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Applied and Environmental Microbiology, June 2001, p. 2571-2577, Vol. 67, No. 6
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.6.2571-2577.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Roy D. Sleator, Cormac G. M. Gahan, and Colin Hill*

Department of Microbiology and National Food Biotechnology Centre, University College Cork, Cork, Ireland

Received 6 November 2000/Accepted 7 March 2001

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.

* Corresponding author. Mailing address: Department of Microbiology, University College Cork, Cork, Ireland. Phone: 353-21-4902397. Fax: 353-21-4903101. E-mail: c.hill{at}ucc.ie.

Applied and Environmental Microbiology, June 2001, p. 2571-2577, Vol. 67, No. 6
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.6.2571-2577.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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