| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 281, Issue 32, 22761-22772, August 11, 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Laboratoire des Enveloppes Bactériennes et Antibiotiques, UMR 8619 CNRS, Université Paris-Sud, 91405 Orsay, France
Received for publication, March 27, 2006 , and in revised form, June 14, 2006.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Various bacterial cell envelope polysaccharides (peptidoglycan, O-antigen, teichoic acid, capsular polysaccharide) in both Gram-negative and Gram-positive bacteria have a lipid-linked intermediary stage in their biosynthesis that is dependent on the essential carrier lipid undecaprenyl phosphate (C55-P)4 (4-15) (Scheme 1). In the peptidoglycan pathway, this lipid is needed for the synthesis and transport of hydrophilic GlcNAc-MurNAc-peptide monomeric motifs across the cytoplasmic membrane to the external sites of polymerization. In other pathways, it can serve as an acceptor for oligosaccharide repeating units or, in some cases, possibly for completed polymers. C55-P is generated from the dephosphorylation of undecaprenyl pyrophosphate (C55-PP), which itself is the product of eight sequential condensations of isopentenyl pyrophosphate with farnesyl pyrophosphate, a reaction catalyzed by the C55-PP synthase UppS (16, 17). Several integral membrane proteins exhibiting a C55-PP phosphatase (UppP) activity were recently identified in E. coli (18, 19): the BacA protein and three members from the PAP2 superfamily of phosphatases, YbjG, YeiU, and PgpB. Only the inactivation of these multiple genes was lethal and therefore required to completely deplete cells of the latter essential activity (19). C55-P phosphatase and undecaprenol kinase activities that catalyzed the interconversion of C55-P into undecaprenol (C55-OH) were also earlier detected and purified from extracts of some Gram-positive bacteria but their genes have not been identified to date (20-23).
|
In the present article, the effects of colicin M on peptidoglycan metabolism were reinvestigated. Colicin M was shown to exhibit both in vitro and in vivo enzymatic properties of degradation of the lipid I and lipid II peptidoglycan intermediates. The depletion of the pools of these precursors results in an inhibition of peptidoglycan polymerization steps and cell lysis. To the best of our knowledge, this is the first time such an activity has been described to date.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
(supE44 
lacU169 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 
80 dlacZ M15) (Invitrogen) was used as the host for plasmids. The FB8r lysA::KmR strain (33) was used to study the incorporation of radiolabeled meso-A2pm into peptidoglycan. The plasmid vector pTrc99A was from Amersham Biosciences and the construction of pTrcHis30 and pTrcHis60 plasmids has been previously described (34). The pTO4 plasmid carrying the cma gene encoding colicin M and the cmi immunity gene has been previously described (35). The BW25113 strain and the pKD3 and pKD46 plasmids used for gene disruption experiments (36) were kindly provided by B. Wanner via the E. coli Genetic Stock Center (Yale University, New Haven). 2YT medium or minimal medium M63 (37) was used for growing cells. Ampicillin, kanamycin, and chloramphenicol were used at 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 were performed in a Thermocycler 60 apparatus (Bio-med) using the Expand-Fidelity polymerase from Roche. The DNA fragments were purified using the Wizard PCR Preps DNA purification kit (Promega). Standard procedures for endonuclease digestions, ligation and agarose electrophoresis were used (38). Small and large scale plasmid isolations were carried out by the alkaline lysis method (38). E. coli cells were made competent for transformation with plasmid DNA by the method of Dagert and Ehrlich (39) or by electroporation.
Construction of Expression Plasmids—A plasmid allowing overexpression of the colicin M cma gene was constructed as follows: PCR primers Cma1 and Cma2 (see Table 1) were designed to incorporate an NcoI site 5' to the initiation codon of the gene and a PstI site 3' to the gene after the stop codon, respectively. The pTO4 plasmid (35) was used as the source of cma gene for the amplification and the resulting DNA fragment was treated with NcoI and PstI and ligated between the same sites of vector pTrc99A. The resulting plasmid, pMLD188, allowed expression of the cma gene under the control of the strong isopropyl-
-D-thiogalactopyranoside (IPTG)-inducible trc promoter.
|
A plasmid carrying both the cma gene and the cmi immunity gene was also constructed: in that case oligonucleotides Cma1 and Cmi were used for PCR amplification from the pTO4 plasmid and the 1.3-kb resulting fragment was cut by NcoI and PstI and inserted between the same sites of the vector pTrc99A, generating pMLD170. In all cases DNA sequencing was performed to determine that the sequence of the cloned fragments was correct.
Construction of a Null fhuA Mutant—The E. coli mutant strain BW25113 fhuA::CmR, carrying a deletion of the chromosomal fhuA gene (an internal 1,819-bp fragment of the 2,244-bp gene was deleted and replaced by the chloramphenicol resistance gene), was created by following the method of Datsenko and Wanner (36). The FhuA-Inact1 and FhuA-Inact2 oligonucleotides (Table 1) were used for PCR amplification of the antibiotic resistance gene from pKD3 flanked by sequences designed for specific disruption of the fhuA gene. The 1.1-kb PCR product was transformed by electroporation into BW25113(pKD46) cells that express phage lambda Red recombinase (36). Chloramphenicol-resistant clones were isolated, and the inactivation of the fhuA gene was verified by PCR using the FhuA1 and FhuA2 primers.
Preparation of Crude Extracts and Purification of Colicin M— BW25113 fhuA::CmR cells carrying plasmids pMLD189 or pMLD190 were grown at 37 °C in 2YT-ampicillin medium (2-liter cultures). When the optical density of the culture reached 1, IPTG was added at a final concentration of 1 mM, and growth was continued for 3 h. Cells were harvested and washed with 40 ml of cold 20 mM potassium phosphate buffer, pH 7.4, containing 0.5 mM MgCl2, 2 mM -mercaptoethanol, and 150 mM NaCl (buffer A). The cell pellet (
8 g wet wt) was suspended in 25 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 in a TL100 Beckman centrifuge. The supernatant (400 mg of proteins) was stored at -20 °C.
The His6-tagged colicin M was purified basically following the manufacturer's recommendations (Qiagen). Soluble fractions described above were incubated for 1 h at 4 °C with nickel-nitrilotriacetate (Ni2+-NTA)-agarose pre-equilibrated in buffer A. The polymer was then washed extensively with buffer A containing 20 mM imidazole and elution of the protein was obtained by increasing step by step the concentration of imidazole, from 40 to 300 mM, in buffer A. Pure protein-containing fractions (100-200 mM imidazole) were concentrated to 8 ml on a Millipore PM10 membrane and then dialyzed overnight against 100 volumes of buffer A. The final preparations were stored at -20 °C after addition of 15% glycerol.
Determination of Colicin M Activity—The bacteriolytic activity of the crude soluble extracts prepared from the colicin M-expressing BW25113 fhuA strain and of the purified His6-tagged colicin M was tested both on plates and in liquid medium. 2YT-agar plates were overlaid with 3 ml of soft nutrient agar containing 108 indicator bacteria (BW25113 or DH5). Serial dilutions (x3) of colicin M-containing solutions were made in buffer A containing 150 mM NaCl and 2-µl samples were spotted on the overlay. Plates were incubated overnight at 37 °C and the titer of the colicin was expressed as the reciprocal of the final dilution still giving a clear spot. When assayed in liquid medium, dilutions of colicin M were added to exponentially growing cells when the optical density at 600 nm reached the value of 0.6.
Preparation of UDP-MurNAc-pentapeptide and Derivatives—Unlabeled and radiolabeled forms of UDP-MurNAc-L-[14C]Ala-
-D-Glu-meso-A2pm-D-Ala-D-Ala were prepared as previously described (40). 1-Pyrophospho-MurNAc-pentapeptide and 1-phospho-MurNAc-pentapeptide were obtained by treatment of UDP-MurNAc-pentapeptide with periodate (41, 42) and nucleotide pyrophosphatase (43), respectively. MurNAc-pentapeptide was obtained by mild-acid hydrolysis of UDP-MurNAc-pentapeptide (44). These compounds were purified by reverse-phase high performance liquid chromatography (HPLC) and analyzed by mass spectrometry. 1-Pyrophospho-MurNAc-tetrapeptide and 1-pyrophospho-MurNAc-tripeptide were generated by treatments of 1-pyrophospho-MurNAc-pentapeptide with purified penicillin-binding protein PBP5 (45) and L,D-carboxypeptidase LdcA (46), respectively.
Synthesis of Radiolabeled C55-PP, Lipid I, and Lipid II—Radiolabeled C55-PP was produced enzymatically by incubating farnesyl pyrophosphate and [14C]isopentenyl pyrophosphate in the presence of pure UppS synthase, as previously described (18). [14C]C55-P was obtained by treatment of [14C]C55-PP with purified BacA phosphatase (18). The reaction mixture (200 µl) containing 100 mM Tris-HCl buffer, pH 7.5, 10 mM MgCl2, 3.9 mM n-dodecyl--D-maltoside (DDM), 5 µM [14C]C55-PP (20 kBq), and BacA enzyme (5 µg). The radiolabeled C55-P was recovered by extraction with 1-butyl alcohol and dried under vacuum. The synthesis of lipid I was performed in a reaction mixture (400 µl) consisting in 100 mM Tris-HCl, pH 7.5, 40 mM MgCl2, 1.25 mM C55-P, 150 mM NaCl, 2 mM UDP-MurNAc-pentapeptide, and 8.4 mM N-lauroyl sarcosine. The reaction was initiated by the addition of pure MraY enzyme (40) (10 µg), and the mixture was incubated for 2 h at 37°C under shaking with a thermomixer (Eppendorf). [14C]lipid I labeled either in the pentapeptide moiety or the C55-P moiety were synthesized as described above for the unlabeled lipid I except that the reaction volume was 20 µl and the concentrations of radiolabeled [14C]C-P and UDP-MurNAc-[14C55]pentapeptide were 0.3 mM and 0.6 mM, respectively. The synthesis of lipid II was performed in a reaction mixture (200 µl) containing 200 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM lipid I, 2 mM UDP-GlcNAc, and 35% (v/v) Me2SO (47). The reaction was initiated by the addition of MurG enzyme (48)(10 µg), and the mixture was incubated for 2 h at 37 °C under shaking with a thermomixer (Eppendorf). [14C]Lipid II labeled in the GlcNAc residue was synthesized as described above for the unlabeled lipid II except that the reaction volume was 20 µl, and the concentrations of lipid I and radiolabeled UDP-[14C]GlcNAc were 0.3 mM and 0.6 mM, respectively. In all cases, the extraction, analysis and quantification of lipid I and lipid II were performed as described previously (40).
Enzymatic Assays—(i) C55-PP Synthase Assay: the assay 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, and 50 µM [14C]isopentenyl pyrophosphate (2 kBq). The reaction was initiated by the addition of pure UppS enzyme (1 µg) and when required, colicin M (2.7 µg), and the mixture was incubated for 3 h at 25 °C. (ii) C55-PP 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: 5 µl of an appropriate dilution (in buffer A supplemented with 3.9 mM DDM) of membranes or pure BacA protein (0.1 µg). When required, colicin M (2.7 µg) was added and the mixture was incubated for 1 h at 37°C. (iii) MraY Translocase Assay: the assay was performed in a reaction mixture (20 µl) containing 100 mM Tris-HCl, pH 7.5, 40 mM MgCl2, 1.1 mM C55-P, 150 mM NaCl, 12.5 µM UDP-MurNAc-[14C]pentapeptide (800 Bq), and 0.2% DDM. The reaction was initiated by the addition of pure MraY enzyme (0.01 µg). When required, 2.7 µg of colicin M was added and the mixture was incubated for 30 min at 37 °C under shaking with a thermomixer (Eppendorf). When E. coli membranes instead of pure MraY enzyme were used, C55-P was omitted. (iv) Standard assay for Colicin M activity: unless otherwise noted, the activity of colicin M was tested in a reaction mixture (20 µl) containing 100 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 150 mM NaCl, 40 µM of 14C-radiolabeled substrate (400 Bq) and 0.2% DDM. The reaction was initiated by the addition of pure colicin M (2.7 µg), and the mixture was incubated for 30 min at 37 °C under shaking with a thermomixer (Eppendorf).
In all cases, the reaction was stopped by heating at 100 °C for 1 min, and the mixture was analyzed by thin-layer chromatography (TLC) on silica gel plates LK6D (Whatman) using either 1-propyl alcohol/ammonium hydroxide/water (6:3:1; v/v/v) (solvent system I) or diisobutyl ketone/acetic acid/water (8:5:1, v/v/v) (solvent system II) as a mobile phase. The radioactive spots were located and quantified with a radioactivity scanner (model Multi-Tracemaster LB285; Berthold France).
Purification of Lipid I and Lipid II Degradation Products by HPLC—Standard colicin M assays were performed as described above except that lipid I and lipid II (1 nmol) were used in unlabeled form. The reaction mixtures were diluted into 1 ml of 50 mM sodium phosphate buffer, pH 4.5, and applied to a Nucleosil 100C18 5 µm (4.6 x 250 mm, Alltech) column. Elution was performed with the phosphate buffer for 30 min, followed by a linear gradient of methanol from 0 to 20% during the next 30 min, at a flow rate of 0.6 ml·min-1. Peaks were detected at 215 nm. In these conditions the 1-pyrophospho-MurNAc-pentapeptide and 1-pyrophospho-MurNAc-(pentapeptide)-GlcNAc degradation products were eluted at 18 and 40 min, respectively. In the same conditions, pure 1-phospho-Mur-NAc-pentapeptide and the two and anomers of MurNAc-pentapeptide were eluted at 28, 34, and 50 min, respectively. The two degradation products were collected, lyophilized, and desalted by gel-filtration on a column of Sephadex G-25 fine. After lyophilization, these compounds were taken up in 10 µlof water and analyzed by mass spectrometry as described below.
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry—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 laser. (i) 1-Pyrophospho derivatives: 1 µl of matrix solution (10 mg·ml-1 2,5-dihydroxybenzoic acid in 0.1 M citric acid) was deposited on the plate followed with 2 µl of sample solution. After evaporation of water, spectra were recorded in the negative mode at an acceleration voltage of -20 kV and an extraction delay time of 100 ns. A mixture of UDP-MurNAc, UDP-MurNAc-dipeptide, and UDP-MurNAc-pentapeptide was used as an external calibrant. (ii) C55-OH: the sample solution was mixed with an equal volume of 0.5 M 2,5-dihydroxybenzoic acid in methanol containing 0.1% trifluoroacetic acid (49). 1 µl of the mixture was deposited on the plate. After evaporation of the solvents, spectra were recorded in the positive mode at an acceleration voltage of +20 kV and an extraction delay time of 100 ns. External calibration was performed using the calibration mixture 1 of the SequazymeTM peptide mass standards kit (Applied Biosystems).
Uptake of Radiolabeled meso-Diaminopimelic Acid (A2pm) and Its Incorporation into Peptidoglycan—The incorporation of meso-[3H]A2pm into the peptidoglycan of strain FB8r lysA was followed essentially as described earlier (33). The strain was grown exponentially in minimal M63 medium (50 ml cultures) supplemented with 0.4% glucose, and 100 µg·ml-1 each of lysine, threonine, and methionine (33). At an optical density of 0.2, cultures were either treated or not with colicin M at 50 ng·ml-1, and [3H]A2pm was added 10 min later (2.5 kBq·ml-1). After designated time intervals, 500-µl samples were removed and centrifuged for 2 min with an Eppendorf centrifuge. The supernatants were analyzed for radioactivity to estimate the A2pm uptake. In parallel, other 500-µl samples were added to 9.5 ml of ice-cold 5.5% trichloroacetic acid. Suspensions were kept at 0 °C for 60 min and labeled peptidoglycan (trichloroacetic acid-insoluble material) was filtered over glass fiber filters (GF/C, Whatman). The filters were washed with 5.5% trichloroacetic acid, dried, and immersed in 2 ml of 0.1 M NaOH. 13 ml of Unisafe 1 mixture (Zinsser Analytic) were added, and radioactivity was counted in a liquid scintillation spectrophotometer.
Cellular Distribution of [3H]A2pm—To identify the step in peptidoglycan metabolism that was blocked by colicin M, the cellular distribution of [3H]A2pm incorporated in strain FB8r lysA was determined. The strain was grown, treated or not with colicin M (at t = 0), and labeled with [3H]A2pm (at t = 10 min) as described above, except that more radioactivity was used (50 kBq·ml-1). Cells were harvested 15 min later, i.e. 25 min after the addition of colicin M and just before the onset of cell lysis. The pellet of cells was resuspended in 2 ml of boiling water and maintained at 100 °C for 15 min. After centrifugation at 200,000 x g for 20 min, the pellet was resuspended in 800 µl of water, and the supernatant was lyophilized and resuspended into 400 µl of water. As earlier demonstrated (33, 50, 51), the pellet fraction contained the peptidoglycan and lipid intermediates I and II and the supernatant contained A2pm and the UDP-MurNAc-peptides as labeled compounds. To determine the amounts of peptidoglycan and lipid intermediates, aliquots of the pellet fraction were analyzed by TLC (solvent system I). Under these conditions, peptidoglycan remains at the origin and the mixture of lipids I and II migrates with an Rf value of 0.6. The analysis of the soluble fractions by TLC allowed the separation of A2pm from the pool of UDP-MurNAc-peptides and from the mixture of lipid I and II degradation products (see "Results"). An additional step of HPLC was then required to determine the relative amounts of the different UDP-MurNAc-peptides as well as the ratio of lipid I and II degradation products. This was achieved by using the Nucleosil column and isocratic elution with a mixture of 50 mM triethylammonium formate, pH 4.6, and methanol (94:6; v/v), at a flow rate of 0.6 ml·min-1. Under these conditions, A2pm, UDP-MurNAc-tripeptide, UDP-MurNAc-tetrapeptide, and UDP-MurNAc-pentapeptide were eluted at 5, 35, 47, and 62 min, respectively. 1-Pyrophospho-MurNAc-tripeptide, 1-pyrophospho-MurNAc-pentapeptide, and 1-pyrophospho-MurNAc-(pentapeptide)-GlcNAc, which were detected in extracts from colicin M-treated cells, eluted at 22, 38, and 60 min, respectively. Detection of radioactivity in HPLC effluents was performed with a radioactive flow detector (model LB-506-C1, Berthold France) using the Quicksafe Flow 2 scintillator (Zinsser Analytic) at 0.6 ml·min-1. Quantitation was carried out with the Winflow software (Berthold France).
Analysis and Quantitation of C55-OH in E. coli Cell Membranes—Cultures (300 ml) of BW25113 were treated or not with colicin M (at OD = 0.8) as described above, and cells were harvested 40 min thereafter, just before the onset of cell lysis. The cell pellets were resuspended in 0.1 M Tris-HCl, pH 7.4, and disrupted by sonication in the cold. The resulting suspensions were centrifuged at 4 °C for 30 min at 200,000 x g. Membrane pellets were resuspended in phosphate-buffer saline, pH 7.4, and the lipids were extracted by the chloroform-methanol method (52). The chloroform extracts containing C55-OH and its phosphorylated derivatives were dried, dissolved in the HPLC eluent, and analyzed on the Nucleosil column. Elution was performed with methyl alcohol-2-propyl alcohol (4:1, v/v) containing 10 mM phosphoric acid (17), at a flow rate of 0.6 ml·min-1. Peaks were detected at 210 nm. Quantitation of C55-OH and its phosphorylated derivatives was performed with respect to commercial standards injected in the same conditions. The C55-OH peak was collected, and the solvents were evaporated. The residue was taken up in ether, and the organic solution was washed with water to remove phosphoric acid. After evaporation of ether and lyophilization to remove residual water, the material was dissolved in 20 µl of methyl alcohol-2-propyl alcohol (1:1, v/v) and analyzed by mass spectrometry as described above.
Protein Monitoring—SDS-PAGE analysis of proteins was performed as described by Laemmli and Favre (53). Protein concentrations were determined using the Bradford procedure (54) 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.
Chemicals—DNA restriction enzymes were obtained from New England Biolabs. Oligonucleotides and DNA sequencing were done by MWG-Biotech, and 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, [4-14C]isopentenyl-pyrophosphate (1.5-2.2 GBq·mmol-1) from PerkinElmer Life Science Products, and [3H]A2pm (1.11-2.22 TBq·mmol-1) from ARC. N-Lauroyl sarcosine was purchased from USB, DDM was from Fluka, and Ni2+-NTA-agarose from Qiagen. UDP-MurNAc-peptides were prepared as previously described (55). Pure MraY translocase (40), MurG transferase (48), LdcA L,D-carboxypeptidase (46), PBP5 D,D-carboxypeptidase (45), BacA C55-PP phosphatase, and UppS C55-PP synthase (18) were prepared as described earlier. Antibiotics and reagents were from Sigma.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
) as only transformants carrying the empty vectors were isolated on 2YT-ampicillin plates, and complete lysis of the cell population occurred when transformation mixtures were grown in liquid medium. This toxic effect reflected the sensitivity of host cells to the colicin M they release in the growth medium. It indicated that a significant expression of the cma gene already occurred in the absence of inducer (IPTG) and further suggested that all three forms of colicin M were active and that the presence of a His6 tag at the N- or C-terminal extremity did not abolish its biological activity. The problem of toxicity was overcome either by co-expressing the cmi immunity gene on the same plasmid (pMLD170), or by using BW25113 fhuA::CmR that does not express the colicin M-outer membrane receptor as the host strain. Plasmids pMLD188, pMLD189, and pMLD190 were effectively successfully isolated in the latter genetic background, in total agreement with previous data from Harkness and Braun (56), which showed that colicin M should enter cells from the outside to exert its lethal effect.
BW25113 fhuA::CmR cells carrying the different expression plasmids were grown in 2YT-ampicillin medium and induced with IPTG for 3 h. Induced cells exhibited some morphological differences when observed by phase-contrast microscopy: they appeared as greatly enlarged rods and cell lysis frequently occurred after prolonged incubation. Accumulation in the cell content of a protein species of about 30 kDa was detected (Fig. 1), a value in agreement with that (29, 453) calculated from the DNA sequence of cma (29). A typical fractionation procedure of cell extracts showed that colicin M was found mainly in the soluble fraction, but significant amounts (10-20%) were also detected in the particulate fraction. The N-terminal- and C-terminal His6-tagged versions of colicin M were apparently overproduced to similar levels (Fig. 1) and their migrations on SDS-PAGE gels were consistent with their slightly higher molecular mass, 30,580 (Met-His6-Gly-Ser-extension) and 30,548 (-Arg-Ser-His6 extension), respectively.
|
8 ng of proteins of crude extracts), confirming that the presence of a His tag on the protein had no effect on its activity.
A large scale preparation of the two His6-tagged versions of colicin M was made from 2-liter cultures of BW25113 fhuA::CmR cells carrying either the pMLD189 or pMLD190 plasmid. Crude soluble extracts were purified on Ni2+-NTA-agarose and pure fractions, as judged by SDS-PAGE (Fig. 1), were dialyzed, concentrated to 1 mg·ml-1 of protein, and then conserved at -20 °C in the presence of 15% glycerol. This procedure yielded about 10 mg of either form of pure His6-tagged colicin M.
Pure colicin M preparations were assayed for bacteriolytic activity on E. coli growing cells. Both His6-tagged forms behaved similarly and induced rapid lysis of BW25113 cells at a concentration of about 10 ng·ml-1 (Fig. 2). It confirmed that the presence of the His tag at either the N-terminal or C-terminal end of the protein had no significant effect on the activity of the colicin.
|
We thus envisaged that the downstream steps leading to the formation of peptidoglycan lipid intermediates I and II could be targeted by the colicin. Interestingly, when a MraY translocase assay that consists in following the formation of radiolabeled lipid I from C55-P and UDP-MurNAc-[14C]pentapeptide was performed, an additional radioactive spot of unidentified product was observed when colicin M was present (Fig. 3B). Its absence when either the enzyme or the C55-P substrate was omitted from the reaction mixture, strongly suggested that it was a degradation product of lipid I. Incubation of purified [14C]lipid I (radiolabeled in the peptide moiety) with colicin M confirmed this assumption as a complete conversion of lipid I into the latter product was observed (Fig. 3C). The low migration of this product in the TLC conditions used suggested the loss of the C55 lipid moiety. To localize the site of cleavage, MurNAc-pentapeptide and its 1-phospho and 1-pyrophospho derivatives were generated by mild acid hydrolysis or treatments with pyrophosphatase or periodate of UDP-MurNAc-[14C]pentapeptide, respectively. From the behavior of these different compounds on HPLC (details under "Experimental Procedures"), it was concluded that the degradation product was 1-pyrophospho-MurNAc-pentapeptide. The other product resulting from the degradation of lipid I by colicin M was therefore C55-OH but this product was not labeled and consequently could not be detected in these experiments. To confirm the data, [14C]lipid I labeled this time in the lipid moiety was synthesized. Incubation of this substrate, [14C]C55-PP-MurNAc-pentapeptide, with colicin M effectively resulted in its quantitative conversion into [14C]C55-OH, as unambiguously confirmed by TLC analysis (in solvent system II, the Rf values of lipid I and its product were 0.20 and 0.98, and those of authentic standards of C55-PP, C55-P, and C55-OH were 0.36, 0.50, and 0.98, respectively).
|
-phosphate group should not be free. The fact that UDP-MurNAc-pentapeptide was not a substrate either clearly showed that colicin M was specific of C55-linked peptidoglycan precursors. We also analyzed the effects of colicin M on [14C]lipid II synthesized from lipid I and UDP-[14C]GlcNAc by purified MurG transferase. The same phenomena were observed, namely the cleavage of this lipid into two products, which were identified in that case as C55-OH and 1-pyrophospho-MurNAc(pentapeptide)-GlcNAc, and the absence of hydrolysis of the nucleotide precursor UDP-GlcNAc (data not shown). Under the in vitro assay conditions used, the specific activities of colicin M for lipid I and lipid II substrates were estimated at about 0.2 and 0.4 nmol·min·mg-1 of protein, respectively. To further confirm the nature of the degradation products, pure unlabeled lipids I and II were treated by colicin M and the degradation products were purified by HPLC ("Experimental Procedures") and analyzed by MALDI-TOF mass spectrometry. One main peak was observed in each case (Fig. 4), with m/z ratios of 967.2 and 1170.3, that perfectly matched the expected values for the [M-H]- ions of 1-pyrophospho-MurNAc-pentapeptide (C32H55N7O23P2, average molecular mass of 967.8 g·mol-1) and 1-pyrophospho-MurNAc(pentapeptide)-GlcNAc (C40H68N8O28P2, average molecular mass of 1171.0 g·mol-1), respectively. In both spectra, smaller peaks corresponding to cation adducts were present.
In Vivo Activity of Colicin M—Our data suggested that the effects of colicin M on E. coli cells, i.e. the inhibition of peptidoglycan synthesis followed by cell lysis, might result from the specific degradation of lipid intermediates I and II. To control the physiological significance of the phenomena observed in vitro, cell labeling experiments were performed that used radiolabeled A2pm as a specific marker of peptidoglycan metabolism. As previously reported, to ensure a specific and efficient labeling, a lysA mutant strain and particular growth conditions in minimal medium were used to reduce the internal pool of A2pm and block its conversion into lysine (33, 57). A rapid uptake of [3H]A2pm was observed after its addition to cultures of FB8r lysA cells. This was followed by a rapid incorporation of radioactivity into trichloroacetic acid-precipitable material (peptidoglycan), which was completed in about 30 min (Fig. 5). When colicin M at a final concentration of 50 ng·ml-1 was added to the culture 10 min before the addition of [3H]A2pm, the incorporation of radioactivity into peptidoglycan was first similar to that detected in untreated cells during the first 5 min of labeling but it rapidly stopped thereafter. The arrest of peptidoglycan synthesis was followed by the onset of cell lysis about 10 min later (Fig. 5).
The cellular distribution of [3H]A2pm was then determined in cells treated or not with colicin (see "Experimental Procedures" for details). In control cells UDP-MurNAc-pentapeptide appeared as the main labeled compound, accompanied by small amounts of UDP-MurNAc-tripeptide and lipid intermediates (Table 2). A very low amount of UDP-MurNAc-tetrapeptide was also detected. This pattern typical of E. coli cells had been consistently observed previously (51, 58, 59). In colicin M-treated cells, an accumulation of UDP-MurNAc-pentapeptide and the concomitant depletion of both lipid intermediates were observed. This was consistent with the previous assumption that a membrane step leading to the formation of lipid I was blocked by colicin M (27, 31). More interestingly, the additional spot corresponding to the mixture of lipid I and lipid II degradation products was also detected here (Fig. 6B). The resolution of this mixture by HPLC revealed that both degradation products were effectively present (Fig. 7A) but that the lipid I derivative was largely predominant. A third radiolabeled product was also detected (peak X in Fig. 7A). We hypothesized that the latter compound could be a degradation product of 1-pyrophospho-MurNAc-pentapeptide by the D,D- and L,D-carboxypeptidase activities that are known to be present in the cell content. These enzymes, which are involved in the remodeling and recycling of the peptidoglycan structure, remove the fifth and fourth residues of pentapeptide chains, respectively, yielding tetrapeptide- and tripeptide-containing "muropeptides." HPLC analysis of 1-pyrophospho-MurNAc-pentapeptide before and after treatment with purified PBP5 (D,D-carboxypeptidase) and LdcA (L,D-carboxypeptidase) confirmed that compound in peak X was 1-pyrophospho-MurNAc-tripeptide (Fig. 7B).
|
|
200 nmol·g-1 cell dry weight. Its identity was further confirmed by the MALDI-TOF mass spectrum of the collected material: although the molecular ion peak (calculated m/z ratio 768) was not visible, a peak of m/z ratio of 750.9, corresponding to the molecular ion after loss of water, was present (Fig. 4C). Such an elimination reaction in mass spectrometry conditions has already been observed for C55-OH (20, 60). This peak was absent in the spectrum of the similar fraction collected from the control membranes (data not shown). All these data clearly validated the physiological significance of the in vitro established enzymatic properties of colicin M and definitely demonstrated that this antibiotic protein acts by destroying lipid intermediates involved in peptidoglycan biosynthesis. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-lactam antibiotics that inhibit polymerization steps catalyzed by penicillin-binding proteins (31). As lipopolysaccharide O-antigen synthesis was concomitantly blocked, the metabolism of the carrier lipid C55-P that is shared by the two pathways clearly appeared as the target for colicin M (27, 32). However, the inhibited metabolic step was not precisely identified and the mechanism of action of colicin M remained unknown. We now revisited this question by purifying this protein to near homogeneity and analyzing in details its effects on cell wall peptidoglycan metabolism, using both in vitro and in vivo experiments. Our data show that purified colicin M exhibits enzymatic properties and catalyzes in vitro the hydrolysis of peptidoglycan lipid intermediates I and II. This is clearly the mode of action of colicin M as the degradation of the latter lipid intermediates was confirmed in E. coli growing cells by appropriate radiolabeling experiments. The enzymatic cleavage of the peptidoglycan precursors occurs between the undecaprenyl and 1-pyrophospho-MurNAc moieties. This is to our knowledge the first time such an activity is reported. The degradation products, namely C55-OH and either 1-pyrophospho-MurNAc-pentapeptide or 1-pyrophospho-MurNAc-(pentapeptide)-GlcNAc, are expected to be end products as they a priori could not be directly reused for de novo peptidoglycan synthesis. The fact that no C55-OH kinase activity has been identified to date in E. coli suggests that C55-OH either does not normally exist or could not be recycled into C55-P in this bacterial species. This could explain why C55-OH readily accumulates in colicin M-treated cells. However, a further degradation of the pyrophosphorylated degradation products into free MurNAc and peptides by specific glucosaminidase (NagZ), amidase (AmpD), and carboxypeptidase (LdcA) activities involved in the peptidoglycan recycling process (see Ref. 62 for references) was expected to occur. The present identification of 1-pyrophospho-MurNAc-tripeptide as a secondary degradation product supports this assumption.
|
|
|
The pool level of C55-P is generally assumed to be low (17, 51, 63) although it is required for the synthesis of multiple cell envelope components, and although huge amounts of precursors (several millions molecules per generation time) are translocated via this lipid across the cytoplasmic membrane. It was earlier considered as a potential regulatory factor controlling the flow of metabolites going through these different pathways (64, 65). The pool levels of the peptidoglycan lipid intermediates I and II are also quite low (51, 58, 59). Both the low size pool of the C55-P carrier lipid and the enzymatic mode of action of colicin M that results in the interruption of its recycling likely explain the powerful effect of colicin M. We effectively observed that pure colicin M provoked lysis of sensitive E. coli cells at extremely low concentrations, in the ng·ml-1 range (50-200 molecules per cell). It should be noted that Braun and co-workers (61) previously showed that colicin M rapidly lost activity (within 20 min) after its dilution in cell growth medium, because of a denaturation that could be partly overcome by addition of bovine serum albumin or nonionic detergents such as Triton X-100. As we did not check the stability of colicin M in our growth conditions and performed all our experiments in the absence of any additives, the minimal number of molecules needed to kill a single cell could probably still be lower. This is perfectly consistent with the earlier estimate that a single lethal unit, deduced from single hit kinetics, corresponded to 10 molecules of added colicin M (61). Amounts of colicin M that are accumulated in the cell content of the fhuA mutant following expression from the pMLD188 to pMLD190 plasmids are enormous as compared with those needed to kill cells when externally supplied in the growth medium. It confirms the previous observation that colicin M exhibits its bactericidal properties only when entering the cell from outside (56). The lipid I and lipid II intermediates, the colicin M targets, are synthesized by the MraY and MurG enzymes on the inner side of the cytoplasmic membrane and then translocated to the outer side of the membrane where the polymerization steps occur (10). The absence of deleterious effects observed for colicin M molecules produced inside cells is therefore surprising. This could be interpreted as a non accessibility of this enzyme to its targets from the cytoplasm side, suggesting that the latter could be either protected within MraY and MurG enzymes active sites or rapidly transferred to the outer leaflet of the membrane once synthesized.
Neither the nucleotide precursors UDP-MurNAc-pentapeptide and UDP-GlcNAc nor C55-PP are cleaved by colicin M, indicating a requirement for both the lipid and sugar moieties present in the structure of lipids I and II. Interestingly, colicin M was earlier shown to interfere with the biosynthesis of the O-antigen moiety of lipopolysaccharides (32). This could simply be an indirect effect of the depletion of the C55-P pool that itself results from the degradation of the peptidoglycan lipid intermediates by colicin M. Alternatively, it is tempting to speculate that the arrest of O-antigen synthesis also results from the degradation by colicin M of its respective lipid-linked precursor, C55-PP-GlcNAc, the wecA gene product (66). Experiments previously reported by Harkness and Braun (32) did not allow to discriminate between the two hypotheses. Further experiments with the purified protein and different synthetic C55-PP-sugar derivatives are underway to analyze in more details the kinetic properties and substrate specificity of colicin M.
As is the case for all colicins, colicin M is organized in three distinct domains, the C-terminal one being responsible of its lethal activity (28). Interestingly, an alignment of amino acid sequences had revealed some homology between the C-terminal domain of colicin M (215-249 region) and the region around the active-site serine residue in the penicillin-binding proteins (29). Whether this discrete homology could be related to the presently identified activity of colicin M remains to be demonstrated. It is noteworthy that these two classes of proteins bind to (and compete for) the same substrate, lipid II, but however they catalyze quite different reactions of degradation and polymerization, respectively. The local sequence homologies could then reflect some evolutionary conserved binding domain for this complex substrate. Interestingly in this respect, multiple hydroxylamine-generated inactive forms of colicin-M were previously described that carried mutations on residues 193-197 and 223-252, suggesting that residues important for substrate binding or catalysis might effectively reside in the latter regions. The deletion of the two terminal amino acid residues (Lys-Arg) at the C-terminal extremity of the protein was shown to abolish its activity (67), confirming the importance of the integrity of the C-terminal domain. Addition of a His tag at this extremity had however no apparent effect on its activity, as shown in the present report. All of this available information will be useful in future work aimed at characterizing this protein in more detail, both biochemically and structurally, in the light of its newly identified activity.
|
FOOTNOTES |
|---|
1 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).
2 Supported by the European Community.
3 To whom correspondence should be addressed: Laboratoire des Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR8619 CNRS, Université Paris-Sud, Bâtiment 430, 91405 Orsay Cedex, France. Tel.: 33-1-69-15-48-41; Fax: 33-1-69-85-37-15; E-mail: dominique.mengin-lecreulx{at}ebp.u-psud.fr.
4 The abbreviations used are: C55-P, undecaprenyl phosphate; GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid; C55-PP, undecaprenyl pyrophosphate; C55-OH, undecaprenol; PP, pyrophosphoryl; A2pm, diaminopimelic acid; lipid I, C55-PP-MurNAc-pentapeptide; lipid II, C55-PP-MurNAc-(pentapeptide)-GlcNAc; Ni2+-NTA-agarose, nickel-nitrilotriacetate-agarose; DDM, n-dodecyl--D-maltoside; IPTG, isopropyl--D-thiogalactopyranoside; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
