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Journal of Bacteriology, December 2002, p . 6434-6436, Vol . 184, No . 23
Substrate Specificity of the AmpG Permease Required for Recycling of Cell Wall Anhydro-Muropeptides
Qiaomei Cheng and James T . Park*
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
Received 5 June 2002/
Accepted 6 September 2002
AmpG was originally identified as a gene required for induction of ß-lactamase . Subsequently, we found AmpG to be a permease required for recycling of murein tripeptide and uptake of anhydro-muropeptides . We have now studied the specificity of the AmpG permease . The principal requirement is for the presence of the disaccharide, N-acetylglucosaminyl-ß-1,4-anhydro-N-acetylmuramic acid (GlcNAc-anhMurNAc) . These unique substrates for AmpG, which contain murein peptides linked to GlcNAc-anhMurNAc, are produced by turnover of the cell wall during logarithmic growth . AmpG permease is sensitive to carbonylcyanide m-chlorophenylhydrazone, demonstrating that AmpG permease is a single-component permease and that transport is dependent on the proton motive force .
AmpG is a cytoplasmic membrane protein required for recycling of murein tripeptide as well as induction of Citrobacter freundii ß-lactamase (4, 6, 7) . AmpG has been presumed to be the permease for N-acetylglucosaminyl-ß-1,4-anhydro-N-acetylmuramic acid (GlcNAc-anhMurNAc)-peptides, since anhydro-N-acetylmuramyl-L-alanyl- -D-glutamyl-meso-diaminopimelic acid (anhMurNAc-tripeptide) accumulates in the cytoplasm of ampD cells but not in ampG, ampD cells (4) and since the only ß-N-acetylglucosaminidase in Escherichia coli is cytoplasmic (12, 13) . GlcNAc-anhMurNAc-peptides are the products of breakdown of the murein sacculus of E . coli by multiple lytic transglycosylases (11) . During active growth, well over half of the side wall of the sacculus is broken down each generation (1) . To determine the specificity of the AmpG permease, a number of radioactive ligands were prepared from E . coli cells labeled with D-[6-3H(N)]glucosamine (except for three ligands labeled with 3H-diaminopimelic acid [3H-Dap] as noted) . These ligands were used to compare uptake by freeze-thawed cells of E . coli ampG+ with those of ampG freeze-thawed cells .
Bacterial strain, plasmids, and growth conditions.
The E . coli K-12 strains and plasmids used in this study are listed in Table 1 . Cells were grown aerobically at 37°C in L broth, which is Luria-Bertani broth (10) modified to contain only 5 g of NaCl per liter . Ampicillin (100 µg/ml) and chloramphenicol (10 µg/ml) were used as required .
| TABLE 1 . E . coli K-12 strains and plasmids used in this work
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Preparation of freeze-thawed cells.
Cells from 40 ml of overnight culture were harvested, washed with 0.1 M phosphate buffer (pH 7.0) in the cold, and resuspended in 1.3 ml of the same buffer to give a suspension usually containing 3 to 5 mg of protein/ml . Stationary-phase cells were used because they were found to be more active than mid-log-phase cells . The cell suspension was adjusted to contain 1 mg of protein/ml . Two-hundred-microliter aliquots were rapidly frozen in a dry ice alcohol bath and were then thawed in a water bath and held at room temperature for 15 to 30 min . After this period, radioactive substrate was added and the incubation was continued for 50 min . An incubation time of 50 min at room temperature was chosen since significant uptake continued for that length of time . Thereafter, the sample was diluted with 1.4 ml of stop solution (0.1 M LiCl in 0.1 M KPO4 [pH 5.5]), filtered immediately through a 24-mm, 0.7-µm-pore-size fiberglass filter (Whatman International Ltd., Maidstone, England), and washed once with another 1.5 ml of stop solution . The dilution, filtration, and washing procedures were conducted in less than 30 s . The filter was removed immediately, dried, and counted .
HPLC analysis.
High-performance liquid chromatography (HPLC) was performed with Rainin Rabbit HP pumps and mixer equipment (Rainin Instrument Co., Woburn, Mass.) by two different methods (1) . In method 1, the column used was a LiChrosphere RP-18 column (250 by 4.6 mm, 3-µm particle size; E . Merck) . Isocratic elution with 50 mM sodium phosphate (pH 4.31) at a flow rate of 0.5 ml/min for 20 min was followed by a linear gradient of 0 to 35% 75 mM sodium phosphate (pH 4.95) in 15% methanol over 40 min and then by isocratic treatment for 60 min . The samples were desalted by method 2 . In method 2, the sample was adjusted to a pH of
 2.5 with trifluoroacetic acid, adsorbed on a 150- by 4.6-mm X-Terra RP-18 column (Waters Corp., Milford, Mass.) and was eluted at 0.5 ml/min with 0.05% trifluoroacetic acid for 35 min, followed by a gradient from 0.05% trifluoroacetic acid to 50% acetonitrile-containing 0.035% trifluoroacetic acid over a period of 15 min and then by isocratic treatment for 40 min .
Preparation of radioactive substrates.
In general, a strain of E . coli was labeled for about five generations during growth in L broth supplemented with 1 µCi of D-[6-3H(N)]glucosamine (21.6 Ci/mmol; NEN Life Science Products, Boston, Mass.)/ml . Some substrates were derived from well-washed sacculi recovered after the cells had been boiled in 4% sodium dodecyl sulfate for 30 min . Radioactive muropeptide monomers and dimers were obtained by digestion of murein sacculi with Chalaropsis muramidase . These muropeptides contained the native muramic acid present in murein . Muropeptide monomers and dimers containing anhydromuramic acid in the disaccharide (GlcNAc-anhMurNAc) were obtained by digestion of radiolabeled sacculi with a partially purified preparation of E . coli soluble lytic transglycosylase (1) . Radioactive N-acetylglucosaminyl-ß-1,4-anhydro-N-acetylmuramyl-L-alanyl--D-glutamyl-meso-diaminopimelyl-D-alanine (GlcNAc-anhMurNAc-tetrapeptide) was then isolated from the digest by HPLC . The retention time was 85 min . Radioactive GlcNAc-anhMurNAc-tripeptide was isolated from hot-water extracts of E . coli TP78B labeled as described earlier (1) . This compound accumulates in large amounts in TP78B, which lacks nagZ, the structural gene for ß-N-acetylglucosaminidase, as well as the ampD anhMurNAc-L-alanine amidase (1) . Radioactive N-acetylglucosaminyl-ß-1,4-anhydro-N-acetylmuramyl-L-alanyl--D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine (GlcNAc-anhMurNAc-pentapeptide) was obtained from late-log-phase cells of E . coli TP78B treated with 0.02 µg (MIC/3) of imipenem/ml for 80 min . The washed cells were extracted with water at 95°C for 5 min . The extract was concentrated, and GlcNAc-anhMurNAc-pentapeptide was recovered by HPLC . The retention time was 100 min . The identities of GlcNAc-anhMurNAc-tetrapeptide and GlcNAc-anhMurNAc-pentapeptide were confirmed by mass spectrometry . Radioactive anhMurNAc-tripeptide labeled with either 3H-Dap or 3H-GlcNH2 was obtained by HPLC fractionation of hot-water extracts of TP73( ampDE) labeled as described above . Radioactive anhMurNAc and free murein tripeptide (L-alanyl--D-glutamyl-meso-diaminopimelic acid) were isolated by HPLC following treatment of the anhMurNAc-tripeptide with AmpD amidase (3, 5) in 10 mM phosphate buffer (pH 7.0) as described earlier (9) . The radioactive disaccharide GlcNAc-anhMurNAc was obtained as described earlier (1) from hot-water extracts of TP77B(nagZ::Cm) .
Other methods.
The protein content of cell suspensions was determined by the Bradford Protein Assay (Bio-Rad, Hercules, Calif.) with bovine serum albumin as a standard . Transformations were performed as described elsewhere (8, 10) . Mass spectrometry was performed at the Tufts Protein Chemistry Facility utilizing a PE Biosystems Voyager Maldi Mass Spectrometer . The concentration of GlcNAc-anhMurNAc-tripeptide was determined by an amino acid analysis performed with a Waters Picotag System at the Tufts Protein Chemistry Facility .
Specificity of the AmpG permease.
The data in Fig . 1 compare the uptake by strain TP73 with the amount taken up by the isogenic ampG strain TP74 . The concentration of ligand used in these experiments was approximately 10 µM . This is well below an apparent Km of 100 µM as determined for GlcNAc-anhMurNAc-tripeptide under non-steady-state conditions . In terms of radioactivity, usually 5,000 to 20,000 cpm of ligand was added . In cells lacking AmpG, 1 to 3% of the added ligand was retained by the cells . As can be seen from examination of Fig . 1, at the low concentrations employed, AmpG permease only transports anhydro-muropeptides . The principal requirement for uptake is the presence of the disaccharide GlcNAc-anhMurNAc . Muropeptides lacking either GlcNAc or anhMurNAc are not transported . A parallel set of experiments comparing uptake by TP74/pGKS273-3(ampG+) with TP74 confirmed these results (data not shown) . TP72(ampG::kan) also converted to active uptake upon introduction of pGKS273-3(ampG+) . Although duplicate samples gave reproducible results, the uptake by individual batches of freeze-thawed cells was variable . This is reflected in the rather wide range of values shown in Fig . 1 . Uptake of GlcNAc-anhMurNAc by intact whole cells of TP78(ampDE, nagZ) (3.5%) was much greater than by the isogenic strain TP78G(ampDE, nagZ, ampG) (0.4%) . This compares favorably with the results from using freeze-thawed cells .
 | FIG . 1 . Uptake of various ligands by freeze-thawed cells of wild-type and ampG strains . *, labeled with 3H-Dap; all other ligands labeled with 3H-GlcNH2.
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Figure 1 also shows that 20 µM carbonylcyanide m-chlorophenylhydrazone (CCCP) prevented the uptake of GlcNAc-anhMurNAc and GlcNAc-anhMurNAc-peptides .
The method for measuring uptake requires some explanation . We developed this method because of difficulty demonstrating measurable uptake by membrane vesicles . In contrast, each batch of freeze-thawed cells gave significant uptake though there was significant variation from batch to batch . Freeze-thawed cells have been used in the past to make cells permeable to substrates that normally cannot cross the cytoplasmic membrane . Since we consistently observed more uptake by freeze-thawed wild-type cells than by freeze-thawed ampG cells, its evident that some freeze-thawed cells must reseal their cytoplasmic membrane . It follows also that a fraction of the population resealed their cytoplasmic membrane but not their outer membrane, since several muropeptides studied here are too large to pass through an intact outer membrane . Although the amount was quite variable, from 1 to 3% of the added substrate appeared to be taken up by the ampG cells that presumably should be impermeable to the ligands . Because E . coli possesses only a cytoplasmic ß-N-acetylglucosaminidase, we believe that this apparent uptake is the result of utilization of GlcNAc that is released from the muropeptides by that fraction of the freeze-thawed cells whose cytoplasmic membrane remained defective . Consistent with this interpretation, note that ampG cells retained a much lower percentage of 3H-Dap-labeled ligands than of 3H-GlcNH2-labeled ligands (Fig . 1) .
Since we found that the permease was sensitive to 20 µM CCCP, this suggests that AmpG permease is a single-component permease dependent on the proton motive force .
Our results indicate that, for a muropeptide to be imported by the AmpG permease, it must contain the disaccharide GlcNAc-anhMurNAc . Muropeptides lacking either GlcNAc or anhMurNAc were not imported under the conditions employed here . For example, anhMurNAc-tripeptide and GlcNAc-MurNAc-muropeptides were not taken up via AmpG . Uptake was independent of the length of the peptide side chain (Fig . 1) . Dietz et al., based on the observation that various anhydro-muropeptides accumulated in ampD cells, also concluded that peptide chain length was not critical (2) . Interestingly, our results demonstrate that the disaccharide itself was readily transported and that the N-acetylmuramic acid moiety must be present in the 1,6-anhydro form . Thus, gram-negative bacteria have evolved to produce a permease exquisitely specific for high- molecular-weight degradation products from their own cell wall . Remarkably, these products are further degraded and efficiently recycled (1, 4) .
This work was supported in part by Public Health Service grant GM51610 from the National Institute of General Medical Sciences .
We thank the Digestive Disease Center (NIDDK, P30 DK34928) for production of E . coli cells .
* Corresponding author . Mailing address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111 . Phone: (617) 636-6753 . Fax: (617) 636-0337 . E-mail: James.Park{at}tufts.edu .
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