monocytogenesinvasion of host cells, intracellular growth, and cell-to-cell spread, and it is absolutely essential for bacterial virulence (Freitag, 2006;Scorttiet al., 2007). Based on sequence and structural homology, PrfA has been identified as a member of the Crp/Fnr family of transcriptional activators (Eitinget al., 2005;Korneret al., 2003;Ripioet al., 1997). to form dimers in solution appeared to inversely correlate with levels of PrfA-dependent gene expression. Based on comparisons of protein activity and structural similarities with UC-1728 PrfA family members Crp and CooA, theprfA*mutations modify distinct aspects of PrfA activity that include DNA binding and protein-protein interactions. == INTRODUCTION == The transcriptional regulator PrfA (positive regulatory factor A) is responsible for regulating the gene expression of nearly all known virulence factors ofListeria monocytogenes(Chakrabortyet al., 1992;Grayet al., 2006;Leimeister-Wachteret al., 1990;Mineret al., 2008;Pizarro-Cerda & Cossart, 2006;Scorttiet al., 2007). PrfA is a 27 kD protein that recognizes and binds a 14 base pair DNA palindrome present in the promoters of its target genes (Freitaget al., 1992;Mengaudet al., 1989). PrfA regulates the expression of gene products required forL. monocytogenesinvasion of host cells, intracellular growth, and cell-to-cell spread, and it is absolutely essential for bacterial virulence (Freitag, 2006;Scorttiet al., 2007). Based on sequence and structural homology, PrfA has been identified as a member of the Crp/Fnr family of transcriptional activators (Eitinget al., 2005;Korneret al., 2003;Ripioet al., 1997). Proteins within this family generally become activated following the binding of small molecule cofactors. Crp, for example, undergoes an allosteric change after binding cAMP and becomes a site-specific DNA binding protein that recognizes target promoters and interacts with RNA polymerase (RNAP) (Busby & Ebright, 1999;Kimet al., 1992;Kolbet al., 1993;Lawsonet al., 2004). Crp appears to exist in an equilibrium between an active form that efficiently binds DNA target sequences and an inactive form that does not. Co-factor cAMP binding by Crp shifts the equilibrium toward the active form, either by stabilizing this form or by destabilizing the inactive form of the protein (Younet al., 2007). PrfA may exist in an analogous equilibrium state such that binding of a co-factor is required to shift PrfA to a high activity form capable of high affinity DNA binding. Although it is generally believed that a PrfA co-factor exists, this co-factor has MMP7 not yet been identified. Mutations incrphave been identified that result in an active form of Crp in the absence of cAMP cofactor (Garges & Adhya, 1985;Harmanet al., 1986;Kimet al., 1992;Younet al., 2006;Younet al., 2007). Structural and functional studies of these mutants (known as Crp* mutants) have led to the identification of regions of Crp that are important for activity, and it has been observed that Crp* mutants exhibit a conformation that resembles that of wild type Crp bound to cofactor (Harmanet al., UC-1728 1986). Similar tocrp*, severalprfAmutations have been identified that appear to result in activation of PrfA in the absence of cofactor (known asprfA* mutants) (Mineret al., 2008;Mueller & Freitag, 2005;Ripioet al., 1997;Shetron-Ramaet al., 2003;Vegaet UC-1728 al., 2004;Wong & Freitag, 2004). Strains withprfA* mutations express high levels of PrfA-dependent gene products under conditions in which gene expression is usually repressed. TheprfA* mutations identified thus far are not functionally equivalent, and significant differences in bacterial virulence have been reported forL. monocytogenesstrains containing differentprfA* alleles (Mineret al., 2008;Mueller & Freitag, 2005;Scorttiet al., 2007;Shetron-Ramaet al., 2003). This study describes a biochemical comparison of wild-type PrfA with five different PrfA* mutants (including a novelprfA* mutation) to elucidate the effects of specific amino acid substitutions on distinct aspects of PrfA function. == METHODS == == Bacterial strains and plasmids == The strains and plasmids used in this study are listed inTable 1.L. monocytogenesstrains were grown in brain heart infusion (BHI) medium andE. colistrains were grown in Luria Broth (LB) at 37 C with shaking. Strains containing high activityprfA* alleles such asprfAL140F andprfAG145S have previously been proven difficult to construct using standard allelic exchange techniques [(Port & Freitag, 2007;Wong & Freitag, 2004) and unpublished observations]. However, a modified approach for generating isogenic mutants UC-1728 was developed and used successfully as follows:prfAL140F andprfAG145S were introduced intoL. monocytogenesNF-L1124 containing a transcriptional fusion ofgusandneo(Karow & Piggot, 1995) downstream ofactAin the bacterial chromosome. Selection for the mutant strains was then facilitated based on an increased level of neomycin resistance in the presence of theprfA* allele as conferred by the PrfA-dependent promoteractA. In addition, to prevent expression of the introducedprfA*mutations from the plasmid vector used for allelic exchange,prfAcoding sequences missing the ATG start codon were amplified by PCR and inserted into UC-1728 the temperature sensitive plasmid shuttle vector pKSV7 (generating plasmid pNF1147) and the L140F and G145S mutations were then separately introduced via QuikChange Site-Directed Mutagenesis.