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Biochemistry. Author manuscript; available in PMC 2011 May 23.
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PMCID: PMC3100163

Purification and Functional Characterization of the ϕX174 Lysis Protein E


Two classes of bacteriophages, the single-stranded DNA Microviridae, and the single-stranded RNA Alloleviviridae, accomplish lysis by expressing “protein antibiotics”, or polypeptides that inhibit cell wall biosynthesis. Previously, we have provided genetic and physiological evidence that E, a 91 aa membrane protein encoded by the prototype microvirus, ϕX174, is a specific inhibitor of the translocase MraY, an essential membrane-embedded enzyme that catalyzes the formation of the murein precursor, Lipid I, from UDP-N-acetylmuramic acid-pentapeptide and the lipid carrier, undecaprenol phosphate. Here we report the first purification of E, which has been refractory to over-expression because of its lethality to E. coli. Moreover, using a fluorescently-labeled analog of the sugar-nucleotide substrate, we demonstrate that E acts as a non-competitive inhibitor of detergent-solubilized MraY, with respect to both soluble and lipid substrates. In addition, we show that the E-sensitivity of five MraY mutant proteins, produced from alleles selected for resistance to E, can be correlated to the apparent affinities determined by in vivo multicopy suppression experiments. These results are inconsistent with previous reports that E inhibited membrane-embedded MraY but not the detergent-solubilized enzyme, which led to a model in which E functions by binding MraY and blocking the formation of an essential hetero-multimeric complex involving MraY and other murein biosynthesis enzymes. We discuss a new model in which E binds to MraY at a site composed of the two transmembrane domains within which the E-resistance mutations map and that the result of this binding is a conformational change that inactivates the enzyme.

Small, single-strand nucleic acid bacteriophages effect host lysis by expression of a single gene and without the elaboration of a muralytic enzyme, in contrast to the larger, double-stranded DNA phages, which invariably employ a holin and an endolysin for lysis (1). The best-studied example is the gene E of the classic coliphage and prototype of the ubiquitous Microviridae, ϕX174. E, entirely embedded in the +1 reading frame of the unrelated essential gene D, encodes a membrane protein of 91 residues (Fig. 1) (24). Previously, we have shown that protein E causes lysis by inhibiting MraY, the enzyme which catalyzes the synthesis of the murein precursor, Lipid I, and thus blocking cell wall synthesis (5, 6). E has been shown to function in a number of Gram-negative hosts (7, 8). Gene fusion experiments have shown that only its N-terminal 35 amino acids, including the sole predicted transmembrane domain (TMD1), are required for lytic activity (5, 9, 10), which fits well with the multi-spanning integral membrane protein character of MraY (Fig. 1).

Figure 1
Features of E and MraY

Because there are no useful antibiotics that target MraY, the mechanism by which E functions as an inhibitor has been of interest. In vivo, MraY catalyzes the formation of Lipid I by transferring phosphate-N-acetylmuramic acid-pentapeptide (in E. coli, phosphate-N-acetylmuramic acid-L-Ala-γ-D-Glu-meso-diaminopimelic acid (DAP)-D-Ala-D-Ala) (P-MurNAc-pentapeptide) from UDP-MurNAc-pentapeptide to undecaprenol-phosphate (undecaprenol-P). A two-step reaction pathway involving a covalent MraY-P-MurNAc-pentapeptide has been proposed (11) (Fig. 2).

Figure 2
Proposed reaction mechanism of MraY

The mechanism of E-mediated inhibition of MraY function was initially addressed by genetics and physiological experiments. In vivo, lysis by the wt E protein, but not E fusions where the C-terminal domain is replaced by a heterologous domain (9), requires SlyD, a cytoplasmic FKBP (FK506 binding protein)-type peptidyl-prolyl cis-trans isomerase (12). However, this requirement has been shown to be purely a matter of the stability of E and is unrelated to E function (13). Lysis is completely restored in a slyD knockout background by mutations, designated as Epos (plates on slyD), that increase the translation rate of the E mRNA and thus return the E protein to normal levels, despite its continued instability (13). Thus SlyD has no role in inhibition of MraY, despite the strict slyD-dependent lysis phenotype of ϕX174.

The original studies identifying MraY as the target of E were based on the isolation of dominant mutants in mraY resistant to the expression of a cloned E gene (5). To date, five missense mutants of mraY providing resistance to E-mediated lysis, and impaired in plaque-formation by ϕX174, have been isolated. These mutations map to TMDs 5 and 9 of the proposed topological map of MraY (Fig. 1) (14). Experiments in which inductions of plasmid-borne mraY genes have been used to protect the host from E lysis have indicated that E binds to MraY in vivo and that the five E-resistant mraY alleles can be separated into three classes based on apparent affinities for E (14). Moreover, E was found to be incapable of causing lysis when the active form of B. subtilis MraY (BsMraY) was produced in E. coli, indicating that the great disparity between the sequences of BsMraY and EcMraY prevents E binding.

In vitro studies of E inhibition has been hampered by the fact that E is lethal to E. coli. Experiments with quantifiable EΦlacZ gene fusions indicated that lysis is brought about when only a few hundred chimeric molecules were present, suggesting that the in vivo levels of MraY must also be very low (9). Purification of E has not been reported. However, Mendel et al. (15) prepared a synthetic polypeptide, Epep, corresponding to the N-terminal 37 residues of the predicted E sequence. Consistent with the earlier finding that only the N-terminal portion of E is required for lysis, inhibition was observed when SDS-solubilized Epep was added to membranes containing over-expressed MraY. Unexpectedly, it was found that Epep was not able to inhibit the MraY activity in detergent-solubilized membrane extracts. This led to a model in which E effects lysis by preventing the assembly of MraY into an essential hetero-multimeric integral membrane complex with high MraY activity, a complex which would not be formed in detergent. Here we report the first purification of full-length E protein and characterize its ability to inhibit MraY; the results are discussed in terms of a different model for E action.


Media, chemicals, strains and culture methods

Growth and induction conditions for bacterial cultures have been described, including the use of Luria-Bertani (LB) broth, supplemented as appropriate with these antibiotics: ampicillin (100 μg/ml), chloramphenicol (10μg/ml), and kanamycin (40 μg/ml) or the inducers isopropyl-β-D-thiogalactopyranoside (IPTG) and arabinose (16). The hosts for over-expression of E and mraY were, respectively, BL21(DE3) and BL21(DE3)plysS (Novagen). Tunicamycin (mixture of isomers A, B, C, and D), phytol and phospray, a reagent for detection of phospholipid, were purchased from Sigma. The sources of detergents were as follows: Tween 20, SDS, and Empigen BB (EBB) from Sigma; cholic acid, saponin, and Triton X-100 from EMD; Nonidet P40 from Bethesda Research Laboratories; 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) from Avanti Polar Lipids; n-dodecyl-β-D-maltoside (DDM) from Anatrace.


Plasmids were constructed using standard PCR, digestion and ligation methods, as previously described; all final constructs were verified by sequencing at the Laboratory for Plant Genome Technology at the Texas Agricultural Experiment Station. The plasmids pETE6his and pETMY contain, respectively, the ϕX174 E gene extended with 6 histidine codons (13) or the wild type E. coli mraY gene (5), inserted between the NdeI and BamHI sites of pET11a vector (Novagen). The plasmid pBsMraYKan contains BsmraY under the control of the PBAD promoter and was derived from pBAD30-BsMraY (14) by deleting the bla gene and inserting a kanamycin-resistance cassette from pZS*24-MCS-1 (17) into the unique ClaI site.

Over-production and purification of E

To determine optimum conditions for over-production of E6his, BL21(DE3) cells harboring either the pETE6his plasmid alone or both the pBsMraYKan and pETE6his plasmids were induced at A550 ~ 0.6 with either 1mM IPTG or 0.2% arabinose and 1mM IPTG (added 2 minutes after arabinose), respectively. At various times, the cells from 1 ml aliquots were chilled, collected by centrifugation in the cold, resuspended in SDS-PAGE buffer in volumes chosen to normalize for constant cell mass, and analyzed by SDS-PAGE and immunoblotting, as described previously (16).

A total of 5 L of culture of BL21(DE3) cells harboring the pBSMraYKan and pETE6his plasmids was induced at A550 = 0.6 with 0.2% arabinose and 1mM IPTG (added 2 minutes after arabinose) for 30 min and then harvested by centrifugation (rotor JA-10, Beckman) at 8K rpm (7000 × g) for 15 min in the cold. The cells were resuspended in 1/200 volume of cold French press buffer (50 mM Tris pH 8.0, 170 mM KCl, 5 mM EDTA, 1 mM PMSF, 1 mM DTT) and disrupted by French press. Whole cells and debris were removed by centrifugation at 5000 × g in a JA-20 rotor (Beckman), and membranes were collected from the supernatant by centrifugation at 130,000 × g for 1 hour in the cold, using a Type 50.2 Ti rotor (Beckman). Membrane pellets were resuspended in buffer containing 50 mM Tris pH8.0, 170 mM KCl, 10 mM MgCl2 (Buffer R) plus various detergents, as indicated, and shaken gently overnight at 4°C. Insoluble material was removed by centrifugation at 130,000xg for 1 hour in the cold, using a Type 50.2 Ti rotor (Beckman).

The E6his polypeptide, extracted in 2% EBB, was purified by immobilized metal affinity chromatography (IMAC) with 250 μl Talon Metal Affinity resins (Clontech) in a Poly-Prep column (Bio-Rad) using gravity elution. The elution buffer was the Buffer R supplemented with 100 mM imidazole and 0.06% EBB. Fractions containing E6his protein were identified by western blot, as described (16). A mock E extract was prepared in the same way using a strain carrying the pET11a vector. The concentration of purified E6his was determined by two independent methods: A280, using a molar absorption coefficient of E calculated as defined by Pace et al. (18); or by densitometry using Coomassie-blue stained SDS-PAGE gels, with egg white lysozyme (Sigma) as a standard. The two methods gave the same results.

Quantification of E in vivo

As described previously (16) MDS12 lacIQ tonA : : Tn10 carrying plasmids pQ and indicating pRW derivatives was grown in LB-kanamycin-ampicillin at 37 °C, induced with IPTG and arabinose at A550 = 0.5, and aerated for 25 min before 20 ml samples were harvested by centrifugation in the cold. The cells were resuspended in 2 ml of cold French press buffer and lysed by French press. Whole cells and debris were removed by centrifugation at 5000 × g in a JA-20 rotor (Beckman). Membranes were collected from 1.5 ml of the supernatant by centrifugation at 100,000 × g for 1 hour in the cold, using a TLA-100.3 rotor (Beckman). Membranes were resuspended in 100μl sample loading buffer and processed for for immunoblotting was as described previously (16).

Substrates for the in vitro reaction

UDP-MurNAc-pentapeptide was isolated from B. subtilis W23 as described (6). The dansylated UDP-MurNAc-pentapeptide, UDP-MurNAc-L-γD-Glu-m-DAP(Nε-dansyl)-D-Ala-D-Ala (UDP-MurNAc-pentapeptide-DNS), was prepared by the reaction of UDP-MurNAc-pentapeptide with dansyl chloride as described (19). Phytol -phosphate (phytol-P), a C-20 analog of undecaprenol-P was used as the lipid substrate and was prepared by the chemical phosphorylation of phytol following the method by Danilov et al. (20). The phospholipid product is detected by thin layer chromatography (TLC) using chloroform: methanol: water (60:25:4) as the mobile phase, and confirmed as a single species using both I2 and phospray staining (Silica Gel TLC plates, Whatman). The concentration of phytol-P was determined by measuring free phosphate after hydrolysis with 4N HCl at 90°C, as described (21). Using this lipid substrate, tunicamycin, a competitive inhibitor of the sugar-nucleotide substrate for this class of enzymes (2224), exhibited efficient competitive inhibition of UDP-MurNAc-pentapeptide-DNS (data not shown).

In vitro reaction catalyzed by MraY

MraY activity was measured using a 10 μl reaction mixture containing Buffer R, 2.5 μL of MraY extract or membranes, and the substrates or substrates plus E protein or mock E extract (see below), as indicated. Phytol-P was provided in Buffer R plus 4% DDM; the final concentration of DDM in the total reaction was 1.25%. The reaction was started by addition of the UDP-MurNAc-pentapeptide-DNS substrate. The reaction mixture was incubated at 37°C for various times. For determination of initial rates, reactions were terminated at 5 min, which was determined to be within the linear phase of the reaction. After termination by boiling for 2 min, the reaction mixture was spotted on a TLC plate (Silica Gel, EMD Chemicals). The plate was developed for 6 to 7 hours using isobutyric acid: NH4OH: water (66:1:33) as the mobile phase. Quantification of the product was done by scraping the spot, extracting with methanol, and measuring the fluorescent intensity. Fluorescence measurements were done in a Koala spectrofluorometer (ISS) (excitation = 340nm, emission = 535nm), with a volume of 100 μl in a 130-μl fluorescence micro cell (Hellma).

Preparation of an MraY-enriched membrane fraction

A total of 10 L of culture of BL21(DE3)plysS cells harboring the pETMY plasmid were induced at A550 = 0.6 with 1 mM IPTG for one hour and then harvested by centrifugation (rotor JA-10, Beckman) at 8K rpm (7000 × g) for 15 min in the cold. The cells were resuspended in 1/100 volume of cold French press buffer and disrupted by French press. The lysate was cleared of whole cells by centrifugation in a JA-20 rotor (Beckman) at 5000 × g for 10 min in the cold. Membranes were collected by centrifugation at 38K rpm (130,000 × g) for 1 hour in the cold, using a Type 50.2 Ti rotor (Beckman). To extract MraY activity, membrane pellets were resuspended in Buffer R plus 1% DDM and stirred for 1 hour in the cold. Insoluble material was removed by centrifugation at 50K (100,000 × g) for 1 hour in a TLA-100.3 rotor (Beckman). The supernatant from this clearing step was used without further treatment for MraY reactions.


Over-production and purification of E

Purification of E protein is especially challenging since it is lethal to E. coli, and many other Gram-negative bacteria, and thus not readily overproduced. Induction of an E allele encoding a C-terminal oligohistidine tag caused lysis within 15 minutes (Fig. 3), making recovery of membrane material from the large culture volume difficult and allowing only a small amount of E to accumulate in the membranes (Fig. 4A, lane 4). To overcome this obstacle, we took advantage of the insensitivity of BsMraY to E inhibition. In the presence of the heterologous enzyme, lysis did not occur, making recovery of the membrane material much more efficient (Fig. 3), and E6his protein accumulated to a much higher level (Fig. 4B). E6his could be efficiently extracted from membranes of these induced cells with the zwitterionic detergent EBB or with SDS, but not with other commonly-used detergents (Table 1). The EBB-solubilized material was purified by IMAC, yielding a preparation 54 μM E6his protein (Fig. 4C) that was 84% pure. This corresponds to a yield of 27μg per liter of induced culture. A mock purification using the empty vector yielded the same background species (compare lanes 3 -8 with 9 - 15 in Fig. 4C) and was used as the negative control in all experiments with E.

Figure 3
BsMraY prevents lysis from over-expression of E6his
Figure 4
Over-production and purification of E6his
Table 1
Extraction of E6his from E. coli membranesa

Quantification of E in vivo

The lysis function of E depends on it having at least a stoichiometric relationship with MraY. Previous attempts at quantifying E were based on indirect in vivo approaches, one involving relative incorporation of label into E versus virion proteins (25) and the other based on the β-galactosidase activity of E-βgal chimeras (9), yielding estimates of 100–300 molecules and ~1000 molecules per cell, respectively. Here, using the purified E6his as a standard for quantitative immunoblotting, a direct quantification of the amount of E in membranes could be attempted. Although the large amounts of lysate needed tended to distort visualization of the E species in SDS-PAGE (Fig. 5), nevertheless reproducible results were obtained that allowed an estimate of ~ 500 molecules of E at the time of lysis (Table 2). This confirms the more indirect estimates made previously and puts an upper limit on the number of MraY molecules that can be present in vivo. At 500 molecules/cell, each functional MraY molecule would have to have kcat ~200 min−1 to account for the level of murein synthesis in E. coli(26). This is in reasonable agreement with kcat ~320 min−1 measured by Al-Dabbagh et al. (27) for purified E. coli MraY.

Figure 5
Quantification of E in vivo
Table 2
E amount at lysis in vivob

MraY preparation and fluorescence assay

Initially we constructed H6mraY an allele of mraY encoding an N-terminal oligohistidine-tagged variant, to facilitate enrichment of MraY activity by IMAC, as reported by Bouhss et al. (28). However, this allele failed a stringent complementation test with a chromosomal ΔmraY (data not shown) (14). This result was not surprising, since the N terminus of MraY is predicted to be periplasmic, and an appended oligopeptide tag could interfere with proper folding in the membrane. Since our goal was biochemical characterization of E-mediated inhibition, which might have stringent requirements for MraY folding, we decided to use the wt mraY allele for over-production and enrichment.

To measure MraY activity, we developed a fluorescence-based transfer assay for measuring MraY enzymatic activity in both crude membrane preparations and detergent-solubilized forms. The substrates used are UDP-MurNAc-pentapeptide-DNS, a fluorescent substrate analogue, and phytol-P, a twenty-carbon analogue of undecaprenol-P (Fig. 2). The fluorescently-labeled product, phytol-P-P-MurNAc-pentapeptide-DNS, is separated from the reaction mixture by TLC (Fig. 6) and quantified by fluorescence spectroscopy. Using this assay, Michaelis-Menten kinetics was observed for both substrates. Using Ping Pong Bi Bi formalism (29, 30), the Km for UDP-MurNAc-pentapeptide-DNS was found to be 0.2 ± .09 mM, consistent with the estimated in vivo concentration of this substrate (31), and the apparent Km for phytol-P was 0.84 ± 0.2 mM (Fig. 7). We also determined the Km parameters for the MraYF288L mutant protein, which has been shown to be the most extreme mutant, in terms of E-resistance, among the five (14), and obtained the same values (Fig. 7). This indicates that E-resistance is not due to an altered substrate affinity.

Figure 6
Fluorescence-based assay for MraY
Figure 7
Determination of Km values

E-mediated inhibition of MraY

Next, the purified detergent-solubilized E6his protein was examined for its ability to inhibit MraY in vitro. In contrast to the findings of Mendel et al. (15) with a synthetic peptide corresponding to the first 37 residues of E, we found the purified E6his protein inhibited solubilized MraY efficiently (Fig. 8). We also observed inhibition by E6his with membranes containing MraY, consistent with both the findings of Mendel et al. (15) and our original demonstration that E inhibits MraY specifically when both are present in the same membranes (6). For a given concentration of E, the extent of inhibition is lower than for the solubilized enzyme, which is not surprising considering that, in the assays with particulate MraY, the E protein must somehow enter the membrane from its detergent-solubilized state. Next, the mode by which E inhibited the solubilized MraY was determined with respect to both the lipid and sugar-nucleotide substrates (Fig. 9) by measuring MraY activity in the presence of varying concentrations of E6his. Experiments to obtain the kinetic data were performed with crude MraY sample containing membrane detergent extracts. Kinetic analysis revealed that the Km parameters for both UDP-MurNAc-pentapeptide-DNS and phytol-P were unchanged in the presence of E6his, whereas in both cases Vmax was decreased. Thus, E is a non-competitive inhibitor of MraY with respect to both lipid and sugar-nucleotide substrates, with an average Ki = 0.53 ± 0.12μM (Fig. 9).

Figure 8
Inhibition of both particulate and detergent-solubilized MraY by E6his
Figure 9
E6his is a non-competitive inhibitor of MraY with respect to both soluble and lipid substrates

Sensitivity of MraY mutant alleles

We then investigated the ability of E to inhibit the MraY proteins from the five mutant alleles isolated by selecting for resistance to the lysis protein. The five mutants fall into 3 classes according to the degree of inhibition at [E6his] = 2.7 μM: MraYG186S and MraYV291 are almost as sensitive to E as the wt protein; MraYF288L is, like BsMraY, not detectably inhibited; and MraYP170L and MraYΔL172 are inhibited to a degree intermediate between the first two classes (Fig. 10). These results match the classes of apparent affinities determined in vivo by comparing the ability of multicopy plasmids carrying these alleles to protect the wt protein from E inhibition (14).

Figure 10
Inhibition of MraY mutants by E6his


In the more than 40 years since E was defined as the lysis gene of the paradigm Microvirus ϕX174 (32), many hypotheses have been advanced for the molecular basis of its ability to effect lysis in the absence of detectable muralytic activity (3237). Recently, genetic and physiological studies from our laboratory provided evidence that E acts as a specific inhibitor of MraY, the first membrane-embedded enzyme in the pathway of murein precursor biosynthesis (5, 6). Specifically, mutations that conferred resistance to E-mediated lysis mapped to two TMDs of MraY, and MraY activity, but not the activity of an unrelated member of the translocase super-family, was inhibited in membranes containing E (6). In vitro studies needed to extend these studies have been stymied by the inability to obtain purified E protein.

Here we report over-production of E achieved by providing an E-insensitive heterologous MraY protein in trans. This is the first purification of any of the single-gene phage lysis proteins and opens the way for the structural and biophysical characterization of E. Moreover, the availability of E protein makes it possible now to study the E-SlyD interaction with purified components. PPIases are ubiquitous, found in all cells and in all major cellular compartments, but their biological roles, at least in the absence of drugs, have been elusive. Although SlyD is irrelevant to E function, its interaction with E remains the most genetically tractable PPIase-protein substrate phenotype known in biology. SlyD is easily purified and in fact has been inadvertently purified by many laboratories, since it possesses a histidine-rich C-terminal tail and thus contaminates IMAC purifications of soluble (but not membrane) proteins (3840).

In the work reported here, the purified E protein was used to investigate its mode of inhibition of MraY. Using solubilized extracts of membranes enriched in MraY, we have demonstrated that E protein acts as a non-competitive inhibitor with respect to both the lipid and sugar-nucleotide substrates of MraY. Our results are significantly different from those of Mendel et al. (15), who reported that Epep, a synthetic peptide corresponding to the first 37 residues of E, inhibited membrane-embedded MraY but not detergent-solubilized MraY. The discrepancy may arise from differences in the assays used. Mendel et al. used SDS-solubilized Epep for their inhibition studies, resulting in SDS concentrations at or above its CMC (critical micelle concentration) in the final reaction mixes. Although the authors report that the detergent alone at this concentration had no effect on MraY activity, it is possible that it affects E-MraY interactions. In our hands, 1% SDS completely destroys MraY activity (data not shown). Although the authors reported CD measurements indicating alpha-helical character, it is also possible that the synthetic 37 residue polypeptide used was not correctly folded, perhaps due to the lack of a C-terminal domain. The C-terminal hydrophilic domain of E can be replaced by some, but not all, heterologous protein folding domains (9, 10), and there have been no reports that simple C-terminal truncations of E are lytic in vivo. Most importantly, here we have shown that the E-sensitivity of solubilized MraY proteins in vitro correlates with the allelic state of mraY and with our previous assessments of apparent E-MraY binding in vivo (5, 14). In the absence of commensurate genetic validation, results obtained with the synthetic polypeptide must be interpreted with caution.

We have now demonstrated that E inhibits MraY specifically in vivo (5), in membranes (6), and in solubilized extracts. In addition, we have found that E acts in a non-competitive fashion with respect to both its lipidic and soluble substrates. Taken together, these results obviate the need to invoke the existence of a detergent-sensitive, E-sensitive hetero-multimeric complex of membrane proteins required for the biological activity of MraY (15). The existence of such a complex was difficult to reconcile with the fact that the MraY enzymes from two Gram-positive bacteria, B. subtilis and S. aureus, were found to complement E. coli mraY defects in vivo (14, 41). The ability of these diverged proteins (43% similarity between EcMraY and BsMraY; 40% between EcMraY and SaMraY; 54% between BsMraY and SaMraY) to complement would not seem compatible with a model requiring intimate interactions with other E. coli cell division and murein synthesis proteins. The data presented here, taken with the genetic studies and the in vivo protection experiments reported previously (5, 14), suggest a simpler model in which E binds to MraY by interactions between the single transmembrane domain (TMD) of E and TMDs 5 and 9 of MraY, and this binding results in non-competitive inhibition of the enzyme by causing a conformational change. The catalytically important aspartate residues of MraY are associated with cytoplasmic loops terminating in TMDs 4 (Asp115, 116) and 8 (Asp267) (Fig. 1). TMDs almost invariably interact with their adjacent neighbors in primary structure (42, 43), which in this case would include the TMDs 5 and 9 that appear to define the E binding site, so an E-dependent conformational change based on transmembrane-helix interactions would not be difficult to conceive. Alternatively, it is worth noting that E has two basic residues, Lys33 and Arg34, predicted to be at the cytoplasmic interface of the membrane; binding of E to TMD9 of MraY may localize these residues near to the catalytic Asp267, predicted to be at the cytoplasmic interface of TMD8.

Protein inhibitors of biosynthetic enzymes are rare, so in this respect alone the ability of E to inhibit MraY is of interest. Moreover, MraY is universally conserved in bacteria, so understanding how E mediates non-competitive inhibition may be useful in the development of new antibacterial agents. E is particularly attractive as a probe for the mechanistic investigation of MraY because, unlike small molecule inhibitors, such as mureidomycin and tunicamycin, E is a genetic system on its own. This offers many advantages, especially since E can be tagged with any number of C-terminal protein moieties without affecting its inhibitory function. Also, the existence of the three classes of E-resistant mutants of MraY indicates that the affinity of E could be tuned by manipulation of the E sequence too. Indeed, a suppressor analysis looking for E missense changes that overcome the E-resistance mutations in TMDs 5 and 9 may allow a point-to-point interaction map to be generated, if allele-specificity can be demonstrated. Recently, the MraY of B. subtilis has been purified substantially on a small scale (28). Similar progress with the E. coli enzyme might allow the use of the genetically tractable E lysis protein system to probe MraY at the structural and mechanistic level.


We thank the members of the Young laboratory, past and present, for their helpful criticisms and suggestions.


This work was supported by Public Health Service grant GM27099 to R.Y., the Robert A. Welch Foundation, and the Program for Membrane Structure and Function, a Program of Excellence grant from the Office of the Vice President for Research at Texas A&M University.

1The abbreviations used are: TMD, transmembrane domain; P-MurNAc-pentapeptide, phosphate-N-acetylmuramic acid-L-Ala-γ-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala; undecaprenol-P, undecaprenol-phosphate; FKBP, FK506 binding protein; LB, Luria-Bertani; IPTG, isopropyl-β-D-thiogalactopyranoside; UDP-MurNAc-pentapeptide-DNS, UDP-MurNAc-L-γ-D-Glu-meso-diaminopimelic acid (Nε-dansyl)-D-Ala-DAla; phytol-P, phytol-phosphate; TLC, thin layer chromatography; DDM, n-dodecyl-β-D-maltoside; EBB, Empigen BB; IMAC, immobilized metal affinity chromatography; CMC, critical micelle concentration; DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine


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