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中国科技论文在线 Pusillimonas Oleiphila Sp. Nov., a New Member Of

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中国科技论文在线 Pusillimonas Oleiphila Sp. Nov., a New Member Of 中国科技论文在线 http://www.paper.edu.cn Pusillimonas oleiphila sp. nov., a new member of the genus Pusillimonas that degrades fuel oils at low temperature# Ma Ting1, Huang Lei2, Li Guoqiang1* (1. Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College 5 of Life Sciences, Nankai University, TianJin 300071; 2. College of Chemistry and Chemical engineering, Tianjin University of Technology, TianJin 300191) Abstract: The taxonomic position of a novel strain, designated T7-7T, was investigated. This strain was isolated at 10ºC as a diesel-oil-degrading organism from seabed mud in the Bohai Sea. Previously, 10 a polar lipid pattern with phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and unknown aminolipids AL1 as the primary components was found. Partial 16S rRNA gene sequence analysis indicated that this strain was related most closely to Pusillimonas ginsengisoli DCY25T (98.4% sequence similarity), Pusillimonas soli MJ07T(97.5%) and Pusillimonas noertemannii BN9T (96.7%). The levels of 16S rRNA gene sequence similarity between strain T7-7T and other recognized 15 species of the family Alcaligenaceae were below 95.3%. This suggested that strain T7-7T represented a member of the genus Pusillimonas. Different methods for the construction of phylogenetic dendrograms separated this strain from other previously known strains of Pusillimonas. Fatty acid analysis demonstrated the presence of high concentrations of C16:0 and C17:0 cyclo. Strain T7-7T exhibited relatively low levels of DNA–DNA relatedness with P. ginsengisoli DCY25T(47.1%), P. 20 soli MJ07T(44.5%) and P. noertemannii BN9T (38.6%). Based on the 16S rRNA gene sequence similarity to the strains of Pusillimonas, and other unique phenotypic properties such as fatty acid composition, polar lipid profiles, and DNA-DNA hybridization, T7-7T was classified as a new species with the name Pusillimonas oleiphila sp. nov. The strain type was designated as T7-7T (=CGMCC 1.6148 T =DSM 18250T). 25 Keywords:Microbiology; Pusillimonas oleiphila; Fuel oils degradation 0 Introduction The genera Alcaligenes, Achromobacter, Bordetella, Pigmentiphaga, Advenella, and Pusillimonas belong to the β-Proteobacteria and are grouped together in the family 30 Alcaligenaceae (De Ley et al., 1986; Yabuuchi et al., 1998; Blümel et al., 2001; Coenye et al., 2005; Stolz et al., 2005). The genus Pusillimonas was first defined by Stolz et al., 2005. At the time of writing, the genus contains only three recognized species, P. noertemannii (Stolz et al., 2005), P. ginsengisoli (Srinivasan et al., 2010) and P. soli MJ07T (Lee et al., 2010). Strain T7-7T was isolated at 10ºC from seabed mud in the Bohai Sea at E118º26΄36˝, 35 W38º50΄30˝. At this location an oil tanker leaked on 23 November 2002, this polluted the beach nearby. Samples of seabed mud from the polluted areas were collected for further study to remediate the oil spill. From the samples several strains were obtained, including T7-2 and T7-5, which were identified as Rhodococcus erythropolis and Pseudomonas putida. Interestingly, there were many small, weak growing colonies, which were later named T7-7 on Luria–Bertani (LB) 40 agar at 10ºC when purifying the degradation mixtures. It was subsequently demonstrated that this strain could easily degrade fuel oils like diesel oil, which is one of the main pollutants of the sea. Analysis of the metabolic profiles of strain T7-7 showed that it could metabolize saturated alkanes, from C14 to C32. For example, alkane C22 (1% alkane in medium) was degraded 30% at 25ºC within 7 days. However, aromatic hydrocarbons could not be degraded using this strain. This 45 strain appeared to represent a novel taxon based on preliminary investigations of its phylogenetic Foundations: This work was supported by Research Fund for new teacher of the Doctoral Program of Higher Education of China (20070055049) Brief author introduction:Ma Ting, (1977-), Male, associate professor, study on petroleum microbiology. E-mail: [email protected] - 1 - 中国科技论文在线 http://www.paper.edu.cn and phenotypic characteristics. Here, we describe the physiological, chemotaxonomic, and phylogenetic characteristics of strain T7-7T and propose a novel species of the genus Pusillimonas, Pusillimonas oleiphila sp. nov., for this strain. 1 Strain description 50 Generally, growth of strain T7-7T was observed on all complex media [LB or nutrient broth (NB medium)] within 3 days incubation at 10, 16, 30 and 37ºC. The optimal growth temperature of strain T7-7T was found to be 30ºC, which resulted in a colony diameter of approximately 2–3 mm. Moreover, T7-7T showed only weak growth at 4ºC within 15 d, and no growth was found at temperatures higher than 37ºC. In the presence of increased CO2 concentrations (5%) no visible 55 differences compared with growth at normal O2 and CO2 pressures were observed (at 15ºC). Cells of strain T7-7T were rod-shape, approximately 1–1.5μm long and 0.3-0.6μm in diameter, as observed using an electron-micrograph (Fig. 1). The phenotypic and chemotaxonomic characteristics that differentiate strain T7-7T from P. ginsengisoli DCY25T, P. soli MJ07T and P. noertemannii BN9T are listed in Table 1. 60 图 1 T7-7 菌株的电镜照片 Fig.1 An electron-micrograph of cells of strain T7-7T 表1 T7-7以及与它相近菌株的不同表型特征 65 Tab. 1 Different phenotypic characteristics of strain T7-7T from its phylogenetically closest relatives Taxa: 1, Pusillimonas oleiphila sp. nov. T7-7T; 2, P. ginsengisoli DCY25T; 3, P. soli MJ07T; 4, P. noertemannii BN9T. Data were obtained in this study unless indicated otherwise. +, All strains are positive; –, all strains are negative. In API ZYM, API ID 32GN, API 20NE and API 50 CHB tests, all strains showed identical biochemical characteristics except those indicated here. Characteristics 1 2dcy 3mj 4 Growth at 42ºC – + + + Enzyme activities (API ZYM) N-Acetyl-β-glucosaminidase + – – + Acid phosphatase – + + – α-Chymotrypsin – + – – Cystine arylamidase – + – – Leucine arylamidase + + + – Valine arylamidase – + – – Naphthol-AS-BI-phosphohydrolase – + + – Trypsin – + – – Urease + – – + Assimilation of (API ID 32GN, API 20NE): Utilization of saturated hydrocarbons C14~C32 – – – 3-Hydroxybenzoate + – + + 4-Hydroxybenzoate + – + + Adipate + + + – Caprate + – – + Citrate – + + – Malonate + + + – Phenyl acetate – + + – Propionate + + + – Suberate + – + + Valerate + + + – - 2 - 中国科技论文在线 http://www.paper.edu.cn D-Glucose + – + – D-Ribose – – + – myo-Inositol – – + – L-Alanine + + + – Acid production from (API 50 CHB): L-Arabinose + – + – Arbutin – – + – Gluconate – – + – D-Glucose + – + – D-Fucose, L-fucose – – – + Maltose + – – + D-Ribose – – + – Trehalose + + – – β-D-Xylopyranoside – – + – production of H2S + – + – DNA G+C content (mol%) 52.2 57.3a 59.4b 61.8c 70 a Data taken from Srinivasan et al., 2010. b Data taken from Lee et al., 2010. c Data taken from Stolz et al., 2005. For the phylogenetic analysis of strain T7-7T, Genomic DNA of this strain was prepared by 75 the method of Marmur (1961) and the purity was checked spectrometrically; the 16S rRNA gene was amplified with the described method (Zhang et al., 2002); sequencing of the PCR products and determination of the phylogenetic position were also performed with the method described previously (Zhang et al., 2002). The 16S rRNA sequence of strain T7-7T, containing a continuous stretch of 1493 bp (Brosius et al., 1978), was used to search the GenBank library. The results 80 showed that strain T7-7T was most closely related phylogenetically to representatives of the β-Proteobacteria. Phylogenetic trees were generated by using the Neighbor-Joining method from the PHYLIP program package (Felsenstein et al., 1993; Stolz et al., 2005). A closer relationship was suggested by Mega 6.0 software between strain T7-7T and members of the genus Pusillimonas. The results of the sequence similarity calculations indicated that the closest relatives of strain T7-7 85 T were P. ginsengisoli DCY25T (98.4%), P. soli MJ07T(97.5%) and P. noertemannii BN9T (96.7%). The levels of 16S rRNA gene sequence similarity between strain T7-7T and other recognized species of the family Alcaligenaceae were below 95.3% (Fig. 2). Strain T7-7T, P. ginsengisoli DCY25T, P. soli MJ07T and P. noertemannii BN9T formed a coherent cluster. This suggested that strain T7-7T represented a member of the genus Pusillimonas. To differentiate 90 strain T7-7T from its phylogenetically closest relatives, the following experiments were performed. - 3 - 中国科技论文在线 http://www.paper.edu.cn 91 Achromobacter piechaudii DSM 10342T (AB010841) 66 Achromobacter ruhlandii DSM 653T (AB010840) 97 Achromobacter xylosoxidans subsp. denitrificans DSM 30026T (AJ278451) Achromobacter xylosoxidans subsp. xylosoxidans DSM 2402T (D88005) Bordetella avium DSM 11332T (AF177666) Bordetella trematum DSM 11334T (AJ277798) Bordetella hinzii LMG13501T (AF177667) 96 Bordetella pertussis DSM 5571T (U04950) 83 T Bordetella holmesii CCUG 34073 (U04820) T 99 Bordetella bronchiseptica CCUG 219 (U04948) 76 T 87 Bordetella parapertussis DSM 4922 (AJ278450) T 75 Bordetella petrii DSM 12804 (AJ249861) Kerstersia gyiorum LMG 5906T (AY131213) 41 Pigmentiphaga kullae DSM 13608T (AF282916) 100 Pigmentiphaga daeguensis K110T (EF100696) Castellaniella defragans DSM12141T (AJ005447) T 100 Castellaniella denitrificans NKNTAU (U82826) 92 Castellaniella denitrificans TJ4T (AF508102) 100 Taylorella equigenitalis DSM 10668T (X68645) 98 Pelistega europaea LMG 10982T (Y11890) Advenella incenata R-18191T (AY569459) 40 Pusillimonas noertemannii
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