Identification and Disruption of the proBA Locus in Listeria monocytogenes : Role of Proline Biosynthesis in Salt Tolerance and Murine Infection

Abstract

Intracellular accumulation of the amino acid proline has previously been linked 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 glutamyl kinase (GK) and glutamylphosphate reductase (GPR) enzyme complex which catalyzes the first and second steps of proline biosynthesis in E. coli. The first gene of the operon, proB, is predicted to encode GK, a 276-residue protein with a calculated molecular mass of 30.03 kDa and pl of 5.2. Distal to the promoter and overlapping the 3 end of proB by 17 bp is proA, which 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 deletion 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 virulence in mouse assays (either perorally or intraperitoneally), growth at low (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.

Genome Diversification in Phylogenetic Lineages I and II ofListeria monocytogenes: Identification of Segments Unique to Lineage II Populations

“…The proline biosynthetic pathway encoded by the proAB genes catalyzes de novo synthesis of proline from intracellular pools of glutamate via gammaglutamyl kinase (proA) and gammaglutamyl phosphate reductase (proB). In the serotype 1/2c laboratory strain LO28, this pathway participates in osmotic protection in the absence of preferred osmoprotectants betaine and carnitine (53). Although both the microarray and southern blot data indicate that the proAB genes are absent in all lineage I strains that we tested, four different restriction fragment length polymorphism patterns were observed among the lineage II strains and three lineage II strains were missing the genes.…”

Identification ofLactobacillus plantarumGenes That Are Induced in the Gastrointestinal Tract of Mice

“…Moreover, in vivo approaches have demonstrated that genes involved in arginine, biotin, and proline metabolism in Vibrio cholerae (7), E. coli (26) and Helicobacter pylori (25), respectively, are induced during mouse infection. In one of the closest relatives of L. plantarum, Listeria monocytogenes, proline metabolism is induced under high-osmolarity conditions (47). Such conditions could potentially be found in the colon and suggest differential colonic expression of lp0017 (Table 2).…”

Mixed-Genome Microarrays Reveal Multiple Serotype and Lineage-Specific Differences among Strains of Listeria monocytogenes

“…Several of our microarray probe sequences have close BLASTx matches for proteins that might confer differential fitness in different food processing or natural environ- ments. These include a protein related to the repair of UV damage to the chromosome (probe 961), two proteins implicated in salt tolerance (probes 774 and 1085) (30), one gene related to exopolysaccharide synthesis (biofilm formation; probe 1094), and one heavy-metal transporter (probe 289). Additionally, probe 955 is most similar to beta-glucoside-specific enzyme II (bvrB) of the L. monocytogenes phosphotransferase system.…”