Substrate Specificity of the AmpG Permease Required for Recycling of Cell Wall Anhydro-Muropeptides

AmpG was originally identified as a gene required for inductionof ß-lactamase. Subsequently, we found AmpG to bea permease required for recycling of murein tripeptide and uptakeof anhydro-muropeptides. We have now studied the specificityof the AmpG permease. The principal requirement is for the presenceof the disaccharide, N-acetylglucosaminyl-ß-1,4-anhydro-N-acetylmuramicacid (GlcNAc-anhMurNAc). These unique substrates for AmpG, whichcontain murein peptides linked to GlcNAc-anhMurNAc, are producedby turnover of the cell wall during logarithmic growth. AmpGpermease is sensitive to carbonylcyanide m-chlorophenylhydrazone,demonstrating that AmpG permease is a single-component permeaseand that transport is dependent on the proton motive force.

INTRODUCTION

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Introduction

AmpG is a cytoplasmic membrane protein required for recyclingof murein tripeptide as well as induction of Citrobacter freundiiß-lactamase (4, 6, 7). AmpG has been presumed to bethe permease for N-acetylglucosaminyl-ß-1,4-anhydro-N-acetylmuramicacid (GlcNAc-anhMurNAc)-peptides, since anhydro-N-acetylmuramyl-L-alanyl- ${gamma}$ -D-glutamyl-meso-diaminopimelicacid (anhMurNAc-tripeptide) accumulates in the cytoplasm ofampD cells but not in ampG, ampD cells (4) and since the onlyß-N-acetylglucosaminidase in Escherichia coli is cytoplasmic(12, 13). GlcNAc-anhMurNAc-peptides are the products of breakdownof the murein sacculus of E. coli by multiple lytic transglycosylases(11). During active growth, well over half of the side wallof the sacculus is broken down each generation (1). To determinethe specificity of the AmpG permease, a number of radioactiveligands 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 uptakeby freeze-thawed cells of E. coli ampG⁺ with those of ampG freeze-thawedcells.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strain, plasmids, and growth conditions. The E. coli K-12 strains and plasmids used in this study arelisted in Table 1. Cells were grown aerobically at 37°Cin L broth, which is Luria-Bertani broth (10) modified to containonly 5 g of NaCl per liter. Ampicillin (100 µg/ml) andchloramphenicol (10 µg/ml) were used as required.

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TABLE 1. E. coli K-12 strains and plasmids used in this work

Preparation of freeze-thawed cells. Cells from 40 ml of overnight culture were harvested, washedwith 0.1 M phosphate buffer (pH 7.0) in the cold, and resuspendedin 1.3 ml of the same buffer to give a suspension usually containing3 to 5 mg of protein/ml. Stationary-phase cells were used becausethey 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 dryice alcohol bath and were then thawed in a water bath and heldat room temperature for 15 to 30 min. After this period, radioactivesubstrate was added and the incubation was continued for 50min. An incubation time of 50 min at room temperature was chosensince 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 througha 24-mm, 0.7-µm-pore-size fiberglass filter (Whatman InternationalLtd., Maidstone, England), and washed once with another 1.5ml of stop solution. The dilution, filtration, and washing procedureswere conducted in less than 30 s. The filter was removed immediately,dried, and counted.

HPLC analysis. High-performance liquid chromatography (HPLC) was performedwith Rainin Rabbit HP pumps and mixer equipment (Rainin InstrumentCo., Woburn, Mass.) by two different methods (1). In method1, the column used was a LiChrosphere RP-18 column (250 by 4.6mm, 3-µm particle size; E. Merck). Isocratic elution with50 mM sodium phosphate (pH 4.31) at a flow rate of 0.5 ml/minfor 20 min was followed by a linear gradient of 0 to 35% 75mM sodium phosphate (pH 4.95) in 15% methanol over 40 min andthen by isocratic treatment for 60 min. The samples were desaltedby 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-mmX-Terra RP-18 column (Waters Corp., Milford, Mass.) and waseluted at 0.5 ml/min with 0.05% trifluoroacetic acid for 35min, followed by a gradient from 0.05% trifluoroacetic acidto 50% acetonitrile-containing 0.035% trifluoroacetic acid overa 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 generationsduring 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 recoveredafter the cells had been boiled in 4% sodium dodecyl sulfatefor 30 min. Radioactive muropeptide monomers and dimers wereobtained by digestion of murein sacculi with Chalaropsis muramidase.These muropeptides contained the native muramic acid presentin murein. Muropeptide monomers and dimers containing anhydromuramicacid in the disaccharide (GlcNAc-anhMurNAc) were obtained bydigestion of radiolabeled sacculi with a partially purifiedpreparation of E. coli soluble lytic transglycosylase (1). RadioactiveN-acetylglucosaminyl-ß-1,4-anhydro-N-acetylmuramyl-L-alanyl- ${gamma}$ -D-glutamyl-meso-diaminopimelyl-D-alanine(GlcNAc-anhMurNAc-tetrapeptide) was then isolated from the digestby HPLC. The retention time was 85 min. Radioactive GlcNAc-anhMurNAc-tripeptidewas isolated from hot-water extracts of E. coli TP78B labeledas described earlier (1). This compound accumulates in largeamounts in TP78B, which lacks nagZ, the structural gene forß-N-acetylglucosaminidase, as well as the ampD anhMurNAc-L-alanineamidase (1). Radioactive N-acetylglucosaminyl-ß-1,4-anhydro-N-acetylmuramyl-L-alanyl- ${gamma}$ -D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine(GlcNAc-anhMurNAc-pentapeptide) was obtained from late-log-phasecells of E. coli TP78B treated with 0.02 µg (MIC/3) ofimipenem/ml for 80 min. The washed cells were extracted withwater at 95°C for 5 min. The extract was concentrated, andGlcNAc-anhMurNAc-pentapeptide was recovered by HPLC. The retentiontime was 100 min. The identities of GlcNAc-anhMurNAc-tetrapeptideand GlcNAc-anhMurNAc-pentapeptide were confirmed by mass spectrometry.Radioactive anhMurNAc-tripeptide labeled with either ³H-Dapor ³H-GlcNH₂ was obtained by HPLC fractionation of hot-waterextracts of TP73( ${Delta}$ ampDE) labeled as described above. RadioactiveanhMurNAc and free murein tripeptide (L-alanyl- ${gamma}$ -D-glutamyl-meso-diaminopimelicacid) were isolated by HPLC following treatment of the anhMurNAc-tripeptidewith AmpD amidase (3, 5) in 10 mM phosphate buffer (pH 7.0)as described earlier (9). The radioactive disaccharide GlcNAc-anhMurNAcwas obtained as described earlier (1) from hot-water extractsof TP77B(nagZ::Cm).

Other methods. The protein content of cell suspensions was determined by theBradford Protein Assay (Bio-Rad, Hercules, Calif.) with bovineserum albumin as a standard. Transformations were performedas described elsewhere (8, 10). Mass spectrometry was performedat the Tufts Protein Chemistry Facility utilizing a PE BiosystemsVoyager Maldi Mass Spectrometer. The concentration of GlcNAc-anhMurNAc-tripeptidewas determined by an amino acid analysis performed with a WatersPicotag System at the Tufts Protein Chemistry Facility.

RESULTS

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Results

Specificity of the AmpG permease. The data in Fig. 1 compare the uptake by strain TP73 with theamount taken up by the isogenic ampG strain TP74. The concentrationof ligand used in these experiments was approximately 10 µM.This is well below an apparent K_m of 100 µM as determinedfor GlcNAc-anhMurNAc-tripeptide under non-steady-state conditions.In terms of radioactivity, usually 5,000 to 20,000 cpm of ligandwas added. In cells lacking AmpG, 1 to 3% of the added ligandwas retained by the cells. As can be seen from examination ofFig. 1, at the low concentrations employed, AmpG permease onlytransports anhydro-muropeptides. The principal requirement foruptake 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 uptakeby individual batches of freeze-thawed cells was variable. Thisis reflected in the rather wide range of values shown in Fig.1. Uptake of GlcNAc-anhMurNAc by intact whole cells of TP78( ${Delta}$ ampDE,nagZ) (3.5%) was much greater than by the isogenic strain TP78G( ${Delta}$ ampDE,nagZ, ampG) (0.4%). This compares favorably with the resultsfrom using freeze-thawed cells.

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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.

Figure 1 also shows that 20 µM carbonylcyanide m-chlorophenylhydrazone(CCCP) prevented the uptake of GlcNAc-anhMurNAc and GlcNAc-anhMurNAc-peptides.

DISCUSSION

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Discussion

The method for measuring uptake requires some explanation. Wedeveloped this method because of difficulty demonstrating measurableuptake by membrane vesicles. In contrast, each batch of freeze-thawedcells gave significant uptake though there was significant variationfrom batch to batch. Freeze-thawed cells have been used in thepast to make cells permeable to substrates that normally cannotcross the cytoplasmic membrane. Since we consistently observedmore uptake by freeze-thawed wild-type cells than by freeze-thawedampG cells, its evident that some freeze-thawed cells must resealtheir cytoplasmic membrane. It follows also that a fractionof the population resealed their cytoplasmic membrane but nottheir outer membrane, since several muropeptides studied hereare too large to pass through an intact outer membrane. Althoughthe amount was quite variable, from 1 to 3% of the added substrateappeared to be taken up by the ampG cells that presumably shouldbe impermeable to the ligands. Because E. coli possesses onlya cytoplasmic ß-N-acetylglucosaminidase, we believethat this apparent uptake is the result of utilization of GlcNActhat is released from the muropeptides by that fraction of thefreeze-thawed cells whose cytoplasmic membrane remained defective.Consistent with this interpretation, note that ampG cells retaineda much lower percentage of ³H-Dap-labeled ligands than of ³H-GlcNH₂-labeledligands (Fig. 1).

Since we found that the permease was sensitive to 20 µMCCCP, this suggests that AmpG permease is a single-componentpermease dependent on the proton motive force.

Our results indicate that, for a muropeptide to be importedby the AmpG permease, it must contain the disaccharide GlcNAc-anhMurNAc.Muropeptides lacking either GlcNAc or anhMurNAc were not importedunder the conditions employed here. For example, anhMurNAc-tripeptideand GlcNAc-MurNAc-muropeptides were not taken up via AmpG. Uptakewas independent of the length of the peptide side chain (Fig.1). Dietz et al., based on the observation that various anhydro-muropeptidesaccumulated in ampD cells, also concluded that peptide chainlength was not critical (2). Interestingly, our results demonstratethat the disaccharide itself was readily transported and thatthe N-acetylmuramic acid moiety must be present in the 1,6-anhydroform. Thus, gram-negative bacteria have evolved to produce apermease exquisitely specific for high- molecular-weight degradationproducts from their own cell wall. Remarkably, these productsare further degraded and efficiently recycled (1, 4).

ACKNOWLEDGMENTS

This work was supported in part by Public Health Service grantGM51610 from the National Institute of General Medical Sciences.

We thank the Digestive Disease Center (NIDDK, P30 DK34928) forproduction of E. coli cells.

FOOTNOTES

* 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.

REFERENCES

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