Mutations in the listerial proB gene leading to proline overproduction: Effects on salt tolerance and murine infection

The observed sensitivity of Listeria monocytogenes to the toxic proline ana logue L-azetidine-2-carboxylic acid (AZ) suggested that proline synthesis i n Listeria may be regulated by feedback inhibition of gamma -glutamyl kinas e (GK), the first enzyme of the proline biosynthesis pathway, encoded by th e proB gene. Taking advantage of the Epicurian coli mutator strain XL1-Red, we performed random mutagenesis of the recently described proBA operon and generated three independent mutations in the listerial proB homologue, lea ding to proline overproduction and salt tolerance when expressed in an E. c oli (Delta proBA) background. While each of the mutations (located within a conserved 26-amino-acid region of GK) was shown to confer AZ resistance (A Z(r)) on an L. monocytogenes proBA mutant, listerial transformants failed t o exhibit the salt-tolerant phenotype observed in E. coli. Since proline ac cumulation has previously been linked to the virulence potential of a numbe r of pathogenic bacteria, we analyzed the effect of proline overproduction on Listeria pathogenesis. However, our results suggest that as previously d escribed for proline auxotrophy, proline hyperproduction has no apparent im pact on the virulence potential of Listeria.