decreasing DNA-binding affinity

AU - Velge, Philippe

AU - Herler, M.

AU - Johansson, J.

AU - Roche, S. M.

AU - Témoin, S.

AU - Fedorov, A. A.

AU - Gracieux, P.

AU - Almo, Steven C.

AU - Goebel, W.

AU - Cossart, P.

PY - 2007/4

Y1 - 2007/4

N2 - 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 EGDΔprfA 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 αH. 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 Cl 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.

AB - 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 EGDΔprfA 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 αH. 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 Cl 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.

UR - http://www.scopus.com/inward/record.url?scp=34247278297&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34247278297&partnerID=8YFLogxK

U2 - 10.1099/mic.0.2006/002238-0

DO - 10.1099/mic.0.2006/002238-0

M3 - Article

VL - 153

SP - 995

EP - 1005

JO - Microbiology (United Kingdom)

JF - Microbiology (United Kingdom)

SN - 1350-0872

IS - 4

ER -