Three transporters mediate uptake of glycine betaine and carnitine by Listeria monocytogenes in response to hyperosmotic stress

Abstract

The uptake and accumulation of the potent osmolytes glycine betaine and carnitine enable the food-borne pathogen Listeria monocytogenes to proliferate in environments of elevated osmotic stress, often rendering salt-based food preservation inadequate. To date, three osmolyte transport systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and a carnitine transporter OpuC. We investigated the specificity of each transporter towards each osmolyte by creating mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state osmolyte accumulation data together with growth rate experiments demonstrated that osmotically activated glycine betaine transport is readily and effectively mediated by Gbu and BetL and to a lesser extent by OpuC. Osmotically stimulated carnitine transport was demonstrated for OpuC and Gbu regardless of the nature of stressing salt. BetL can mediate weak carnitine uptake in response to NaCl stress but not KCl stress. No other transporter in L. monocytogenes 10403S appears to be involved in osmotically stimulated transport of either osmolyte, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown under elevated osmotic stress.

FIG. 1.

DNA sizes after PCR amplification of chromosomal DNA extracted from wild-type L. monocytogenes 10403S; the double-mutant derivatives ASA4 (ΔbetS Δgbu), ASA5 (ΔbetL ΔopuC), and ASA6 (Δgbu ΔopuC); and the triple mutant ASA7 (ΔbetL Δgbu ΔopuC) with the gene specific SOE-F and SOE-R primers. Lanes 1, PCR with betL primers; lanes 2, PCR with opuC primers; lanes 3, PCR with gbu primers. DNA ladder positions (in base pairs) from bottom to top: 200, 400, 600, 800, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 6,000, 8,000, and 10,000.

FIG. 2.

Osmolyte transport activity of L. monocytogenes 10403S and mutant derivatives ASA4, ASA5, ASA6, and ASA7. Uptake of 100 μM [¹⁴C]glycine betaine (A, C, E, G, and I) or carnitine (B, D, F, H, and J) was measured in strain 10403S (A and B), ASA5 (C and D), ASA4 (E and F), ASA6 (G and H), and ASA7 (I and J) grown to late log phase in modified Pine's medium at 30°C with 0.7 M NaCl (triangles), 0.72 M KCl (squares), or no added salt (circles). Transport was assayed as described in Materials and Methods. Error bars indicate the range of duplicate values. Note that the scales on the time axes vary.

FIG. 2.

Osmolyte transport activity of L. monocytogenes 10403S and mutant derivatives ASA4, ASA5, ASA6, and ASA7. Uptake of 100 μM [¹⁴C]glycine betaine (A, C, E, G, and I) or carnitine (B, D, F, H, and J) was measured in strain 10403S (A and B), ASA5 (C and D), ASA4 (E and F), ASA6 (G and H), and ASA7 (I and J) grown to late log phase in modified Pine's medium at 30°C with 0.7 M NaCl (triangles), 0.72 M KCl (squares), or no added salt (circles). Transport was assayed as described in Materials and Methods. Error bars indicate the range of duplicate values. Note that the scales on the time axes vary.

FIG. 2.

Osmolyte transport activity of L. monocytogenes 10403S and mutant derivatives ASA4, ASA5, ASA6, and ASA7. Uptake of 100 μM [¹⁴C]glycine betaine (A, C, E, G, and I) or carnitine (B, D, F, H, and J) was measured in strain 10403S (A and B), ASA5 (C and D), ASA4 (E and F), ASA6 (G and H), and ASA7 (I and J) grown to late log phase in modified Pine's medium at 30°C with 0.7 M NaCl (triangles), 0.72 M KCl (squares), or no added salt (circles). Transport was assayed as described in Materials and Methods. Error bars indicate the range of duplicate values. Note that the scales on the time axes vary.

FIG. 2.

Osmolyte transport activity of L. monocytogenes 10403S and mutant derivatives ASA4, ASA5, ASA6, and ASA7. Uptake of 100 μM [¹⁴C]glycine betaine (A, C, E, G, and I) or carnitine (B, D, F, H, and J) was measured in strain 10403S (A and B), ASA5 (C and D), ASA4 (E and F), ASA6 (G and H), and ASA7 (I and J) grown to late log phase in modified Pine's medium at 30°C with 0.7 M NaCl (triangles), 0.72 M KCl (squares), or no added salt (circles). Transport was assayed as described in Materials and Methods. Error bars indicate the range of duplicate values. Note that the scales on the time axes vary.

FIG. 3.

Compatible solute accumulation by L. monocytogenes 10403S and mutant L. monocytogenes ASA5, ASA4, ASA6, and ASA7 during balanced growth in modified Pine's medium with 0.5 mM glycine betaine and carnitine and under 0.7 M NaCl stress. Exponentially growing cultures were harvested and washed, and cytoplasmic contents were extracted with perchloric acid. Alanine (50 mM) was added to each extract as an internal standard, and compatible solutes were quantitated by using natural-abundance ¹³C-NMR spectroscopy.

FIG. 4.

Growth characteristics of L. monocytogenes 10403S (wild type), ASA5 (containing only Gbu), ASA4 (containing only OpuC), ASA6 (containing only BetL), and ASA7 (triple deletion). Cultures were grown in BHI medium, washed, and used to inoculate (1%) modified Pine's medium. These cultures were grown to late log phase, 10-fold diluted, and used to inoculate (1%) modified Pine's medium containing 0.7 M NaCl (A) or 0.72 M KCl (B), in the presence of 1 mM glycine betaine (GB), 1 mM carnitine (CAR), or a 0.5 mM concentration of each osmolyte (GB+CAR) or in the absence of osmolytes (−). Error bars indicate ±1 standard deviation of triplicate values.

FIG. 4.

Growth characteristics of L. monocytogenes 10403S (wild type), ASA5 (containing only Gbu), ASA4 (containing only OpuC), ASA6 (containing only BetL), and ASA7 (triple deletion). Cultures were grown in BHI medium, washed, and used to inoculate (1%) modified Pine's medium. These cultures were grown to late log phase, 10-fold diluted, and used to inoculate (1%) modified Pine's medium containing 0.7 M NaCl (A) or 0.72 M KCl (B), in the presence of 1 mM glycine betaine (GB), 1 mM carnitine (CAR), or a 0.5 mM concentration of each osmolyte (GB+CAR) or in the absence of osmolytes (−). Error bars indicate ±1 standard deviation of triplicate values.