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Phylogenetic Analysis of Erwinia Species Based on 16S Rrna Gene Sequences?

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Phylogenetic Analysis of Erwinia Species Based on 16S Rrna Gene Sequences? INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1997, p. 1061-1067 Voi. 47, No. 4 0020-771 3/97/$04.00+0 Copyright 0 1997, International Union of Microbiological Societies Phylogenetic Analysis of Erwinia Species Based on 16s rRNA Gene Sequences? SOON-WO KWON,'" SEUNG-JOO GO,' HEE-WAN KANG,l JIN-CHANG RYU,l AND JIN-KI J02 National Institute of Agricultural Science & Technology, Suwon, and Department of Animal Science, Kyungpook National University, Daegu, Karea The phylogenetic relationships of the type strains of 16 Erwinia species were investigated by performing a comparative analysis of the sequences of the 16s rRNA genes of these organisms. The sequence data were analyzed by the neighbor-joining method, and each branch was supported by moderate bootstrap values. The phylogenetic tree and sequence analyses confirmed that the genus Erwinia is composed of species that exhibit considerable heterogeneity and form four clades that are intermixed with members of other genera, such as Escherichia coli, Klebsiella pneumoniae, and Serratia marcescens. Cluster I includes the type strains of Envinia herbicola, Erwinia milletiae, Erwinia ananas, Erwinia uredovora, and Erwinia stewartii and corresponds to Dye's herbicola group. Cluster I1 consists of Erwinia persicinus, Erwinia rhapontici, Erwinia amylovora, and Erwinia cypripedii. Cluster I11 consists of Erwinia carotovora subspecies and Erwinia chrysanthemi and is characterized by the production of pectate lyases and cellulases. Envinia salicis, Erwinia rubrifaciens, and Erwinia nigrijluens form the cluster that is most distantly related to other Erwinia species. The data from the sequence analyses are discussed in the context of biochemical and DNA-DNA hybridization data. The genus Erwinia was proposed by Winslow et al. (51) for obacter agglomerans, a taxon which Ewing and Fife (20, 21) gram-negative, non-spore-forming, peritrichous, fermentative, proposed for clinical isolates. Recently, some strains belonging rod-shaped bacteria, and it belongs to the family Enterobacte- to the Enterobacter agglomerans-Erwinia herbicola complex, a riaceae. This genus was proposed for plant-associated bacteria heterogeneous group that includes strains of the herbicola that are pathogens, saprophytes, and epiphytes. group and Enterobacter agglomerans, were placed in a new Although the heterogeneous taxonomic structure of the ge- genus, the genus Pantoea (23, 29, 36). Despite the various nus Erwinia has been discussed by using phenotypic data (13, taxonomic reevaluations of the genus Erwinia, the taxonomic 15-19, 31, 22, 35, 42, 44) and genotypic data (3, 6-10), the complexity of this taxon has not been completely resolved, and taxonomic position of this genus remains problematic (5, 32, a dual system of nomenclature is in use (9, 10, 15, 23, 29, 36, 40, 41). Previously, Dye (15-18) classified the members of the 48). genus Erwinia into four natural clusters. The carotovora group Analysis of 16s rRNA sequences has been demonstrated to is characterized by soft-rot-causing and biochemically active be one of the most powerful methods for investigating the species. Although some authors (10, 45) have proposed that natural relationships of microorganisms (52). In this study, we this group should be designated the genus Pectobacterium and determined 16s rRNA gene (rDNA) sequences of 16 Erwinia differentiated from other Erwinia species on the basis of dis- species and obtained the sequences of members of represen- tinct pathogenic and biochemical properties and this view was tative genera of the family Enterobacteriaceae from the EMBL partially supported by DNA-DNA hybridization studies (9, and GenBank databases in order to further clarify the taxon- lo), it has not been generally accepted. The amylovora group omy of the heterogeneous genus Erwinia on the intrageneric consists of pathogens that cause dry necrosis or wilt in their and intergeneric levels. specific host plants, and the taxonomic position of this group as a true Erwinia group has rarely been questioned. Furthermore, MATERIALS AND METHODS Dye (15) considered members of this group subspecies (or varieties) of Erwinia amylovora. However, each species belong- Organisms and culture conditions. Bacterial strains were obtained from the American Type Culture Collection. Table 1 shows the strains whose sequences ing to the amylovora group forms a distinct phenon, as shown were determined in this study and the reference strains used for comparison in the numerical analyses of Verdonck et al. (44) and Mergaert when phylogenetic trees were constructed. Table 1 includes the strain designa- et al. (35). In addition, DNA-DNA hybridization studies (3, 6, tions and the GenBank accession numbers for the 16s rDNA sequences. The 7, 23, 37) indicated that Erwinia amylovora had low levels of culture media and conditions used were the media and conditions recommended relatedness to other species of the amylovora group, as well as in the American Type Culture Collection Catalogue of Bacteria and Bacteriophages (1). other Erwinia species, and the DNA-DNA relatedness values DNA extraction. Chromosomal DNA was isolated by the method of Ausubel for Erwinia salicis, Erwinia rubrifaciens, and Erwinia nign@ens et al. (2), except that the lysates were extracted two times with chloroform to are moderately high. The herbicola group of the genus Erwinia remove residual phenol. PCR amplification of 16s rDNA. The 16s rDNAs were amplified by using is taxonomically rather complex. Most members of this group universal primers fD1 and rP2 (47). Each PCR mixture (50 1.1) contained primers produce a yellow pigment and are closely related to Enter- (each at a concentration of 20 pmol), a mixture of deoxynucleaside triphosphates (Promega Co., Southampton, England) (each at a concentration of 200 pM), Taq polymerase buffer, and chromosomal DNA (ca. 100 ng). Taq polymerase (2.5 U) (Promega Co.) and 1 drop of mineral oil were added to each of the reaction * Corresponding author. Mailing address: Division of Molecular solutions. The DNA thermal cycler (Perkin-Elmer Co., Nonvalk, Conn.) used for amplification was programmed as follows: (i) an initial extensive denaturation Genetics, National Institute of Agricultural Science & Technology, step consisting of 94°C for 4 min; (ii) 35 cycles, with each cycle consisting of 94°C RDA, Suwon 441-707, Korea. Phone: 82-331-290-0338. Fax: 82-331- for 1 min, 58°C for 1 min, and 72°C for 3 min; and (iii) a final extension step 290-0392. E-mail: brmg@ sun20. asti. re. kr. consisting of 72°C for 10 min. iThis paper is a contribution from the National Institute of Agri- Isolation and cloning of amplified 16s rDNA. The PCR solutions were elec- cultural Science and Technology, Suwon, Korea. trophoresed on 0.8% agarose gels, and then 16s rDNAs were purified with a 1061 1062 KWON ET AL. INT.J. SYST.BACTERIOL. TABLE 1. Strains used and their nucleotide sequence accession numbers Species or subspecies“ Strainb Accession no. Reference Erwinia amylovora ATCC 15580T US0195 This study Erwinia ananas (Pantoea ananas) ATCC 33244Tc US0196 This study Erwinia carotovora subsp. betavasculorum ATCC 43762T US0198 This study Erwinia carotovora subsp. carotovora ATCC 15713T US0197 This study Erwinia carotovora subsp. wasabiae ATCC 43316T US0199 This study Erwinia chtysanthemi ATCC 11663T US0200 This study Erwinia cypripedii ATCC 29267T US0201 This study Erwinia herbicola (Pantoea agglomerans) ATCC 33243T US0202 This study Erwinia milletiae (Pantoea agglomerans) ATCC 33261T US0183 This study Erwinia nignJEuens ATCC 1302ST US0203 This study Erwinia persicinus ATCC 3599ST US0205 This study Erwinia rhapontici ATCC 292S3T US0206 This study Erwinia rubrifaciens ATCC 29291T US0207 This study Erwinia salicis ATCC 15712T US0210 This study Erwinia stewartii (Pantoea stewartii subsp. stewartii) ATCC 8199Tc US0208 This study Erwinia uredovora (Pantoea ananas) ATCC 19321T US0209 This study Escherichia coli 501695 11 Hafnia alvei ATCC 13337T M59155 Unpublished data Klebsiella pneumoniae DSM 30104 X87276 Unpublished data Proteus vulgaris 501874 12 Serratia marcescens ATCC 13880T M59160 Unpublished data Yersinia intermedia ER 3854 X75279 27 a The names in parentheses are subjective synonyms. ATCC, American Type Culture Collection, Rockville, Md.; DSM, German Collection of Microorganisms, Braunschweig, Germany; ER, Yersinia Reference Center, Toronto, Canada. Type strains of proposed Pantoea species (23, 36). QIAquick gel extraction kit (Qiagene GmbH, Hilden, Germany). Purified rD- regions can be useful in identifying Erwinia species. The vari- NAs were ligated into pUCll9 vectors. Ligated plasmids were then transformed ation in the sequence homology values (range, 92.5 to 99.9%) into Eschen‘chia coli DH5aF‘ cells, and transformants were selected by the blue-white screening procedure (39). for different Erwinia species (Table 2) indicates that there is Sequencing of 16s rDNAs. Plasmids containing the 16s rDNA fragments were substantial intrageneric heterogeneity. isolated by using a QIAquick plasmid minikit (Qiagene GmbH). Purified plas- Interspecific relationships within clusters. One phyletic mids were manually sequenced by using both a T7 sequencing kit (Pharmacia line, corresponding to the herbicola group of Dye (17), consists Biotech, Inc., Piscataway, N.J.) and the fino1 DNA sequencing system (Promega Co.) according to the suppliers’ instructions. The ends of 16s rDNAs were of the type strains of Erwinia herbicola, Erwinia milletiae, Er- sequenced by using forward sequencing primer
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