Fire Blight is a destructive disease of apple and pear caused by the enteric bacterial pathogen, Erwinia amylovora. E. amylovora initiates infection by colonizing the stigmata of apple and pear trees, and entering the plants through natural openings. Epiphytic populations of the related enteric bacterium, Pantoea, reduce the incidence of disease through competition and antibiotic production. In this study, we identify an antibiotic from Pantoea ananatis BRT175, which is effective against E. amylovora and select species of Pantoea. We used transposon mutagenesis to create a mutant library, screened approximately 5,000 mutants for loss of antibiotic production, and recovered 29 mutants. Sequencing of the transposon insertion sites of these mutants revealed multiple independent disruptions of an 8.2 kb cluster consisting of seven genes, which appear to be coregulated. An analysis of the distribution of this cluster revealed that it was not present in any other of our 115 Pantoea isolates, or in any of the fully sequenced Pantoea genomes, and is most closely related to antibiotic biosynthetic clusters found in three different species of Pseudomonas. This identification of this biosynthetic cluster highlights the diversity of natural products produced by Pantoea.
References
[1]
Beer SV, Rundle JR (1984) Interaction between Erwinia amylovora and Erwinia herbicola in vitro, in immature pear fruits and in apple blossums. Acta Horti 151: 203–204.
[2]
Jin M, Liu L, Wright SAI, Beer SV, Clardy J (2003) Structural and functional analysis of pantocin A: An antibiotic from Pantoea agglomerans discovered by heterologous expression of cloned genes. Angew Chem Int Ed 42: 2898–2901. doi: 10.1002/anie.200351053
[3]
Johnson KB, Stockwell VO (2000) Fire blight: the disease and its causative agent, Erwinia amylovora. Fire Blight: The Disease and Its Causative Agent, Erwinia amylovora. Wallingford: CAB International. pp. 319–338.
[4]
Giddens SR, Houliston GJ, Mahanty HK (2003) The influence of antibiotic production and pre-emptive colonization on the population dynamics of Pantoea agglomerans (Erwinia herbicola) Eh1087 and Erwinia amylovora in planta. Environ Microbiol 5: 1016–1021. doi: 10.1046/j.1462-2920.2003.00506.x
[5]
Bonn WG, van der Zwet T (2000) Distribution and economic importance of fire blight. Fire Blight: The Disease and Its Causative Agent, Erwinia amylovora. Wallingford: CAB International. pp. 37–53.
[6]
Pusey PL, Stockwell VO, Reardon CL, Smits THM, Duffy B (2011) Antibiosis activity of Pantoea agglomerans biocontrol strain E325 against Erwinia amylovora on apple flower stigmas. Phytopathology 101: 1234–1241. doi: 10.1094/phyto-09-10-0253
[7]
Kamber T, Smits THM, Rezzonico F, Duffy B (2011) Genomics and current genetic understanding of Erwinia amylovora and the fire blight antagonist Pantoea vagans. Trees 26: 227–238. doi: 10.1007/s00468-011-0619-x
[8]
Johnson KB, Stockwell VO, McLaughlin RJ, Sugar D, Loper JE, et al. (1993) Effect of antagonistic bacteria on establishment of honey bee-dispersed Erwinia amylovora in pear blossums and on fire blight control. Phytopathology 83: 995–1002. doi: 10.1094/phyto-83-995
[9]
Ozaktan H, Bora T (2004) Biological control of fire blight in pear orchards with a formulation of Pantoea agglomerans strain Eh 24. Braz J Microbiol 35: 224–229. doi: 10.1590/s1517-83822004000200010
[10]
Riggle JH, Klos EJ (1972) Relationship of Erwinia herbicola to Erwinia amylovora. Can J Bot 50 : 1077-&.
[11]
Chatterjee AK, Gibbins LN, Carpente Ja (1969) Some observations on physiology of Erwinia herbicola and its possible implication as a factor antagonistic to Erwinia amylovora in fire blight syndrome. Can J Microbiol 15: : 640-&.
[12]
Thomson SV, Gouk SC, New Zealand Plant Protect SOC (1992) Interactions between an antagonist, Erwinia herbicola, and E. amylovora and potential for biological control of fire blight. Proceedings of the Forty-Fifth New Zealand Plant Protection Conference: 295–300.
[13]
Sholberg PL, Boule J (2008) Evaluation of antibiotics and plant extracts for control of streptomycin-resistant Erwinia amylovora. In: Johnson KB, Stockwell VO, editors. Proceedings of the Eleventh International Workshop on Fire Blight.pp. 423–428.
[14]
Pusey PL, Stockwell VO, Rudell DR (2008) Antibiosis and acidification by Pantoea agglomerans strain E325 may contribute to suppression of Erwinia amylovora. Phytopathology 98: 1136–1143. doi: 10.1094/phyto-98-10-1136
[15]
Wright SA, Beer SV (1996) The role of antibiotics in biological control of fire blight by Erwinia herbicola strain EH318. Acta Horti 411: 309–311.
[16]
Wright SAI, Beer SV (2002) Genes for biosynthesis of pantocin A and B by Pantoea agglomerans Eh318. In: Hale C, Mitchell R, editors. Proceedings of the IX International Workshop on Fire Blight.pp. 237–241.
[17]
Wright SAI, Jin M, Clardy J, Beer SV (2006) The Biosynthetic genes of pantocin A and pantocin B of Pantoea agglomerans Eh318. In: Bazzi C, Mazzucchi U, editors. Proceedings of the X International Workshop on Fire Blight.pp. 313–319.
[18]
Wright SAI, Zumoff CH, Schneider L, Beer SV (2001) Pantoea agglomerans strain EH318 produces two antibiotics that inhibit Erwinia amylovora in vitro. Appl Environ Microbiol 67: 284–292. doi: 10.1128/aem.67.1.284-292.2001
[19]
Smits THM, Rezzonico F, Kamber T, Blom J, Goesmann A, et al. (2011) Metabolic versatility and antibacterial metabolite biosynthesis are distinguishing genomic features of the fire blight antagonist Pantoea vagans C9-1. PloS One 6.
[20]
Smits THM, Rezzonico F, Kamber T, Goesmann A, Ishimaru CA, et al. (2010) Genome sequence of the biocontrol agent Pantoea vagans strain C9-1. J Bac 192: 6486–6487. doi: 10.1128/jb.01122-10
[21]
Vanneste JL, Cornish DA, Yu J, Voyle MD (2002) The peptide antibiotic produced by Pantoea agglomerans Eh252 is a microcin. In: Hale C, Mitchell R, editors. Proceedings of the X International Workshop on Fire Blight. pp. 285–290.
[22]
Vanneste JL, Cornish DA, Yu J, Voyle MD (2000) A microcin produced by a strain of Erwinia herbicola is involved in biological control of fire blight and soft rot caused by Erwinia sp. In: Fokkema NJ, Beek MA, VanSteekelenburg NAM, Samyn G, Maas JL, et al., editors. Proceedings of the XXV International Horticultural Congress, Pt 3: Culture Techniques with Special Emphasis on Environmental Implications. pp. 39–46.
[23]
Vanneste JL, Yu J, Cornish DA (2008) Presence of genes homologous to those necessary for synthesis of microcin MccEh252 in strains of Pantoea agglomerans. In: Johnson KB, Stockwell VO, editors. Proceedings of the Eleventh International Workshop on Fire Blight. pp. 391–396.
[24]
Giddens SR, Feng YJ, Mahanty HK (2002) Characterization of a novel phenazine antibiotic gene cluster in Erwinia herbicola Eh1087. Mol Microbiol 45: 769–783. doi: 10.1046/j.1365-2958.2002.03048.x
[25]
Elgoorani MA, Hassanein FM, Shoeib AA (1992) Antibacterial and antifungal spectra of antibiotics produced by different strains of Erwinia herbicola (Pantoea agglomerans). J Phytopathology 136: 335–339. doi: 10.1111/j.1439-0434.1992.tb01316.x
[26]
Brady SF, Wright SA, Lee JC, Sutton AE, Zumoff CH, et al. (1999) Pantocin B, an antibiotic from Erwinia herbicola discovered by heterologous expression of cloned genes. J Am Chem Soc 121: 11912–11913. doi: 10.1021/ja992790m
[27]
Jin M, Wright SAI, Beer SV, Clardy J (2003) The biosynthetic gene cluster of pantocin A provides insights into biosynthesis and a tool for screening. Angew Chem Int Ed 42: 2902–2905. doi: 10.1002/anie.200351054
[28]
Smith DDN, Kirzinger MWB, Stavrinides J (2013) Draft genome sequence of the antibiotic-producing epiphytic isolate Pantoea ananatis BRT175. Genome Announc 1.
[29]
Sanchez S, Chavez A, Forero A, Garcia-Huante Y, Romero A, et al. (2010) Carbon source regulation of antibiotic production. J Antibiot 63: 442–459. doi: 10.1038/ja.2010.78
[30]
Schatz A, Bugie E, Waksman SA (2005) Streptomycin, a substance exhibiting antibiotic activity against gram positive and gram-negative bacteria. Clin Orthop Relat Res 437: 3–6. doi: 10.1097/01.blo.0000190391.55638.b0
[31]
Slininger PJ, Jackson MA (1992) Nutritional factors regulating growth and accumulation of phenazine-1-carboxylic acid by Pseudomonas fluorescens 2-79. Appl Microbiol Biotechnol 37: 388–392. doi: 10.1007/bf00210998
[32]
Brady CL, Venter SN, Cleenwerck I, Engelbeen K, Vancanneyt M, et al. (2009) Pantoea vagans sp nov., Pantoea eucalypti sp nov., Pantoea deleyi sp nov and Pantoea anthophila sp nov. Int J Syst Evol Microbiol 59: 2339–2345. doi: 10.1099/ijs.0.009241-0
[33]
Coutinho TA, Preisig O, Mergaert J, Cnockaert MC, Riedel KH, et al. (2002) Bacterial blight and dieback of Eucalyptus species, hybrids, and clones in South Africa. Plant Dis 86: 20–25. doi: 10.1094/pdis.2002.86.1.20
[34]
Kido K, Adachi R, Hasegawa M, Yano K, Hikichi Y, et al. (2008) Internal fruit rot of netted melon caused by Pantoea ananatis ( = Erwinia ananas) in Japan. J Gen Plant Path 74: 302–312. doi: 10.1007/s10327-008-0107-3
[35]
Walcott RR, Gitaitis RD, Castro AC, Sanders FH, Diaz-Perez JC (2002) Natural infestation of onion seed by Pantoea ananatis, causal agent of center rot. Plant Dis 86: 106–111. doi: 10.1094/pdis.2002.86.2.106
[36]
Gitaitis RD, Gay JD (1997) First report of a leaf blight, seed stalk rot, and bulb decay of onion by Pantoea ananas in Georgia. Plant Dis 81: 1096–1096. doi: 10.1094/pdis.1997.81.9.1096c
[37]
Fucikovsky L, Aranda S (2006) Pantoea ananas a new pathogen of agave in Mexico. Phytopathology 96: S37–S37.
[38]
Bell AA, Medrano EG, Jones MA. Frequency and pathogenicity of microorganisms associated with cotton seed rot in South Carolina; 2004; San Antonio, TX .
[39]
Cother EJ, Reinke R, McKenzie C, Lanoiselet VM, Noble DH (2004) An unusual stem necrosis of rice caused by Pantoea ananas and the first record of this pathogen on rice in Australia. Australas Plant Path 33: 495–503. doi: 10.1071/ap04053
[40]
Roper MC (2011) Pantoea stewartii subsp. stewartii: lessons learned from a xylem-dwelling pathogen of sweet corn. Mol Plant Path 12: 628–637. doi: 10.1111/j.1364-3703.2010.00698.x
[41]
Pérez-y-Terrón R, Villegas MC, Cuellar A, Mu?oz-Rojas J, Casta?eda-Lucio M, et al. (2009) Detection of Pantoea ananatis, causal agent of leaf spot disease of maize, in Mexico. Australas Plant Dis Notes 4: 96–99.
[42]
Azad HR, Holmes GJ, Cooksey DA (2000) A new leaf blotch disease of sudangrass caused by Pantoea ananas and Pantoea stewartii. Plant Dis 84: 973–979. doi: 10.1094/pdis.2000.84.9.973
[43]
Cha JS, Pujol C, Ducusin AR, Macion EA, Hubbard CH, et al. (1997) Studies on Pantoea citrea, the causal agent of pink disease of pineapple. J Phytopathology 145: 313–319. doi: 10.1111/j.1439-0434.1997.tb00407.x
[44]
Marin-Cevada V, Vargas VH, Juarez M, Lopez VG, Zagada G, et al. (2006) Presence of Pantoea citrea, causal agent of pink disease, in pineapple fields in Mexico. Plant Path 55: 294–294. doi: 10.1111/j.1365-3059.2005.01331.x
[45]
Makoff AJ, Radford A (1978) Genetics and biochemistry of carbamoyal phosphate biosynthesis and its utilization in the pyrimidine biosynthetic pathway. Microbiol Rev 42: 307–328.
[46]
Gu ZM, Martindale DW, Lee BH (1992) Isolation and complete sequence of the purL gene encoding FGAM synthase II in Lactobacillus casei. Gene 119: 123–126. doi: 10.1016/0378-1119(92)90076-2
[47]
Resch M, Schiltz E, Titgemeyer F, Muller YA (2012) Insight into the induction mechanism of the GntR/HutC bacterial transcriptional regulator YvoA. Nuc Acids Res 38: 2485–2497. doi: 10.1093/nar/gkp1191
[48]
Magarvey N, He J, Aidoo KA, Vining LC (2001) The pdx genetic marker adjacent to the chloramphenicol biosynthesis gene cluster in Streptomyces venezuelae ISP5230: functional characterization. Microbiol 147: 2103–2112.
[49]
Akoh CC, Lee GC, Liaw YC, Huang TH, Shaw JF (2004) GDSL family of serine esterases/lipases. Prog Lip Res 43: 534–552. doi: 10.1016/j.plipres.2004.09.002
[50]
Martin JR (1998) New aspects of genes and enzymes for β-lactam antibiotic synthesis. Appl Microbiol Biotechnol 50: 1–15.
[51]
Marchler-Bauer A, Zheng C, Chitsaz F, Derbyshire MK, Geer LY, et al. (2013) CDD: conserved domains and protein three-dimensional structure. Nuc Acids Res 41: 348–352. doi: 10.1093/nar/gks1243
[52]
Vrljic M, Garg J, Bellmann A, Wachi S, Freudl R, et al. (1999) The LysE superfamily: topology of the lysine exporter LysE of Corynebacterium glutamicum, a paradyme for a novel superfamily of transmembrane solute translocators. J Mol Microbiol Biotechnol 1: 327–336.
[53]
Aleshin VV, Zakataeva NP, Livshits VA (1999) A new family of amino-acid-efflux proteins. Trends Biochem Sci 24: 133–135. doi: 10.1016/s0968-0004(99)01367-5
[54]
Price-Whelan A, Dietrich LEP, Newman DK (2006) Rethinking ‘secondary’ metabolism: physiological roles for phenazine antibiotics. Nat Chem Biol 2: 71–78. doi: 10.1038/nchembio764
[55]
Mavrodi DV, Parejko JA, Mavrodi OV, Kwak YS, Weller DM, et al. (2013) Recent insights into the diversity, frequency and ecological roles of phenazines in fluorescent Pseudomonas spp. Environ Microbiol 15: 675–686. doi: 10.1111/j.1462-2920.2012.02846.x
[56]
Chin-A-Woeng TFC, Bloemberg GV, Mulders IHM, Dekkers LC, Lugtenberg BJJ (2000) Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot. Mol Plant Microbe Interact 13: 1340–1345. doi: 10.1094/mpmi.2000.13.12.1340
[57]
Girard G, Rigaliz S (2011) Role of the phanzine-inducing protein Pip in stress resistance of Pseudomonas chlororaphis. Microbiol 157: 393–407. doi: 10.1099/mic.0.043075-0
[58]
Kearns LP, Mahanty HK (1998) Antibiotic production by Erwinia herbicola Eh1087: Its role in inhibition of Erwinia amylovora and partial characterization of antibiotic biosynthesis genes. Appl Environ Microbiol 64: 1837–1844.
[59]
Nadarasah G, Stavrinides J (2014) Quantitative evaluation of the host-colonizing capabilites of the of the enteric bacterium Pantoea using plant and insect hosts. Microbiol 160: 602–615. doi: 10.1099/mic.0.073452-0
[60]
Alexeyev M, Shokolenko I, Croughan T (1995) New mini-Tn5 derivatives for insertion mutagenesis and genetic engineering in Gram-negative bacteria. Can J Microbiol 41: 1053–1055. doi: 10.1139/m95-147
[61]
Coplin DL, Majerczak DR, Zhang YX, Kim WS, Jock S, et al. (2002) Identification of Pantoea stewartii subsp stewartii by PCR and strain differentiation by PFGE. Plant Dis 86: 304–311. doi: 10.1094/pdis.2002.86.3.304
[62]
Smith D, Kirzinger M, Stavrinides J (2013) Draft genome sequence of the antibiotic-producing cystic fibrosis isolate Pantoea agglomerans Tx10. Genome Announcements 1.
[63]
Coplin D, Frederick R, Majerczak D, ES H (1986) Molecular cloning of virulence genes from Erwinia stewartii J Bacteriol. 168: 619–623.