XIN Yanjuan, QU Junge, XU Junyi, WU Peichun, CAO Xupeng, and XUE Song, *

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sp. nov., Isolated from a Crude Oil-Contaminated Marine Sponge

XIN Yanjuan1), QU Junge2), XU Junyi3), WU Peichun1), CAO Xupeng1), and XUE Song1), *

1) Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, P.R. China?2)Department of Biology and Pharmacy, Zhejiang Pharmaceutical College, Ningbo315100, P.R. China?3)Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian 116001,P.R. China

An aerobic, Gram-negative bacterium, strain 2-5T, was isolated from a crude oil-contaminated marine sponge collected near Dalian Bay, China, and subjected to a polyphasic taxonomic investigation. Cells of strain 2-5Twere non-spore forming, non-motile, rods 0.2–0.3μm wide and 1.1–1.2μm long. Strain 2-5Tgrew well on nutrient agar, TSA, R2A agar and LB agar. Colonies of strain 2-5Ton LB agar were circular, smooth with entire margins, non-transparent and pale yellow after 3d of incubation at 30℃. Growth of strain 2-5Toccurred in LN medium with 0–6% NaCl; no growth occurred in the presence of 8.0% NaCl. Strain 2-5Tgrew at 15–42℃ and at pH 6.0–8.0. Comparative 16S rRNA gene sequence analysis showed that strain 2-5Tclustered with the species of the genus. Its closet neighbors were the type strains ofKCTC 12205T(97% similarity),ZS79T(96%), andAPB-9T(96%). The value for DNA-DNA relatedness between strain 2-5TandKCTC 12205Twas 23%. Branched fatty acids iso-C16: 0, iso-C15: 0, iso-C11: 03-OH, iso-C17: 1ω9and iso-C11: 0were found to be predominant. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Strain 2-5Thad a DNA G+C content of 63.8mol%. On the basis of the phenotypic, chemotaxonomic, DNA-DNA hybridization and phylogenetic data, strain 2-5Trepresents a novel species of the genus, for which the namesp. nov. is proposed. The type strain is 2-5T(=CGMCC 1.12190T= JCM 18137T).

sp. nov.; taxonomy; phylogenetic analysis

1 Introduction

The genuswas first described by Christensen and Cook (1978), and the description was emended by Park. (2008). The genus, grouped in the family, is classified in the class(Christensen and Cook, 1978) based on non-fruiting bodies, lack of flagella, gliding nature and high genomic DNA G+C content (typically ranging between 65.4 and 70.1mol%) (Aslam., 2009). At the time of writing, the genuscomprises 27 species with validly published names (http: //www. bacterio. cict. fr/). Recently, three novel species of the genus,ZS79Tfrom iron-mined soil (Luo., 2012),107-E2Tfrom an Antarctic freshwater lake (Fukuda., 2013) andCJ29Tfrom ginsengsoil (Choi., 2014) have been described. Members of the genus were of great potential for the de-velopment of biocontrol agents against plant fungal pathogens (Islam., 2005; Park., 2008) and antibiotic bioactivities against human pathogens (Ahmed., 2003; Hashizume., 2004). Here we report the characterization of a novel marine bacterium, strain 2-5T, of the genus,which was isolated from a crude oil-contaminated marine sponge.

2 Materials and Methods

2.1 Bacterial Strains

Strain 2-5Twas isolated from a crude oil-contaminated sponge specimen (), collected at the inter-tidal beach of Dalian, on the Chinese Yellow Sea, located in northern China (38?52′N 121?41′E). Freshly collected sponge specimens were rinsed five times in sterile seawater to remove unassociated bacteria and then thoroughly homogenized in a sterile mortar. A 10-fold dilution series of sponge homogenate was made and plated on 2216E plates (Difco, USA) in triplicate. After incubating the plates at 28℃ for 7d, an isolate, designated 2-5T, was picked and sub-cultured on 2216E plates, repeating the above steps until a pure culture was obtained. The novel strain was deposited into the CGM CC (China General Microbiological Culture Collection Center) as CGMCC 1.12190Tand the JCM (Japan Collection of Microorganisms) as JCM 18137T. The reference strain used for the DNA-DNA homology tests wasKCTC 12205T, obtained from the KCTC (Korean Collection for Type Cultures).

2.2 Morphology and Physiological Characteristics

To investigate the morphological and physiological characteristics, cell grown on R2A agar plates at 30℃ for 2d. Cell morphology and motility were examined using light microscopy (Olympus; ×1000) and transmission electron microscopy (H-7650; Hitachi, Japan) using cells from an exponentially growing culture. The strain Gram straining reaction was carried out according to the classical procedure described by Doetsch (1981). Gliding motility was determined as described by Bowman (2000). The physiological properties of strain 2-5Twere determined by the CGMCC using established procedures described by Gordon. (1974) and Yokota. (1993). Catalase activity, oxidase activity, enzyme activity and acid production from different carbohydrates were determined by the CGMCC with Biolog GN2, API 20E, API 20NE kits according to the manufacturer’s instructions. The assimilation of single carbon substrates was determined by the CGMCC with Biolog GN2 and API 20NE strips cultured at 28℃ for 24 h. Hydrolysis of casein and chitin was determined using previously described test (Smibert and Krieg, 1994; Brown, 2007). Growth at 4, 10, 15, 25, 30, 37, 42 and 45℃ and at pH 4.0–10.0 (at intervals of 1.0pH unit) was assessed after 5d of incubation on 2216E agar. Growth on nutrient agar, trypticase soy agar (TSA; Difco), R2A agar (Difco, USA) and LB agar (Difco, USA) was also evaluated at 28℃. Salt tolerance was tested in LN medium (LB without NaCl) supplemented with 0–10% NaCl after 10d of incubation.

2.3 G+C Content and Analysis of Cellular Fatty Acids

Cell grown on R2A agar plates at 30℃ for 2d were used for the analysis of cellular fatty acid and polar lipid. The fatty acids were extracted, methylated and analysed using the standard Sherlock MIDI (Microbial Identification) system (Sasser, 1990; K?mpfer and Kroppenstedt, 1996). Polar lipids were extracted and analyzed as described by Tindall (1990). A 6.75mL portion of chloroform/methanol/0.3% aqueous NaCl (1: 2: 0.8) was added to 100 mg freeze-dried cell material. The preparation was stirred overnight and cell debris was pelleted by centrifugation. Polar lipids were recovered into the chloroform phase by adjusting the chloroform/methanol/0.3% aqueous NaCl mixture to a ratio of 1: 1: 0.9 and then dried under nitrogen. The dried polar lipids were resuspended in chloroform/methanol (2:1) and separated by two-dimensional TLC.

The G+C content of the genomic DNA was determined by the CGMCC by thermal denaturation (Mandel and Marmur, 1968), for whichK-12 (CGMCC 1.365) was used as a standard.

2.4 Phylogenetic Analysis and DNA-DNA Hybridization

Genomic DNA from strain 2-5Twas extracted and purified according to standard procedures (Sambrook and Russell, 2001). The 16S rRNA gene cloned into pMD- 18T (Takara, Japan) was sequenced using an automated sequencer (Applied Biosystems model 3730). The 16S rRNA gene sequence of strain 2-5Twas compared with known sequences found in the GenBank database using the BLAST program (http://blast.ncbi.nlm.nih.gov/ Blast.cgi). Phylogenetic analysis was performed with MEGA5 program (Tamura., 2011) after multiple alignments of the data via CLUSTAL_X program (Thom- pson., 1997). A distance matrix method (distance options according to the Kimura two-parameter model), including clustering using the neighbor-joining and maximum-likelihood (Kluge and Farris, 1969) method. In each case, bootstrap values were calculated based on 1000 replications (Felsenstein, 1985). The taxonomic relationship between strain 2-5Tand its phylogenetic relative was further examined using DNA-DNA hybridization. DNA–DNA hybridization values between 2-5TandKCTC 12205Twas performed fluorometrically, according to the method developed by Ezaki. (1989) using photobiotin-labelled DNA probes and micro dilution wells. Hybridization was conducted in five replications for each sample. The highest and lowest values obtained for each sample were excluded, and the remaining three values were utilized in the calculation of hybridization values.

3 Results and Discussion

3.1 Morphological and Physiological Characteristics

Cells of strain 2-5Twere Gram-negative, non-spore forming, non-motile (but showing gliding activity), aerobic rods 0.2–0.3μm wide and 1.1–1.2μm long (Supplementary Fig.S1-2). Strain 2-5Tgrew well on nutrient agar, TSA, R2A agar and LB agar. Colonies of strain 2-5Ton LB agar were circular, smooth with entire margins, non- transparent and pale yellow after 3d of incubation at 30℃. Growth of strain 2-5Toccurred in LN medium with 0–6% NaCl; no growth occurred in the presence of 8.0% NaCl. Strain 2-5Tgrew at 15–42℃ (optimal 28–30℃) and at pH 6.0–8.0 (optimum pH 7.0). In the Biolog GN2, API 20E and API 20NE kits, strain 2-5Tassimilated glycogen,-acetyl-D-galactosamine,-acetylglucosamine, ribitol, L-arabinose, D-arabitol, D-cellobiose,D-fructose, alpha-D-glucose, M-inositol,D-galactonic acid lactone,D-galacturonic acid, D, L-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, succinamic acid, glucuronamide, L-ala- ninamide, L-alanine, L-alanyl-glycine, L-asparagine, L- aspartic acid, glycyl-L-aspartic acid, glycyl-L-glutamic acid, hydroxy-L-proline.The phenotypic characteristics of strain 2-5Tare summarized in the species description and a comparison of selective characteristics with refe- rence strains are summarized in Table 1.

Table 1 Physiological and biochemical characteristics that differentiate strain 2-5T from related type strains of the genus Lysobacter

Notes: Strains: 1, 2-5T(data from this study); 2,KCTC 12205T(Bae., 2005); 3,ZS79T(Lue., 2012); 4,DSM 18482T(Yassin., 2007). +, positive; -, negative; ND, no data available.

3.2 G+C content and Analysis of Cellar Fatty Acids

Table 2 Cellular fatty acid compositions (%) of strain 2-5T and related type strains of the genus Lysobacter.

Notes: Strains: sames as those in Table 1. -, <1% or not detected.

The major cellular fatty acids of strain 2-5Twere iso- C16: 0(15.1%), iso-C15: 0(14.4%), iso-C17: 1ω9(9.9%), iso-C11: 03-OH (8.4%) and iso-C11: 0(7.7%) (Table 2). The presence of branched fatty acids, namely iso-C16:0, iso-C15: 0, iso-C17:1ω19and iso-C17: 0, was consistent with the placement of strain 2-5Twithin the genus(Bae., 2005; Weon., 2006, 2007; Romanenko., 2008). However, strain 2-5Tcontained C16:0ω7/16:1 ω6and C19:0cyclo ω8, which differences with the reference strains. The predominant polar lipid of strain 2-5Twere phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and diphosphatidylglyceroal (DPG) (Supplementary Fig.S3). The DNA G+C content of strain 2-5Twas 63.8 mol%. This value is within the range reported for the genus(Christensen and Cook, 1978; Aslam., 2009).

3.3 Phylogenetic Analysis and DNA-DNA Hybridization

An almost-complete 16S rRNA gene sequence of strain 2-5Tconsisting of 1436bp was obtained. The phylogenetic tree shows that strain 2-5Tclusters within the genusin the class Gammaproteobacteria(Fig.1). 16S rRNA gene sequence analysis revealed that strain 2-5Twas related most closely to.Ko07T(97%similarity),.ZS79T(96%), and.APB-9T(96%). Based on the above results, DNA- DNA hybridization experiments were carried out between strain 2-5TandKo07T. The value obtained was 23%, which is significantly below the value of 70% proposed by Wayne. (1987) for species discrimination.

3.4 Toxonomic Conclusion

It is clear from the 16S rRNA gene sequence comparison and DNA–DNA hybridization data that strain 2-5Trepresents a novel species of the genus(Wayne., 1987). In addition, strain 2-5Twas positive for L-Arabinose, D-Mannitol, N-Acetylglucosamine, Maltose, and Trisodium citrate, differs from the type strain.Ko07T. Based on the phenotypic, phylogenetic and genomic evidence, strain 2-5Twas identified as a novel species of the genus, for which the name.sp. nov. is proposed.

3.5 Description ofsp. nov.

(hy.me.ni.a.ci?do.nis. N.L. gen. n.of, the generic name of the marine sponge, the source of the type strain).

Cells are Gram-negative, aerobic, non-spore forming, non-motile (but show gliding activity) and rod-shaped,about 0.2–0.3μm wide and 1.1–1.2μmlong. Colonies grown on LB agar are convex, circular, smooth, non- transparent and pale yellow after 3d of incubation at 30℃. Grows at 15–37℃ (optimal 28–30℃); no growth occurs below 4℃ or above 45℃. The pH range for growth is 6.0–8.0 (optimal pH 7.0). Growth occurs in the absence of NaCl and no growth occurs in 8.0% NaCl. Oxidase and catalase-positive. Positive for acid production from glucose, but negative for reduction of nitrates to nitrites. Hydrolyses aesculin, gelatin and casein, but not starch and chitin. Positive for citric acid utilization andacetoin production (Voges–Proskauer reaction). Activities of beta-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease and tryptophan deaminase are negative (API 20 E).Negative for assimilation of dextrin, tween40, tween80, I-erythritol, L-fruc- tose, D-galactose, dextrinose, D-mannose, D-allulose, sucrose, methyl pyruvate, succinic acid methyl, betaphenylglycollic acid, itaconic acid, alpha-oxo acid, sebacic acid, succinic acid, bromosuccinic acid, D-alanine, L-histidine, D-serine, gamma-amino butyric acid, rocanic acid, thymidine, phenyl ethylamine, putrescine, 2-amino ethanol, 2, 3-butanediol, glycerol, glycerol phosphate, D-glucose-1-phosphate, D-glucose-6-phosphate. Utilizes alpha-Cyclodextrin, glycogen, N-acetylgalactosamine, N- acetylglucosamine, ribitol, L-arabinose, D-arabitol, D- cellobiose, D-fructose, D-glucose, m-inositol,alpha-D- lactose, lactulose, maltose, D-mannitol, D-melibiose, beta-methyl-D-glucoside, D-raffinose, L-rhamnose, D- sorbitol, D-trehalose, turanose, xylitol, acetic acid, cis-aconitic acid, citric acid, formic acid, D-galactose acid lactone, D-galacturonic acid, D-gluconic acid, beta- methyl glucoside, D-glucuronic acid, alpha-hydroxy butyric acid, beta-hydroxy butyric acid, gamma-hydroxy butyric acid,alpha-oxo-butanoic acid,alpha-oxo-penta- nedioic acid,D,L-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharinic acid, succinamic acid, glucuronamide, L-alanyl amine, L-alanine, L-alanylglycine, L-asparagine, glycyl aspartic acid, glycyl glutamic acid, hydroxy proline, L-leucine, L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid, L-serine, L-threonine, D, L-carnitine, inosine, uridine(API 20 NE and Biolog GN2).

Fig.1 Phylogenetic tree of strain 2-5T and the type strains of related taxa based on 16S rRNA gene sequences. The tree wasreconstructed using the neighbor-joining (NJ) and maximum-likelihood (ML) methods and numbers at nodes representbootstrap percentages (NJ/ML, based on 1000 resamplings). Filled circles indicate genetic branches that were present in boththe NJ and ML trees. GenBank accession numbers are given in parentheses. Bootstrap values over 50% are shown at branching points. Bar, 0.005 substitutions per nucleotide position.

The major cellular fatty acids of strain 2-5Twere iso- C16:0, iso-C15:0, iso-C17:1ω9and iso-C11:03-OH; detailed fatty acid compositions are given in Table 2. The polar lipids consist of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglyceroal. The genomic DNA G+C content of the type strain is 63.8 mol%. The type strain 2-5T(=CGMCC 1.12190T= JCM 18137T), was isolated from a crude oil-contaminated marine spon- ge in Dalian, China.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31100092), the Hundred Talent Program of the Chinese Academy of Sciences (No. A1097) and the Ningbo Natural Science Foundation of China (2011A610028).

Supplementary

Fig.S1 Transmission electron micrograph of strain 2-5.

Fig.S2 Scanning electron micrograph of strain 2-5.

Fig.S3 Two-dimensional TLC of polar lipids of strains 2-5Tstained with 5% ethanolic molybdophosphoric acid. PE, phosphatidylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol.

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(Edited by Ji Dechun)

DOI 10.1007/s11802-015-2789-4

ISSN 1672-5182, 2015 14 (6): 1019-1024

? Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2015

(October 27, 2014; revised August 14, 2015; accepted August 30, 2015)

* Corresponding author. Tel: 0086-411-84379069 E-mail:xuesong@dicp.ac.cn