IJSEM Papers in Press. Published March 12, 2014 as doi:10.1099/ijs.0.059667-0
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Burkholderia cordobensis sp. nov. from agricultural soils in Argentina
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Walter O. Draghi12; Charlotte Peeters2; Margo Cnockaert2; Cindy Snauwaert3; Luis G. Wall4;
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Angeles Zorreguieta1 and Peter Vandamme2*
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1
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Tecnológicas, Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina.
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2
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Ledeganckstraat 35, B-9000 Ghent, Belgium
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3
Fundación Instituto Leloir and IIBA - Consejo Nacional de Investigaciones Científicas y
Laboratory of Microbiology, Dept. Biochemistry and Microbiology, Ghent University, K. L.
BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, K. L.
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Ledeganckstraat 35, B-9000 Ghent, Belgium.
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4
12
Bernal, B1876BXD, Buenos Aires, Argentina
Science and Technology Dept. National University of Quilmes. Roque Sáenz Peña 352,
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*
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e-mail: [email protected]
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Keywords: Burkholderia, Burkholderia cordobensis sp. nov., Burkholderia glathei,
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Burkholderia sp. YI23
Author for correspondence: Peter Vandamme, Tel : +32 9 264 5113, Fax : +32 9 264 5092,
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Running title: Burkholderia cordobensis sp. nov.
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The Contents Category: New Taxa, subsection Proteobacteria
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and gyrB gene sequences
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of Burkholderia cordobensis sp. nov. LMG 27620T and LMG 27621 are HG324048 and
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HG324055, and HG324049 and HG324056, respectively. The GenBank/EMBL/DDBJ
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accession number for the gyrB gene sequence of Burkholderia grimmiae LMG 27580T is
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HG324054.
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Abstract
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Two gram-negative, rod-shaped bacteria were isolated from agricultural soils in the Córdoba
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province in central Argentina. Their 16S rRNA gene sequences demonstrated that they belong
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to the genus Burkholderia, with Burkholderia zhejiangensis as most closely related, formally
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named species which was confirmed through comparative gyrB sequence analysis. Whole cell
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fatty acid analysis supported their assignment to the genus Burkholderia. Burkholderia sp.
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strain YI23 for which a whole genome sequence is available represents the same taxon as
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demonstrated by its highly similar 16S rRNA (100%) and gyrB (99.1-99.7%) gene sequences.
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The results of DNA-DNA hybridization experiments and the physiological and biochemical
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characterization further substantiated the genotypic and phenotypic distinctiveness of the
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Argentinian soil isolates for which the name Burkholderia cordobensis sp. nov. is proposed,
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with strain LMG 27620T (=CCUG 64368T) as the type strain.
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Burkholderia species belong to the β-proteobacteria and are aerobic, chemoorganotrophic and
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motile (except for Burkholderia mallei) Gram negative rods, which display a wide
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physiological versatility allowing them to inhabit extremely different ecological niches
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(Coenye & Vandamme, 2003; Compant et al., 2008). Several Burkholderia species are human
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or animal pathogens; these include members of the Burkholderia cepacia complex, a group of
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closely related species commonly isolated from respiratory specimens of cystic fibrosis
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patients (Vandamme & Dawyndt, 2011) and species belonging to the Burkholderia
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pseudomallei group which includes B. pseudomallei, the etiological agent of melioidosis in
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humans and B. mallei, the causative agent of glanders in animals (Galyov et al., 2010).
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However, Burkholderia strains including B. cepacia complex bacteria (Parke & Gurian-
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Sherman, 2001) may have beneficial characteristics as well, which include biological nitrogen
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fixation, phytohormone synthesis, plant defense induction and xenobiotic activity (Suárez-
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Moreno et al., 2011; Vial et al., 2011). Also, a growing number of Burkholderia species lives
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associated with other organisms: these include endophytes from plant tissues including root
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nodules, and inhabitants of insect guts or fungal mycelia and spores (Gyaneshwar et al., 2011;
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Kikuchi et al., 2005; Levy et al., 2003; Mahenthiralingam et al., 2008; Verstraete et al.,
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2013).
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Burkholderia strains occur commonly in pristine, agricultural or polluted soils in which their
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population density can reach levels of 103-105 cfu/g soil (Pallud et al., 2001; Salles et al.,
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2006b) but antagonistic Burkholderia species can be lost when soils are continuously used
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under arable management schemes (Salles et al., 2006a). As part of the BIOSPAS Consortium
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(www.biospas.org/en), we analyzed the composition of Burkholderia populations across
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Argentinian agricultural soils which were subjected to different agricultural managements. A
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thorough description of site characteristics and treatment definitions was published elsewhere
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(Figuerola et al., 2012). From these samples, 1 g of soil was suspended into 10 ml saline
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solution (0.85% w/v NaCl), shaken at 240 rpm for 30 min, vortexed for 1 min, sonicated in a
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water bath during 30 sec and centrifuged for 2 min at 500 rpm. Two hundred µl of soil
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suspension was plated onto modified semi-selective medium PCAT (but using citrulline
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instead of tryptamine as the main nitrogen source; PCAT refers to Pseudomonas cepacia,
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azelaic acid and tryptamine) composed of, in gl-1, MgSO4, 0.1; citrulline, 0.2; azelaic acid, 2;
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K2HPO4, 4; KH2PO4, 4; yeast extract, 0.02 and agar, 15. The pH was set at 5.7. The medium
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was supplemented with cycloheximide (0.2 gl-1) and crystal violet (0.002 gl-1) to inhibit
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growth of fungi and Gram-positive bacteria, respectively (Burbage et al., 1982). After 5 days
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of incubation at 28 °C, colonies were randomly selected, subcultured on LB medium
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(composed of, in gl-1: Tryptone, 10; Yeast Extract, 5; NaCl,
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15) to check purity, and maintained at -80 °C as a suspension with 20% (v/v) glycerol. Two
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isolates from Monte Buey soils, in the Córdoba province (32°58′14″S; 62°27′06″W),
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MMP81T (= LMG 27620T, isolated in 2010) and MAN52 (= LMG 27621, isolated in 2011)
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were further characterized in the present study. When analyzing our data we found highly
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similar 16S rRNA and gyrB sequences in the whole genome sequence of strain Burkholderia
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sp. YI23, which was isolated from a golf course soil in South Korea and which showed the
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ability to degrade the pesticide fenitrothion (GenBank accession numbers CP003087,
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CP003088, CP003089, CP003090, CP003091, and CP003092 (Lim et al., 2012).
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For PCR, genomic DNA was prepared using the method as described by (Niemann et al.,
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1997). Random amplified polymorphic DNA patterns were generated using the primers
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TGCGCGCGGG and AGCGGGCCAA, as previously described (Williams et al., 1990) and
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demonstrated that isolates LMG 27620T and LMG 27621 represent genetically distinct strains
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(Figure S1).
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The nearly complete sequences of the 16S rRNA gene of strains LMG 27620T and LMG
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27621 were obtained as described previously (Peeters et al., 2013). The Mothur software
10 and agar,
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package (Schloss et al., 2009) was used to align the 16S rRNA gene sequences of strains
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LMG 27620T, LMG 27621 and YI23 (1484 bp) with those of type strains of phylogenetically
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related Burkholderia species (1388–1525 bp) against the Silva reference database (www.arb-
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silva.de). Phylogenetic analysis was conducted in MEGA5 (Tamura et al., 2011) (Figure 1).
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Uncorrected pairwise distances were calculated using MEGA5 (Tamura et al., 2011) and all
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ambiguous positions were removed for each sequence pair. Strains LMG 27620T, LMG 27621
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and YI23 had identical 16S rRNA gene sequences, which were highly similar to those of
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Burkholderia zhejiangensis OP-1T (99.7%) and Burkholderia grimmiae R27T (99.3%) (Figure
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1).
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Partial sequences of the gyrB gene of strains LMG 27620T and LMG 27621 and their nearest
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neighbors as determined by 16S rRNA sequence proximity were obtained using the method
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described by (Spilker et al., 2009) as modified by (De Meyer et al., 2013). Sequence
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assembly was performed using BioNumerics v5.10 (Applied Maths). Sequences (589-704 bp)
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were aligned based on amino acid sequences using Muscle (Edgar, 2004) in MEGA5 (Tamura
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et al., 2011). Phylogenetic analysis of nucleotide sequences was conducted in MEGA5
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(Tamura et al., 2011) (Figure 2). Uncorrected pairwise distances were calculated using
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MEGA5 (Tamura et al., 2011). Strains LMG 27620T, LMG 27621 and YI23 formed a
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homogeneous cluster (sequences were >99% similar), which was supported by a 75%
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bootstrap value (Figure 2) and grouped with B. zhejiangensis LMG 27258T (95.3-95.9%) and
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B. grimmiae LMG 27580T (90.8-91.6%) as nearest neighbors.
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For DNA-DNA hybridization and for the determination of the DNA G+C content, high-
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molecular weight DNA was prepared as described by (Pitcher et al., 1989). DNA-DNA
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hybridizations were performed using a microplate method and biotinylated probe DNA (Ezaki
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et al., 1989). The hybridization temperature was 45°C ± 1°C. Reciprocal reactions (A x B and
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B x A) were performed and the variation was within the limits of this method (Goris et al.,
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1998). DNA-DNA hybridization experiments were performed between strain LMG 27620T
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and B. zhejiangensis LMG 27258T, its nearest named phylogenetic neighbor (Figure 1 and 2).
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The DNA-DNA hybridization value between both strains was 44%. The G+C content of DNA
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was determined by HPLC according to the method of (Mesbah & Whitman, 1989) using a
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Waters Breeze HPLC system and XBridge Shield RP18 column thermostabilised at 37°C. The
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solvent was 0.02 M NH4H2PO4 (pH 4.0) with 1.5% (v/v) acetonitrile. Non-methylated lambda
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phage (Sigma) and E. coli DNA were used as calibration reference and control, respectively.
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The DNA G+C content of strain LMG 27620T was 63.6 mol%, which is within the range
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reported for Burkholderia genus (59-69.9 mol%) (Gillis et al., 1995; Yabuuchi et al., 1992)
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and which corresponds to the G+C content of 63.5%, reported for the Burkholderia sp. strain
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YI23 whole genome sequence (Lim et al., 2012).
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Phenotypic analysis of strains LMG 27620T and LMG 27621 and of B. zhejiangensis LMG
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27258T was performed on Tryptone Soya Agar (TSA) at 28°C unless indicated otherwise
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(LMG medium 14; http://bccm.belspo.be/db/media_search_form.php). Cell morphology and
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motility were observed by phase contrast microscopy. Oxidase activity was detected by
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immersion of cells in 1% N,N,N′,N′-tetramethyl-p-phenylenediamine solution and catalase
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activity was determined by bubbling through flooding a colony with 10% H2O2. Lipase
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activity was determined according to the method described by (Sierra, 1957). Growth on
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MacConkey medium was observed after 48h of incubation at 28 °C. Starch hydrolysis was
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observed after 48h of incubation at 28 °C in LMG14 medium amended with 2% of starch.
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Deoxyribonuclease activity (DNase) was observed after 48h of incubation at 28 °C on BD
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Difco™ DNase Test Agar, according to the method of (Jeffries et al., 1957). Casein hydrolysis
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was observed after 48 h of incubation at 28 °C on TSA plates amended with 1.3% of skim
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milk, through the observation of clear halos around colonies. Other biochemical tests were
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performed by inoculating API 20NE and API ZYM strips (BioMérieux) according to the
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manufacturer’s instructions and incubating for 48h at 28°C, or for 4 h at 28 °C, respectively.
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Growth was tested at 28°C on nutrient broth (NB, from BD Difco™) at pH4 - pH9 using
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appropriate biological buffers (acetate, citrate-Na2HPO4, phosphate buffer and Tris-
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hydrochloride). Growth on LMG 14 medium was tested at 4 °C, 15 °C, 28 °C, 30 °C, 37 °C,
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40 °C and 45°C on LMG medium14. The results of the phenotypic and biochemical tests are
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given in the species description below and in Table 1. They allow a clear differentiation of the
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taxon represented by strains LMG 27620T and LMG 27621 and its nearest phylogenetic
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neighbor, B. zhejiangensis: the former exhibits oxidase but not arginine dihydrolase or urease
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activity, reduces nitrate, hydrolyses Tween 80, and does not assimilate caprate; opposite test
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results were obtained for the latter. In addition, on the basis of phenotypic data published by
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(Tian et al., 2013) using the same test gallery, strains LMG 27620T and LMG 27621 can be
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differentiated from B. grimmiae R27T: the former exhibits no arginine dihydrolase or urease
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activity, and assimilates phenyl acetate. In addition, it does not hydrolyse starch and grows on
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MacConkey agar; opposite test results were obtained for B. grimmiae R27T. Differential
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characteristics towards other Burkholderia species in this same lineage are presented in Table
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1.
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Whole-cell fatty acid methyl esters were extracted according to the MIDI protocol
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(http://www.microbialid.com/PDF/TechNote_101.pdf).
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temperature, medium, and physiological age (overlap area of the second and third quadrant
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from a quadrant streak for late log phase growing cells) were as in the MIDI protocol. The
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profiles were generated using an Agilent Technologies 6890N gas chromatograph (Santa
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Clara, CA USA), identified and clustered using the Microbial Identification System software
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and MIDI TSBA database version 5.0. The most abundant fatty acids of strains LMG 27620T
All
characteristics
such
as
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and LMG 27621 were: C18:1 ω7c, summed feature 3 (most likely C16:1 ω7c) and C16:0. Moderate
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to low amounts of summed feature 2 (most likely C14:0 3OH), cyclo-C17:0, C16:0 3-OH, C14:0,
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cyclo-C19:0 ω8c, C16:1 2-OH and C16:0 2-OH have been detected. The presence of C16:0 3-OH
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supports the placement of these strains in the genus Burkholderia (Yabuuchi et al., 1992) but
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the overall profile is very similar to that of its nearest neighbors (Table 2).
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The present phenotypic, chemotaxonomic and genotypic data demonstrate that strains LMG
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27620T, LMG 27621 and YI23 represent a novel Burkholderia species that can be
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distinguished from its nearest phylogenetic neighbors both phenotypically as well as
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genotypically. We therefore propose to classify these bacteria as a new species for which the
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name Burkholderia cordobensis sp. nov. is proposed, with strain LMG 27620T
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(=CCUG64368T) as the type strain.
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Description of Burkholderia cordobensis sp. nov.
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Burkholderia cordobensis (cor.do.ben´sis. N. L. fem. adj. cordobensis pertaining to the
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Argentinian province Córdoba.
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Gram negative, aerobic, motile, non-spore forming rods about 0.4-0.7 in width and 1.2-1.8 in
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length. Colonies are round, with entire margins, a convex elevation, a white-creamy color, and
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a moist appearance and are 1-2 mm in diameter after 48 h of growth on TSA medium at 28
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°C. Growth on MacConkey agar. Growth occurs between 28°C and 37 °C, and from pH 6 to
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pH 8 in liquid nutrient broth at 28 °C. Catalase and oxidase activity is present, but no arginine
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dihydrolase, urease, β-glucosidase, gelatinase or β-galactosidase activity. When tested by
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using API ZYM strips, activities of the following enzymes are present: alkaline and acid
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phospatase, leucyl arylamidase, and phosphoamidase; enzymatic activities were absent for C4-
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lipase, C8-lipase, C14-lipase, valine and cystine arylamidase, trypsin, chymotrypsin, α-
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galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-
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glucosaminidase, α- mannosidase and α-fucosidase. Tween 20, Tween 40, Tween 60 and
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Tween 80 are degraded, but casein and starch were not hydrolysed. When tested by using API
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20NE strips nitrate was reduced; glucose, arabinose (except for strain LMG 27621), mannose,
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mannitol, N-acetyl-glucosamine, gluconate, malate and phenyl-acetate were assimilated, but
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not maltose, caprate, adipate or citrate. No fermentation of glucose, tryptophanase, arginine
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dihydrolase, urease, or PNPG beta-galactosidase activity; no hydrolysis of aesculin or gelatin
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liquefaction. The most abundant fatty acids were C18:1 ω7c, summed feature 3 (probably C16:1
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ω7c) and C16:0.
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The type strain, LMG 27620T(=CCUG 64368T), was isolated from agricultural soils at
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Córdoba province, Argentina. The DNA G+C content is 63.6%. The whole genome sequence
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of strain YI23 is 8.89 Mb and consists of three chromosomes and three plasmids (Lim et al.,
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2012).
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Acknowledgements
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This work was partially supported by grants PAE 36976-PID 52 and PICT 2010/2691
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(Agencia Nacional de Promoción Científica y Tecnológica, Argentina). W.O.D., A.Z. and
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L.G.W. are members of Research Career at CONICET, Argentina.
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Verstraete, B., Janssens, S., Smets, E. & Dessein, S. (2013). Symbiotic β-proteobacteria beyond legumes: Burkholderia in Rubiaceae. PLoS One 8, e55260.
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Vial, L., Chapalain, A., Groleau, M. C. & Deziel, E. (2011). The various lifestyles of the
318
Burkholderia cepacia complex species: a tribute to adaptation. Environ Microbiol 13,
319
1-12.
320
Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A. & Tingey, S. V. (1990).
321
DNA polymorphisms amplified by arbitrary primers are useful as genetic markers.
322
Nucleic Acids Res 18, 6531-6535.
323
Yabuuchi, E., Kosako, Y., Oyaizu, H., Yano, I., Hotta, H., Hashimoto, Y., Ezaki, T. &
324
Arakawa, M. (1992). Proposal of Burkholderia gen. nov. and transfer of seven
325
species of the genus Pseudomonas homology group II to the new genus, with the type
326
species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol
327
Immunol 36, 1251-1275.
328 329
330
Table 1. Phenotypic characteristics distinguishing B. cordobensis sp. nov. from its nearest
331
phylogenetic neighbor species.
332
Species: 1. B. cordobensis sp. nov. (LMG 26720T, LMG 27621); 2: B. zhejiangensis (LMG
333
27258T, LMG 26180, LMG 26181); 3, B. grimmiae (R27T); 4, B. choica (LMG 22940T); 5, B.
334
glathei (LMG 14190T); 6, B. humi (LMG 22934T);7, B. sordidicola (LMG 22029T); 8, B.
335
telluris (LMG 22936T); 9, B. terrestris (LMG 22937T); 10, B. udeis (LMG 27134T). Results
336
from B. cordobensis sp. nov. and its nearest neighbor B. zhejiangensis are from the present
337
study. Test results of the type strain are given first, followed by remaining strains in order as
338
presented in the title. Data from B. grimmiae are extracted from (Tian et al., 2013). Data from
339
the remaining species were taken from (Vandamme et al., 2013). +, Present; 2, absent; W,
340
weak reaction; ND, Not determined.
341
1 Growth at: 37 °C 40 °C pH 8 Hydrolysis of Tween 60 Nitrate reduction (API -20NE) β-Galactosidase activity (API -20NE) Assimilation of (API -20NE): Arabinose Mannose Mannitol N-Acetylglucosamine Gluconate Caprate Malate Citrate Phenylacetate Enzyme activity (API ZYM) C4-Lipase C8-Lipase Valine arylamidase Cystine arylamidase β-Galactosidase 342
+-ww ++ w+
2
3
4 5 6 7 8 9 10
+++ + w +++ + +++ + +++ ND + +++ + -
+ -
+ -
w +
w + +
-
+ +
--
---
-
- - - + - - w
+++ ++ ++ ++ -++ -++
+++ +++ --+++ +++ +++ +++ --++w
+ + + + + + -
w w w w w -
w + w + + + + + +
+ + + + w + + +
+ + + + + v -
+ + + + + + + + +
+ + + + w + + +
+ + + + + + -
------
-----------
+ + -
w + w + -
+ w w -
+ + -
+ + +
+ w -
+ w w -
+ + + +
343
Table 2. Average fatty acid composition of B. cordobensis sp. nov. and of its nearest
344
phylogenetic neighbor species. Species: 1, B. cordobensis sp. nov. (LMG 27620T and LMG
345
27621); 2, B. zhejiangensis (LMG 27258T, LMG 26180, LMG 26181); 3, B. grimmiae (LMG
346
27258T); 4, B. choica; 5, B. glathei; 6, B. humi; 7, B. sordidicola; 8, B. telluris; 9, B. terrestris;
347
10, B. udeis. Data for B. cordobensis sp. nov. and B. grimmiae are from the present study. The
348
remaining data were extracted from (Vandamme et al., 2013) and were generated in the same
349
cultivation and analysis conditions. Those fatty acids for which the mean amount for all taxa
350
was < 1% are not included, therefore, the percentages may not add up to 100 %. TR, Trace
351
amount (
1
Burkholderia cordobensis sp. nov. from agricultural soils in Argentina
2
Walter O. Draghi12; Charlotte Peeters2; Margo Cnockaert2; Cindy Snauwaert3; Luis G. Wall4;
3
Angeles Zorreguieta1 and Peter Vandamme2*
4 5
1
6
Tecnológicas, Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina.
7
2
8
Ledeganckstraat 35, B-9000 Ghent, Belgium
9
3
Fundación Instituto Leloir and IIBA - Consejo Nacional de Investigaciones Científicas y
Laboratory of Microbiology, Dept. Biochemistry and Microbiology, Ghent University, K. L.
BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, K. L.
10
Ledeganckstraat 35, B-9000 Ghent, Belgium.
11
4
12
Bernal, B1876BXD, Buenos Aires, Argentina
Science and Technology Dept. National University of Quilmes. Roque Sáenz Peña 352,
13 14
*
15
e-mail: [email protected]
16
Keywords: Burkholderia, Burkholderia cordobensis sp. nov., Burkholderia glathei,
17
Burkholderia sp. YI23
Author for correspondence: Peter Vandamme, Tel : +32 9 264 5113, Fax : +32 9 264 5092,
18 19
Running title: Burkholderia cordobensis sp. nov.
20
The Contents Category: New Taxa, subsection Proteobacteria
21 22 23 24 25
26
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and gyrB gene sequences
27
of Burkholderia cordobensis sp. nov. LMG 27620T and LMG 27621 are HG324048 and
28
HG324055, and HG324049 and HG324056, respectively. The GenBank/EMBL/DDBJ
29
accession number for the gyrB gene sequence of Burkholderia grimmiae LMG 27580T is
30
HG324054.
31
Abstract
32
Two gram-negative, rod-shaped bacteria were isolated from agricultural soils in the Córdoba
33
province in central Argentina. Their 16S rRNA gene sequences demonstrated that they belong
34
to the genus Burkholderia, with Burkholderia zhejiangensis as most closely related, formally
35
named species which was confirmed through comparative gyrB sequence analysis. Whole cell
36
fatty acid analysis supported their assignment to the genus Burkholderia. Burkholderia sp.
37
strain YI23 for which a whole genome sequence is available represents the same taxon as
38
demonstrated by its highly similar 16S rRNA (100%) and gyrB (99.1-99.7%) gene sequences.
39
The results of DNA-DNA hybridization experiments and the physiological and biochemical
40
characterization further substantiated the genotypic and phenotypic distinctiveness of the
41
Argentinian soil isolates for which the name Burkholderia cordobensis sp. nov. is proposed,
42
with strain LMG 27620T (=CCUG 64368T) as the type strain.
43
44
Burkholderia species belong to the β-proteobacteria and are aerobic, chemoorganotrophic and
45
motile (except for Burkholderia mallei) Gram negative rods, which display a wide
46
physiological versatility allowing them to inhabit extremely different ecological niches
47
(Coenye & Vandamme, 2003; Compant et al., 2008). Several Burkholderia species are human
48
or animal pathogens; these include members of the Burkholderia cepacia complex, a group of
49
closely related species commonly isolated from respiratory specimens of cystic fibrosis
50
patients (Vandamme & Dawyndt, 2011) and species belonging to the Burkholderia
51
pseudomallei group which includes B. pseudomallei, the etiological agent of melioidosis in
52
humans and B. mallei, the causative agent of glanders in animals (Galyov et al., 2010).
53
However, Burkholderia strains including B. cepacia complex bacteria (Parke & Gurian-
54
Sherman, 2001) may have beneficial characteristics as well, which include biological nitrogen
55
fixation, phytohormone synthesis, plant defense induction and xenobiotic activity (Suárez-
56
Moreno et al., 2011; Vial et al., 2011). Also, a growing number of Burkholderia species lives
57
associated with other organisms: these include endophytes from plant tissues including root
58
nodules, and inhabitants of insect guts or fungal mycelia and spores (Gyaneshwar et al., 2011;
59
Kikuchi et al., 2005; Levy et al., 2003; Mahenthiralingam et al., 2008; Verstraete et al.,
60
2013).
61
Burkholderia strains occur commonly in pristine, agricultural or polluted soils in which their
62
population density can reach levels of 103-105 cfu/g soil (Pallud et al., 2001; Salles et al.,
63
2006b) but antagonistic Burkholderia species can be lost when soils are continuously used
64
under arable management schemes (Salles et al., 2006a). As part of the BIOSPAS Consortium
65
(www.biospas.org/en), we analyzed the composition of Burkholderia populations across
66
Argentinian agricultural soils which were subjected to different agricultural managements. A
67
thorough description of site characteristics and treatment definitions was published elsewhere
68
(Figuerola et al., 2012). From these samples, 1 g of soil was suspended into 10 ml saline
69
solution (0.85% w/v NaCl), shaken at 240 rpm for 30 min, vortexed for 1 min, sonicated in a
70
water bath during 30 sec and centrifuged for 2 min at 500 rpm. Two hundred µl of soil
71
suspension was plated onto modified semi-selective medium PCAT (but using citrulline
72
instead of tryptamine as the main nitrogen source; PCAT refers to Pseudomonas cepacia,
73
azelaic acid and tryptamine) composed of, in gl-1, MgSO4, 0.1; citrulline, 0.2; azelaic acid, 2;
74
K2HPO4, 4; KH2PO4, 4; yeast extract, 0.02 and agar, 15. The pH was set at 5.7. The medium
75
was supplemented with cycloheximide (0.2 gl-1) and crystal violet (0.002 gl-1) to inhibit
76
growth of fungi and Gram-positive bacteria, respectively (Burbage et al., 1982). After 5 days
77
of incubation at 28 °C, colonies were randomly selected, subcultured on LB medium
78
(composed of, in gl-1: Tryptone, 10; Yeast Extract, 5; NaCl,
79
15) to check purity, and maintained at -80 °C as a suspension with 20% (v/v) glycerol. Two
80
isolates from Monte Buey soils, in the Córdoba province (32°58′14″S; 62°27′06″W),
81
MMP81T (= LMG 27620T, isolated in 2010) and MAN52 (= LMG 27621, isolated in 2011)
82
were further characterized in the present study. When analyzing our data we found highly
83
similar 16S rRNA and gyrB sequences in the whole genome sequence of strain Burkholderia
84
sp. YI23, which was isolated from a golf course soil in South Korea and which showed the
85
ability to degrade the pesticide fenitrothion (GenBank accession numbers CP003087,
86
CP003088, CP003089, CP003090, CP003091, and CP003092 (Lim et al., 2012).
87
For PCR, genomic DNA was prepared using the method as described by (Niemann et al.,
88
1997). Random amplified polymorphic DNA patterns were generated using the primers
89
TGCGCGCGGG and AGCGGGCCAA, as previously described (Williams et al., 1990) and
90
demonstrated that isolates LMG 27620T and LMG 27621 represent genetically distinct strains
91
(Figure S1).
92
The nearly complete sequences of the 16S rRNA gene of strains LMG 27620T and LMG
93
27621 were obtained as described previously (Peeters et al., 2013). The Mothur software
10 and agar,
94
package (Schloss et al., 2009) was used to align the 16S rRNA gene sequences of strains
95
LMG 27620T, LMG 27621 and YI23 (1484 bp) with those of type strains of phylogenetically
96
related Burkholderia species (1388–1525 bp) against the Silva reference database (www.arb-
97
silva.de). Phylogenetic analysis was conducted in MEGA5 (Tamura et al., 2011) (Figure 1).
98
Uncorrected pairwise distances were calculated using MEGA5 (Tamura et al., 2011) and all
99
ambiguous positions were removed for each sequence pair. Strains LMG 27620T, LMG 27621
100
and YI23 had identical 16S rRNA gene sequences, which were highly similar to those of
101
Burkholderia zhejiangensis OP-1T (99.7%) and Burkholderia grimmiae R27T (99.3%) (Figure
102
1).
103
Partial sequences of the gyrB gene of strains LMG 27620T and LMG 27621 and their nearest
104
neighbors as determined by 16S rRNA sequence proximity were obtained using the method
105
described by (Spilker et al., 2009) as modified by (De Meyer et al., 2013). Sequence
106
assembly was performed using BioNumerics v5.10 (Applied Maths). Sequences (589-704 bp)
107
were aligned based on amino acid sequences using Muscle (Edgar, 2004) in MEGA5 (Tamura
108
et al., 2011). Phylogenetic analysis of nucleotide sequences was conducted in MEGA5
109
(Tamura et al., 2011) (Figure 2). Uncorrected pairwise distances were calculated using
110
MEGA5 (Tamura et al., 2011). Strains LMG 27620T, LMG 27621 and YI23 formed a
111
homogeneous cluster (sequences were >99% similar), which was supported by a 75%
112
bootstrap value (Figure 2) and grouped with B. zhejiangensis LMG 27258T (95.3-95.9%) and
113
B. grimmiae LMG 27580T (90.8-91.6%) as nearest neighbors.
114
For DNA-DNA hybridization and for the determination of the DNA G+C content, high-
115
molecular weight DNA was prepared as described by (Pitcher et al., 1989). DNA-DNA
116
hybridizations were performed using a microplate method and biotinylated probe DNA (Ezaki
117
et al., 1989). The hybridization temperature was 45°C ± 1°C. Reciprocal reactions (A x B and
118
B x A) were performed and the variation was within the limits of this method (Goris et al.,
119
1998). DNA-DNA hybridization experiments were performed between strain LMG 27620T
120
and B. zhejiangensis LMG 27258T, its nearest named phylogenetic neighbor (Figure 1 and 2).
121
The DNA-DNA hybridization value between both strains was 44%. The G+C content of DNA
122
was determined by HPLC according to the method of (Mesbah & Whitman, 1989) using a
123
Waters Breeze HPLC system and XBridge Shield RP18 column thermostabilised at 37°C. The
124
solvent was 0.02 M NH4H2PO4 (pH 4.0) with 1.5% (v/v) acetonitrile. Non-methylated lambda
125
phage (Sigma) and E. coli DNA were used as calibration reference and control, respectively.
126
The DNA G+C content of strain LMG 27620T was 63.6 mol%, which is within the range
127
reported for Burkholderia genus (59-69.9 mol%) (Gillis et al., 1995; Yabuuchi et al., 1992)
128
and which corresponds to the G+C content of 63.5%, reported for the Burkholderia sp. strain
129
YI23 whole genome sequence (Lim et al., 2012).
130
Phenotypic analysis of strains LMG 27620T and LMG 27621 and of B. zhejiangensis LMG
131
27258T was performed on Tryptone Soya Agar (TSA) at 28°C unless indicated otherwise
132
(LMG medium 14; http://bccm.belspo.be/db/media_search_form.php). Cell morphology and
133
motility were observed by phase contrast microscopy. Oxidase activity was detected by
134
immersion of cells in 1% N,N,N′,N′-tetramethyl-p-phenylenediamine solution and catalase
135
activity was determined by bubbling through flooding a colony with 10% H2O2. Lipase
136
activity was determined according to the method described by (Sierra, 1957). Growth on
137
MacConkey medium was observed after 48h of incubation at 28 °C. Starch hydrolysis was
138
observed after 48h of incubation at 28 °C in LMG14 medium amended with 2% of starch.
139
Deoxyribonuclease activity (DNase) was observed after 48h of incubation at 28 °C on BD
140
Difco™ DNase Test Agar, according to the method of (Jeffries et al., 1957). Casein hydrolysis
141
was observed after 48 h of incubation at 28 °C on TSA plates amended with 1.3% of skim
142
milk, through the observation of clear halos around colonies. Other biochemical tests were
143
performed by inoculating API 20NE and API ZYM strips (BioMérieux) according to the
144
manufacturer’s instructions and incubating for 48h at 28°C, or for 4 h at 28 °C, respectively.
145
Growth was tested at 28°C on nutrient broth (NB, from BD Difco™) at pH4 - pH9 using
146
appropriate biological buffers (acetate, citrate-Na2HPO4, phosphate buffer and Tris-
147
hydrochloride). Growth on LMG 14 medium was tested at 4 °C, 15 °C, 28 °C, 30 °C, 37 °C,
148
40 °C and 45°C on LMG medium14. The results of the phenotypic and biochemical tests are
149
given in the species description below and in Table 1. They allow a clear differentiation of the
150
taxon represented by strains LMG 27620T and LMG 27621 and its nearest phylogenetic
151
neighbor, B. zhejiangensis: the former exhibits oxidase but not arginine dihydrolase or urease
152
activity, reduces nitrate, hydrolyses Tween 80, and does not assimilate caprate; opposite test
153
results were obtained for the latter. In addition, on the basis of phenotypic data published by
154
(Tian et al., 2013) using the same test gallery, strains LMG 27620T and LMG 27621 can be
155
differentiated from B. grimmiae R27T: the former exhibits no arginine dihydrolase or urease
156
activity, and assimilates phenyl acetate. In addition, it does not hydrolyse starch and grows on
157
MacConkey agar; opposite test results were obtained for B. grimmiae R27T. Differential
158
characteristics towards other Burkholderia species in this same lineage are presented in Table
159
1.
160
161
Whole-cell fatty acid methyl esters were extracted according to the MIDI protocol
162
(http://www.microbialid.com/PDF/TechNote_101.pdf).
163
temperature, medium, and physiological age (overlap area of the second and third quadrant
164
from a quadrant streak for late log phase growing cells) were as in the MIDI protocol. The
165
profiles were generated using an Agilent Technologies 6890N gas chromatograph (Santa
166
Clara, CA USA), identified and clustered using the Microbial Identification System software
167
and MIDI TSBA database version 5.0. The most abundant fatty acids of strains LMG 27620T
All
characteristics
such
as
168
and LMG 27621 were: C18:1 ω7c, summed feature 3 (most likely C16:1 ω7c) and C16:0. Moderate
169
to low amounts of summed feature 2 (most likely C14:0 3OH), cyclo-C17:0, C16:0 3-OH, C14:0,
170
cyclo-C19:0 ω8c, C16:1 2-OH and C16:0 2-OH have been detected. The presence of C16:0 3-OH
171
supports the placement of these strains in the genus Burkholderia (Yabuuchi et al., 1992) but
172
the overall profile is very similar to that of its nearest neighbors (Table 2).
173
The present phenotypic, chemotaxonomic and genotypic data demonstrate that strains LMG
174
27620T, LMG 27621 and YI23 represent a novel Burkholderia species that can be
175
distinguished from its nearest phylogenetic neighbors both phenotypically as well as
176
genotypically. We therefore propose to classify these bacteria as a new species for which the
177
name Burkholderia cordobensis sp. nov. is proposed, with strain LMG 27620T
178
(=CCUG64368T) as the type strain.
179
180
Description of Burkholderia cordobensis sp. nov.
181
Burkholderia cordobensis (cor.do.ben´sis. N. L. fem. adj. cordobensis pertaining to the
182
Argentinian province Córdoba.
183
Gram negative, aerobic, motile, non-spore forming rods about 0.4-0.7 in width and 1.2-1.8 in
184
length. Colonies are round, with entire margins, a convex elevation, a white-creamy color, and
185
a moist appearance and are 1-2 mm in diameter after 48 h of growth on TSA medium at 28
186
°C. Growth on MacConkey agar. Growth occurs between 28°C and 37 °C, and from pH 6 to
187
pH 8 in liquid nutrient broth at 28 °C. Catalase and oxidase activity is present, but no arginine
188
dihydrolase, urease, β-glucosidase, gelatinase or β-galactosidase activity. When tested by
189
using API ZYM strips, activities of the following enzymes are present: alkaline and acid
190
phospatase, leucyl arylamidase, and phosphoamidase; enzymatic activities were absent for C4-
191
lipase, C8-lipase, C14-lipase, valine and cystine arylamidase, trypsin, chymotrypsin, α-
192
galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-
193
glucosaminidase, α- mannosidase and α-fucosidase. Tween 20, Tween 40, Tween 60 and
194
Tween 80 are degraded, but casein and starch were not hydrolysed. When tested by using API
195
20NE strips nitrate was reduced; glucose, arabinose (except for strain LMG 27621), mannose,
196
mannitol, N-acetyl-glucosamine, gluconate, malate and phenyl-acetate were assimilated, but
197
not maltose, caprate, adipate or citrate. No fermentation of glucose, tryptophanase, arginine
198
dihydrolase, urease, or PNPG beta-galactosidase activity; no hydrolysis of aesculin or gelatin
199
liquefaction. The most abundant fatty acids were C18:1 ω7c, summed feature 3 (probably C16:1
200
ω7c) and C16:0.
201
The type strain, LMG 27620T(=CCUG 64368T), was isolated from agricultural soils at
202
Córdoba province, Argentina. The DNA G+C content is 63.6%. The whole genome sequence
203
of strain YI23 is 8.89 Mb and consists of three chromosomes and three plasmids (Lim et al.,
204
2012).
205
206
Acknowledgements
207
This work was partially supported by grants PAE 36976-PID 52 and PICT 2010/2691
208
(Agencia Nacional de Promoción Científica y Tecnológica, Argentina). W.O.D., A.Z. and
209
L.G.W. are members of Research Career at CONICET, Argentina.
210
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326
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327
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328 329
330
Table 1. Phenotypic characteristics distinguishing B. cordobensis sp. nov. from its nearest
331
phylogenetic neighbor species.
332
Species: 1. B. cordobensis sp. nov. (LMG 26720T, LMG 27621); 2: B. zhejiangensis (LMG
333
27258T, LMG 26180, LMG 26181); 3, B. grimmiae (R27T); 4, B. choica (LMG 22940T); 5, B.
334
glathei (LMG 14190T); 6, B. humi (LMG 22934T);7, B. sordidicola (LMG 22029T); 8, B.
335
telluris (LMG 22936T); 9, B. terrestris (LMG 22937T); 10, B. udeis (LMG 27134T). Results
336
from B. cordobensis sp. nov. and its nearest neighbor B. zhejiangensis are from the present
337
study. Test results of the type strain are given first, followed by remaining strains in order as
338
presented in the title. Data from B. grimmiae are extracted from (Tian et al., 2013). Data from
339
the remaining species were taken from (Vandamme et al., 2013). +, Present; 2, absent; W,
340
weak reaction; ND, Not determined.
341
1 Growth at: 37 °C 40 °C pH 8 Hydrolysis of Tween 60 Nitrate reduction (API -20NE) β-Galactosidase activity (API -20NE) Assimilation of (API -20NE): Arabinose Mannose Mannitol N-Acetylglucosamine Gluconate Caprate Malate Citrate Phenylacetate Enzyme activity (API ZYM) C4-Lipase C8-Lipase Valine arylamidase Cystine arylamidase β-Galactosidase 342
+-ww ++ w+
2
3
4 5 6 7 8 9 10
+++ + w +++ + +++ + +++ ND + +++ + -
+ -
+ -
w +
w + +
-
+ +
--
---
-
- - - + - - w
+++ ++ ++ ++ -++ -++
+++ +++ --+++ +++ +++ +++ --++w
+ + + + + + -
w w w w w -
w + w + + + + + +
+ + + + w + + +
+ + + + + v -
+ + + + + + + + +
+ + + + w + + +
+ + + + + + -
------
-----------
+ + -
w + w + -
+ w w -
+ + -
+ + +
+ w -
+ w w -
+ + + +
343
Table 2. Average fatty acid composition of B. cordobensis sp. nov. and of its nearest
344
phylogenetic neighbor species. Species: 1, B. cordobensis sp. nov. (LMG 27620T and LMG
345
27621); 2, B. zhejiangensis (LMG 27258T, LMG 26180, LMG 26181); 3, B. grimmiae (LMG
346
27258T); 4, B. choica; 5, B. glathei; 6, B. humi; 7, B. sordidicola; 8, B. telluris; 9, B. terrestris;
347
10, B. udeis. Data for B. cordobensis sp. nov. and B. grimmiae are from the present study. The
348
remaining data were extracted from (Vandamme et al., 2013) and were generated in the same
349
cultivation and analysis conditions. Those fatty acids for which the mean amount for all taxa
350
was < 1% are not included, therefore, the percentages may not add up to 100 %. TR, Trace
351
amount (
