From the Department of Chemistry, University of
Alberta, Edmonton, Alberta T6G 2G2, Canada and the
§ Department of Food Science and Technology, New York State
Agricultural Experiment Station, Cornell University, Geneva,
New York 14456
Received for publication, December 4, 2002
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ABSTRACT |
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Mattacin is a nonribosomally synthesized,
decapeptide antibiotic produced by Paenibacillus kobensis
M. The producing strain was isolated from a soil/manure sample and
identified using 16 S rRNA sequence homology along with chemical and
morphological characterization. An efficient production and isolation
procedure was developed to afford pure mattacin. Structure elucidation
using a combination of chemical degradation, multidimensional NMR
studies (COSY, HMBC, HMQC, ROESY), and mass spectrometric (MALDI MS/MS) analyses showed that mattacin is identical to polymyxin M, an uncommon
antibiotic reported previously in certain Bacillus species by Russian investigators. Mattacin (polymyxin M) is cyclic and possesses an amide linkage between the C-terminal threonine and the
side chain amino group of the diaminobutyric acid residue at position
4. It contains an (S)-6-methyloctanoic acid moiety attached
as an amide at the N-terminal amino group, one D-leucine, six L- To survive in the natural environment and compete with other
microorganisms for resources, many bacteria produce antimicrobial compounds to inhibit or kill other competing strains, including human
and animal pathogens. One subclass of these antimicrobial compounds is
the antibacterial peptides, which can be divided into two categories
based on the biosynthetic pathways by which they are generated. One
group consists of gene-encoded, ribosomally synthesized peptides
(bacteriocins) that typically have 30-60 residues, may be either
unmodified or extensively post-translationally altered (i.e.
lantibiotics), and are active against closely related bacteria (1-3).
Peptides in the second class are nonribosomal in origin and are
produced by a series of condensations catalyzed by specific
nonribosomal peptide synthetases using a templated multienzyme
mechanism (4, 5). These synthetases are large, multifunctional proteins
composed of different modules, each of which has different domains
capable of performing one step in the condensation of an amino acid
onto a growing peptide chain (6). The resulting peptidic
compounds often contain nonproteinaceous amino acids, including D-amino acids, hydroxy acids,
or other unusual constituents (7). The peptide portion of antibiotics produced in this fashion is generally smaller than in ribosomal bacteriocins and usually has fewer than 20 amino acids (8) (Fig.
1).
,
-diaminobutyric acid, and three
L-threonine residues. Transfer NOE experiments on the
conformational preferences of mattacin when bound to lipid A and
microcalorimetry studies on binding to lipopolysaccharide showed that
its behavior was very similar to that observed in previous studies of
polymyxin B (a commercial antibiotic), suggesting an identical
mechanism of action. It was capable of inhibiting the growth of a wide
variety of Gram-positive and Gram-negative bacteria, including several
human and plant pathogens with activity comparable with purified
polymyxin B. The biosynthesis of mattacin was also examined briefly
using transpositional mutagenesis by which 10 production mutants were
obtained, revealing a set of genes involved in production.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
General structure of polymyxins.
DAB, A2bu.
Our interest in bacteriocins from lactic acid bacteria, both unmodified
(9, 10) and multicomponent, post-translationally modified lantibiotics
(11), has led us to examine other species of Gram-positive bacteria,
such as Bacillus (12), for novel peptidic antimicrobial
agents. During a screening program we found that a Paenibacillus
kobensis strain isolated from a soil/manure sample produced an
active modified peptide with broad activity against both Gram-positive
and Gram-negative organisms, including a number of human and animal
pathogens. We now report that the structure of the principal
antimicrobial compound formed by this strain, mattacin, is identical to
polymyxin M, an uncommon antibiotic reported previously in the Russian
literature (13-16; all reports in Russian)
(Fig. 2). Although polymyxins represent
one of the earliest classes of commercially important antibiotics to be
identified (17), and at least 15 unique polymyxins have been described (18), only polymyxin B is currently widely used and studied. In
addition to efficient production and purification of mattacin, the
present study describes its NMR solution structure and transfer NOE1 determination of
conformational changes that occur upon binding to lipid A and compares
these with previous results reported by others (19) with polymyxin B. Isothermal titration calorimetry was also employed to compare
the binding of mattacin and polymyxin B to lipopolysaccharide (LPS),
the major antigen of the outer membrane of Gram-negative bacteria.
Finally, the biological potency of mattacin was assessed compared with
that of polymyxin B, and the biosynthesis of mattacin was briefly
examined.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and Culture Conditions-- The producer strain, P. kobensis M, was isolated from a soil/manure sample mix and grown aerobically at 30 °C on tryptic soy agar (TSA) or in broth (TSB) with shaking (250 rpm). Escherichia coli BF2, a laboratory strain, was used as the standard sensitive strain. E. coli Jm2r' was used as a gene cloning host for the recovery of the transposon-interrupted mutants. E. coli strains were grown aerobically at 37 °C on Luria-Bertani agar or in broth with shaking (250 rpm). All other strains used for the inhibition spectrum assay were grown in both broth and agar culture under their established optimum conditions and media. 1.5 µg/ml erythromycin, 20 µg/ml lincomycin, and 5 µg/ml tetracycline were used for selection of producer strain transformants. 20 µg/ml kanamycin was used for selection of E. coli Jm2r' transformants.
Producer Strain Identification-- A pair of degenerate primers was designed to amplify the 16 S rRNA gene (20). The PCR product was purified from agarose using a Qiagen gel extraction kit (Qiagen Corp., Valencia, CA) and sequenced using an ABI Prism 373 DNA sequencer (Applied Biosystems, Foster City, CA). The resulting sequence was analyzed by homology comparison using the NCBI nucleotide Blast search data base. Biochemical and morphological assays were then performed as described by Reva et al. (21) and Shida et al. (22) to confirm the identity of the bacterium determined by 16 S rRNA gene sequence comparison down to the species level (Table I).
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Antimicrobial Activity Monitoring during Purification-- Antimicrobial activity was monitored by inhibition of indicator strain growth on agar plates. Plates were prepared by inoculating 200 ml of molten (48 °C) TSA (40 g/liter) with 1.0 ml of a culture of the indicator organism E. coli BF2 (0.5% inoculum). The molten agar was swirled gently and then dispensed in 20-ml aliquots onto sterile Petri plates, allowed to cool, and then stored at 4 °C. When performing activity assays, small wells (4.6-mm diameter) were made in the seeded agar plates, and 50-µl aliquots of the solutions to be tested were dispensed into each well. The plates were then incubated at 30 °C, with growth of the indicator being visible in as few as 3 h.
Spectrum of Activity-- The antimicrobial spectrum was determined using a deferred inhibition assay described previously by Ahn and Stiles (23). Briefly, the producer strain was spotted onto TSA plates and incubated 24 h at 30 °C. Molten 0.75% TSA, Luria Burtani, or MRS soft agar (medium used was optimum for each indicator strain) was then inoculated with 80 µl of a late log phase indicator broth culture (~106 viable cells/ml) and poured onto the surface of the plate containing the producer strain colonies. These agar overlays were then incubated overnight at either 30 or 37 °C (according to the indicator's optimum temperature), and the zones of inhibition were then measured. Alternatively, 10 µl of a purified, antimicrobial peptide solution with a known concentration was spotted onto the surface of a TSA plate, allowed to dry, and overlaid as described above.
Antimicrobial Peptide Production during Growth and Arbitrary Unit Definition-- 10 ml of a fresh, overnight producer strain culture was inoculated into 1 liter of TSB and incubated at 30 °C with stirring. At various time intervals during this incubation, 1 ml of culture was collected to determine growth phase and antimicrobial peptide production. These samples were centrifuged immediately (14,000 rpm, 20 min, 4 °C) to remove the cells. The supernatant was heat treated at 65 °C for 20 min to inactivate any protease activity. The activity of each supernatant was then determined using a 2-fold dilution agar diffusion test. The samples were diluted serially in 2-fold increments, and 20 µl of each dilution was spotted onto a TSA plate. These spots were dried, and the plate was overlaid and read as described above with E. coli BF2 as the indicator strain. An arbitrary activity unit, defined as the 20-µl sample from the highest dilution that had a clear inhibition zone, was then determined for each fraction.
DNA Preparation and Transformation-- Plasmids from E. coli were isolated using the Qiagen plasmid mini kit as described by the manufacturer's instructions. Plasmids from the producer strain were isolated using the method of Sambrook et al. (24), except that cells were treated with 10 mg/ml lysozyme for 30 min at 37 °C before the SDS lysis step. Transformation of E. coli Jm2r' was performed using CaCl2 E. coli competent cell preparation according to the method of Sambrook et al. (24), and transformation of the producer strain was performed with the transposon delivery plasmid pLTV3 (25) following the protocol of Dramsi et al. (26).
Transpositional Mutagenesis and DNA Cloning-- Producer strain transformants were inoculated into TSB and grown to an A600 of ~0.2. The cultures were then shifted to 41 °C for 4 h to force random chromosomal insertion of the transposon, creating a transpositional mutagenesis library. This library was then screened for loss of antibiotic production by a colony-deferred inhibition assay as described above. Confirmation of the transposon insertion of each mutant was performed by a PCR using primers derived from the Tn917 sequence. Mutant chromosomal extractions showing the Tn917 PCR fragment were then digested with XbaI (Promega Corp., Madison, WI) followed by ligation with high concentration T4 DNA ligase (Invitrogen). The ligation mixtures were then used to transform E. coli Jm2r'. Plasmid preparations were performed on all Jm2r' transformants and were sequenced in both the forward and reverse directions. Two primers were designed for plasmid sequencing. The first was derived from the sequence of the monoclonal site of the pLTV3 plasmid (5'-CCG GGG ATC CTC TAG A-3'), and the second was derived from 70 bp upstream of the lacZ gene on the pLTV3 plasmid (5'-GTT AAA TGT ACA AAA TAA CAG CGA-3'). The self-ligated plasmids were sequenced using an ABI Prism 373 DNA sequencer and the results analyzed by NCBI Blast homology searches (Table II).
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Isolation of Mattacin-- In a 10-ml culture tube of TSB, a preculture of P. kobensis M was grown for 24 h with shaking (200 rpm) at 30 °C. A 1-liter batch of TSB was then prepared by first passing through a column (2.5 × 30 cm) packed with 40 g of Amberlite XAD-16 resin (Sigma) to remove hydrophobic components from the medium which would otherwise interfere with the isolation of the hydrophobic peptide. After sterilization (15 min at 121 °C) and cooling, this modified TSB was inoculated with the entire 10-ml P. kobensis M preculture (1% inoculum). After a total growth time of 16-24 h at 30 °C with shaking (200 rpm), the cells were removed by centrifugation (20 min, 8,000 rpm). The supernatant was then passed through a column (2.5 × 50 cm) containing 60 g of Amberlite XAD-16 resin at a flow rate of 15 ml/min with the aid of a peristaltic pump. The column was then washed with 500 ml of 30% ethanol. The active peptide was then removed from the Amberlite column by washing with 500 ml of 70% acid and isopropyl alcohol (pH 2 by addition of 1 M HCl). All fractions were assessed for activity using the well plate assay described above. The active 70% acid and isopropyl alcohol (pH 2) fraction was evaporated to dryness by rotary evaporation, and the yellow residue was redissolved/suspended in 5.0 ml of purified (Milli-Q system, Millipore, Bedford, MA) water. This concentrated solution was next applied to a column (2.5 × 50 cm) containing Sephadex G-25 superfine (Amersham Biosciences) at a flow rate of 1.0 ml/min. The column was eluted with purified (Milli-Q) water overnight, and 10-ml fractions were collected. Each fraction was assayed again for activity as described above. The active fractions 17-20 were then pooled, evaporated, and redissolved in 10 ml of 20% isopropyl alcohol in preparation for reverse phase HPLC. Complete isolation of mattacin required two separate HPLC methods, each employing a C18 steel-walled column (Vydac, 10 × 250 mm, 5 µm). During the initial HPLC work, all isolatable peaks were assessed for antimicrobial activity using the aforementioned well plate assay until the retention time of mattacin was well established. In the first method, a 1.0-ml injection was applied, and a gradient of water and isopropyl alcohol (0.1% trifluoroacetic acid), starting at 20% and climbing to 30% isopropyl alcohol over 25 min, was used (flow rate = 2.5 ml/min, detection at 225 nm). Using this method, most of the polar impurities were removed with mattacin eluting in a broad peak (Rt = 18-20 min) somewhat later. The active fractions were pooled, evaporated to dryness, and redissolved in 6.0 ml of 45% methanol. The second method employed the same C18 column using a gradient of water and methanol (0.1% trifluoroacetic acid), starting at 45% and climbing to 60% methanol over 15 min (flow rate = 4.0 ml/min, detection at 225 nm). Under these conditions, mattacin was isolated as a single peak (Rt = 10-11 min). Using 1.0-ml sample injections, the entire sample was purified, and after pooling, evaporation of the methanol, and lyophilization, as much as 5 mg of pure mattacin was obtained as white powder from a 1-liter culture.
Amino Acid Analysis-- 100 µg of mattacin was hydrolyzed at 160 °C for 1 h with 100 µl of 5.7 M HCl and 0.1% phenol in a sealed, evacuated tube. The solvent was removed by vacuum centrifugation (Speed-Vac), and the dried hydrolysate was redissolved in 0.2 M sodium citrate buffer (pH 2.0). Analysis was achieved through cation exchange chromatography with a Beckman 6300 amino acid analysis instrument using a 120 × 2.5-mm (inner diameter) column with postcolumn detection/quantitation by reaction with ninhydrin at 135 °C.
Acetylation of Mattacin-- With the knowledge that mattacin contained threonine, it was hoped that a crystalline product, suitable for x-ray analysis, might be obtained by chemically modifying nucleophilic residues. To this end, 500 µg of mattacin was treated with 1.0 ml of pyridine/acetic anhydride (1:1) on ice. The mixture was allowed to warm to room temperature, and after 4 h a 10-µl aliquot was removed for mass spectrometric (MS) analysis.
Mass Spectrometry-- Samples for MALDI-MS analysis were prepared using sinnapinic acid as the matrix. Solutions containing the sample peptide were mixed in even part with a stock solution of sinnapinic acid (10 mg/ml) in 60% acetonitrile (0.1% trifluoroacetic acid). A thin layer of sinnapinic acid was deposited on the surface of the gold target plate by delivery of a 0.7-µl droplet of a solution containing sinnapinic acid (4 mg/ml) in 50% acetone and 50% methanol. After evaporation of the acetone/methanol, a 0.3-µl droplet of the solution containing the sample peptide/matrix mixture was deposited on top of the fresh matrix layer on the plate. The solvent was evaporated at 1 atm prior to analysis. Mass spectra were recorded with a singlestage reflectron, MALDI-TOF mass spectrometer (Applied Biosystems API QSTAR Pulsar with a MALDI source) (27). Tandem MS (MS/MS) was performed using two different instruments. The QSTAR instrument, described above, was of the geometry QqTOF, where MS/MS analysis was achieved through collision-induced dissociation in the RF-only section of the mass spectrometer (Q2) after mass selection with the Q1 resolving quadrupole. Fragment ions were detected in the orthogonal TOF section of the mass spectrometer. Ion generation was achieved through MALDI ionization using sinnapinic acid as the matrix. The QSTAR was equipped with a 20-Hz pulsed nitrogen laser operating at 337 nm. Collision-induced dissociation MS/MS analysis was completed using argon as the collision gas in Q2. Also used in a second MS/MS analysis was a ThermoFinnegan (San Jose, CA) LCQ XP ion trap instrument equipped with a nanospray source. The peptide sample was dissolved in 1:1 methanol/water acidified with 0.2% formic acid and loaded into a PicoTip (New Objective, Woburn, MA) nanospray tip. Static nanospray was achieved by applying ~800 V to the nanospray tip. Ions were introduced to the mass spectrometer, and before MS/MS analysis all ions except the parent ion of interest were ejected from the ion trap. MS/MS and MS3 analyses were completed using resonance excitation with the mass range up to m/z 1,200 for the fragment ions.
NMR Spectrometry--
NMR spectra were obtained in a 90%
H2O and 10% D2O solution at 27 °C and at a
peptide concentration of 2 mM. Spectra were acquired on a
Varian Inova 600 spectrometer; data matrices of 2048 detected and 512 (1024 for DQF-COSY) indirect data points with 64 (96 for ROESY) scans
were recorded and processed using a 90 shifted sine bell window
function (unshifted for DQF-COSY). Water signal suppression was
achieved by transmitter presaturation. All experiments were performed
at pH 2 (H2O contained 0.1% trifluoroacetic acid);
under these conditions, the possibility of peptide aggregation was
reduced because of A2bu--amino group protonation. The
assignment of 1H resonances was performed using standard
two-dimensional DQF-COSY (28), TOCSY (29) (mixing time 70 ms), and
two-dimensional NOE experiments (NOESY (30) and ROESY (31), mixing
times 200 ms). The temperature coefficients of the amide proton
chemical shifts were calculated using a series of one-dimensional
experiments performed at four different temperatures in the range of
27-42 °C. Two-dimensional transferred NOE (TRNOE) experiments (32, 33) with mixing times of 200 ms were done using a mixture of mattacin
and LPS which corresponded to an 8:1 w/w ratio of both components;
these conditions yielded moderately broadened lines in the amide region
of the mattacin one-dimensional spectrum. A three-dimensional structure
of mattacin was computed based on NOE restraints derived from the TRNOE
experiment using a dynamic annealing protocol in CNS 1.1 (©Yale
University). 50 representative structures were calculated based on 62 NOEs. The NOEs were classified as strong, medium, and weak,
corresponding to maximum distances of 3.0, 4.0, and 5.0 Å,
respectively, based on the volumes of the assigned cross-peaks in the
NOESY spectrum.
Stereochemical Analysis of Mattacin--
1 mg of mattacin was
hydrolyzed (1 ml of 6 N HCl, 110 °C, 18 h), and the
hydrolysate was dried under a nitrogen stream and then derivatized
using an Alltech (Deerfield, IL) pentafluoropropyl amide-isopropyl
ester amino acid derivatization kit. The dried hydrolysate was treated
with 0.2 N HCl (5 min at 110 °C) and dried under an
argon stream. To this, 150 µl of acetylchloride and 500 µl of
isopropyl alcohol were added, and the mixture was heated at 110 °C
for 45 min. After drying with an argon stream, the derivatizing agent,
pentafluoropropyl propionic anhydride (1 ml dissolved in 2 ml of
CH2Cl2), was added, and the solution was heated
at 115 °C for 15 min, blown dry with argon, and then solubilized in
CH2Cl2. For standards, 10 mg each of
D/L-threonine, leucine, and
,
-A2bu were subjected to the identical derivatization
sequence. Also, 1 mg of purified polymyxin B was hydrolyzed and treated
in the same fashion to serve as standard. All samples were analyzed by gas chromatography-MS under identical conditions, using a Heliflex Chirasil-Val, 50 m × 0.25 mm × 0.16 µm column (Alltech),
helium as carrier gas (0.6 ml/min), and a temperature gradient
beginning at 90 °C (5-min hold) and ramping to 160 °C
(3 °C/min) followed by a 12-min hold.
Isothermal Titration Calorimetry--
LPS from E. coli strain 055:B5 was obtained from Sigma, and polymyxin B
sulfate was purchased from Fluka. The polymxyin B was purified further
by reverse phase HPLC (using methods identical to those described above
for mattacin), and the LPS preparation was used as purchased. Using a
molecular mass estimate of 20,000 Da for the LPS monomer (34) a
0.05 mM LPS solution was prepared by dissolving LPS in 50 mM sodium phosphate buffer (pH 6.8) along with equimolar
quantities of triethylamine with respect to the anionic groups in the
LPS monomer (4 eq). The solution was vortexed vigorously for 15 min and
sonicated for 5 min prior to use. Titrations were performed using the
OMEGA high sensitivity microcalorimeter manufactured by MicroCal Inc.
(Northampton, MA) as described previously (35). For measurement of heat
exchanges accompanying the binding of polymyxin B or mattacin to LPS,
the LPS solution was loaded into the sample cell of the calorimeter
(volume = 1.4423 ml), and the reference cell was filled with water
containing 0.05% sodium azide. Next, polymyxin B or mattacin, in the
same buffer, was placed in a 250-µl syringe at a concentration of
1.25 mM (25-fold higher than that of the LPS). The system
was allowed to equilibrate at 20.0 °C, and a stable base line was
recorded before initiating an automated titration. A titration sequence
involved 7.2-µl aliquot injections of polymyxin B or mattacin
delivered over 10 s at 5-min intervals into the sample cell.
Throughout the titration, the cell was stirred continuously at 400 rpm.
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RESULTS |
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Producer Strain Identification-- NCBI nucleotide Blast homology search results of the 16 S rRNA gene sequence revealed high homology to the Paenibacillus genus. Specific biochemical and morphological results, shown in Table I, revealed the bacterium to be P. kobensis, later named strain M.
Spectrum of Activity-- Live cell deferred inhibition assays showed that P. kobensis M inhibited numerous Gram-positive and Gram-negative species including among others E. coli O157:H7 ATCC 33150, Salmonella enterica serovar Rubislaw, and Listeria monocytogenes, but failed to inhibit Pediococcus acidilactici. Purified mattacin and polymyxin B showed the same inhibition spectrum as the live P. kobensis M cells with the exception that they both failed to inhibit the strains of Listeria and Bacillus tested. Furthermore, mattacin showed a consistently higher level of activity against all strains tested in this study, including activity against Vibrio parahemeolyticus G1-166, against which polymyxin B was inactive. Table III shows the complete antimicrobial spectrum elucidated in this study as well as the inhibition spectrum of polymyxin B against the same organisms for comparative purposes.
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Antimicrobial Peptide Production during Growth--
Fig.
3 shows the relationship between P. kobensis M growth and antimicrobial peptide production. As can be
seen from this figure, production started at the exponential phase and
reached its highest point during the stationary phase.
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Transpositional Mutagenesis of Mattacin Biosynthetic Genes-- To identify the genes related to polymyxin M production, we performed transposon insertional mutagenesis with Tn917. Transformation of P. kobensis M with the plasmid pLTV3 produced seven transformants, of which one was used to produce our insertional mutagenesis library. Nine partial production mutants and one nonproduction mutant were obtained from a screen of ~7,000 colonies. PCR amplification of these mutants revealed all to contain the Tn917 transposon sequence with the wild-type producer strain lacking any endogenous transposon sequence. Plasmid sequencing from the resultant E. coli Jm2r' transformants showed a variety of homologies to known genes as summarized in Table II.
Mattacin Structure Elucidation--
Initial investigations into
the structure of mattacin did not immediately lead to the polymyxin
family of peptide antibiotics. MALDI MS analysis suggested a molecular
mass of 1157 Da (Fig. 4A).
Standard peptide analyses were then performed consisting of Edman
degradation and amino acid analysis after HCl hydrolysis. Attempts at
Edman sequencing of the peptide failed, thereby indicating the presence
of an N-terminal blocking moiety. Upon strong acid hydrolysis of the
peptide, detectable amounts of Leu, Thr, and a third nonproteinaceous
amino acid (later identified as ,
-A2bu) were
observed. Attempts at partial hydrolysis of the peptide with either
mild base or acid failed to yield fragments amenable to Edman
sequencing. With the knowledge that the peptide contained threonine,
acetylation experiments were performed in the hope that the derivatized
peptide might yield material suitable for x-ray crystallographic
analysis. Although the acetylated peptide is not crystalline, it was
determined that eight acetyl groups were incorporated (shown by
MALDI-TOF MS) indicating eight nucleophilic moieties in the structure
(Fig. 4B).
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Extensive MS was done on the native peptide using MALDI-TOF MS/MS
techniques in an attempt to gain sequence information. This MS work
showed the presence of a number of amino acid residues with an
experimental mass of 100.064 Da having a molecular formula of
C4H8N2O, thereby suggesting the
presence of ,
-A2bu moieties in the peptide. A high
resolution molecular ion MH+ value of 1,157.7370 (monoisotopic) indicated a molecular formula of
C51H97N16O14. Thus it
became apparent that mattacin was likely a member of the polymyxin
class of antibiotics.
NMR Spectrometry--
Complete structure elucidation of mattacin
was achieved by use of NMR spectrometry. Initial one-dimensional and
HH-COSY experiments confirmed that mattacin belonged to the polymyxin
group of antibiotics by revealing the presence of 11 amide protons and
10 alpha protons (Fig. 5, A
and B) as well as the fatty acid side chain. The spin systems in the TOCSY spectra of mattacin were assigned to the respective residues by taking into consideration the characteristic frequencies and numbers of resonances (Fig.
6). Sequential assignment of the peptide
is achieved using dN(i,i + 1) connectivities in the ROESY spectrum as well as the
connectivities determined by the HH-COSY spectrum (Fig.
7). A series of one-dimensional NMR
experiments at various temperatures (ranging from 27 to 42 °C) with
mattacin show that two of the ring-amide protons are shielded from
solvent by intramolecular H bonding (Table
IV). The transferred NOE experiments
performed with the mattacin/LPS mixture revealed a number of NOEs for
the heptacyclic region of the peptide which were not visible before the
addition of LPS. These NOEs (62 total) were used to produce a
conformational model for comparison with that of polymyxin B (19).
Matticin adopted a chair-like conformation with the side chains of
A2bu-4 and -8 pointing downward from those of Thr-6 and
Leu-7 (Fig. 8).
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Stereochemical Analysis--
To verify the stereochemistry of each
residue present in mattacin, a sample was hydrolyzed, and the resulting
amino acid mixture was derivatized to corresponding pentafluoropropyl
amide-isopropyl esters for analysis using phase gas chromatography-MS.
From the analysis it was clear that all three threonines were of the
L configuration (D-isomer
Rt = 10.8 min, L-isomer
Rt = 11.2 min) as were all six
,
-A2bu residues (D-isomer
Rt = 30.6 min, L-isomer Rt = 31.1 min). The leucine residue had a
D configuration (D-isomer Rt = 15.5 min, L-isomer
Rt = 16.3 min). A sample of polymyxin B,
derivatized and analyzed in the same manner (to serve as a secondary
standard), gave results in complete agreement with the known
stereochemistry for each residue. Thus the sequence, constituent amino
acids, lipid portion, and connectivity of mattacin were identical to
the structure proposed earlier by Russian workers for polymyxin M
(13-16), although no conformational information or detailed comparison
with polymyxin B was reported.
Isothermal Titration Calorimetry--
To investigate whether
mattacin behaved in a fashion similar to that ascribed to polymyxin B
in its binding to LPS (31), a calorimetric investigation was performed.
Both peptides were titrated into a solution of LPS and the heats of
binding monitored. The results of these binding assays were complex.
However, it was evident that both peptides bound LPS in almost
identical fashion. The binding isotherms were virtually
indistinguishable (Fig. 9) and suggested
that both peptides were interacting with the same receptor site(s)
present in LPS. The results did not support a simple 1:1 interaction,
but rather a model with a high probability of identical stoichiometry
and a series of sequential binding events.
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DISCUSSION |
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In recent years a large number of antimicrobial peptides from Gram-positive bacteria have been discovered, including ribosomally produced bacteriocins (36-38). The nonribosomally generated polymyxins represent one of the earliest classes of structurally unique peptide antibiotics to be identified (17). Paenibacillus spp. are Gram-positive, spore-forming bacteria from which polymyxins have been isolated, and their in vitro biosynthesis by cell-free enzyme systems has been successfully demonstrated (39). Although the first member of the polymyxin family, polymyxin B, was discovered in 1947 (17), the genetic control of their biosynthesis has not been described. We utilized the Tn917 transposon to study the mattacin (polymyxin M) biosynthesis genes and screened ~7,000 colonies for production mutants.
The majority of the production mutants exhibited decreased levels of production as opposed to a complete loss of production. This is not consistent with other studies using Tn917 for iturin and fengycin biosynthesis genes (40, 41). In those studies, the transposon disrupted the nonribosomal peptide synthetase genes and blocked the antimicrobial peptide synthesis completely. The identified genes in our study show low homology to other known genes in the NCBI data base. The biosynthesis genes of mattacin in Paenibacillus spp. may be quite different from other peptide synthases in DNA sequence, which may be the result of different evolutionary gene origin. Despite all of the differences, our study did show some similarities between mattacin biosynthesis and the synthetic processes of other peptide antibiotics, including the use of nonribosomal peptide synthetases for peptide growth and ABC transporters for secretion.
Structural analysis of mattacin utilized a combination of chemical degradations, mass spectrometry, and multidimensional NMR analyses to obtain primary sequence, identity of the lipid side chain, connectivity, stereochemistry, and conformational preferences. As is common for small peptides, mattacin shows considerable conformational flexibility when pure in solution, as shown by the absence of extensive long range NOE interactions. Recent NMR investigations by Pristovsek and Kidric (19) have shown that in solution, two of the ring-amide protons in polymyxin B participate in intramolecular hydrogen bonding. This is detected by the temperature dependences of the amide proton chemical shifts; specifically, amide protons that are least affected by temperature change are likely shielded from solvent by participation in H bonding. In mattacin, it was observed that the amide protons of A2bu-8 and the side chain of A2bu-4 are both shielded, as in polymyxin B. These results suggest that mattacin and polymyxin B, though containing structural differences, are both highly flexible in solution and can adopt similar conformations.
It has long been believed that polymyxins elicit their bactericidal
effects by binding to and disrupting the action of LPS, the major
antigen of the outer membrane of Gram-negative bacteria (42). LPS
contains three major structural components: lipid A, a core
oligosaccharide, and an outer polysaccharide composed of repeating
hetero-oligosaccharide subunits. Lipid A is a hydrophobic, lipid-rich
moiety that harbors the endotoxic principle of LPS and is the most
highly conserved part of the structure, containing two glucosamines,
two phosphate esters, and six fatty acid chains (43). The proposed
binding model for the polymyxin-LPS conjugate involves ionic
interactions between the side chain amino groups of the polymyxin
peptide cycle (positively charged in acidic medium) and the negatively
charged phosphate groups of the lipid A disaccharide (44). Also
proposed to contribute to binding is the hydrophobic interaction
between the nine-carbon fatty acid side chain of the polymyxin and the
fatty acid portion of lipid A (Fig.
10).
|
Using transferred NOE two-dimensional NMR techniques, Pristovsek and
Kidric (19) recently investigated the preferred conformation(s) induced
in polymyxin B when in solution with LPS from E. coli. Their
results indicate that although the peptide is highly flexible, the side
chain of A2bu-8 and the -amide NH of A2bu-4
as well as the amide NH of A2bu-8 with the side chain of
A2bu-4 are in close proximity while bound to LPS. In
performing these experiments with mattacin and the same LPS
preparation, however, we did not detect the same correlations. Our
conformational model in fact is somewhat different from that of
polymyxin B (Fig. 8). Although the side chains of all three
A2bu residues contained in the heptacycle were on the
opposite side of the molecule from the hydrophobic Phe and Leu side
chains in the polymyxin B structure, the bend in the mattacin
heptacycle was in the opposite direction. This resulted in a less
dramatic separation of hydrophobic and hydrophilic side chains. Whether
this was because of our experiments not being able to detect the
critical NOEs seen for polymyxin B or whether the conformational
differences have an explanation resulting from the structural
differences is not clear. Mattacin was significantly different at
residues 6 and 7 (D-Leu, L-Thr) compared with
the corresponding residues in polymyxin B (D-Phe,
L-Leu). It was in this region of the heptacycle which the
greatest conformational variation in the two models was observed, and
it seems reasonable to suggest that the significantly diverse
hydrophobic/hydrophilic residues present in the two peptides contribute
to these differences.
To ascertain whether mattacin behaved in a manner similar to polymyxin B in its interaction with LPS, isothermal titration calorimetry was employed. The thermodynamics of polymyxin B binding to LPS have been investigated previously using isothermal titration calorimetry by the group of Surolia (34). In these studies, a highly processed LPS preparation from E. coli was used (extensive treatments with proteases, chelating agents, and purifications via dialysis and size exclusion chromatography). Using these conditions, which lead to smaller fragments of LPS, Surolia and co-workers obtained results supporting a simple 1:1 binding between polymyxin B and LPS. We chose to use an LPS from the same E. coli strain, but without extensive processing because our primary interest was the comparison of mattacin with polymyxin B and their respective binding to LPS. The same stock LPS solution and concentrations were used for isothermal titration calorimetry experiments with both mattacin and polymyxin B. As seen in Fig. 9, the binding isotherms for both peptide titrations were almost indistinguishable. The data do not support a simple binding model, and the ORIGIN analysis software could only fit the data using a complex sequential binding model. Initial interaction of the polymyxins with unprocessed LPS appears to lead to disruption of tertiary structural arrangements, thereby exposing additional lipid A binding sites. The simple 1:1 binding of polymyxin B seen in previous experiments (34) with highly processed lipid A may be caused by the predominance of smaller unconglomerated units. Whether this form provides a more accurate model of what occurs with living bacterial cells remains uncertain. However, our results clearly suggest that mattacin binds to LPS in a manner similar to that of polymyxin B.
Results from the activity assays for both mattacin and polymyxin B
showed that the two peptides have virtually indistinguishable spectra
of activity. Mattacin did appear to be slightly more active in most
cases, but these differences are not very significant (less than 1 order of magnitude). An interesting observation in the activity assays
was that all strains of Listeria and Bacillus were inhibited by the live cells of P. kobensis M, but the
purified polymyxins B and M had no effect. This suggests that another
compound(s) is produced by this organism which is either lethal to
Listeria and Bacillus on its own or acts in
synergy with another compound(s), possibly the polymyxin, to elicit its
killing effects. Many multiple component bacteriocin systems are now
known to be produced by Gram-positive organisms and have been reviewed
recently (11, 45). Future investigations aimed at determining
whether P. kobensis M produces other novel antimicrobial
compounds and at elucidating the details of polymyxin biosynthesis are
in progress.
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ACKNOWLEDGEMENTS |
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We thank Michael Carpenter (Department of Biochemistry, University of Alberta) for peptide sequencing and amino acid analysis; Bernd Keller and Liang Li (Department of Chemistry, University of Alberta) for use of the QSTAR MALDI MS instrument; Albin Otter (Department of Chemistry, University of Alberta) for extensive assistance with NMR studies; and Tara Sprules (Department of Chemistry, University of Alberta) for aid in the NOE modeling.
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FOOTNOTES |
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* This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada, the Alberta Heritage Foundation for Medical Research, the Canada Foundation for Innovation, and the Canada Research Chair in Bioorganic and Medicinal Chemistry (to J. C. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence may be addressed. Tel.: 315-787-2279; Fax: 315-787-2284; E-mail: rww8@nysaes.cornell.edu.
To whom correspondence may be addressed. Tel.: 780-492-5475;
Fax: 780-492-8231; E-mail: john.vederas@ualberta.ca.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M212364200
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ABBREVIATIONS |
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The abbreviations used are: NOE, nuclear Overhauser effect; A2bu, diaminobutyric acid; DQF-COSY, double quantum filtered correlation spectrometry; HH-COSY, proton-proton correlation spectrometry; HPLC, high performance liquid chromatography; LPS, lipopolysaccharide; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NOESY, NOE spectrometry; ROESY, rotational nuclear Overhauser effect spectrometry; Rt, retention time; Tn, transposon; TOCSY, total correlation spectrometry; TOF, time-of-flight; TRNOE, two-dimensional transferred NOE; TSA, tryptic soy agar; TSB, tryptic soy broth.
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REFERENCES |
---|
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---|
1. | Twomey, D., Ross, R. P., Ryan, M., Meaney, B., and Hill, C. (2002) Antonie Leeuwenhoek 82, 165-185[CrossRef][Medline] [Order article via Infotrieve] |
2. | Klaenhammer, T. R. (1988) Biochimie (Paris) 70, 337-349 |
3. | Tagg, J. R., Dajani, A. S., and Wannamaker, L. W. (1976) Bacteriol. Rev. 40, 722-756[Medline] [Order article via Infotrieve] |
4. | Marahiel, M. A., Stachelhaus, T., and Mootz, H. D. (1997) Chem. Rev. 97, 2651-2673[CrossRef][Medline] [Order article via Infotrieve] |
5. | Doekel, S., and Marahiel, M. A. (2001) Metab. Eng. 6, 64-77[CrossRef] |
6. | Du, L., and Shen, B. (1999) Chem. Biol. 6, 507-517[CrossRef][Medline] [Order article via Infotrieve] |
7. | Kleinkauf, H., and von Dorhren, H. (1990) Eur. J. Biochem. 192, 1-15[Abstract] |
8. | Kleinkauf, H., and von Dohren, H. (1988) Crit. Rev. Biochem. 8, 1-32 |
9. | van Belkum, M. J., Worobo, R. W., and Stiles, M. E. (1997) Mol. Microbiol. 2, 1293-1301 |
10. | Wang, Y., Henz, M. E., Fregeau Gallagher, N. L., Chai, S., Yan, L. Z., Stiles, M. E., Wishart, D. S., and Vederas, J. C. (1999) Biochemistry 38, 15438-15447[CrossRef][Medline] [Order article via Infotrieve] |
11. | Garneau, S., Martin, N. I., and Vederas, J. C. (2002) Biochimie (Paris) 84, 577-592 |
12. |
Zheng, G.,
Yan, L. Z.,
Vederas, J. C.,
and Zuber, P.
(1999)
J. Bacteriol.
181,
7346-7355 |
13. | Silaev, A. B., Maevskaya, S. N., Trifonova, Z. P., and Yulikova, E. P. (1975) Zh. Obshch. Khim. 45, 2331-2337 |
14. | Okhanov, V. V., Dubovskii, P. V., Trakhanova, M. N., and Bairamashvili, D. I. (1987) Antibiot. Med. Biotekhnol. 32, 738-743[Medline] [Order article via Infotrieve] |
15. | Trakhanova, M. N., Zinchenko, A. A., Okhanov, V. V., and Dubovskii, P. V. (1989) Antibiot. Khimioter. 34, 20-24[Medline] [Order article via Infotrieve] |
16. | Okhanov, V. V., Dubovskii, P. V., and Miroshnikov, A. I. (1991) Bioorg. Khim. 17, 1689-1693[Medline] [Order article via Infotrieve] |
17. | Ainsworth, G. C., Brown, A. M., and Brownlee, G. (1947) Nature 160, 263-264 |
18. | Storm, D. R., Rosenthal, K. S., and Swanson, P. E. (1977) Annu. Rev. Biochem. 46, 723-763[CrossRef][Medline] [Order article via Infotrieve] |
19. | Pristovsek, P., and Kidric, J. (1999) J. Med. Chem. 42, 4604-4613[CrossRef][Medline] [Order article via Infotrieve] |
20. | Edwards, U., Rogall, T., Blocker, H., Emde, M., and Bottger, E. C. (1989) Nucleic Acids Res. 17, 7843-7853[Abstract] |
21. |
Reva, O. N.,
Sorokulova, I. B.,
and Smirnov, V. V.
(2001)
Int. J. Syst. Evol. Microbiol.
51,
1361-1371 |
22. |
Shida, O.,
Takagi, H.,
Kadowaki, K.,
Nakamura, L. K.,
and Komagata, K.
(1997)
Int. J. Syst. Bacteriol.
47,
289-298 |
23. | Ahn, C., and Stiles, M. E. (1990) J. Appl. Bacteriol. 69, 302-310[Medline] [Order article via Infotrieve] |
24. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , pp. 1.25-1.28, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
25. | Camille, A., Portnoy, D. A., and Youngman, P. (1990) J. Bacteriol. 172, 3738-3744[Medline] [Order article via Infotrieve] |
26. | Dramsi, S., Biswas, I., Maguin, E., Braun, L., Mastroeni, P., and Cossart, P. (1995) Mol. Microbiol. 16, 251-261[Medline] [Order article via Infotrieve] |
27. | Baldwin, M. A., Medzihradszky, K. F., Lock, C. M., Settineri, T. A., and Burlingame, A. L. (2001) Anal. Chem. 73, 1707-1720[CrossRef][Medline] [Order article via Infotrieve] |
28. | Piantini, U., Sorensen, O. W., and Ernst, R. R. (1982) J. Am. Chem. Soc. 104, 6800-6801 |
29. | Braunschweiler, L., and Ernst, R. R. (1983) J. Magn. Reson. 53, 521-528 |
30. | Jeneer, J., Meier, B. H., Bachman, P., and Ernst, R. R. (1979) J. Chem. Phys. 71, 4546-4553[CrossRef] |
31. | Bothner-By, A. A., Stephens, R. L., Lee, J., Warren, C. D., and Jeanloz, R. W. (1984) J. Am. Chem. Soc. 106, 811-813 |
32. | Clore, G. M., and Gronenborn, A. M. (1982) J. Magn. Reson. 48, 402-417 |
33. | Gronenborn, A. M., and Clore, G. M. (1982) J. Mol. Biol. 157, 155-160[Medline] [Order article via Infotrieve] |
34. | Srimal, S., Surolia, N., Balasubramanian, S., and Surolia, A. (1996) Biochem. J. 315, 679-686[Medline] [Order article via Infotrieve] |
35. | Wiseman, T., Williston, S., Brandts, J. F., and Lin, L. N. (1989) Anal. Biochem. 179, 131-137[Medline] [Order article via Infotrieve] |
36. | Nissen-Meyer, J., and Nes, I. F. (1997) Arch. Microbiol. 167, 67-77[CrossRef] |
37. | Riley, R. A., and Wertz, J. E. (2002) Annu. Rev. Microbiol. 56, 117-137[CrossRef][Medline] [Order article via Infotrieve] |
38. | Sahl, H.-G., and Bierbaum, G. (1998) Annu. Rev. Microbiol. 52, 41-79[CrossRef][Medline] [Order article via Infotrieve] |
39. | Komura, S., and Kurahashi, K. (1985) J. Biochem. 97, 1409-1417[Abstract] |
40. |
Tsuge, K.,
Akiyama, T.,
and Shoda, M.
(2001)
J. Bacteriol.
183,
6265-6273 |
41. | Chen, C.-L., Chang, L.-K., Chang, Y.-S., Liu, S.-T., and Tschen, J. S.-M. (1995) Mol. Gen. Genet. 248, 121-125[Medline] [Order article via Infotrieve] |
42. | Tsubery, H., Ofek, I., Cohen, S., and Fridkin, M. (2000) J. Med. Chem. 43, 3085-3092[CrossRef][Medline] [Order article via Infotrieve] |
43. | Rietschel, E. T., Brade, L., Holst, O., Kulshin, V. A., Lindner, B., Moran, A. P., Schade, U. F., Zaehringer, U., and Brade, H. (1990) in Cellular and Molecular Aspects of Endotoxin Reactions (Nowotny, A. , Spitzer, J. J. , and Ziegler, E. J., eds) , pp. 15-32, Elsevier Science Publishers B. V., Amsterdam |
44. | Koch, P. J., Frank, J., Schuler, J., Kahle, C., and Bradaczek, H. (1999) Colloid Interface Sci. 213, 557-564[CrossRef] |
45. | van Belkum, M. J., and Stiles, M. E. (2000) Nat. Prod. Rep. 17, 323-335[CrossRef][Medline] [Order article via Infotrieve] |