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Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA

Journal of Bacteriology publish this investigation article

December 1st, 1998

Most Listeria monocytogenes virulence genes are positively regulated by the PrfA protein, a transcription factor sharing sequence similarities with cyclic AMP (cAMP) receptor protein (CRP). Its coding gene, prfA, is regulated by PrfA itself via an autoregulatory loop mediated by the upstream PrfA-dependent plcA promoter. We have recently characterized prfA* mutants from L. monocytogenes which, as a result of a single amino acid substitution in PrfA, Gly145Ser, constitutively overexpress prfA and the genes of the PrfA virulence regulon. Here, we show that about 10 times more PrfA protein is produced in a prfA* strain than in the wild type. Thus, the phenotype of prfA* mutants is presumably due to the synthesis of a PrfA protein with higher promoter-activating activity (PrfA*), which keeps its intracellular levels constantly elevated by positive feedback. We investigated the interaction of PrfA and PrfA* (Gly145Ser) with target DNA. Gel retardation assays performed with a DNA fragment carrying the PrfA binding site of the plcA promoter demonstrated that the PrfA* mutant form is much more efficient than wild-type PrfA at forming specific DNA-protein complexes. In footprinting experiments, the two purified PrfA forms interacted with the same nucleotides at the target site, although the minimum amount required for protection was 6 to 7 times lower with PrfA*. These results show that the primary functional consequence of the Gly145Ser mutation is an increase in the affinity of PrfA for its target sequence. Interestingly, similar mutations at the equivalent position in CRP result in a transcriptionally active, CRP* mutant form which binds with high affinity to target DNA in the absence of the activating cofactor, cAMP. Our observations suggest that the structural similarities between PrfA and CRP are also functionally relevant and support a model in which the PrfA protein, like CRP, shifts from transcriptionally inactive to active conformations by interaction with a cofactor




Vega Y., Dickneite C., Ripio MT., Bockmann R., Gonzalez-Zorn B., Novella S., Dominguez-Bernal G., Goebel W. and Vazquez-Boland JA.




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Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA

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Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA



Participants:

Universidad ComplutenseDepartamento de Sanidad Animal. Facultad de Veterinaria. Universidad Complutense (UCM).

Universidad ComplutenseServicio de Zoonosis de Transmisión Alimentaria y Resistencia a Antimicrobianos (ZTA). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM).

Universidad ComplutenseGrupo de Patogénesis Molecular Bacteriana. Facultad de Veterinaria. Universidad Complutense (UCM).

Lehrstuhl für Mikrobiologie, Biozentrum. Universität Würzburg.







Journal of Bacteriology
FACTOR YEAR Q
3.805 1998

NLMID: 2985120R

PMID: 9852011

ISSN: 0021-9193



TITLE: Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA


JOURNAL: J Bacteriol


NUMERACIÓN: 180(24):6655-60


AÑO: 1998


PUBLISHER: American Society for Microbiology


AUTHORS: Vega Y., Dickneite C., Ripio MT., Bockmann R., Gonzalez-Zorn B., Novella S., Dominguez-Bernal G., Goebel W. and Vazquez-Boland JA.


Bruno González Zorn

DOI: https://doi.org/10.1128/JB.180.24.6655-6660.1998


CITE THIS PUBLICATION:

Vega Y., Dickneite C., Ripio MT., Bockmann R., Gonzalez-Zorn B., Novella S., Dominguez-Bernal G., Goebel W. and Vazquez-Boland JA. Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA. Journal of Bacteriology. 180(24):6655-60. 1998. (A). ISSN: 0021-9193. DOI: 10.1128/JB.180.24.6655-6660.1998


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