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Isolation, Structural Characterization, and Properties of Mattacin (Polymyxin M), a Cyclic Peptide Antibiotic Produced byPaenibacillus kobensis M

2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês

10.1074/jbc.m212364200

1083-351X

Nathaniel I. Martin, Hai-Jing Hu, Matthew M. Moake, John J. Churey, Randy M. Whittal, Randy W. Worobo, John C. Vederas,

Bacteriophages and microbial interactions

Mattacin is a nonribosomally synthesized, decapeptide antibiotic produced by Paenibacillus kobensisM. 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-α,γ-diaminobutyric acid, and threel-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. Mattacin is a nonribosomally synthesized, decapeptide antibiotic produced by Paenibacillus kobensisM. 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-α,γ-diaminobutyric acid, and threel-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. nuclear Overhauser effect diaminobutyric acid double quantum filtered correlation spectrometry proton-proton correlation spectrometry high performance liquid chromatography lipopolysaccharide matrix-assisted laser desorption ionization mass spectrometry tandem mass spectrometry NOE spectrometry rotational nuclear Overhauser effect spectrometry retention time transposon total correlation spectrometry time-of-flight two-dimensional transferred NOE tryptic soy agar tryptic soy broth 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 (1Twomey D. Ross R.P. Ryan M. Meaney B. Hill C. Antonie Leeuwenhoek. 2002; 82: 165-185Crossref PubMed Scopus (152) Google Scholar, 2Klaenhammer T.R. Biochimie (Paris). 1988; 70: 337-349Crossref PubMed Scopus (950) Google Scholar, 3Tagg J.R. Dajani A.S. Wannamaker L.W. Bacteriol. Rev. 1976; 40: 722-756Crossref PubMed Google Scholar). 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 (4Marahiel M.A. Stachelhaus T. Mootz H.D. Chem. Rev. 1997; 97: 2651-2673Crossref PubMed Scopus (920) Google Scholar, 5Doekel S. Marahiel M.A. Metab. Eng. 2001; 6: 64-77Crossref Scopus (67) Google Scholar). 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 (6Du L. Shen B. Chem. Biol. 1999; 6: 507-517Abstract Full Text PDF PubMed Scopus (41) Google Scholar). The resulting peptidic compounds often contain nonproteinaceous amino acids, including d-amino acids, hydroxy acids, or other unusual constituents (7Kleinkauf H. von Dorhren H. Eur. J. Biochem. 1990; 192: 1-15Crossref PubMed Scopus (240) Google Scholar). The peptide portion of antibiotics produced in this fashion is generally smaller than in ribosomal bacteriocins and usually has fewer than 20 amino acids (8Kleinkauf H. von Dohren H. Crit. Rev. Biochem. 1988; 8: 1-32Google Scholar) (Fig.1). Our interest in bacteriocins from lactic acid bacteria, both unmodified (9van Belkum M.J. Worobo R.W. Stiles M.E. Mol. Microbiol. 1997; 2: 1293-1301Crossref Scopus (118) Google Scholar, 10Wang Y. Henz M.E. Fregeau Gallagher N.L. Chai S. Yan L.Z. Stiles M.E. Wishart D.S. Vederas J.C. Biochemistry. 1999; 38: 15438-15447Crossref PubMed Scopus (100) Google Scholar) and multicomponent, post-translationally modified lantibiotics (11Garneau S. Martin N.I. Vederas J.C. Biochimie (Paris). 2002; 84: 577-592Crossref PubMed Scopus (190) Google Scholar), has led us to examine other species of Gram-positive bacteria, such as Bacillus (12Zheng G. Yan L.Z. Vederas J.C. Zuber P. J. Bacteriol. 1999; 181: 7346-7355Crossref PubMed Google Scholar), 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 (17Ainsworth G.C. Brown A.M. Brownlee G. Nature. 1947; 160: 263-264Crossref Scopus (106) Google Scholar), and at least 15 unique polymyxins have been described (18Storm D.R. Rosenthal K.S. Swanson P.E. Annu. Rev. Biochem. 1977; 46: 723-763Crossref PubMed Google Scholar), 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 (19Pristovsek P. Kidric J. J. Med. Chem. 1999; 42: 4604-4613Crossref PubMed Scopus (153) Google Scholar) 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. 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 coliBF2, 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. colistrains 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. A pair of degenerate primers was designed to amplify the 16 S rRNA gene (20Edwards U. Rogall T. Blocker H. Emde M. Bottger E.C. Nucleic Acids Res. 1989; 17: 7843-7853Crossref PubMed Scopus (2240) Google Scholar). 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. (21Reva O.N. Sorokulova I.B. Smirnov V.V. Int. J. Syst. Evol. Microbiol. 2001; 51: 1361-1371Crossref PubMed Scopus (44) Google Scholar) and Shida et al.(22Shida O. Takagi H. Kadowaki K. Nakamura L.K. Komagata K. Int. J. Syst. Bacteriol. 1997; 47: 289-298Crossref PubMed Scopus (399) Google Scholar) to confirm the identity of the bacterium determined by 16 S rRNA gene sequence comparison down to the species level (TableI).Table IChemical and morphological characteristics of the producer strainCharacteristicResultSpore shapeOvalVegetative cell volume5 μm × 1 μmResistance to polymyxin BNoGrowth at 50 °CNoGrowth in presence of 0.001% lysozymeYesGrowth in presence of 5% NaClNoGrowth at pH6Yes Open table in a new tab 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. The antimicrobial spectrum was determined using a deferred inhibition assay described previously by Ahn and Stiles (23Ahn C. Stiles M.E. J. Appl. Bacteriol. 1990; 69: 302-310Crossref PubMed Scopus (110) Google Scholar). 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. 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. 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. (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1.25-1.28Google Scholar), 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 CaCl2E. coli competent cell preparation according to the method of Sambrook et al. (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1.25-1.28Google Scholar), and transformation of the producer strain was performed with the transposon delivery plasmid pLTV3 (25Camille A. Portnoy D.A. Youngman P. J. Bacteriol. 1990; 172: 3738-3744Crossref PubMed Google Scholar) following the protocol of Dramsi et al. (26Dramsi S. Biswas I. Maguin E. Braun L. Mastroeni P. Cossart P. Mol. Microbiol. 1995; 16: 251-261Crossref PubMed Scopus (390) Google Scholar). Producer strain transformants were inoculated into TSB and grown to anA600 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 withXbaI (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 thelacZ 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).Table IINCBI Blast homology search results of Tn917-flanking fragmentsPlasmid no.Mutant typeHomologous gene from LacZ directionHomologous gene from MCS direction33-1Lower level productionVector sequenceResponse regulator-like gene and vector sequence33-2Lower level productionPeptide antibiotic synthetase geneABC transporter gene in tyrocidine biosynthesis operon34Lower level productionVector sequenceAcyl-CoA synthase55Lower level productionPeptide antibiotic synthetase geneABC transporter gene in tyrocidine biosynthesis operon56Lower level productionPeptide antibiotic synthetase geneABC transporter gene in tyrocidine biosynthesis operon239Lower level productionProbable transcriptional regulatorProbable transcriptional regulator301NonproductionNo significant alignmentSensor histine kinase in two-component regulator system410Lower level productionABC transporterTranscription regulator411Lower level productionABC transporter16 S rRNA gene460NonproductionPeptide antibiotic synthetase genePeptide antibiotic synthetase gene Open table in a new tab 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. 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. 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. 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) (27Baldwin M.A. Medzihradszky K.F. Lock C.M. Settineri T.A. Burlingame A.L. Anal. Chem. 2001; 73: 1707-1720Crossref PubMed Scopus (84) Google Scholar). 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 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-γ-amino group protonation. The assignment of 1H resonances was performed using standard two-dimensional DQF-COSY (28Piantini U. Sorensen O.W. Ernst R.R. J. Am. Chem. Soc. 1982; 104: 6800-6801Crossref Scopus (1888) Google Scholar), TOCSY (29Braunschweiler L. Ernst R.R. J. Magn. Reson. 1983; 53: 521-528Crossref Scopus (3112) Google Scholar) (mixing time 70 ms), and two-dimensional NOE experiments (NOESY (30Jeneer J. Meier B.H. Bachman P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4845) Google Scholar) and ROESY (31Bothner-By A.A. Stephens R.L. Lee J. Warren C.D. Jeanloz R.W. J. Am. Chem. Soc. 1984; 106: 811-813Crossref Scopus (1971) Google Scholar), 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 (32Clore G.M. Gronenborn A.M. J. Magn. Reson. 1982; 48: 402-417Google Scholar,33Gronenborn A.M. Clore G.M. J. Mol. Biol. 1982; 157: 155-160Crossref PubMed Scopus (43) Google Scholar) 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. 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 ofd/l-threonine, leucine, and α,γ-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. 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 (34Srimal S. Surolia N. Balasubramanian S. Surolia A. Biochem. J. 1996; 315: 679-686Crossref PubMed Scopus (141) Google Scholar) 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 (35Wiseman T. Williston S. Brandts J.F. Lin L.N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2447) Google Scholar). 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. 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 beP. kobensis, later named strain M. Live cell deferred inhibition assays showed that P. kobensis M inhibited numerous Gram-positive and Gram-negative species including among others E. coliO157: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 liveP. 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 parahemeolyticusG1–166, against which polymyxin B was inactive. TableIII 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.Table IIIAntimicrobial spectrum of live P. koben