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Pseudoxanthomonas Composti Sp. Nov., Isolated from Compost

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Pseudoxanthomonas Composti Sp. Nov., Isolated from Compost Antonie van Leeuwenhoek https://doi.org/10.1007/s10482-019-01253-z (0123456789().,-volV)(0123456789().,-volV) ORIGINAL PAPER Pseudoxanthomonas composti sp. nov., isolated from compost Jun Lin . Guiqin Yang . Jia Tang . Zhen Li . Zhen Yu . Li Zhuang Received: 17 January 2019 / Accepted: 27 February 2019 Ó Springer Nature Switzerland AG 2019 Abstract A Gram-staining negative bacterium, des- respiratory quinone. The major cellular fatty acids T ignated as GSS15 , was isolated from compost in ( [ 5%) were iso-C15:0 (18.7%), C16:1x7c (18.6%), T Guangzhou, China. Cells of strain GSS15 were rod- anteiso-C15:0 (13.2%), C16:0 (9.8%), and iso-C16:0 shaped and non-motile. The isolate was able to grow at (8.8%). The polar lipids consisted of phos- 15–42 °C (optimum 30 °C) and pH 6.0–11.0 (optimum phatidylethanolamine, phosphatidylglycerol, and pH 8.0), and tolerate up to 6.0% NaCl (w/v). When the diphosphatidylglycerol. Based on its phenotypic, 16S rRNA gene sequence of the isolate was compared chemotaxonomic and genotypic data, strain GSS15T with those of other bacteria, the highest similarity was (= KCTC 52974T =MCCC1K03334T)isdesignated observed with Pseudoxanthomonas helianthi roo10T as the type strain of a novel species of the genus (96.9%). Furthermore, strain GSS15T showed low ANI Pseudoxanthomonas, for which the name Pseudoxan- (75.7–79.5%) and DDH (24.2–18.3%) values to the thomonas composti sp. nov. is proposed. closely related species. Q-8 was the predominant Keywords Pseudoxanthomonas composti Á Novel species Á Polyphasic taxonomy Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10482-019-01253-z) con- tains supplementary material, which is available to authorized users. Introduction J. Lin Á G. Yang Á L. Zhuang (&) Guangdong Key Laboratory of Environmental Pollution The genus Pseudoxanthomonas, belonging to the family and Health, School of Environment, Jinan University, Xanthomonadaceae, was first described by Finkmann Guangzhou 510632, China e-mail: [email protected] et al. (2000) when the type species Pseudoxanthomonas broegbernensis was isolated from ammonia-supplied J. Tang Á Z. Yu (&) biofilters. Emended descriptions of the genus were later Guangdong Key Laboratory of Integrated Agro- made by Thierry et al. (2004)andLeeetal.(2008). The Environmental Pollution Control and Management, Guangdong Institute of Eco-Environmental Science and genus Pseudoxanthomonas comprises Gram-staining Technology, Guangzhou 510650, China negative and rod-shaped bacteria that are characterized e-mail: [email protected] chemotaxonomically by the presence of ubiquinone 8 (Q- 8) as the major isoprenoid quinone and iso-C as the Z. Li 15:0 College of Packaging and Materials Engineering, Hunan predominant cellular fatty acid (Finkmann et al. 2000; University of Technology, Zhuzhou 412007, China Thierry et al. 2004; Lee et al. 2008). The DNA G ? C 123 Antonie van Leeuwenhoek content ranged from 60.1 to 71.1 mol% among different gene was amplified by PCR using the universal species of this genus (Kittiwongwattana and Thawai primers 27F and 1492R (Baker et al. 2003), and then 2016). At the time of writing, the genus Pseudoxan- double-checked by sequencing both strands. The thomonas comprised 19 species which were isolated pairwise sequence similarity was calculated using from various sources such as roots of Jerusalem the EzBiocloud server (https://www.ezbiocloud.net/; artichoke (Kittiwongwattana and Thawai 2016), a Yoon et al. 2017a). Phylogenetic analysis was carried hexachlorocyclohexane dumpsite (Kumari et al. 2011), out using MEGA version 7.0 with the neighbor-join- oil-contaminated soil (Young et al. 2007), North Pacific ing (NJ), maximum-likelihood (ML) and maximum- Ocean (Harada et al. 2006), cotton waste composts parimony (MP) models after multiple alignments of (Weon et al. 2006), and hot springs (Chen et al. 2002). the sequence data with Clustal_W (Kumar et al. 2016). In this study, we characterized the taxonomic Statistical support for the branches of the phylogenetic position of a bacterial strain GSS15T, and demon- trees was determined using bootstrap analysis (based strated, using a polyphasic approach, that it is repre- on 1000 re-samplings) (Felsenstein 1985). sentative of a novel species of the genus The draft genome of strain GSS15T was sequenced Pseudoxanthomonas. using paired-end sequencing method with the Illumina PE150 platform by Novogene Bioinformatics Tech- nology Co., Ltd., China. Reads of each data set were Materials and methods filtered and high quanlity paired-end reads were assembled using SOAP denovo (version 2.04). Con- Bacterial strain and culture conditions tigs with length greater than 500 bp were kept for gene prediction by GeneMarkS (version 4.17). In silico The sample for isolation of strain GSS15T was DNA-DNA hybridization (DDH) values were calcu- obtained from a composting demonstration plant in lated by genome-to-genome distance calculator Guangzhou City, Guangdong Province, China. The (GGDC) (Meier-Kolthoff et al. 2013). Values of compost windrow consisted of sewage sludge and crop average nucleotide identity (ANI) were calculated by straw. The samples were collected after composting using ANI Calculator (https://www.ezbiocloud.net/ for 13 days. Strain GSS15T was obtained during tools/ani) (Yoon et al. 2017b). Further multilocus investigating the cultured aerobic bacteria in the sequence analysis (MLSA) based on sequences of compost samples using LB agar (10.0 g peptone, housekeeping genes (recA, recombinase A; gyrA, 5.0 g yeast extract, 10.0 g NaCl and 20.0 g agar; pH DNA gyrase A subunit; rpoD, RNA polymerase, 7.2) at 30 °C. The new isolate was cryopreserved at sigma 70) was performed with NJ, ML and ME - 80 °C in 15% (v/v) glycerol. The experiments on methods supported with bootstrap values based on the new isolate were carried out at 30 °C, pH 8.0 and 1000 replications. The genome sequences for DDH with 0.5% NaCl (w/v) unless indicated else. and ANI calculation and the sequences of the house- ThecloselyrelatedtypestrainsPseudoxanthomonas keeping genes were downloaded from the NCBI helianthi roo10T, Stenotrophomonas panacihumi JCM database. 16536T, Pseudoxanthomonas broegbernensis B1616/ 1T,andP. spadix IMMIB AFH-5T were selected as Morphological, physiological and biochemical references. These references were obtained from the characterization German Collection of Microorganisms and Cell Cul- tures (DSMZ) and Japan Collection of Microorganisms Cell morphology was investigated with a transmission (JCM), and were cultured in the media and conditions electron microscope (JEOL, Japan) after cultivation recommended by these collections. on LB plate at 30 °C for 24 h (Yang et al. 2013). Cell motility was tested by observing the growth spread in a Phylogenies and average nucleotide identity test tube containing semi-solid LB medium. The Gram analyzes reaction was determined by a Gram staining kit HB8278 (Qingdao Hope-Bio Technology Co., Ltd; Genomic DNA of strain GSS15T was extracted using a China). Oxidase activity was determined using an DNA Extraction Kit (Aidlab; China). The 16S rRNA oxidase reagent (BioMe´rieux), and catalase activity 123 Antonie van Leeuwenhoek was determined by observing bubble production in 3% genomic DNA was determined by HPLC according to (v/v) hydrogen peroxide solution. The temperature the method of Mesbah et al. (1989). range for growth was determined at 10, 15, 25, 30, 37, 42, 45, and 50 °C. The pH range for growth was determined at pH 4.0–11.0 (at intervals of 0.5 pH unit) Results and discussion using the following buffer systems: pH 4.0–5.0, 0.1 M citric acid/0.1 M sodium citrate; pH 6.0–8.0, 0.1 M Phylogenetic analysis and ANI values KH2PO4/0.1 M NaOH; pH 9.0–10.0, 0.1 M NaHCO3/ 0.1 M Na2CO3; pH 11.0, 0.05 M Na2HPO4/0.1 M A nearly complete 16S rRNA gene sequence (1443nt) NaOH (Zhang et al. 2009). The tolerance for NaCl was determined for strain GSS15T. Comparison concentrations was determined with 0–8.0% (w/v) between the 16S rRNA gene sequence of strain NaCl (with increments of 0.5%). Growth at different GSS15T and those of the validly named species temperature, pH and NaCl concentration was investi- showed that, strain GSS15T shares high sequence gated for up to 1 week. Hydrolysis of gelatin and similarity with P. helianthi roo10T (96.9%), S. starch was tested as described by Dong and Cai (2001). panacihumi JCM 16536T (96.7%) and Xanthomonas Other enzyme activities and acid production charac- maliensis M97T (96.4%). The NJ, ML and MP trees teristics were characterized with the API 20NE and depicting the phylogenetic relationships between API 50CH systems (BioMe´rieux) according to the strain GSS15T and its near phylogenetic neighbours manufacturer’s instructions. are shown in Fig. 1 and supplementary Fig. S1 and S2, respectively. In addition, strain GSS15T formed a Chemotaxonomic characterization subcluster with P. spadix in the neighbor-joining tree. The phylogenetic analysis indicated that the new For respiratory quinone and polar lipids analysis, isolate is closely related to the genus Pseudoxan- strain GSS15T was cultured in LB medium at 30 °Cto thomonas and is relatively distant from the genera the exponential growth phase. Cells were collected by Stenotrophomona and Xanthomonas. centrifugation at 12,000 rpm at 4 °C, and free-dried The draft genome of strain GSS15T was deposited using the vacuum freeze drying apparatus (FD-1A-50, at DDBJ/EMBL/GenBank under the accession Beijing Biocool, China). The quinones were extracted SAWZ00000000. The assembled genome sequence with methanol according to Collins et al. (1977) and consists of 4,340,084 bp with a DNA G ? C content analyzed by HPLC as described by Groth et al. (1997). of 68.2%, which falls within the range observed for the Polar lipids were extracted from the freeze-dried cells, other members of the genus Pseudoxanthomonas separated by two-dimensional thin layer chromatog- (60.1–71.1 mol%) (Kim et al.
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