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Originally published In Press as doi:10.1074/jbc.M400228200 on April 21, 2004

J. Biol. Chem., Vol. 279, Issue 27, 28233-28242, July 2, 2004
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Siderophore Peptide, a New Type of Post-translationally Modified Antibacterial Peptide with Potent Activity*

Xavier Thomas{ddagger}, Delphine Destoumieux-Garzón{ddagger}, Jean Peduzzi{ddagger}, Carlos Afonso§, Alain Blond{ddagger}, Nicolas Birlirakis¶, Christophe Goulard{ddagger}, Lionel Dubost{ddagger}, Robert Thai||, Jean-Claude Tabet§, and Sylvie Rebuffat{ddagger}**

From the {ddagger}Laboratoire de Chimie et Biochimie des Substances Naturelles, UMR 5154 CNRS USM 502, the Département Régulations, Développement et Diversité Moléculaire, Muséum National d'Histoire Naturelle, 63 Rue Buffon, 75005 Paris, the §Laboratoire de Chimie Structurale Organique et Biologique, UMR 7613 CNRS, Université Pierre et Marie Curie, 4 Place Jussieu, 75252 Paris Cedex 5, the CNRS, Laboratoire de RMN à Haut Champ, Institut de Chimie des Substances Naturelles, 91190 Gif-sur-Yvette, and the ||Département d'Etude et d'Ingénierie des Protéines, CEA Saclay, 91190 Gif-sur-Yvette, France

Received for publication, January 9, 2004 , and in revised form, April 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Microcin E492 (MccE492, 7886 Da), the 84-amino acid antimicrobial peptide from Klebsiella pneumoniae, was purified in a post-translationally modified form, MccE492m (8717 Da), from culture supernatants of either the recombinant Escherichia coli VCS257 strain harboring the pJAM229 plasmid or the K. pneumoniae RYC492 strain. Chymotrypsin digestion of MccE492m led to the MccE492m-(74–84) C-terminal fragment that carries the modification and that was analyzed by mass spectrometry and nuclear magnetic resonance at natural abundance. The 831-Da post-translational modification consists of a trimer of N-(2,3-dihydroxybenzoyl)-L-serine linked via a C-glycosidic linkage to a {beta}-D-glucose moiety, itself linked to the MccE492m Ser-84-carboxyl through an O-glycosidic bond. This modification, which mimics a catechol-type siderophore, was shown to bind ferric ions by analysis of the collision-induced dissociation pattern obtained for MccE492m-(74–84) by electrospray ion trap mass spectrometry experiments in the presence of FeCl3. By using a series of wild-type and mutant isogenic strains, the three catechol-type siderophore receptors Fiu, Cir, and FepA were shown to be responsible for the recognition of MccE492m at the outer membrane of sensitive bacteria. Because MccE492m shows a broader spectrum of antibacterial activity and is more potent than MccE492, we propose that by increasing the microcin/receptor affinity, the modification leads to a better recognition and subsequently to a higher antimicrobial activity of the microcin. Therefore, MccE492m is the first member of a new class of antimicrobial peptides carrying a siderophore-like post-translational modification and showing potent activity, which we term siderophore-peptides.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Microcins are low molecular weight antibacterial peptides secreted by enterobacteria, mostly Escherichia coli. As they are active against phylogenetically related Gram-negative bacteria, it has been suggested that they balance the intestinal microbial flora (1). Although many antimicrobial peptides of microbial origin are produced by large multidomain enzyme complexes, the peptide synthetases, microcins are synthesized through the ribosomal pathway. The microcin genetic systems include a series of genes encoding the microcin precursor, modification enzymes, secretion factors, and immunity proteins. These gene clusters give rise to a broad diversity of microcin structures and mechanisms of action (2, 3).

Microcin E492 (MccE492) was found for the first time in the culture medium of the Klebsiella pneumoniae RYC492 fecal strain (4). It was shown to be chromosomally encoded, and the genetic determinants necessary for its production, secretion, and immunity were cloned into the pJAM229 plasmid (5, 6). MccE492 was expressed in the recombinant E. coli VCS257 strain harboring the pJAM229 plasmid (5) and characterized as an 84-residue peptide (GETDPNTQLLNDLGNNMAWGAALGAPGGLGSAALGAAGGALQTVGQGLIDHGPVNVPIPVLIGPSWNGSSSGYNSATSSSGSGS) of 7886 Da (7). MccE492 is a membrane-active antibacterial peptide (8, 9), which depolarizes the E. coli cytoplasmic membrane (9, 10). However, this membrane activity alone is not believed to account for the peptide antibacterial properties, which are thought instead to rely on a receptor-mediated mechanism (9, 11). In agreement with this hypothesis, enterobactin (12), also named enterochelin (13), was shown to be an MccE492 antagonist (14). Enterobactin is an iron-chelating compound (siderophore) produced by enterobacteria (15) and also by some Streptomyces species (16). This tricatechol derivative of a cyclic triserine lactone (cyclic trimer of N-(2,3-dihydroxybenzoyl)-L-serine, DHBS)1 as well as its breakdown products, the linear DHBS trimer, dimer, and monomer, are able to transport ferric ions into enterobacteria (12, 17–19). The catecholate iron-siderophore receptors involved, Fiu, Cir, and FepA, have been suggested to recognize MccE492 (9, 11, 20). In addition, this recognition was found to be TonB-dependent (9).

From the first description of the MccE492 structure (7), it was thought that together with ColV, MccH47, and MccL, MccE492 belonged to a class of unmodified microcins (2). However, the lack of post-translational modification in the MccE492 structure (7) was questionable because of the presence within the large genetic system of MccE492 of several genes, three of which displayed homologies with genes encoding glycosyltransferases (mceC), acyltranferases (mceI), and enterobactin esterases (mceD), whereas no homology was found for mceJ and mceE (21). In recent work, inactivation of the mceC, mceI, or mceJ genes was shown to result in loss of MccE492 antibacterial activity. Because such inactivation did not result in any modification of the primary structure, the genes concerned were proposed to encode molecular chaperones involved in the acquisition of the MccE492 active conformation rather than modification enzymes (11).

In this study, we report for the first time the isolation and characterization of a modified form of MccE492. The structure of this post-translational modification was determined by mass spectrometry and nuclear magnetic resonance. It consists of a sugar moiety carrying a linear trimer of DHBS that is shown to bind a ferric ion. The antibacterial activity of MccE492m was examined against wild-type and mutant strains, and the membrane receptors involved in the microcin recognition at the outer membrane of the sensitive strains were determined. We show here the advantage in terms of activity of the modification carried by MccE492, which thus appears as the first member of a new class of post-translationally modified antibacterial peptides that we name siderophore-peptides.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Plasmids—Microcin production was performed in E. coli VCS257 (Stratagene), E. coli C600, E. coli C600 aroB– harboring the pJAM229 plasmid (5), and K. pneumoniae RYC492 (4). The bacteria used to establish the spectrum of antibacterial activity were those described previously (9). Micrococcus luteus CIP5345 was from the Pasteur Institute collection (Paris, France). Studies of the MccE492m mechanism of action included a series of wild-type and mutant E. coli strains listed in Table I. Complementation of E. coli W3110-KP1344 (tonB–) was performed with the pMS7 plasmid (KanR, encoding TonB) (9). Similarly, complementation of E. coli C600 was performed with the pHX405 plasmid (AmpR, encoding FhuA) (22).


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  		TABLE I
Wild type and mutant E. coli strains used in the study of the MccE492m mechanism of action

 
Large Scale Purification of MccE492 and MccE492m—The microcin-producing strains E. coli VCS257 and E. coli C600 harboring the pJAM229 plasmid as well as K. pneumoniae RYC492 were grown for 16 h at 37 °C with vigorous shaking in 2 liters of M63 minimum medium containing 0.25 g/liter MgSO4 and 1 mg/liter thiamine. Alternatively, glucose or trisodium citrate (0.25%) was used as a source of carbon, and casamino acids (Difco) or bactotryptone (BD Biosciences) was used at 1 g/liter as a source of amino acids. After cell removal by centrifugation (5000 x g, 15 min, 4 °C), the supernatant was loaded onto a Sep-Pak C8 cartridge (Waters) previously equilibrated with 0.1% aqueous trifluoroacetic acid (pH 2). The cartridge was washed once with 0.1% aqueous trifluoroacetic acid, and three stepwise elutions were performed with 30, 35, and 40% ACN in 0.1% aqueous trifluoroacetic acid. The absorbance was monitored at 226 nm, and the fractionation was performed at a flow rate of 10 ml/min. The 40% ACN Sep-Pak fraction was concentrated under vacuum (Rotavapor, Büchi Labortechnik) before being lyophilized. The lyophilized fraction was resuspended in 40% ACN in 0.1% aqueous trifluoroacetic acid and subjected to RP-HPLC on an analytical Inertsil ODS2 column (5 µm, 250 x 4.6 mm, Interchim, France). Isocratic elution with 39% ACN in 0.1% aqueous trifluoroacetic acid at a flow rate of 1 ml/min allowed successive elution of MccE492 and MccE492m. Absorbance was monitored at 226 nm.

Chymotrypsin Digestion and Isolation of the MccE492m-(74–84) Fragment—MccE492 or MccE492m (150 nmol) dissolved in 25 mM NH4HCO3 (pH 7.8) was digested with chymotrypsin (Sigma) at an enzyme to substrate ratio of 1:50 (w/w) for 90 min at 25 °C. The reaction was stopped by acidification with glacial acetic acid, and samples were dried under vacuum (SpeedVac, Savant). The proteolytic fragments were separated on an Inertsil ODS2 column (5 µm, 250 x 4.6 mm), with a linear gradient of 0–60% ACN in 0.1% aqueous trifluoroacetic acid over 90 min at a flow rate of 1 ml/min. Absorbance was monitored at 226 nm.

SDS-PAGE—Purified MccE492 and MccE492m were loaded onto a 16.5% SDS-Tricine polyacrylamide gel (23) without boiling and detected by silver staining. The molecular weight marker was the ultra-low range color marker (Sigma).

Amino Acid Composition and Microsequence Analysis—Peptides (0.5 nmol) were hydrolyzed under vacuum for 24 h at 110 °Cin6 M constant boiling HCl (Sigma). Separation by ion-exchange HPLC of the resulting amino acids and post-column derivatization with ninhydrin were performed as described previously (9). Lyophilized purified samples (10 nmol) of MccE492 and MccE492m and of selected chymotrypsin digest fragments of MccE492 and MccE492m were resuspended in MilliQTM water (Millipore) or methanol. Each sample was loaded and argon-dried on a Biobrene-coated filter and then subjected to N-terminal sequencing according to Edman degradation on a Procise 492 automatic protein sequencer (Applied Biosystems, PerkinElmer Life Sciences).

Liquid Growth Inhibition Assay—Antibacterial assays were performed as described previously (9). Briefly, serial dilutions of MccE492 or MccE492m (10 µl) were incubated in a 96-well microtiter plate with a 90-µl suspension of bacteria in midlogarithmic growth phase diluted in poor-broth nutrient medium (1% bactotryptone (BD Biosciences), 0.5% NaCl) to a starting absorbance of 0.001 at 620 nm. Inhibition of growth was determined by measuring the absorbance at 620 nm with a Ceres 900 microplate recorder (Bioteck Instruments) after a 16-h incubation at 30 °C. Minimal inhibitory concentrations (MICs) were defined as the lowest concentrations that cause 100% growth inhibition and were determined in triplicate.

Mass Spectrometry—The purified MccE492, MccE492m, and the chymotryptic peptides were analyzed for molecular mass determination by MALDI-TOF MS with a Voyager-de-Pro spectrometer (Applied Biosystems) operating in the linear or reflectron mode with positive ion detection. Samples were loaded on matrix crystals obtained by rapid evaporation of a saturated solution of {alpha}-cyano-4-hydroxycinnamic acid in 50% ACN. The dry droplet method was chosen for target preparation. Bovine insulin (MH+ at m/z 5734.59), thioredoxin (MH+ at m/z 11674.48), and apomyoglobin (MH+ at m/z 16952.56) were used for calibration. Sequencing of the purified MccE492-(74–84) and MccE492m-(74–84) and analysis of the structure of the modification were performed by using hybrid ESI-Qq-TOF (Q-Star, Applied Biosystems) and nano-ESI-IT (ESQUIRE 3000, Bruker Daltonics) instruments operating in the positive and negative ion detection modes. Peptide samples (10 pmol/µl) were prepared in 50% ACN. Formic acid (0.5%) was added for positive ion mode analysis. In Fe3+/peptide binding experiments, a solution of FeCl3 was added to obtain metal to peptide ratios varying from 1:1 to 8:1. CID experiments (MS2 and MS3) were performed from selected ions submitted to resonant excitation amplitude from 0.5 to 1.5 VP-P. 2 µl of sample were loaded into Proxeon (Odense, Denmark) nano-electrospray tips. Accurate mass measurement was performed on the ESI-Qq-TOF instrument. Internal calibration was applied from the theoretical m/z ratio of identified b ions. The Roepstorff nomenclature (24) was used to describe peptide fragmentations.

NMR Spectroscopy—MccE492m-(74–84) was dissolved at a final 2.5 mM concentration either in CD3OH/H2O (50:50, v/v) or CD3OD/D2O (50:50, v/v). One- and two-dimensional NMR spectra were recorded on Bruker Avance 400 and DRX 800 spectrometers (Wissembourg, France) using a BBI probe and a TXI cryoprobe, respectively, both equipped with shielded gradients z. 1H and 13C chemical shifts were measured using the methanol methyl resonance as internal reference taken at 3.313 and 49.3 ppm, respectively. Conventional two-dimensional experiments 1H-1H double quantum filtered correlation spectroscopy, TOCSY (an 80-ms z-filtered DIPSI-2 sequence was used for Hartmann-Hahn mixing), incredible natural abundance double quantum transfer, NOESY (200 ms mixing time), as well as natural abundance 1H-13C HSQC (using adiabatic decoupling during acquisition) and HMBC (containing a low-pass filter) were performed for each sample. In both spectrometers, solvent suppression was obtained either by presaturation or by means of pulsed field gradients used in a water suppression by gradient tailored excitation scheme (25) or for coherence selection. Data were processed with the XWINNMR 3.1 software. Typically, matrixes were zero filled and forward linear prediction at the indirect dimension was applied prior to square sine bell apodization (shifted by {pi}/3) and Fourier transformation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of a Modified Form of MccE492
E. coli VCS257 harboring pJAM229 was grown in M63 minimal medium containing either glucose or citrate as a source of carbon and casamino acids or bactotryptone as a source of amino acids. No major difference in terms of antibacterial activity was observed upon carbon source replacement (data not shown), glucose thus being maintained for subsequent cultures. Conversely, significantly higher activities were detected in the supernatants of cultures using bactotryptone instead of the conventionally used casamino acids-supplemented medium (data not shown). This correlated with a much faster growth of the MccE492-producing strain in the bactotryptone-containing medium, as compared with the casamino acids strain (Fig. 1A). Therefore, when the cultures were stopped after a 16-h incubation at 37 °C, the bacteria were at the end and beginning of the exponential phase of growth, respectively. MccE492 was purified by RP-HPLC from both supernatants as described under "Experimental Procedures." The purified fractions were both active against E. coli ML35p. Surprisingly, although the microcin purified from the casamino acid-containing medium showed the expected electrophoretic behavior (9) on a silver-stained SDS-polyacrylamide gel (Fig. 1B), the peptide issued from the bactotryptone-containing medium exhibited a higher apparent molecular mass (Fig. 1B). MALDI-TOF MS measurements showed that this peptide exhibited a molecular mass 831 Da higher (MH+ at m/z 8718) than the expected value (MH+ at m/z 7887) (Fig. 1C). Both peptides displayed an identical 37-residue N-terminal amino acid sequence by automatic Edman sequencing and a similar amino acid composition, with the exception of three extra serine residues in the higher molecular weight peptide (Table II). These results indicated the unknown peptide was a modified MccE492 bearing an 831-Da post-translational modification. It was consequently named MccE492m. Both the unmodified and modified peptides were obtained in high yield, using their optimized culture medium (2.1 mg/liter MccE492 and 9.5 mg/liter MccE492m for M63-casamino acids and M63-bactotryptone media, respectively). MccE492m was also purified from the culture supernatant of the wild-type K. pneumoniae RYC492 and was characterized by MALDI-TOF MS.



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  		FIG. 1.
Isolation and characterization of a novel form of MccE492. A, E. coli VCS257 harboring the pJAM229 plasmid was cultured at 37 °C in casamino acid () or bactotryptone ({square})-containing M63 minimal medium; the growth curves were obtained by A620 measurement. B, silver-stained Tris-Tricine SDS-PAGE analysis of the active peptides purified from the culture supernatants after a 16-h incubation (lane 1, casamino acids medium; lane 2, bactotryptone medium). C, MALDI-TOF MS analysis of MccE492 and MccE492m, showing the MH+ ions.

 


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  		TABLE II
Amino acid composition of MccE492, MccE492m, and their chymotryptic fragments MccE492-(74–84) and MccE492m-(74–84)

The results are expressed as the number of residues/mol. Compositions are calculated from duplicate (MccE492-(74–84) and MccE492m-(74–84) fragments) or quintuplicate (microcins) 24-h HCl hydrolysates.

 
Antibacterial Activity of MccE492m
The antibacterial activity of MccE492m was compared with that of the unmodified microcin against Gram-negative and Gram-positive bacteria. Like MccE492, MccE492m was highly active against all the E. coli and the Salmonella enteritidis strains tested, with MIC values in the 40–80 nM range and at 150 nM, respectively (Table III). Moreover, MccE492m was 4–8 times more active than MccE492 against these strains. In addition, MccE492m was active against Enterobacter cloacae and K. pneumoniae, whereas no activity could be detected for the unmodified microcin in the range of 0.02–10 µM (Table III). As observed previously for MccE492 (9), MccE492m was inactive in the same concentration range against Gram-positive bacteria and Gram-negative bacteria not belonging to enterobacteria, such as Vibrio species. Therefore, MccE492m is a more potent antibacterial peptide than MccE492, characterized by a broader spectrum of activity against enterobacteria and by lower MICs.


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  		TABLE III
Comparative antibacterial activity of MccE492 and MccE492m

 
Localization of the MccE492m Modification
In order to localize the modification in the MccE492 sequence, both MccE492 and MccE492m samples were subjected to chymotrypsin digestion. The RP-HPLCs of the resulting digests were identical (data not shown), with the exception of (i) one fast eluting peak present in the MccE492 digest exclusively, and (ii) a group of three slower eluting peaks observed in the MccE492m digest, which was absent from the MccE492 chromatogram. The MALDI-TOF and ESI-Qq-TOF mass spectra in positive mode of the corresponding fractions showed similar [M + H]+ ions at m/z 1772.6 and [M + 2H]2+ ions at m/z 886.8 for the three MccE492m-specific peaks, whereas the MccE492-specific peak led to an [M + H]+ ion at m/z 941.4, which appeared shifted to lower m/z ratios consistent with a mass difference of 831.2 Da (data not shown). An identical amino acid sequence (NSATSSSGSGS), corresponding to the 74–84 C-terminal residues of MccE492, was determined by Edman degradation for all the MccE492- and MccE492m-specific peptides. Because the three peaks in the MccE492m digest showed identical MS and Edman sequencing results, they were assumed to be three isomers or conformers. Therefore, further analyses were performed with the most abundant peptide form. The calculated mass for the 11-residue peptide sequence perfectly matched the 940.4-Da molecular mass determined for the MccE492-specific peptide, which was termed MccE492-(74–84). Because the molecular mass of the peptide isolated from the MccE492m digest (1771.6 Da) was 831.2 Da higher than that of MccE492-(74–84), it was deduced that the whole post-translational modification was carried by the 74–84 C-terminal peptide, which was named MccE492m-(74–84). CID of both MccE492-(74–84) and MccE492m-(74–84) was performed by ESI-MS in the positive ion mode (Fig. 2), generating two independent series of acylium (b-type) and ammonium (y-type) ions. The b ion series of both peptides, as well as the y ion series of MccE492-(74–84), unambiguously described the NSATSS-SGSGS sequence. By contrast, the MccE492m-(74–84) y ion series was shifted to upper m/z ratios consistent with a mass increase of 831.2 Da as compared with the MccE492-(74–84) y ion series (Fig. 2A). The ion at m/z 937.2, which is consistent with a protonated serine with an additional m/z ratio of 831.2, was attributed to the MccE492m-(74–84) y1 ion. The y2 to y8 ion series showed systematically this same shift in m/z ratio. Altogether, these results unambiguously indicated that the entire modification of MccE492m was carried by the microcin Ser-84 C-terminal residue.



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  		FIG. 2.
MS data on MccE492m-(74–84) showing the localization and the structure of the modification. A, ESI-MS2 spectrum of [M + 2H]2+ (m/z 886.8) in the positive mode, showing the two b- and y-type ion series: y-type ions that are shifted by an m/z ratio of 831.2, as compared with the expected values, are indicated by stars. B, ESI-MS2 spectrum of [M–2H]2– (m/z 884.8) in the negative mode; inset shows an enlargement of the m/z 600–950 region. C, ESI-MS3 spectrum of m/z 848.2 in the negative mode, showing the loss of 2 DHBS units. D, CID pattern of the 831.2-Da modification obtained by MSn, showing the main ions in agreement with the proposed structure.

 
Characterization of the Modification Carried by MccE492m
Characterization of a DHBS Trimer—In addition to the b- and y-type ion series observed as positive product ions, the CID spectrum of MccE492m-(74–84) obtained with the Qq-TOF instrument showed an intense ion at m/z 224, indicative of DHBS (data not shown) (26), which resulted in the corresponding fragment ions. Identification of DHBS as a component of the modification was consistent with the observation of three extra serine residues in the modified fragment as compared with the unmodified one (Table II). The ESI-IT mass spectrum of MccE492m-(74–84) recorded in the negative mode showed the [M – H]– and [M–2H]2– ions at m/z 1770.6 and 884.8, respectively (data not shown). When submitted to CID experiments, they led to a common deprotonated product ion at m/z 848.2 (Fig. 2B), which corresponded to the mass of the modification with an additional water molecule. Consecutive losses of 241.1 and 223.0 mass units were observed from (i) the [M – H]– ion leading to ions at m/z 1529.5 and 1306.4 (data not shown), and (ii) the m/z 848.2 ion (Fig. 2C). Such losses indicated that 2 serially linked DHBS units were located at the end of the modification. The CID pattern resulting from sequential MSn experiments thus showed that the modification contained 2 DHBS units and consisted of a single block linked to the C-terminal serine (Fig. 2, B and C).

An NMR study of MccE492m-(74–84) was conducted at 800 MHz, in order to determine the remaining part of the modification structure (Fig. 3). Spin system identification of the MccE492m-(74–84) peptide moiety was obtained by a combination of COSY-DQF and TOCSY experiments in CD3OH/H2O mixed solvent, using the amide proton resonances as starting point. Sequential assignment then resulted from the NOESY experiment (27), and the NSATSSSGSGS sequence was easily confirmed from dNN(i, i + 1) and d{alpha}N(i, i + 1) inter-residual NOE cross-peaks observed all along the sequence (data not shown). The three remaining low field amide protons in the TOCSY spectrum were assigned unambiguously to the serine parts of 3 DHBS units (Fig. 4A), consistent with the three extra serines characterized in MccE492m. The aromatic moieties of the three DHBS were further assigned by the combined use of TOCSY, HSQC, and HMBC data. In each DHBS unit, linkage of the dihydroxybenzoate ring to the serine part resulted from HMBC 2J cross-peaks involving H8'/C7', H8''/C7'', and H8'''/C7''' and intra-residue NOE correlations between the H6', H6'', and H6''' aromatic protons and their respective amides H8', H8'', and H8'''. The presence of a DHBS trimer in the modification was clearly evidenced first by the identification of the CO11', CO11'', and CO11''' carbonyls (Fig. 3) using the H{alpha}9'/CO11', H{alpha}9''/CO11'', and H{alpha}9'''/CO11''' 2J cross-peaks, followed by sequencing via the two 3J correlations involving the C11'' and C11''' carbonyl groups, and the methylene protons H10' and H10'' in the HMBC spectrum. It should be mentioned that inspection of the amide coupling constants (given in supplemental Table A) suggests that any particular secondary structure should be negligible under the conditions of these studies.



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  		FIG. 3.
Structure of the MccE492m post-translational modification. The main 2JC-H and 3JC-H HMBC correlations affording evidence of the structure are shown as arrows.

 



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  		FIG. 4.
MccE492m-(74– 84) NMR data affording evidence of the structure of the MccE492m post-translational modification. A, TOCSY spectrum of MccE492m-(74–84) showing the spin systems of the 11 amino acids and the additional spin systems of the 3 DHBS units. B, parts of the 1H-13C HMBC (F2, 7.4–6.9 ppm; F1, 84–81 ppm) and NOESY (F2, 7.4–6.9 ppm; F1, 4.2–3.4 ppm) spectra showing the linkage of {beta}-D-Glc to DHBS1 through a C-glycosylation. C, part of the HMBC spectrum (F2, 4.7–3.8 ppm; F1, 174–169 ppm) showing the linkage of {beta}-D-Glc to Ser-11 through an O-glycosylation.

 
Identification of a {beta}-D-Glucose—In order to analyze the remaining part of the modification, the NMR experiments were repeated with a sample in perdeuterated solvent CD3OD/D2O. A sugar unit was identified, attributed to {beta}-D-Glc, mainly from the 1H and 13C chemical shifts (supplemental Tables A and B) and its proton NOE pattern (Table IV). The H1 and H2 protons were in a trans-diaxial position, as shown by their characteristic 9.7-Hz coupling constant. The H1, H3, and H5 protons occupied axial positions on the same side of the sugar ring, because H1/H3, H1/H5, and H3/H5 NOE correlations were observed. As the H4 and H2 protons both showed NOE correlations with H4' and H6' of the DHBS1 unit, H4 was also in an axial position and on the same face of the sugar ring as H2. The attachment of the {beta}-D-Glc C-1 to the C-5' of DHBS1 was deduced from the following observations: (i) 3J couplings between the C-1 and both the H4' and H6' protons in the HMBC spectra, (ii) a 2J coupling between the C-5' and H1, and (iii) a series of NOEs involving the H4' and H6' DHBS1 protons and the {beta}-D-Glc protons (Fig. 3 and Table IV). Complete 1H and 13C assignments were made for the modified fragment MccE492m-(74–84) (supplemental Tables A and B).


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  		TABLE IV
NMR correlations affording complete evidence of the modification present in MccE492m-(74–84)

 
Determination of the Overall Structure of the Modification— The 831.2-Da modification was found to be linked to the C-terminal carboxyl group of the MccE492m-(74–84) Ser-11 by the HMBC correlation between the {beta}-D-Glc methylene proton H6 and the carbonyl group of Ser-11, itself identified through HMBC cross-peaks to the serine H{alpha} and H{beta} protons (Fig. 4C and Table IV). Finally, the 3J correlation involving CH2{beta}10' and the free CO11' carboxyl group unambiguously assigned the direction of linking of the string of 3 DHBS units, with regard to the {beta}-D-Glc.

The proposed structure was totally confirmed by complementary analysis of the MSn data. The presence of a C-glycosidic bond between {beta}-D-Glc and DHBS1 was in complete agreement with the CID pattern of the selected [M – H]– ion, which showed consecutive losses leading to ions at m/z 384, 340, and 220, together with the absence of an ion at m/z 179, typical of a deprotonated glucose entity which would have been formed upon cleavage of an O-glycosidic bond (data not shown). The consecutive loss of a CO2 molecule from the ion at m/z 384 (Fig. 2D) also totally confirmed the connection of the 3 DHBS units in regard to the {beta}-D-Glc. Finally, accurate mass measurements performed on the Qq-TOF instrument confirmed the mass of the modification with a relative error between the theoretical and experimental mass of 6 ppm ([M – H]– of the modification: theoretical mass calculated for C36H38N3O21, 848.200; experimental mass, 848.205). Taken together these data account for a post-translational modification consisting of 3 connected DHBS units linked to the Ser-84 C-terminal residue of MccE492 through a {beta}-D-Glc moiety, which is attached to DHBS1 by a C-glycosidic bond (Fig. 3).

IT-MS Study of the Fe3+-binding Properties of MccE492m-(74–84)
The ferric ion-binding properties of MccE492m were assayed by ESI-MS (Fig. 5), which is considered a powerful method for studying the ion-binding properties of peptides or proteins (28–30). In order to observe metal binding specificity, MS spectra of MccE492 and MccE492m recorded in the presence of Fe3+ ions were compared. Adding up to 8 eq of FeCl3 to MccE492-(74–84) did not modify the mass spectrum recorded in positive mode, which only displayed sodiated [M + Na]+ and [M + 2Na–H]+ ions at m/z 963.3 and 985.3 as major species (data not shown). By contrast, addition of 2 eq of FeCl3 to MccE492m-(74–84) resulted in a mass spectrum obtained in positive mode that exhibited a major [(M – H) + FeIII]2+ species at m/z 913.3 accompanied by an [(M – 2H) + FeIII]+ ion at m/z 1826.6 (data not shown), both indicative of the specific binding of one Fe3+ ion. Furthermore, CID of the cationized ions was carried out in order to locate the metal-binding sites. CID of the m/z 913.3 ion (Fig. 5A) resulted in the series of b-ions that was previously shown to characterize the 74–84-residue sequence (described above under "Localization of the MccE492 Modification"), whereas the y-type ion series was shifted to higher masses by an m/z ratio consistent with a 52.9-Da mass increase. The resulting (y – 3H + FeIII) ion series indicated that Fe3+ was bound to the MccE492m-(74–84) C terminus. The mass spectrum of MccE492m-(74–84) recorded in negative mode in the presence of 2 eq of FeCl3 showed two ions at m/z 911.6 and 1823.6 (data not shown). These were attributed to [(M – 5H) + FeIII]2– and [(M – 4H) + FeIII]– respectively, thus confirming the attachment of one ferric ion to the modified fragment. CID of [(M – 4H) + FeIII]– showed consecutive losses of 241.1 and 222.7 mass units (data not shown), which corresponded to the uncationized DHBS3 and DHBS2 units, respectively, leading to the m/z 1359.3 ion (data not shown), which was attributed to [M – 2(DHBS – 2H) + FeIII]. The CID of [(M – 5H) + FeIII]2– led to (i) an [(M – 4H) – DHBS + FeIII]– ion at m/z 1582.4, and (ii) two complementary ions at m/z 516.0 and 1306.5 attributed to [2(DHBS – 2H) + FeIII]– and [(M – H) – 2DHBS]–, respectively (Fig. 5B). This CID pattern shows on the one hand the direct loss of 1 DHBS unit from [(M – 5H) + FeIII]2–, leading to the m/z 1582.4 ion that carries Fe3+, and on the other hand the occurrence of two competitive dissociations leading either to 2 DHBS units carrying Fe3+ or to the loss of 2 DHBS units from [M – H]–. As expected, this CID pattern indicates a preferential binding of Fe3+ to the region of MccE492m-(74–84) that contains the DHBS units.



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  		FIG. 5.
Characterization of the Fe3+-binding properties of MccE492m-(74–84) by ESI-MS. A, CID mass spectrum of the selected [(M – H) + FeIII]2+ ion recorded in positive mode in the presence of 2 eq of FeCl3, showing the unshifted b ion series and the y ion series shifted by 52.9 units (see stars), typical of the specific binding of FeIII. B, CID mass spectrum in negative mode of the selected [(M – 5H) + FeIII]2– (m/z 911.6), showing the two complementary ions at m/z 1306.5, [(M – H) – 2DHBS]–, and 516.0, [2(DHBS – 2H) + FeIII]–, which do not carry and which carry FeIII, respectively.

 
MccE492m Biosynthesis
In order to understand better the pathways leading to the post-translational modification of MccE492, and due to the structural similarities between the microcin modification and catecholate-type siderophores, MccE492 was expressed in an enterobactin-deficient strain. The E. coli C600 aroB– used in this study is impaired at a very early stage in the enterobactin biosynthetic pathway and is therefore unable to synthesize 2,3-dihydroxybenzoate, and consequently enterobactin and DHBS. The pJAM229 plasmid was transfected into both the wild-type and aroB– mutant of E. coli C600. The recombinant bacteria were cultured in bactotryptone-containing M63 medium until the end of the exponential growth phase. The 40% ACN fractions issued from the Sep-Pak fractionation of the culture supernatants were then analyzed by ESI-Qq-TOF. MccE492m was found to be predominantly expressed in the wild-type aroB+ strain, together with lower amounts of unmodified microcin (Fig. 6A), but was totally absent from the aroB– strain cultures where only unmodified MccE492 was detected (Fig. 6B). Thus, the modified MccE492 is not expressed in strains impaired in DHBS biosynthesis.



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  		FIG. 6.
Characterization of MccE492 expressed in aroB– bacteria. Wild-type E. coli C600 (A) or aroB– E. coli C600 (B) harboring the pJAM229 plasmid was cultured until the end of the exponential phase of growth. The 40% ACN Sep-Pak fraction from the culture supernatants was analyzed by Qq-TOF MS. Masses corresponding to the multicharged ions of MccE492 ([ME + nH]n+) and MccE492m ([MEm + nH]n+) are presented.

 
In order to identify possible intermediates involved in the synthesis of the post-translational modification, the 40% ACN fractions issued from the Sep-Pak fractionation of the culture supernatants obtained at different times (5, 8, 16, 24, and 48 h) on bactotryptone-containing culture medium were analyzed by MALDI-TOF MS. The spectrum in Fig. 7, obtained for the fraction arising from a 16-h culture, shows that the [M + H]+ ions of MccE492m and MccE492 at m/z 8718 and 7887, respectively, are accompanied by two additional [M + H]+ ions at m/z 8272 and 8495, which are attributed to MccE492 carrying a {beta}-D-Glc and a DHBS unit and a {beta}-D-Glc and 2 DHBS units, respectively.



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  		FIG. 7.
Characterization of the additional compounds present in the MccE492m cultures. MALDI-TOF spectrum of the 40% ACN fraction arising from a culture stopped at 16 h of the E. coli VCS257 strain harboring the pJAM229 plasmid on the bactotryptone-supplemented M63 medium, which shows the additional [M + H]+ species at m/z 8272 and 8495.

 
Role of Membrane Receptors in the MccE492m Mechanism of Action
To determine the involvement of outer membrane receptors in the recognition and transport of MccE492m by the target strains, we examined the susceptibility to MccE492m of E. coli strains that possessed mutations in the FepA, Cir, and Fiu catecholate-type receptors or lacked the hydroxamate-type receptor FhuA (Table V). The susceptibility of E. coli strains lacking the associated inner membrane proteins TonB, ExbB, and ExbD was also examined. The fepA–, cir–, or fiu– single mutants or the cir–, fiu– or fepA–, cir– double mutants remained susceptible to MccE492m. By contrast, the fepA–, fiu– double mutant H1877 exhibited an 8-fold increase in resistance to MccE492m, whereas the triple cir–, fiu–, fepA– mutant H1876 was 200-fold more resistant to MccE492m than the parent strain H1443 (Table V). Furthermore, both E. coli C600 (fhuA–) and E. coli C600 harboring the pHX405 plasmid that encodes FhuA were highly susceptible to MccE492m. In addition, and as described previously for MccE492 (9), the tonB– mutation resulted in a high resistance to MccE492m, whereas deletion of both exbB and exbD was without noticeable effect. Moreover, restoration of the TonB expression in E. coli transformed with the TonB-encoding pMS7 plasmid re-established the susceptibility of the strain to MccE492m.


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  		TABLE V
Antibacterial activity of MccE492m against wild-type or mutant E. coli strains

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have isolated from culture supernatants of either the wild-type K. pneumoniae RYC492 or the recombinant E. coli VCS257 harboring the pJAM229 plasmid a modified form of MccE492, named MccE492m, which presents improved antibacterial activity as compared with the unmodified MccE492. MccE492m was shown to differ from the already described MccE492 (7) by the presence of an 831-Da post-translational modification. Electrospray mass spectrometry and CID sequencing of MccE492m-(74–84), the peptide fragment derived from MccE492m chymotrypsin digestion, revealed that the modification was carried by the C-terminal serine residue. Analysis of MccE492m-(74–84) by both ESI-IT-MS and 800-MHz NMR identified the modification as a trimer of DHBS linked to the microcin Ser-84 carboxyl group via a sugar moiety (Fig. 3). Such a structure is reminiscent of salmochelins, linear catecholate-type siderophores recently isolated from Salmonella enterica (31), that differ from the MccE492m modification by the number and order of the {beta}-D-Glc and DHBS units. Covalent bonds between (i) the {beta}-D-Glc C-1 and the C-5' carbon of the DHBS1 unit through a C-glycosylation, and (ii) the {beta}-D-Glc C-6 and the Ser-84 carboxyl through an O-glycosylation were identified from both long range 1H-13C couplings (HMBC data) and NOE data. As for the naturally occurring DHBS trimer, the two remaining DHBS were shown to be linked through their serine carboxyl groups. The unusual C-glycosylation that occurs in MccE492m has been described previously in aryl-C-glycosyl polyketides produced by Streptomyces species, such as the antitumor antibiotics urdamycin, (32) medermycin (33), and gilvocarcins (34), or in RNase Us found in human urine (35). It is presumably responsible for the high stability of the first glycosylated DHBS, which cannot be dissociated from Ser-84 by CID experiments without disrupting the entire molecule.

In order to understand better the biosynthetic pathways leading to MccE492 modification, the peptide was expressed in an aroB– strain unable to synthesize 2,3-dihydroxybenzoate, the precursor of enterobactin and DHBS, as well as in the aroB+ isogenic strain. The absence of MccE492m in cultures of the aroB– strain, as compared with the predominant modified form obtained in its aroB+ counterpart, unambiguously showed that DHBS in a monomeric or oligomeric form is a precursor for MccE492 post-translational modification. During the course of MccE492m purification, additional MccE492m-related compounds, bearing either 1 or 2 DHBS units linked to the {beta}-D-Glc moiety, were observed. They are presumed to be intermediates in MccE492m synthesis, thus suggesting that the DHBS monomer rather than its linear dimer or trimer would be the precursor for the MccE492m modification. Conversely, such MccE492m-related products might result from MccE492m degradation. The presence in the microcin gene cluster of the mceD gene, which encodes a protein homologous to an enterobactin esterase, suggests that like Fes esterase (36) the mceD gene product could break circular enterobactin down into its linear trimer or its smaller subunits (dimer and monomer of DHBS) that could further be transferred onto MccE492. However, the expression of mceD gene cannot by itself account for the complete modification of MccE492. The role of mceC and mceI has been studied previously by Lagos et al. who observed by SDS-PAGE (21) and ESI-MS (11) that MccE492 was expressed as an inactive unmodified microcin in strains harboring the mceC– or mceI– mutant plasmids deriving from pJAM229. However, possibly due to culture conditions, the production of MccE492m by the strain carrying the original pJAM229 was evidenced neither in these studies nor in that of Pons et al. (7). The identification of MccE492 post-translational modification strongly suggests that MceC and MceI, which are homologous to glycosyltransferases and acyltransferases, respectively (21), are responsible for acylation of {beta}-D-Glc by the Ser-84 carboxyl group and for C-glycosylation of the 1st DHBS unit, respectively. This last point is supported by the 20–25% amino acid sequence identity shared by MceC and several C-glycosytransferases, such as UrdGT2 and related proteins (37). Further studies will be needed to determine whether MceC and MceI are enzymes involved in the MccE492 post-translational modification or, as proposed by Corsini et al. (11), molecular chaperones involved in the acquisition of MccE492 active conformation.

As described previously (9) for MccE492, MccE492m was found to display a TonB-dependent activity. Our antimicrobial assays on mutant strains lacking the Cir, Fiu, and/or FepA outer membrane receptors have demonstrated that a cir–fiu–fepA– triple mutant is tolerant to MccE492m but that the expression in isogenic strains of one single receptor out of the three is sufficient to induce a dramatic gain in susceptibility to MccE492m. Therefore, FepA, Fiu, and to a lesser extent Cir are unambiguously involved in the MccE492m recognition. These results are in agreement with a recent study by Patzer et al. (38) showing that Cir, Fiu, and FepA from E. coli as well as IroN, Cir, and FepA from Salmonella are receptors for MccM, MccH47, and MccE492. The three receptors recognize the breakdown products of enterobactin, such as the DHBS monomer (19, 39), whereas FepA only is involved in the enterobactin recognition (19, 40). This indicates that the MccE492 post-translational modification mimics linear catecholate siderophores rather than cyclic enterobactin. The increase in antibacterial activity associated with the MccE492 post-translational modification strongly suggests that by acquiring a DHBS-like motif, MccE492 increases its affinity for the three Cir, Fiu, and FepA receptors at the outer membrane of the sensitive cells.

The 831-Da modification carried by MccE492m-(74–84) was shown here to bind ferric ions specifically with a high affinity, similar to enterobactin and its breakdown products. This binding is mediated by the DHBS units, FeIII being preferentially located in the region of the DHBS units. Glc is excluded from the coordination and plays a spectator role that agrees with the preservation of iron in the FeIII oxidation state, although it is usually changed to FeII when associated with oligosaccharides (41). Therefore, a complex involving the catecholate functionalities assumes the iron coordination. The finding that the MccE492m-(74–84) doubly charged ion undergoes a direct loss of 2 DHBS units attached to FeIII, whereas the singly charged ion loses 2 uncationized DHBS units consecutively, indicates that the ion charge state directs the FeIII coordination site by modifying the number of possible deprotonations.

The specific binding of FeIII by MccE492m, but not by its unmodified counterpart, supports the hypothesis that the microcin gains affinity for the iron-siderophore receptors upon post-translational modification. Therefore, not only would microcins parasitize the iron-siderophore receptors, but they would also use outstanding strategies such as mimicking the iron-siderophore complexes themselves to enter their target cells more efficiently.

The post-translationally modified MccE492 is the first example of a siderophore-peptide. The isolation of such a modified peptide questions the principle of two distinct classes of microcins, bearing or not post-translational modifications (2). Considering the sequence homologies between MccE492, MccM, and MccH47 (i) in their serine-rich C terminus, a region that carries the modification in MccE492, and (ii) in their gene clusters, with the presence of highly similar genes encoding modification enzymes (38), it is likely that all of these microcins will be found to carry post-translational modifications, thus augmenting the new class of potent antibacterial siderophore-peptides initiated by MccE492m.


   FOOTNOTES
 
The abbreviations used are: DHBS, N-(2,3-dihydroxybenzoyl)-L-serine; ACN, acetonitrile; {beta}-D-Glc, {beta}-D-glucose; CID, collision-induced dissociation; ESI, electrospray ionization; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum correlation; IT, ion trap; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; Mcc, microcin; MIC, minimum inhibitory concentration; MS, mass spectrometry; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; RP-HPLC, reversed phase high performance liquid chromatography; TOCSY, total correlation spectroscopy; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)]ethylglycine; Qq-TOF, hybrid quadrupole time-of-flight.

* 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. Back

The on-line version of this article (available at http://www.jbc.org) contains Tables A and B. Back

** To whom correspondence should be addressed. Tel.: 33-1-40-79-31-18; Fax: 33-1-40-79-31-35; E-mail: rebuffat{at}mnhn.fr.


   ACKNOWLEDGMENTS
 
We thank Prof. K. Hantke (Mikrobiologie/Menbranphysiologie, Universität Tübingen, Germany) for the generous gift of the catecholate siderophore receptor E. coli mutant strains used in this study and Prof. F. Moreno (Hopital Ramón y Cajal, Madrid, Spain) and Dr. P. Boulanger (Institut de Biochimie et Biophysique Moléculaire, CNRS, Orsay, France) for the K. pneumoniae and the E. coli C600 aroB– strains, respectively. We thank G. Gastine for careful technical assistance in microbiology. We also thank Dr. P. Boistard (Laboratoire des Relations Plantes-Microorganismes, INRA-CNRS, Toulouse, France) for helpful discussions.



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 DISCUSSION
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