Identification and disruption of the proBA locus in Listeria monocytogenes: Role of proline biosynthesis in salt tolerance and murine infection

Intracellular accumulation of the amino acid proline has previously been li nked to the salt tolerance and virulence potential of a number of bacteria. Taking advantage of the proBA mutant Escherichia coli CSH26, we identified a listerial proBA operon coding for enzymes functionally similar to the gl utamyl kinase (GK) and glutamylphosphate reductase (GPR) enzyme complex whi ch catalyzes the first and second steps of proline biosynthesis in E, call, The first gene of the operon, proB, is predicted to encode GK, a 276-resid ue protein with a calculated molecular mass of 30.03 kDa and pi of 5.2, Dis tal to the promoter and overlapping the 3' end of proB by 17 bp is proA, wh ich encodes GPR, a 415-residue protein with a calculated molecular mass of 45.50 kDa (pl 5.3), Using this information, we created a chromosomal deleti on mutant by allelic exchange which is auxotrophic for proline, This mutant was used to assess the contribution of proline anabolism to osmotolerance and virulence. While inactivation of proBA had no significant effect on vir ulence in mouse assays (either perorally or intraperitoneally), growth at l ow (2 to 4% NaCl) and high (>6% NaCl) salt concentrations in complex media was significantly reduced in the absence of efficient proline synthesis. We conclude that while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of Listeria monocytogenes, it can play an important role in survival in osmolyte-depleted environments of elevated osmolarity.