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J. Biol. Chem., Vol. 279, Issue 29, 30106-30113, July 16, 2004

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The bacA Gene of Escherichia coli Encodes an Undecaprenyl Pyrophosphate Phosphatase Activity^*

Meriem El Ghachi, Ahmed Bouhss, Didier Blanot, and Dominique Mengin-Lecreulx

From the Laboratoire des Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619 CNRS, Université Paris-Sud, Bâtiment 430, 91405 Orsay Cedex, France

Received for publication, February 16, 2004 , and in revised form, May 10, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The bacA gene, the overexpression of which results in bacitracin resistance, was inactivated and shown to be non-essential for growth of Escherichia coli. It was proposed earlier that the bacA gene product may confer resistance to the antibiotic by phosphorylation of undecaprenol (Cain, B. D., Norton, P. J., Eubanks, W., Nick, H. S., and Allen, C. M. (1983) J. Bacteriol. 175, 3784–3789). In the present work, this extremely hydrophobic membrane protein was overproduced and purified to near homogeneity. The analysis of its catalytic properties clearly demonstrated that the purified BacA protein exhibited undecaprenyl pyrophosphate phosphatase activity but not undecaprenol phosphokinase activity. This finding was perfectly consistent with the mechanism of action of bacitracin that consists in the sequestration of undecaprenyl pyrophosphate, the BacA enzyme substrate. The level of undecaprenyl pyrophosphate phosphatase was increased by 280-fold in cells carrying bacA on a multicopy expression plasmid. It was decreased by 75% but was not completely abolished in a bacA disruption mutant, suggesting that BacA is the main E. coli undecaprenyl pyrophosphate phosphatase but that other protein(s) exhibiting such an activity should exist to account for the residual activity and viability of the mutant strain. This is the first gene encoding undecaprenyl pyrophosphate phosphatase identified to date. Considering its newly identified function, we propose to rename the bacA gene uppP.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Undecaprenyl phosphate (C₅₅-P)¹ is a key lipid intermediate involved in the synthesis of various bacterial cell wall polymers such as peptidoglycan, lipopolysaccharides, and teichoic acids (1–5). This carrier lipid enables the transport of hydrophilic precursors through the hydrophobic environment of the membrane to the externally located sites of polymerization. Because a single lipid participates in the synthesis of various wall polymers, C₅₅-P has been considered previously to be a potential site of control over the synthesis of these polymers that prevents an imbalance in the formation of the cell envelope as a whole (6). However, our knowledge of C₅₅-P metabolism remains very limited and based on fragmentary data obtained from various bacterial species (Scheme 1). The precursor for C₅₅-P, undecaprenyl pyrophosphate (C₅₅-PP), is synthesized by addition of isoprene units onto farnesyl pyrophosphate (Scheme 1, step 1). This reaction is catalyzed by a cis-prenyl pyrophosphate synthase that has been characterized in detail both biochemically and structurally (7–11). The dephosphorylation of C₅₅-PP (Scheme 1, step 2) is required before the lipid carrier becomes available for use in the various biosynthetic pathways, and this reaction must also occur at the end of each cycle of polymerization reaction (e.g. of peptidoglycan) where the lipid carrier is released in the pyrophosphate form. This reaction of dephosphorylation, which is the site of action of bacitracin, is catalyzed by a membrane-bound phosphatase (12). The intriguing presence of free undecaprenol (C₅₅-OH) in bacterial membranes has been reported (13). It could represent a reserve pool for the regulation of the C₅₅-P pool, a hypothesis that seems to be corroborated by the detection of two membrane-associated enzyme activities, an undecaprenol phosphokinase and an undecaprenyl phosphate phosphatase, catalyzing the interconversion of C₅₅-OH and C₅₅-P (Scheme 1, steps 3 and 4). Few data on genes and enzymes involved in steps 2, 3, and 4 are available (14–17). Recently, as discussed below, the Escherichia coli gene for the undecaprenol phosphokinase was tentatively identified as bacA (18).

View larger version (23K): [in this window] [in a new window]   SCHEME 1. Metabolism of undecaprenyl phosphate. PBPs, penicillin-binding proteins.

Bacitracin is a mixture of related cyclic polypeptide antibiotics produced by some strains of Bacillus licheniformis and Bacillus subtilis. This potent antibiotic is used clinically for treatments of surface tissue infections in combination with other antimicrobial drugs. Its primary mode of action is the inhibition of bacterial cell wall synthesis through sequestration of the essential carrier lipid C₅₅-PP, resulting in the loss of cell integrity and lysis (19–22). An active ATP-dependent ABC-type efflux system comprised of three BcrA, -B, and -C proteins is responsible for the resistance of B. licheniformis to the antibiotic (23, 24). However, little is known on the mechanisms developed by sensitive bacteria to resist bacitracin. In E. coli and some other Gram-negative bacteria, different mutations that block the synthesis of exopolysaccharides have been shown to also lead to bacitracin resistance (25, 26). Presumably the synthesis of these non-essential polymers also requires the same C₅₅-P transporter, and these mutations therefore indirectly provide an increased supply of this transporter for the synthesis of the essential cell wall component, peptidoglycan. Two E. coli genes whose overexpression in plasmids confers bacitracin resistance have been recently identified. The first one, bcrC, encodes a homologue of the BcrC subunit of the bacitracin transport permease from B. licheniformis, but the mechanism by which increased expression of this gene product modulates antibiotic resistance is yet unknown (27). Overexpression of the second gene, bacA, has been shown to confer a moderate 4-fold increase of undecaprenol phosphokinase activity in membranes, and it was thus hypothesized that this gene codes for the latter enzyme activity (18). Homologues of bacA have since been identified in Staphylococcus aureus and Streptococcus pneumoniae (28). Although bacA appears to be non-essential for in vitro growth of these two pathogenic organisms, its inactivation results in increased bacitracin susceptibility and reduced virulence with the maximal effects being observed in S. pneumoniae (28).

In fact, no demonstration that the E. coli bacA gene is also non-essential for growth has been presented to date. Attempts to disrupt its homologue in the B. subtilis chromosome have been reported recently to be unsuccessful, suggesting that bacA is essential in the latter species (29). Furthermore, with only one exception (18), none of the different previous reports on bacA include any data on the biochemical and functional characterization of the gene product. As mentioned by Cain and co-workers (18), although suggestive, the evidence provided in their report was not sufficient to conclude that the bacA gene encodes undecaprenol phosphokinase. It was thus necessary to reinvestigate the function of BacA by performing the complete set of experiments in a single bacterial species. This report describes such experiments performed in E. coli. The bacA gene was proved to be non-essential for growth, and the effects of its expression on bacitracin resistance were described. The BacA membrane protein was overproduced, extracted from membranes with a detergent, and purified to near homogeneity in the histidine-tagged form. Appropriate enzymatic assays showed that BacA does not exhibit undecaprenol phosphokinase activity but does exhibit undecaprenyl pyrophosphate phosphatase activity, a finding that is perfectly consistent with the associated bacitracin resistance phenotype.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bacterial Strains, Plasmids, and Growth Conditions
The E. coli strains DH5 (supE44 lacU169 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 80 dlacZ M15) (Invitrogen) and JM83 (ara [lac-proAB] rpsL thi 80 dlacZ M15) (30) were used as hosts for plasmids. The C43(DE3) strain used for the overproduction of the BacA protein was obtained from Avidis-France. The plasmid vector pTrc99A was obtained from Amersham Biosciences, and the construction of pTrcHis30 has been described previously (31). The construction of plasmid pET2130, a pET21d (Novagen) derivative for expression of proteins with an N-terminal His₆ tag, is detailed below. The BW25113 strain and the pKD3, pKD4, pKD46, and pCP20 plasmids used for gene disruption experiments (32) were kindly provided by B. Wanner via the E. coli Genetic Stock Center (Yale University, New Haven, CT). The construction of the BW25113 bacA::Cm^R strain is described below. 2YT (33) was used as culture medium, and growth was monitored at 600 nm with a Shimadzu UV-1601 spectrophotometer. For strains carrying drug resistance genes, ampicillin, kanamycin, and chloramphenicol were used at the concentrations of 100, 35, and 25 µg·ml^–1, respectively.

General DNA Techniques and E. coli Cell Transformation
PCR amplification of genes from the E. coli chromosome was performed in a Thermocycler 60 apparatus (Bio-med) using the polymerase Expand-Fidelity from Roche Applied Science. The DNA fragments were purified using the Wizard PCR Preps DNA purification kit (Promega). Small and large scale plasmid isolations were carried out by the alkaline lysis method, and standard procedures for endonuclease digestions, ligation, and agarose electrophoresis were used (34). E. coli cells were made competent for transformation with plasmid DNA by the method of Dagert and Ehrlich (35).

Construction of Expression Plasmids
A first plasmid suitable for overproduction of wild-type BacA was constructed as follows. PCR primers (Table I) were designed to incorporate a BspHI site 5' to the initiation codon of bacA (primer Bac1) and a PstI site 3' to the gene after the stop codon (primer Bac2). The DNA fragment amplified from the E. coli chromosome was treated with BspHI and PstI and ligated between the compatible NcoI and PstI sites of vector pTrc99A. The resulting plasmid, pTrcBac1, allowed expression of the wild-type bacA gene under the control of the strong isopropyl--D-thiogalactopyranoside (IPTG)-inducible trc promoter. For expression of BacA in an N-terminal His₆-tagged form, the gene was amplified with primers Bac2 and Bac3 (Table I), and the resulting fragment was cut with BamHI and PstI and inserted between the same sites of vector pTrcHis30, generating plasmid pTrcBac30.

Construction of a Null bacA Mutant
The E. coli mutant strain BW25113 bacA::Cm^R carrying a complete deletion of the bacA gene, which had been replaced by a chloramphenicol resistance gene, was created by following the method of Datsenko and Wanner (32). The Inact1 and Inact2 oligonucleotides (Table I) were used for PCR amplification of the antibiotic resistance gene from pKD3 flanked by sequences designed for specific disruption of the bacA gene. The PCR product was transformed by electroporation into BW25113(pKD46) cells that express phage Red recombinase (32). Chloramphenicol-resistant clones were isolated, and the disruption of the bacA gene in the chromosome was verified by PCR using the Bac1 and Bac2 primers.

Preparation of Crude Extracts and Purification of the BacA Protein
E. coli cells carrying plasmids described in this work were grown at 37 °C in 2YT-ampicillin medium (1-liter cultures). When the optical density of the culture reached 0.15, IPTG was added at a final concentration of 1 mM, and growth was continued for 16 h. Cells were harvested and washed with 40 ml of cold 20 mM potassium phosphate buffer, pH 7.2, containing 1 mM MgCl₂ and 2 mM -mercaptoethanol (buffer A). The cell pellet was suspended in 12 ml of the same buffer, and cells were disrupted by sonication in the cold (Bioblock Vibracell sonicator, model 72412). The resulting suspension was centrifuged at 4 °C for 30 min at 200,000 x g. The pellet consisting of membranes and associated proteins (420 mg of proteins) was washed twice with buffer A and subjected to solubilization by detergent as follows.

Membranes (2 g, wet weight) containing the overexpressed BacA protein were resuspended in 6 ml of buffer A supplemented with 150 mM NaCl and 20% glycerol. n-Dodecyl--D-maltoside (DDM) (0.9%, w/v) was added, and the mixture was incubated for 2 h at 4 °C with shaking. After centrifugation at 200,000 x g for 30 min at 4 °C, a first supernatant (DDM1) was recovered. The pellet of insoluble material was subjected again to three successive cycles of solubilization/centrifugation in the same conditions (except that DDM was used at 1%), yielding supernatants DDM2–DDM4, respectively.

The His₆-tagged BacA protein was purified basically following the manufacturer's recommendations (Qiagen). Solubilized membrane proteins (DDM2 extract, 70 mg of proteins) were mixed and incubated for 2 h at 4 °C with nickel-nitrilotriacetate (Ni²⁺-NTA)-agarose (20 mg of proteins/ml of resin) pre-equilibrated in buffer B (20 mM sodium phosphate, pH 7.2, 300 mM NaCl, 20% glycerol, 0.2% DDM, 1 mM MgCl₂, 2 mM 2-mercaptoethanol). The resin was then washed with buffer B, and protein elution was performed with increasing concentrations of imidazole from 10 to 300 mM in buffer B. Elution of the protein was followed by SDS-PAGE and enzymatic assays. Pure protein-containing fractions were concentrated and dialyzed using a Vivaspin concentrator (Vivascience) against buffer B.

Synthesis of Radiolabeled Undecaprenyl Pyrophosphate
The E. coli uppS gene (8, 10) encoding undecaprenyl pyrophosphate synthase was cloned in the His₆-tagged form in the expression vector pET2130 as described above. High level overexpression and purification of this protein on Ni²⁺-NTA-agarose yielded about 5 mg of pure enzyme/liter of culture (data not shown). Synthesis of radiolabeled C₅₅-PP was performed in a reaction mixture (20 µl) containing 100 mM HEPES buffer, pH 7.5, 50 mM KCl, 0.5 mM MgCl₂, 0.1% Triton X-100, 5 µM farnesyl pyrophosphate, 50 µM [¹⁴C]isopentenyl pyrophosphate (2,035 Bq), and pure UppS enzyme (5 µg). After 3 h of incubation at 25 °C, the reaction was stopped by addition of 80 µl of 0.4 M NaOH. The radiolabeled C₅₅-PP synthesized was recovered by extraction with butanol and dried under vacuum. The identity of the product was confirmed by TLC analysis as described below.

Enzymatic Assays
Undecaprenol Phosphokinase Assay—The assay was performed in a reaction mixture (20 µl) containing, in final concentrations, 100 mM Tris-HCl buffer, pH 7.5, 20 mM MgCl₂, 0.6 mM C₅₅-OH, 0.2% DDM, 0.1 mM [-³²P]ATP (4,500 Bq), and enzyme (150 µg of protein).

Undecaprenyl Pyrophosphate Phosphatase Assay—The assay was performed in a reaction mixture (20 µl) containing 100 mM Tris-HCl buffer, pH 7.5, 10 mM MgCl₂, 3.9 mM DDM, 5 µM [¹⁴C]C₅₅-PP (2,035 Bq), and enzyme.

Coupled Assay with MraY—The assay was performed in a reaction mixture (20 µl) containing 100 mM Tris-HCl buffer, pH 7.5, 10 mM MgCl₂, 3.9 mM DDM, 5 µM [¹⁴C]C₅₅-PP (2,035 Bq), 0.1 mM UDP-Mur-NAc-pentapeptide, 0.4 µg of purified MraY, and BacA enzyme. In all cases, reaction mixtures were incubated for 30 min at 37 °C, and reactions were stopped by heating at 100 °C for 1 min. Substrates and reaction products were separated by TLC on precoated plates of silica gel 60 (Merck) using diisobutyl ketone/acetic acid/water (8:5:1, v/v/v) as a mobile phase. The radioactive spots were located and quantified with a radioactivity scanner (model Multi-Tracermaster LB285, Berthold-France). Commercial unlabeled compounds were detected by ultraviolet absorption or reaction with iodine vapors. The R_F values observed for ATP, MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol (lipid I), C₅₅-PP, C₅₅-P, and C₅₅-OH were 0, 0.2, 0.36, 0.5, and 0.98, respectively. One unit of enzyme activity corresponds to one nanomole of product formed per minute.

Extraction of C₅₅-P Reaction Product by Butanol
A standard C₅₅-PP phosphatase assay was performed as described above except that C₅₅-PP (1 nmol) was unlabeled. After the reaction had been stopped by addition of 20 µl of 6 M pyridinium acetate, 40 µl of 1-butanol were added, and the mixture was vortexed for 2 min and centrifuged at 10,000 x g for 5 min. The organic phase was recovered, evaporated, taken up in 10 µl of 2-propanol/methanol (1:1, v/v), and analyzed by mass spectrometry.

Protein Monitoring
SDS-PAGE analysis of proteins was performed as described by Laemmli and Favre (36). Protein concentrations were determined by using either the Bradford procedure (37) or the QuantiPro BCA assay kit (Sigma) with bovine serum albumin as the standard and/or by quantitative amino acid analysis with a Hitachi model L8800 analyzer (ScienceTec) after hydrolysis of samples in 6 M HCl for 24 h at 105 °C.

Matrix-assisted Laser Desorption/Ionization Time-of-flight (MALDI-TOF) Mass Spectrometry Analysis
MALDI-TOF mass spectra were recorded in the linear mode with delayed extraction on a PerSeptive Voyager-DE STR instrument (Applied Biosystems) equipped with a 337 nm nitrogen laser.

BacA—The samples were prepared according to Grüber et al. (38). 0.5 µl of BacA preparation was deposited on the plate and allowed to dry. Subsequently 0.5 µl of matrix solution (10 mg/ml -cyano-4-hydroxycinnamic acid in 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid) was applied to the dried sample and again allowed to dry. Spectra were recorded in the positive ion mode at an acceleration voltage of +25 kV and an extraction delay time of 300 ns. Carbonic anhydrase was used as an external calibrant.

C₅₅-P—1 µl of matrix solution (10 mg/ml 2,5-dihydroxybenzoic acid in 20 mM diammonium citrate) was deposited on the plate followed by 0.5 µl of sample dissolved in 2-propanol/methanol (1:1, v/v). After evaporation of the solvents, spectra were recorded in the negative ion mode at an acceleration voltage of –20 kV and an extraction delay time of 100 ns. A mixture of UDP-MurNAc, UDP-MurNAc-L-Ala, and UDP-Mur-NAc-L-Ala-D-Glu was used as an external calibrant.

Chemicals
DNA restriction and modification enzymes were obtained from New England Biolabs, and oligonucleotides were from MWG-Biotech. DNA purification kits were from Promega. C₅₅-OH, C₅₅-P, and C₅₅-PP were provided by the Institute of Biochemistry and Biophysics of the Polish Academy of Sciences. UDP-[U-¹⁴C]GlcNAc (9.85–11.1 GBq·mmol^–1) was purchased from Amersham Biosciences, and [4-¹⁴C]isopentenyl pyrophosphate (1.5–2.2 GBq·mmol^–1) and [-³²P]ATP (111 TBq·mmol^–1) were from PerkinElmer Life Sciences. DDM was from Fluka, and Ni²⁺-NTA agarose was from Qiagen. UDP-MurNAc-peptides were prepared as described previously (39), and pure MraY translocase was prepared in the laboratory (40). Antibiotics and reagents were from Sigma.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Overexpression of bacA and Bacitracin Resistance—The wild-type E. coli bacA gene was cloned under control of the strong IPTG-inducible trc promoter in expression vector pTrc99A, yielding plasmid pTrcBac1. The effects of the overexpression of bacA on cell growth and bacitracin resistance were investigated in different E. coli host strains, DH5, JM83, or BW25113, with similar results. Fig. 1 shows the results of experiments performed in DH5 background. In liquid medium, no particular effect on growth rate and cell morphology (as judged by optical microscopy) was observed following induction of expression of bacA. In solid medium, however, colonies of clones harboring pTrcBac1 appeared significantly more mucoid than control cell colonies. E. coli and Gram-negative species in general are not very sensitive to bacitracin. As shown in Fig. 1, lysis of control DH5(pTrc99A) cells only occurred at relatively high concentrations of this antibiotic, i.e. 100–200 µg·ml^–1. In the absence of IPTG, cells harboring the pTrcBac1 plasmid appeared slightly less sensitive than control cells to bacitracin. When IPTG (1 mM) was added 5 min before bacitracin, growth of the latter cells remained unaffected even at the highest concentration of antibiotic used (Fig. 1). This finding clearly established the tight correlation existing between bacA overexpression and bacitracin resistance in E. coli and further indicated that cell resistance to the antibiotic was a process that developed very rapidly.

View larger version (48K): [in this window] [in a new window]   FIG. 1. Overexpression of bacA mediates bacitracin resistance. Cells of DH5(pTrc99A) and DH5(pTrcBac1) were grown at 37 °C in 2YT-ampicillin medium. At the time indicated by the arrow (optical density = 0.1), IPTG was added (1 mM), and bacitracin at different concentrations was added 5 min thereafter. Cell growth was monitored at 600 nm. A, control DH5(pTrc99A) cells untreated () or treated (, , and ) with 50, 100, and 200 µg·ml–1 bacitracin, respectively. B, DH5(pTrcBac1) cells either (i) non-induced with IPTG and untreated () or treated ( and ) with 100 and 200 µg·ml–1 bacitracin, respectively, or (ii) induced with IPTG () and untreated or treated with 200 µg·ml–1 bacitracin (no significant growth difference).

Extraction and Purification of the BacA Protein—DDM appeared to be an efficient detergent to extract the BacA protein from membranes of induced cells as judged by SDS-PAGE analysis and Coomassie Blue or Western blot detection of the protein (data not shown). Four successive treatments with DDM were performed, one with 0.9% followed by three with 1% DDM, resulting in extracts DDM1–DDM4, respectively. The levels of C₅₅-PP phosphatase activity determined in these different extracts are reported in Table II. The stepwise extraction allowed us to remove significant amounts of proteins but only a little of the BacA protein in DDM1. About one-third of the total activity was recovered in DDM2 with a 65% increase in specific activity. Protein amounts in DDM3 and DDM4 were similar with a specific activity of approximately 125 and 30 units/mg of protein, respectively, as compared with 155 units/mg of protein in DDM2 (Table II). The DDM2 extract was chosen for subsequent steps of purification of the BacA protein.

View larger version (109K): [in this window] [in a new window]   FIG. 2. SDS-polyacrylamide gel electrophoresis of purified E. coli BacA protein. The BacA protein was overproduced in E. coli cells in the His6-tagged form (N-terminal Met-His6-Gly-Ser extension). The one-step purification on Ni2+-NTA-agarose was performed as described in the text, and aliquots were analyzed by SDS-PAGE. Lane A, DDM2 extract; lanes B–E, 20, 60, 80, and 100 mM imidazole-containing fractions, respectively. Molecular mass standards indicated on the left are phosphorylase b, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; and soybean trypsin inhibitor, 20 kDa. The arrow points to the purified BacA protein.

View larger version (12K): [in this window] [in a new window]   FIG. 3. MALDI-TOF mass spectrometry analysis of purified BacA protein. Peaks of m/z 31,011, 15,515, 10,353, and 7,773 were observed that were assigned to be the [M + H]+, [M + 2H]2+, [M + 3H]3+, and [M + 4H]4+ ions, respectively. The molecular mass of BacA calculated from the gene sequence is 30,857 Da (N-terminal Met-His6-Gly-Ser extension included).

View larger version (12K): [in this window] [in a new window]   FIG. 4. Thin-layer chromatography separation of BacA substrate (C55-PP) and product (C55-P). A typical assay was carried out in the presence of [14C]C55-PP and purified BacA. The radiolabeled substrate and product were separated by TLC, and the corresponding spots were detected with a radioactivity scanner as detailed in the text (RF values of 0.36 and 0.5, respectively).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The C₅₅-P carrier lipid is essential for the synthesis of various bacterial cell wall polymers such as peptidoglycan, lipopolysaccharides, and teichoic acids (1–5). Its precursor, C₅₅-PP, is formed by addition of isoprene units onto farnesyl pyrophosphate, a reaction catalyzed by the cis-prenyl pyrophosphate synthase UppS, the only step of C₅₅-P metabolism that has been characterized in detail to date (8–11). Genes and enzymes involved in other subsequent steps of C₅₅-P synthesis and recycling (Scheme 1) still remain to be identified. The cyclic polypeptide antibiotic bacitracin has been shown to inhibit the synthesis of the aforementioned cell envelope components through sequestration of the carrier lipid precursor C₅₅-PP (20, 21). The targeting of this specific and essential bacterial lipid and the resulting bacteriolytic effect (Fig. 1) have been the basis for the development and therapeutic use of this compound. Bacterial resistance to this antibiotic could be due to a decreased uptake or an efflux system as observed in B. licheniformis producing strain (23). In E. coli and other Gram-negative bacteria, different mutations have been described that lead to bacitracin resistance, most of them being characterized by a decreased in vivo requirement for the C₅₅-P carrier lipid. For instance, defective synthesis of non-essential polymers such as osmoregulated periplasmic glycans or capsular polysaccharides result in antibiotic resistance (25, 26) presumably because inactivation of these C₅₅-P-dependent pathways indirectly provides an increased supply of carrier lipid for the synthesis of the essential cell wall component, peptidoglycan. As discussed below, not only mutations but also gene overexpression have reported to be associated with bacitracin resistance (18, 27).

Cain et al. (18) have previously reported the identification of an E. coli gene, named bacA, the presence of which on multicopy plasmids resulted in an increased resistance to bacitracin. They have shown that membranes from cells harboring bacA-containing plasmids contained an approximately 4-fold increased level of C₅₅-OH phosphokinase activity as compared with control host cell membranes and therefore concluded that C₅₅-OH phosphokinase activity should be carried out by the bacA gene product, although this was not undoubtedly established. This question was revisited in the present work. First we cloned the E. coli bacA gene into the efficient expression vector pTrc99A and confirmed the correlation between overexpression of this gene and bacitracin resistance. Then, using appropriate enzymatic assays, we showed that crude membranes from cells overexpressing bacA did not contain an increased level of C₅₅-OH phosphokinase activity, contrary to what was observed earlier by Cain et al. (18), but contained instead a 280-fold increased level of C₅₅-PP phosphatase activity. This unexpected result prompted us to purify the BacA protein from membranes to analyze its function in more details. For this purpose the protein was overexpressed in the His₆-tagged form, extracted from membranes using an appropriate detergent, and purified to near homogeneity as judged by SDS-PAGE and mass spectrometry analyses. This final preparation clearly exhibited a high C₅₅-PP phosphatase activity but no detectable C₅₅-OH phosphokinase activity. The C₅₅-OH phosphokinase activity detected in crude membranes was not retained on the column of Ni²⁺-NTA-agarose used for binding and purification of His₆-BacA protein (data not shown), further demonstrating that this activity was carried by a separate protein species. The increased bacitracin resistance of bacA-overexpressing clones was interpreted previously by Cain et al. (18) as an increased supply of C₅₅-P that might result from the overexpression of C₅₅-OH phosphokinase activity. This hypothesis was based on the assumption that a significant pool of free C₅₅-OH may also exist in E. coli membranes as suggested in some Gram-positive bacteria (13) and that this pool was directed toward the formation of C₅₅-P in the presence of an excess of phosphokinase activity (Scheme 1, step 3). In fact, the present identification of BacA as a C₅₅-PP phosphatase activity remains perfectly consistent with a bacitracin resistance phenotype. Indeed the high level overexpression of this enzyme should theoretically cause a dramatic depletion of the internal pool of C₅₅-PP due to its rapid conversion to C₅₅-P. The decreased availability of C₅₅-PP, the target of bacitracin, most likely accounts for the increased cell resistance to the antibiotic. Confirmation of this hypothesis requires the development of efficient techniques allowing the separation and quantification of these different lipids in the bacterial membranes. Competition of bacitracin and BacA protein for the same ligand could also explain the reduced effect of the antibiotic in a BacA-overproducing strain.

The physiological role of BacA is thus to catalyze the dephosphorylation of C₅₅-PP, a reaction that generates (or regenerates, see Scheme 1) the essential C₅₅-P carrier lipid used for the synthesis of various cell wall components. This is to our knowledge the first gene encoding C₅₅-PP phosphatase identified to date. The partial purification and some properties of the C₅₅-PP phosphatase from Micrococcus lysodeikticus were reported about 30 years ago by Goldman and Strominger (12). Four protein bands were still detected by SDS-PAGE in their final preparation that all migrated between 26.0 and 48.5 kDa standards, but the band corresponding to the phosphatase was not identified. This molecular mass range is consistent with the calculated mass of BacA (29,759 Da for the protein without His₆ tag) and the behavior on polyacrylamide gels of the purified membrane protein purified from E. coli observed in the present study. The size of the C₅₅-OH phosphokinase from S. aureus was estimated previously by Sanderman and Strominger to be much lower, about 17 kDa (16, 17), a finding consistent with the present demonstration that BacA does not exhibit this activity.

The bacA gene has recently been demonstrated to be non-essential for in vitro growth of S. aureus and S. pneumoniae (28), but disruption of the gene in the latter pathogenic species resulted in a hypersensitivity to bacitracin and a reduced virulence in a mouse model of infection. The latter finding suggested that some metabolic changes had occurred that were sufficient to compromise infectivity. A decreased C₅₅-PP phosphatase activity should theoretically cause an accumulation of C₅₅-PP (consistent with bacitracin hypersensitivity) and a partial depletion of the pool of the C₅₅-P carrier lipid, resulting in the inhibition of synthesis of some of the different cell envelope components. However, Chalker et al. (28) did not observe in these mutant strains differences in morphology or sensitivity to other antibiotics that could be interpreted as a deficiency in cell wall synthesis. In the present work we provided evidence that the bacA gene is also non-essential for viability in E. coli. We showed that its overexpression resulted in bacitracin resistance and mucoidy of solid medium, suggesting an increased production of some exopolysaccharides. Inactivation of the gene, however, did not result in an hypersensitivity to bacitracin presumably because E. coli is naturally less sensitive than Gram-positive bacteria to this antibiotic due to the presence of the additional outer membrane.

The UppS synthase catalyzing the formation of C₅₅-PP has been shown to be essential for cell viability in E. coli and S. pneumoniae (8, 10). The bacteriolytic effect of the sequestration of C₅₅-PP by bacitracin confirmed this finding. The same was thus expected for any other enzyme participating in the synthesis of the essential C₅₅-P carrier lipid and in particular for the phosphatase catalyzing the subsequent step of dephosphorylation of C₅₅-PP. The fact that the chromosomal bacA gene could be disrupted without apparent effect on cell morphology and growth was thus quite surprising. Different hypotheses could be made to explain this finding, such as the occurrence of multiple phosphatase genes or a putative metabolic bypass of the dephosphorylation step. One can effectively speculate that more than one C₅₅-PP phosphatase gene exists in E. coli and that in a bacA mutant the other phosphatase(s) could sustain the in vivo requirements of C₅₅-P molecules for cell wall polymer synthesis. Such a phosphatase could be specific of C₅₅-PP, or it could have a different metabolic function but also display nonspecific activity on C₅₅-PP. Multiple genes for diacylglycerol pyrophosphate phosphatase and phosphatidylglycerophosphate phosphatase activities have previously been identified in E. coli (43–45), providing an example of such a gene redundancy and functional complementation. In the present study we observed that the C₅₅-PP phosphatase activity was reduced by 75% in the disrupted bacA mutant, confirming the contribution of both BacA and another protein species to the detected activity.

It should be noted that C₅₅-PP is initially synthesized by UppS at the inner side of the cytoplasmic membrane but that it is also regenerated later at the outer periplasmic side of the membrane in the course of the polymerization steps of cell wall peptidoglycan biosynthesis. This raises the question of the site of dephosphorylation of this compound. Does it take place on each membrane side using different phosphatase activities, or does it occur only on the inner side, implying in that case a flip-flop of the released C₅₅-PP prior to dephosphorylation? As no bacA homologous gene could be detected in the genome of E. coli and other bacteria in which this gene was shown to be non-essential, further work is now required to identify this additional phosphatase and to characterize its substrate specificity.

As already reported by Chalker et al. (28) the gene name bacA has also been used previously to designate bacitracin synthetase of B. licheniformis and a transport protein involved in bacteroid synthesis in Rhizobium species. Considering the newly identified function of the bacA gene product, we propose to rename this gene uppP, for undecaprenyl pyrophosphate phosphatase, to follow the nomenclature previously adopted for enzymes involved in C₅₅-P metabolism (uppS for C₅₅-PP synthase (8)).

	FOOTNOTES

 
^* This work was supported by grants from the European Community (FP6, LSHM-CT-2003-503335, "COBRA" project) and from the CNRS (UMR 8619). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a scholarship from the Ministère de l'Education Nationale, de la Recherche et de la Technologie (Ecole Doctorale "Innovation Thérapeutique, du Fondamental à l'Appliqué").

To whom correspondence should be addressed. Tel.: 33-1-69-15-48-41; Fax: 33-1-69-85-37-15; E-mail: dominique.mengin-lecreulx{at}ebp.u-psud.fr.

¹ The abbreviations used are: C₅₅-P, undecaprenyl phosphate; C₅₅-PP, undecaprenyl pyrophosphate; C₅₅-OH, undecaprenol; Ni²⁺-NTA, nickel-nitrilotriacetate; DDM, n-dodecyl--D-maltoside; MurNAc, N-acetylmuramic acid; UDP-MurNAc-pentapeptide, UDP-MurNAc-L-Ala--D-Glu-meso-diaminopimeloyl-D-Ala-D-Ala; lipid I, MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol; IPTG, isopropyl--D-thiogalactopyranoside; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight.

² A. Bouhss et al., unpublished data.

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