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Inhella Inkyongensis Gen. Nov., Sp. Nov., a New Freshwater Bacterium in the Order Burkholderiales

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Inhella Inkyongensis Gen. Nov., Sp. Nov., a New Freshwater Bacterium in the Order Burkholderiales See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/23974676 Inhella inkyongensis gen. nov., sp nov., a New Freshwater Bacterium in the Order Burkholderiales Article in Journal of Microbiology and Biotechnology · February 2009 DOI: 10.4014/jmb.0802.145 · Source: PubMed CITATIONS READS 6 112 5 authors, including: Hyun-Myung Oh Jung-Sook Lee Pukyong National University Korea Research Institute of Bioscience and Biotechnology KRIBB 55 PUBLICATIONS 1,567 CITATIONS 210 PUBLICATIONS 3,452 CITATIONS SEE PROFILE SEE PROFILE Jang-Cheon Cho Inha University 248 PUBLICATIONS 5,139 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: genes and genomics of marine phage-host systems View project MG100K View project All content following this page was uploaded by Jang-Cheon Cho on 15 February 2017. The user has requested enhancement of the downloaded file. J. Microbiol. Biotechnol. (2009), 19(1), 5–10 doi: 10.4014/jmb.0802.145 First published online 18 July 2008 Inhella inkyongensis gen. nov., sp. nov., a New Freshwater Bacterium in the Order Burkholderiales Song, Jaeho1, Hyun-Myung Oh1, Jung-Sook Lee2, Seung-Buhm Woo1, and Jang-Cheon Cho1* 1Division of Biology and Ocean Sciences, Inha University, Incheon 402-751, Korea 2Korean Collection for Type Cultures, Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea Received: February 20, 2008 / Accepted: June 9, 2008 A freshwater bacterium, designated IMCC1713T, was been a well-defined taxonomic hierarchy among the members isolated from a highly eutrophic artificial pond. Cells of of the order Burkholderiales, several genera closely related the strain were Gram-negative, chemoheterotrophic, poly- to the family Comamonadaceae have been known to be β-hydroxybutyrate granule containing and obligately aerobic taxonomically uncertain and placed as genera insertae short rods that were motile with a single polar flagellum. sedis [25]. This phylogenetically uncertain group called the The 16S rRNA gene sequence similarity analysis showed Sphaerotilus-Leptothrix group [22] contains metabolically that the novel strain was most closely related to the species diverse genera, including the bacteriochlorophyll a-containing Roseateles depolymerans (96.3%), Mitsuaria chitosanitabida bacteria Roseateles [24] and Rubrivivax [26], sheathed (96.2%), Ideonella dechloratans (96.2%), and Pelomonas bacteria such as Leptothrix and Sphaerotilus [22], and saccharophila (96.1%) in the Sphaerotilus-Leptothrix group various recalcitrant material-degrading bacteria such as within the order Burkholderiales. Phylogenetic trees based Mitsuaria [1], Paucibacter [17], Methylibium [14], and on 16S rRNA gene sequences indicated that the isolate Aquincola [10]. formed an independent monophyletic clade within the order During the course of a study for revealing bacterial Burkholderiales. The relatively low DNA G+C content diversity in a eutrophic freshwater pond (Inkyong Reservoir) (57.4 mol%), together with several phenotypic characteristics, located at Inha University, several novel strains belonging differentiated the novel strain from other members of the to the Sphaerotilus-Leptothrix group were isolated [21]. Sphaerotilus-Leptothrix group. From the taxonomic data, The present study focuses on the identification and therefore, the strain should be classified as a novel genus characterization of a novel obligately aerobic and non- and species, for which the name Inhella inkyongensis gen. phototrophic strain (designated IMCC1713T) of the nov., sp. nov. is proposed. The type strain of the proposed Sphaerotilus-Leptothrix group. Based on the taxonomic species is strain IMCC1713T (=KCTC 12791T=NBRC data collected in this study, we propose the inclusion of the 103252T=CCUG 54308T). strain in a new genus and novel species named Inhella inkyongensis gen. nov., sp. nov. Keywords: Inhella inkyongensis, Sphaerotilus-Leptothrix group, T freshwater, novel genus, 16S rRNA gene Strain IMCC1713 was isolated using a standard dilution plating method on R2A agar plates. Strain IMCC1713T was purified from the agar plate, which was incubated aerobically at 20oC for 5 days. After the optimum growth The order Burkholderiales [5] in the class Betaproteobacteria conditions of the strain had been determined, bacterial encompasses phenotypically, metabolically, and ecologically cultures were routinely maintained on R2A agar at 25oC diverse members of Gram-negative bacteria. The order and preserved as glycerol suspension (10%) at -70oC. currently contains four families, Burkholderiaceae, PCR amplification of the bacterial 16S rRNA gene was Oxalobacteraceae, Alcaligenaceae, and Comamonadaceae, performed with the slightly modified universal bacterial that have been circumscribed largely based on the 16S primers 27F-B and 1492R [3]. Sequencing of the 16S rRNA rRNA gene sequence-based phylogeny. Although there has gene was carried out as described previously [3], and the almost complete 16S rRNA gene sequence (1,484 bp) was *Corresponding author Phone: 82-32-860-7711; Fax: 82-32-232-0541; determined. To determine the approximate phylogenetic E-mail: [email protected] affiliation of strain IMCC1713T, the 16S rRNA gene 6 Song et al. sequence was compared with the sequence databases in showed 94.5-96.3% 16S rRNA gene sequence similarity RDP-II and GenBank by BLASTN. These comparative to the validly published species of the Sphaerotilus- sequence analyses showed that the strain belonged to the Leptothrix group and 91.2-92.7% to members of the family order Burkholderiales. The 16S rRNA gene sequence of Comamonadaceae. As shown in the phylogenetic tree strain IMCC1713T was aligned with related bacterial (Fig. 1), strain IMCC1713T formed a unique clade with sequences based on the secondary structure of 16S rRNA unidentified bacterium A1004 and uncultured clone 134ds10 using the ARB software package [11]. The 16S rRNA that did not associate significantly with any of the known gene sequence similarities were calculated from distance genera in the Sphaerotilus-Leptothrix group and the family matrices based on the Jukes-Cantor distance formula in the Comamonadaceae. This clade appeared to be monophyletic ARB. A total of 1,117 unambiguously aligned nucleotide in the trees generated by three different treeing algorithms, positions were exported to PAUP* version 4.0 beta 10 for with 100% bootstrap support. These phylogenetic analyses phylogenetic analyses. Phylogenetic trees were inferred by indicated the uniqueness of the 16S rDNA sequence of the neighbor-joining with the Jukes-Cantor distance model, strain; therefore, strain IMCC1713T was considered to maximum parsimony, and maximum likelihood with a represent a new genus and species in the Sphaerotilus- heuristic search, tree bisection-reconnection (TBR)-branching Leptothrix group of the order Burkholderiales. and a Ti/Tv ratio of 1.648. Robustness of tree topologies For phenotypic and chemotaxonomic characterizations, from the neighbor-joining and maximum parsimony were strain IMCC1713T was routinely grown and tested on evaluated by bootstrap analyses based on 1,000 resamplings R2A agar at 25oC, unless otherwise specified. The growth of sequences. temperature range and optimum were tested at 4, 10, 15, Strain IMCC1713T was most closely related to unidentified 20, 25, 30, 37, and 42oC. The pH range and optimum were betaproteobacterium A1004 (AF236009, GenBank description) examined at pH values from pH 4.0 to 12.0, adjusted with and uncultured freshwater clone 134ds10 [19] with 98.8- 0.1 M HCl and 0.1 M NaOH. The optimum NaCl concentration 99.2% 16S rRNA gene sequence similarity. Sequence for growth was monitored on R2A agar supplemented with comparisons with validly published bacteria showed that the 0-15% NaCl (w/v). Cell morphology was observed under strain shared the highest sequence similarity with Roseateles a phase contrast microscope (Nikon 80i; Nikon, Japan) and depolymerans DSM 11813T (96.3%), followed by Mitsuaria transmission electron microscope (TEM; CM200, Philips, chitosanitabida ATCC BAA-476T (96.2%), Ideonella The Netherlands). The presence of intracellular granules and dechloratans CCUG 30898T (96.2%), and Pelomonas flagellation was also observed using TEM. Colony morphology, saccharophila DSM 654T (96.1%). Overall, the strain size, and color were examined from cultures grown Fig. 1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing distant relationships between strain IMCC1713T and representatives of the Sphaerotilus-Leptothrix group and the family Comamonadaceae. Bootstrap percentages (above 50%) from both neighbor-joining (above nodes) and maximum parsimony (below nodes) are shown. Filled and open circles at each node respectively indicate nodes recovered reproducibly by all treeing methods or by two treeing methods. Bar, 0.01 substitutions per nucleotide position. INHELLA INKYONGENSIS GEN. NOV. IN THE BURKHOLDERIALES 7 aerobically for 5 days. Flagellar motility was tested from wet mounts using a 3-day culture. Gliding motility was tested by phase-contrast microscopy with 17-h incubated cells on microscopic slides coated with R2A agar (0.7% agar) according to the method described previously [2]. The Gram reaction was determined by using both nonstaining method using 3% KOH solution and the Gram-staining kit (Scharlau Chemie, Spain). The presence of poly-β- hydroxybutyrate (PHB) granules was examined using Nile
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