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 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- 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]

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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 + + + –

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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.

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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 BN9T (AY695828) 75 Pusillimonas soli MJ07T (GQ241322) T 100 Pusillimonas oleiphila sp. nov. T7-7 (DQ417606) 78 Pusillimonas ginsengisoli DCY25T(EF672088) 100 Pusillimonas ginsengisoli DCY28 (EF672089) Alcaligenes faecalis DSM 30030T (M22508) Sutterella wadsworthensis WAL9799T (L37785)

0.01

图 2 菌株 T7-7 的系统进化树 95 Fig.2 Phylogenetic tree showing the relationship between strain T7-7 T and selected strains from other bacterial taxa. The tree was constructed by using the Neighbor-Joining (NJ) method and the program MEGA. Bootstrap values (expressed as percentages of 1000 replications) greater than 50% are shown at branch points. Bar, 0.01 substitutions per nucleotide position.

100 Carbon source utilization was tested in the medium described previously (Kämpfer et al., 1990). Controls were grown without substrate addition. The methods used for biochemical tests were described previously (Smibert & Krieg, 1981). Fatty acid analysis was performed using standard methods and compared to the database of fatty acids in the MIDI Sherlock Microbial Identification System (Microbial ID). Polar lipids analysis was performed at DSMZ(Deutsche 105 Sammlung von Mikroorganismen und Zellkulturen GmbH, German Collection of Microorganisms and Cell Cultures). Methods of extraction and analysis of polar lipids were described in the note of supplementary Fig. B. The G+C content of the DNA was determined by the thermal denaturation method (Marmur & Doty, 1962). DNA-DNA hybridization was carried out with BECKMAN COULTER DU800 plus program heating module. 110 The fatty acid profiles of strain T7-7T, P. ginsengisoli DCY25T, P. soli MJ07T and P. T noertemannii BN9 are shown in Table 2. All strains contained C16:0, C17:0 cyclo, C19:0 cyclo ω8c,

but their amounts were differences between these strains. C16:0 and C17:0 cyclo were the major fatty T acids in all strains. However, strain T7-7 had only 3.5% C19:0 cyclo ω8c compared to 22.8% in P. ginsengisoli DCY25T, 11.5% in P. soli MJ07T and 17.1% in P. noertemannii BN9T. Fine

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115 qualitative and quantitative differences in fatty acid patterns could distinguish strain T7-7T from the known Pusillimonas strains.

表 2 T7-7 与表型相关菌株脂肪酸结构比较 Tab. 2 Relative fatty acid compositions of strainT7-7 T in comparison to its phylogenetically closest relatives 120 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; strains were grown on TSA at 30 ℃ for 48 h prior to fatty acid analysis. –, Not present; Tr, trace amounts present. Fatty acid abbreviations: C10:0, 3-hydroxydecanoic acid; C12:0, dodecanoic acid=lauric acid; 2-OH C12:0, 2-hydroxydodecanoic acid; C14:0, tetradecanoic acid=myristic acid; C15:0, pentadecanoic acid; C16:0, hexadecanoic acid=palmitic acid; C17:0 cyclo, D-cis-9,10-methylenehexadecanoic acid; 125 3-OH C16:0, 3-hydrohexadecanoic acid; C17:0, heptadecanoic acid; C17:0 cyclo, cycloheptadecanoate; C18:1ω7c, is-11-octadecenoic acid=vaccenic acid; C18:0, octadecanoic acid=stearic acid; C19:0 cyclo ω8c, D-cis-11,12-methyleneoctadecanoic acid; The fatty acid profiles were determined according to Kämpfer & Kroppenstedt (1996). Values characteristic for strain T7-7T are given in bold type. Fatty acid (% w/w) 1 2 3 4 C10:0 0.1 − − Tr C12:0 7.6 Tr 8.4 5.6 2-OH C12:0 − − − 4.4 C14:0 0.7 6.4 − 0.7 C15:0 0.2 − − − C16:0 28.2 16.6 32 15.7 C17:0 cyclo 34.4 8.7 24.7 20.1 C17:0 0.2 2.6 − − 3-OH C16:0 0.4 − − 3.1 C18: 1 ω7c 4.7 3.7 − 4.0 C18 : 1 ω7c 11 methyl Tr − − 1.0

C18:0 0.7 6.2 2.2 3.8 C19 : 0 iso Tr − − 0.9

C19:0 cyclo ω8c 3.5 22.8 11.5 17.1 C20 : 2 ω6,9c Tr − − 2.1

Summed feature 2* 8.7 15.3 5.9 14.1 Summed feature 3* 8.6 8.6 9.3 3.8 * Summed features represent groups of two or three fatty acids that could not be separated by the MIDI 130 system. Summed feature 2 contained iso-C16:1 I and/or C14:0 3-OH; summed feature 3 contained C16:1ω7c and/or iso-C15:0 2-OH.

The polar lipid compositions of strains T7-7T, P. ginsengisoli DCY25T, P. soli MJ07T, P. noertemannii BN9T, Alcaligenes faecalis DSM 30030T(Papen et al., 1989 ) and Castellaniella 135 defragrans (Foss) Kämpfer DSM 12141T (Kämpfer et al., 2006) are shown in Table 3. Each of the six strains showed a distinct polar lipid profile, including the known polar lipids diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine, as well as several unknown aminophospholipids, aminolipids, phospholipids, and other polar lipids. The polar lipid profile of strain T7-7T consisted of four compounds. Phosphatidylethanolamine was the major 140 lipid, large amounts of phosphatidylglycerol, diphosphatidylglycerol and unknown aminolipids AL1 were also present, which was similar to the lipids composition of P. ginsengisoli DCY25T, P. soli MJ07T and P. noertemannii BN9T. However, the characteristic aminolipids of Pusillimonas AL2 [Labeled as AL on supplementary Fig. B in IJSEM Online] was not present after two-dimensional thin layer chromatography (TLC) (Stolz et al., 2005). This compound profile 145 showed that strain T7-7T was a member of Pusillimonas, but not belonged to any known species, and the results agreed with the 16S rRNA analysis. In particular, the presence of the unknown aminolipid AL1 in all reported strains of Pusillimonas is interesting and it should be noted that this trait is specific for the genus Pusillimonas.

150

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表 3 产碱杆菌科菌株的极性脂分析 Tab. 3 Polar lipid profiles in different members of the Alcaligenaceae 1, Alcaligenes faecalis DSM 30030T (data from Stolz et al., 2005); 2, Castellaniella defragrans (Foss) Kämpfer DSM 12141T (data from Stolz et al., 2005); 3, P. noertemannii BN9T (data from Stolz et al., 2005); 4, P. soli 155 MJ07T (data from Lee et al., 2010); 5, P. ginsengisoli DCY25T (data from Srinivasan et al., 2010); 6, T7−7T; DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; AL1–7, unknown aminolipids; PL1–6, unknown phospholipids; APL1–5, unknown aminophospholipids; L1–8, unknown polar lipids. ++++, Major amounts; +++, moderate amounts; ++, minor amounts; +, trace amounts; −, not detected. Polar Lipid 1 2 3 4 5 6 PE ++++ ++++ ++++ ++++ ++++ ++++ PG +++ +++ ++++ +++ ++++ +++ DPG ++ +++ ++++ +++ +++ +++ AL1 ++ ++ +++ ++ +++ +++ AL2 − − ++++ +++ +++ − PL1 + − − − − − PL2 + + + − − +

PL7 − − − + − − APL1 + + − + ++ − APL2 + − − − − − APL3 − − − − − − APL4 − + + − − + APL5 − + − − − − APL6 − − − + − − L1 − − + + ++ − L2 − − + − ++ − L3 + + ++ − − +

L4 − − + − − − L9-L12 − − − + − −

160 The G+C content of strain T7-7T was determined to be 52.2 mol%, which fits into the known range for Alcaligenaceae of 36.5 to 69 mol% (Busse & Stolz, 2004). In contrast, strain T7-7T exhibited relatively low levels of DNA–DNA relatedness with P. ginsengisoli DCY25T(47.1%), P. soli MJ07T(44.5%) and P. noertemannii BN9T(38.6), which is below the reported threshold (70%) used to determine the taxonomic status of a new strain (Wayne et al., 1987). These results indicate 165 that strain T7-7T represents a distinct species of Pusillimonas. On the basis of overall differences observed between strain T7-7T and other known species of Pusillimonas, we consider strain T7-7T to be representative of a novel member of the genus Pusillimonas. Because of the ability of the strain to use fuel oil as a carbon source, the name Pusillimonas oleiphila sp. nov. is proposed. 170 Description of Pusillimonas oleiphila sp. nov. Pusillimonas oleiphila (o.le.i.phi'la. L. n. oleum, oil; Gr. adj. philos, loving; N.L. fem. adj. oleophila, oil-loving, because this taxon utilizes oil). The T7-7T strain (T7-7T=CGMCC 1.6148T=DSM 18250T) was isolated from seabed mud from the Bohai Sea. The G+C DNA content from this strain was determined to be 52.2 mol% 175 (Tm=64.5ºC). Cells were found to be Gram-negative, oxidase-negative, and having an oxidative metabolism. Cells were observed using an electron-micrograph and found to be rod-shaped (2–3 μm in length and 0.5–1 μm in diameter). They are motile by means of polarly inserted flagella. On nutrient agar (LB or NB), colonies were ivory-white, circular, and lightly translucent. The strain examined was 180 capable of growth between 4 and 37ºC, optimal at 30ºC, but not at 42ºC. In addition, a NaCl concentration of 0 to 5.5% was found to be required for growth, with 2% NaCl being the best. The T major fatty acids found in strain T7-7 were C16:0 (28.2) and C17:0 cyclo (34.4). The following compounds were not assimilated by strain T7-7T: α-cyclodextrin, N-acetyl-D-

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galactosamine, D-galactonic acid lactone, α-hydroxybutyric acid, α-ketobutyric acid, 185 glycyl-L-aspartic acid, L-ornithine, D-serine, inosine, uridine, phenylethylamine, putrescine, and α-D-glucose-1-phosphate. No acids were produced from L-fucose and L-rhamnose. The strain examined assimilated the following substrates: saturated hydrocarbons, dextrin, tween 40, tween 80, N-acetyl-D-glucosamine, pyruvic acid methyl ester, succinic acid mono-methyl ester, acetic acid, (cis)-aconitic acid, citric acid, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, 190 D-glucuronic acid, β-hydroxybutyric acid, γ-hydroxybutyric acid, itaconic acid, α-ketoglutaric acid, D,L-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, succinic acid, bromosuccinic acid, succinamic acid, L-alaninamide, D-alanine, L-alanine, L-alanyl-glycine, L-asparagine, L-aspartic acid, L-glutamic acid, L-histidine, hydroxy- L-proline, L-leucine, L-phenylalanine, L-proline, L-pyroglutamic acid, L-serine, D,L-carnitine, γ-aminobutyric acid, 195 2-aminoethanol, D,L-α-glycerol phosphate, and D-glucose-6-phosphate. Acids were produced from adonitol, L-arabinose, D-arabitol, D-cellobiose, D-fructose, D-galactose, gentiobiose, α-D-glucose, lactulose, maltose, D-mannitol, D-mannose, β-methyl-D-glucoside, D-raffinose, D-sorbitol, sucrose, D-trehalose, turanose, and xylitol. 2 Acknowledgements (Optional) 200 This work was supported by Research Fund for new teacher of the Doctoral Program of Higher Education of China (20070055049). The authors also thank Professor Andreas Stolz and Deok-Chun Yang for making strain BN9T, DCY25T and DCY28 available to us and Professor J. P. Euzéby (Toulouse) for his help with the nomenclature.

205 References

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一株低温解烃的极小单胞菌新菌株及其 性能研究 265 马挺1，黄磊2，李国强1 （1. 南开大学生命科学学院，教育部分子微生物与技术教育部重点实验室，天津 300071； 2. 天津理工大学化工学院，天津 300191） 摘要：本文研究了一株新菌——T7-7，这株菌是从渤海海底泥中筛选得到的，具有能在 10 º C 降解柴油的特性。之前做了该菌株的细胞壁极性脂实验，显示它的主要成分包括磷脂酰乙 270 醇胺、磷脂酰甘油和双磷脂酰甘油以及未知的脂肪酸 AL1。16S rRNA基因序列分析显示该菌 与 Pusillimonas ginsengisoli DCY25T（相似度 98.4%）、Pusillimonas soli MJ07T(97.5%) and Pusillimonas noertemannii BN9T (96.7%)最接近。与其他已报道种属菌株相似度均低 于 95.3%。这表明 T7-7 属于 Pusillimonas 属的一个新种。不同方法做成的系统进化树显示 这个菌株区别于之前报道的 Pusillimonas 属的其他菌株。脂肪酸分析显示具有高浓度的 275 C16:0 和 C17:0 cyclo。菌株 T7-7 与 P. ginsengisoli DCY25T(47.1%), P. soli MJ07T(44.5%) 和 P. noertemannii BN9T (38.6%)都表现出了低的 DNA-DNA 杂交相似性。根据 16S rRNA 基因序列的相似性以及其他特有的表型特征例如脂肪酸结构，极性脂特征和 DNA 杂交结果， T7-7 是一株新种，我们命名为 Pusillimonas oleiphila sp. nov. (=CGMCC 1.6148 T =DSM 18250T)。 280 关键词：微生物学；嗜油极小单胞菌；燃料油降解 中图分类号：Q93

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