Identification of an ATP-driven, osmoregulated glycine betaine transport system in Listeria monocytogenes

Abstract

The ability of the gram-positive, food-borne pathogen Listeria monocytogenes to tolerate environments of elevated osmolarity and reduced temperature is due in part to the transport and accumulation of the osmolyte glycine betaine. Previously we showed that glycine betaine transport was the result of Na(+)-glycine betaine symport. In this report, we identify a second glycine betaine transporter from L. monocytogenes which is osmotically activated but does not require a high concentration of Na(+) for activity. By using a pool of Tn917-LTV3 mutants, a salt- and chill-sensitive mutant which was also found to be impaired in its ability to transport glycine betaine was isolated. DNA sequence analysis of the region flanking the site of transposon insertion revealed three open reading frames homologous to opuA from Bacillus subtilis and proU from Escherichia coli, both of which encode glycine betaine transport systems that belong to the superfamily of ATP-dependent transporters. The three open reading frames are closely spaced, suggesting that they are arranged in an operon. Moreover, a region upstream from the first reading frame was found to be homologous to the promoter regions of both opuA and proU. One unusual feature not shared with these other two systems is that the start codons for two of the open reading frames in L. monocytogenes appear to be TTG. That glycine betaine uptake is nearly eliminated in the mutant strain when it is assayed in the absence of Na(+) is an indication that only the ATP-dependent transporter and the Na(+)-glycine betaine symporter occur in L. monocytogenes.

FIG. 1

Growth characteristics of L. monocytogenes LTG59. Cultures of 10403S (■), DP-L1044 (●), and the glycine betaine transport mutant LTG59 (□) were grown in BHI and inoculated into modified Pine’s medium (1% inoculum). These cultures were grown to late log phase and used to inoculate (1%) modified Pine’s medium containing 100 μM glycine betaine. Cultures were grown at 30°C with 8% NaCl (A) or at 7°C without added NaCl (B). The ranges of duplicate values are indicated by error bars.

FIG. 2

Glycine betaine transport activity of L. monocytogenes LTG59. Uptake of 100 μM [¹⁴C]glycine betaine was measured in 10403S (■), DP-L1044 (●), and the mutant LTG59 (□) grown to late log phase in modified Pine’s medium at 30°C with 8% NaCl (A) or at 7°C without added NaCl (B). Transport was assayed as described in Materials and Methods. The ranges of duplicate values are indicated by error bars.

FIG. 3

Effect of Na⁺ and K⁺ deficiency on the uptake of glycine betaine in L. monocytogenes strains. Cultures of 10403S (○, ●) and LTG59 (□, ■) were grown and assayed for [¹⁴C]glycine betaine uptake in either 4% KCl (open symbols) or 4% NaCl (closed symbols) as described in Materials and Methods. The ranges of duplicate values are indicated by error bars.

FIG. 4

Physical map of the cloned L. monocytogenes gbu region. The locations of the three open reading frames (gbuA, gbuB, and gbuC) and the direction of transcription are indicated by horizontal arrows. The start codon of gbuB overlaps with the stop codon of gbuA by 8 bp, and the intergenic distance between gbuB and gbuC is 13 bp. The sequence of the putative promoter is shown with the −35 and −10 regions underlined. The position of the Tn917-LTV3 insertion is indicated by the vertical arrow. Also shown are restriction sites for HindIII (H), BamHI (B), EcoRI (E), and XbaI (X).

FIG. 5

Alignment of the deduced amino acid sequences encoded by gbu from L. monocytogenes with the homologous systems from B. subtilis and E. coli. Identical amino acids are shaded in black, and conservative substitutions are in gray. (A) The sequence of GbuA is compared with those of OpuAA from B. subtilis and ProV from E. coli. The Walker motifs A and B, two highly conserved sequences found in ATP-dependent transporters, are underlined. Another highly conserved region, Loop 3, is indicated by a double underline. (B) Comparison of GbuB with the corresponding OpuAB protein from B. subtilis and ProW protein of E. coli. (C) Comparison of GbuC with the glycine betaine binding protein OpuAC from B. subtilis and ProX protein from E. coli. The position of the predicted cleavage site of the signal peptide is indicated by the vertical line (between residues 20 and 21 of GbuC).

FIG. 5

Alignment of the deduced amino acid sequences encoded by gbu from L. monocytogenes with the homologous systems from B. subtilis and E. coli. Identical amino acids are shaded in black, and conservative substitutions are in gray. (A) The sequence of GbuA is compared with those of OpuAA from B. subtilis and ProV from E. coli. The Walker motifs A and B, two highly conserved sequences found in ATP-dependent transporters, are underlined. Another highly conserved region, Loop 3, is indicated by a double underline. (B) Comparison of GbuB with the corresponding OpuAB protein from B. subtilis and ProW protein of E. coli. (C) Comparison of GbuC with the glycine betaine binding protein OpuAC from B. subtilis and ProX protein from E. coli. The position of the predicted cleavage site of the signal peptide is indicated by the vertical line (between residues 20 and 21 of GbuC).

FIG. 6

Hydropathy profiles of GbuA, GbuB, and GbuC. The hydrophobicity of the protein is represented as a hydropathy index, computed by using the method of Kyte and Doolittle (24). A window of 20 amino acids was used.

FIG. 7

Restoration of the glycine betaine-dependent osmotic tolerance phenotype in E. coli WG439 transformants. For glycine betaine transport experiments (A), cultures grown aerobically at 37°C in M63 medium supplemented with ampicillin were used to inoculate M63 containing 0.4 M NaCl and ampicillin. After several hours of growth, 100 μM [¹⁴C]glycine betaine was added to initiate transport. For growth rate experiments (B), cultures were grown in M63 containing 0.7 M NaCl and 1 mM glycine betaine and ampicillin. The strains used were WG439 (■), WG439(pUC18) (○), and WG439(pGBU18) (□). The ranges of duplicate values are indicated by error bars.