Pantocin B, an Antibiotic from Erwinia herbicola Discovered by Heterologous Expression of Cloned Genes Sean F. Brady, ² Sandra A. Wright, ‡ Julie C. Lee, ² Amanda E. Sutton, ² Cathy H. Zumoff, ‡ Richard S. Wodzinski, § Steven V. Beer, ‡ and Jon Clardy* ,² Department of Chemistry and Chemical Biology Cornell UniVersity, Ithaca, New York 14853-1301 Department of Plant Pathology Cornell UniVersity, Ithaca, New York 14853-4203 Department of Biology Ithaca College, Ithaca, New York 14850 ReceiVed August 5, 1999 Fire blight devastates apples, pears, and other rosaceous plants with such severe symptoms that at one time lightning was suspected as the cause. The real pathogenic agent is a bacterium, Erwinia amyloVora, that colonizes the external surfaces of the plants’ flowers, buds, and leaves. A closely related bacterium, E. herbicola (syn. Pantoea agglomerans), also colonizes the same plant surfaces 1-3 and produces antibiotics that effectively control E. amyloVora. 4,5 E. herbicola has been studied as a biological control agent for E. amyloVora in orchards where trees are currently treated with copper compounds or streptomycin. 2,6-10 Establishing the molecular nature of the E. herbicola antibiotics has been hampered by a complex biosynthetic pattern in which a single strain of E. herbicola produces multiple antibiotics, and different strains produce different multiple antibiotic complexes. Most of the antibiotics seem to share a common feature: antibiotic activity can be suppressed by the addition of an amino acid to the test medium. 11-13 The complexity of the E. herbicola antibiotics is not known since the only characterization available is the pattern of amino acids that suppress antibiotic activity. As a first step, we elected to decipher the molecular structure and genetics of one antibiotic using an approach based on cloning and heterologously expressing individual biosynthetic pathways. Secondary metabolite biosynthetic and resistance genes are often found clustered together on bacterial chromosomes, facilitating the cloning and heterologous expression of bacterial natural products from single continuous fragments of genomic DNA. 14 A cosmid library of E. herbicola strain 318 (Eh318) DNA was constructed in Escherichia coli, and two independent antibiotic producing clones were identified. 15 The two antibiotics, which have been named pantocin A and B, have their antibiotic activity suppressed by histidine and arginine, respectively. In this paper we report the isolation, structure determination, and mechanism of action of pantocin B (1). Large-scale fermentations of the pantocin B (1) producing subclone pCPP719 of Eh318 cosmid pCPP704 produce two related small molecules. Cation- and anion-exchange chromatog- raphy followed by HPLC gave pantocin B as the minor component (1 mg/L of culture) along with a major component 2 (3 mg/L). 16 In minimal media, pantocin B has picomolar activity against E. amyloVora (IC 50 ) 750 pM); compound 2 has no detectable antibiotic activity. Inhibition studies were performed in minimal media because the antibiosis of pantocin B is suppressed by arginine, which is invariably present in other more complex micro- bial culture broths. Pantocin B is a water soluble, optically active ([R] 25 D +31.5°, c 0.32, H 2 O) small molecule; a molecular formula of C 9 H 17 N 3 O 6 S was established with high-resolution FAB MS (m/z [M + H] + 296.0910 obs., 296.0916 calcd for C 9 H 18 N 3 O 6 S). Analysis of one- and two-dimensional 1 H and 13 C NMR spectra led to the planar structure corresponding to 1 (Table 1). Both two-carbon spin systemssC2-C3 and C9-C10swere easily identified. An HMBC correlation from the C8 carbonyl to the C10 methyl protons and the downfield shift of the C9 proton suggested an alanine fragment, which was confirmed by acid hydrolysis. A methylenediamine was readily apparent from the coupling of both amide NH protons to the C6 methylene protons, and an HMBC correlation from C8 to the C6 methylene protons links the methylenediamine to alanine. Additional HMBC cor- relations from C4 to the C6 methylene protons as well as the * Corresponding author. E-mail: jcc12@cornell.edu. ² Department of Chemistry and Chemical Biology, Cornell University. ‡ Department of Plant Pathology, Cornell University. § Department of Biology, Ithaca College. (1) Eriskine, H. M.; Lopatekci, L. E. Can. J. Microbiol. 1975, 21, 35-41. (2) Wrather, J. A.; Kuc, J.; Williams, E. B. Phytopathology 1973, 63, 1075- 1076 (3) Billings, E.; Baker, L. A. E. J. Appl. Bacteriol. 1963, 26, 58-65. (4) Vanneste, J. L.; Yu, J.; Beer, S. V. J. Bacteriol. 1992, 174, 2785- 2796. (5) Wodzinski, R. S.; Umholtz, T. E.; Rundle, J. R.; Beer, S. V. J. Appl. Bacteriol. 1994, 76, 22-29. (6) Beer, S. V.; Rundle, J. R.; Norelli, J. L. Acta Hortic. 1984, 151, 195- 201. (7) Vanneste, J. L.; Yu, J. Acta Hortic. 1990, 273, 409-410. (8) McIntyre, J. L.; Kuc, J.; Williams, E. B. Phytophathology 1973, 63, 872-877. (9) Riggle, J. H.; Klos, E. J. Can. J. Bot. 1972, 50, 1077-1083. (10) Wison, M.; Epton, H. A. S.; Sigee, D. C. Plant Pathol. 1990, 39, 301-308. (11) Wodzinski, R. S.; Paulin, J.-P. J. Appl. Bacteriol. 1994, 76, 603- 607. (12) El-Goorani, M. A.; Beer, S. V. Phytopathology 1991, 81, 121. (13) Ishimaru, C. A.; Klos, E. J.; Brubaker, R. Phytopathology 1988, 78, 746-750. (14) Martin, J. F. J. Ind. Microbiol. 1992, 9, 73-90. (15) Wright, S. A. I. The Genetics of antibiotic production and the role of antibiotics in biological control of Erwinia AmyloVora by Erwinia Herbicola. Ph.D. Thesis, Cornell University, August 1997. (16) Pantocin B was obtained from 10 L cultures of pCPP719 grown at 21 °C for 48 h in minimal media supplemented with 50 µg/mL ampicillin. The cells were removed by centrifugation, and the antibacterial active cell-free culture broth was applied to a Dowex 50Wx4-200 (H + ) cation-exchange column. The active fractions eluted from the cation-exchange column with 0.5 M NH4OH were then applied to an AG1x8-400 (HCO3 - ) anion-exchange column and eluted with CO2 saturated H2O. Compounds 1 and 2, typically in a 1:3 ratio, were then purified by HPLC using a reversed phase cyano column, first with 94:1:5 H2O/0.1 M NH4OH/CH3CN and then with 0.1% CH3CO2H in H2O as the mobile phase. Table 1. 1 H and 13 C NMR data for Pantocin B (1) position 13 C a 1 H b,c 1 172.4 2 70.4 4.30 (dd, 8.0, 6.5) 3 34.8 2.93 (m) 4 174.9 5 NH 6 47.2 4.62 (s and q, d 13.5) 7 NH 8 173.8 9 51.8 4.03 (q, 7.0) 10 19.2 1.50 (d, 7.0) 11 42.2 3.20 (s) a 13 C spectrum was recorded at 100 MHz in D2O (external reference with TSP in D2O). b 1 H spectrum was recorded at 500 MHz in D2O (referenced to HDO at 4.82 ppm). c Assignments are based on 1 H- 13 C HMQC. d AB quartet. 11912 J. Am. Chem. Soc. 1999, 121, 11912-11913 10.1021/ja992790m CCC: $18.00 © 1999 American Chemical Society Published on Web 12/04/1999