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J. Bacteriol., 03 1997, 1533-1540, Vol 179, No. 5
Copyright © 1997, American Society for Microbiology

A Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes

MT Ripio, G Dominguez-Bernal, M Lara, M Suarez and JA Vazquez-Boland
Unidad de Microbiologia e Immunologia, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.

Virulence genes in Listeria monocytogenes are coordinately expressed under the control of the transcriptional activator PrfA, encoded by prfA, a member of the cyclic AMP (cAMP) receptor protein (CRP)/FNR family of bacterial regulators. Strain P14-A is a spontaneous mutant of L. monocytogenes serovar 4b which produces elevated levels of virulence factors (M. T. Ripio, G. Dominguez-Bernal, M. Suarez, K. Brehm, P. Berche, and J. A. Vazquez-Boland, Res. Microbiol. 147:371-384, 1996). Here we report that P14-A and other variant strains with the same phenotype carry a point mutation in codon 145 of prfA, leading to a Gly- ->Ser substitution. trans-complementation experiments with PrfA- deficient mutants demonstrated that the Gly145Ser prfA allele causes overexpression of virulence factors in L. monocytogenes, to the levels found in the virulence factor-overexpressing variants. In strain P14-A with a chromosomal Glyl45Ser prfA background, transcription of prfA and of PrfA-dependent virulence genes remained constitutively high under culture conditions in which virulence factor expression is downregulated in wild-type L. monocytogenes. The Glyl45Ser substitution is located in a PrfA stretch (residues 141 to 151) showing high sequence similarity to the D alpha-helix of CRP. Interestingly, well- characterized crp* mutations, which make CRP functionally active in the absence of cAMP, map in this region (i.e., Gly141Ser and Ala144Thr substitutions). By analogy with the CRP model, the phenotype conferred to L. monocytogenes by the Gly145Ser substitution in PrfA could be due to the mutant regulatory protein being locked in a transcriptionally active, cofactor-independent conformational state. Our observations allow the construction of a model for PrfA-dependent virulence gene regulation in which the levels of virulence factor expression depend primarily on the conformational state of the PrfA protein, which alternates between active and inactive forms according to its interaction with an environmentally regulated signal molecule or cofactor.


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