A GLY145SER SUBSTITUTION IN THE TRANSCRIPTIONAL ACTIVATOR PRFA CAUSESCONSTITUTIVE OVEREXPRESSION OF VIRULENCE FACTORS IN LISTERIA-MONOCYTOGENES

Virulence genes in Listeria monocytogenes are coordinately expressed u nder the control of the transcriptional activator PrfA, encoded by prf A, a member of the cyclic AMP (cAMP) receptor protein (CRP)/FNR family of bacterial regulators. Strain P14-A is a spontaneous mutant of L. m onocytogenes serovar 4b which produces elevated levels of virulence fa ctors (M. T. Ripio, G. Dominguez-Bernal, M. Suarez, K. Brehm, P. Berch e, 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 s ubstitution, trans-complementation experiments with PrfA-deficient mut ants 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 chrom osomal Gly145Ser prfA background, transcription of prfA and of PrfA-de pendent virulence genes remained constitutively high under culture con ditions in which virulence factor expression is downregulated in wild- type L. monocytogenes, The Gly145Ser 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 mutation s, which make CRP functionally active in the absence of cAMP, map in t his region (i.e., Gly141Ser and Ala144Thr substitutions). By analogy w ith the CRP model, the phenotype conferred to L. monocytogenes by the Gly145Ser substitution in PrfA could be due to the mutant regulatory p rotein being locked in a transcriptionally active, cofactor-independen t conformational state. Our observations allow the construction of a m odel 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 inactiv e forms according to its interaction with an environmentally regulated signal molecule or cofactor.