Identification and characterization of an ATP binding cassette L-carnitine transporter in Listeria monocytogenes

Abstract

We identified an operon in Listeria monocytogenes EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from Bacillus subtilis, which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated opuC, consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated proteins presumed to form the permease (OpuCB and OpuCD). The operon is preceded by a potential SigB-dependent promoter. An opuC-defective mutant was generated by the insertional inactivation of the opuCA gene. The mutant was impaired for growth at high osmolarity in brain heart infusion broth and failed to grow in a defined medium. Supplementation of the defined medium with peptone restored the growth of the mutant in this medium. The mutant was found to accumulate the compatible solutes glycine betaine and choline to same extent as the parent strain but was defective in the uptake of L-carnitine. We conclude that the opuC operon in L. monocytogenes encodes an ABC compatible solute transporter which is capable of transporting L-carnitine and which plays an important role in osmoregulation in this pathogen.

FIG. 1

Organization of the opuC operon in L. monocytogenes EGD depicted schematically. ORFs are shown as solid arrows. The numbers in parentheses above the ORFs indicate predicted sizes (in amino acid residues) of the protein products. A potential transcriptional terminator is indicated (TT). The potential SigB-dependent promoter is indicated by an angled arrow. The predicted amino acid sequence for each ORF was used to perform BLASTP searches (1) against the nonredundant databases (performed at http://www.ncbi.nlm.nih.gov/BLAST/). The eight proteins with the highest levels of sequence similarity (lowest E value [Expect value]) are listed beneath the corresponding ORF, followed in parentheses by the organism abbreviation and the percent identity. The organism abbreviations are as follows: Bs, B. subtilis; Mt, Mycobacterium tuberculosis; Ec, E. coli; Sp, Streptococcus pneumoniae; Dr, Deinococcus radiodurans; Af, Archaeoglobus fulgidus; Hp, Helicobacter pylori; Sc, Streptomyces coelicolor. Xxx, no assigned name.

FIG. 2

Features of the OpuC protein subunits. (A) The first 200 amino acid residues of OpuCA from L. monocytogenes (Lm) were aligned by consensus with the corresponding N-terminal regions of OpuCa and OpuBA from B. subtilis (Bs), and identical residues are indicated by shading. Alignments were performed with a Clustal algorithm (14) using MegAlign software (DNASTAR Inc). The conserved “linker peptide” is overlined, and both Walker motifs (35) are overlined with hatched boxes. (B) The first 40 residues of the substrate binding protein (OpuCC) from L. monocytogenes (Lm) were aligned with the corresponding regions of OpuCC and OpuBC from B. subtilis (Bs). The predicted processing site for the pro-OpuCC lipoprotein is indicated with an arrowhead, and the conserved N-terminal cysteine residue is marked with an asterisk. (C) Kyte-Doolittle (23) hydrophobicity profiles for each of the four protein subunits of OpuC. The amino acid residue number is indicated above the plots. The profiles were obtained with a window of nine residues.

FIG. 3

Growth of EGD (open symbols) and EGD opuCA::pAULA (solid symbols). (a) Cultures were grown in BHI in either the presence (squares) or the absence (circles) of 4% (wt/vol; 0.684 M) added NaCl. (b) Cultures were grown in DM without supplementation (triangles), with 0.5% (wt/vol) peptone (circles), or with 0.1% (wt/vol) Casamino Acids (squares).

FIG. 4

Compatible solute uptake in EGD (circles) or in the opuCA::pAULA mutant derivative (squares). Cultures were grown in DMP prior to the assay. The assay was performed with potassium phosphate buffer with (solid symbols) or without (open symbols) 0.5 M added NaCl. The assay was performed in the presence of the protein synthesis inhibitor chloramphenicol (50 μg ml⁻¹) and with 0.4% (wt/vol) added glucose. l-[³H]carnitine hydrochloride (a) or l-[¹⁴C]betaine (b) was added to a final concentration of 20 μM.

FIG. 5

Compatible solute pools measured at steady state. Intracellular pools of carnitine (a), betaine (b), and choline (c) were measured as described in Materials and Methods. Cells of EGD (WT) or the opuCA::pAULA mutant (opuC) were grown in DMP either with (+) or without (−) 0.5 M NaCl or 0.5 M KCl. The error bars represent the standard deviation from the mean (n = 3).

FIG. 6

Alignment of the putative opuC SigB promoter with known SigB promoters from L. monocytogenes and B. subtilis. The sequence identified upstream from opuCA in L. monocytogenes is shown aligned with other known or predicted SigB promoter sequences. Genes indicated with an asterisk have a potential SigB promoter which has not yet been confirmed experimentally. Position is given as the distance of the final base shown in the −10 box from the initiation codon. The alignment of promoters was adapted and updated from the literature (3, 34).