International Journal of Systematic and Evolutionary Microbiology (2014), 64, 16–20 DOI 10.1099/ijs.0.054007-0

Zunongwangia atlantica sp. nov., isolated from deep-sea water

Rui Shao,1,23 Qiliang Lai,1,23 Xiupian Liu,1 Fengqin Sun,1 Yaping Du,1 Guangyu Li1 and Zongze Shao1

Correspondence 1State Key Laboratory Breeding Base of Marine Genetic Resources; Key Laboratory of Marine Zongze Shao Genetic Resources, Third Institute of Oceanography, SOA; Key Laboratory of Marine Genetic [email protected] Resources of Fujian Province, Xiamen 361005, PR China 2Life Science College, Xiamen University, Xiamen 361005, PR China

A taxonomic study was carried out on strain 22II14-10F7T, which was isolated from the deep-sea water of the Atlantic Ocean with oil-degrading enrichment. The bacterium was Gram-stain- negative, oxidase- and catalase-positive and rod-shaped. Growth was observed at salinities from 0.5 to 15 % and at temperatures from 4 to 37 6C; it was unable to hydrolyse Tween 40, 80 or gelatin. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 22II14- 10F7T represented a member of the genus , with highest sequence similarity of 97.3 % to Zunongwangia profunda SM-A87T, while the similarities to other species were all below 94.0 %. The DNA–DNA hybridization estimate of the similarity between strain 22II14- 10F7T and Z. profunda SM-A87T was 27.20±2.43 % according to their genome sequences. The

principal fatty acids were iso-C15 : 0, anteiso-C15 : 0 , iso-C15 : 1 G, iso-C17 : 0 3-OH, summed

feature 3 (C16 : 1v7c/v6c) and summed feature 9 (iso-C17 : 1v9c or C16 : 0 10-methyl). The G+C content of the chromosomal DNA was 35.5 mol%. The major respiratory quinone was determined to be MK-6. Phosphatidylethanolamine (PE), two aminolipids (AL1 and AL2) and five unknown lipids (L1–L5) were present. The combined genotypic and phenotypic data show that strain 22II14-10F7T represents a novel species of the genus Zunongwangia, for which the name Zunongwangia atlantica sp. nov. is proposed, with the type strain 22II14-10F7T (5CGMCC1.12470T5LMG 27421T5MCCC 1A06481T).

During attempts to investigate oil-degrading in the rosette in 2011 during cruise DY-115A of the R/V Da-Yang deep water of the Atlantic Ocean, many bacterial strains Yi-Hao. The sampling site was on the South Atlantic Ocean were isolated and characterized taxonomically. This study ridge, and numbered as 22II-S025-CTD14, the water focuses on one of these isolates, designated strain 22II14- sample from 2927 m depth was used for enrichment of 10F7T. Comparative 16S rRNA gene sequence analysis oil-degrading bacteria with 1 % (v/v) sterilized crude oil. indicated that strain 22II14-10F7T was closely related to the The bacterial isolation on 216L marine agar medium was genus Zunongwangia, which belongs to the family Flavo- done according to the method described by Lai et al. bacteriaceae. The genus Zunongwangia was proposed by (2009). For morphological and biochemical characteriza- Qin et al., (2007) and, at the time of writing, includes only tion, strain 22II14-10F7T was cultivated on marine agar the type species Zunongwangia profunda. Consequently, the 2216 (BD; Difco) medium. aim of the present work is to determine the exact taxo- nomic position of strain 22II14-10F7T by using polyphasic Genomic DNA was prepared according to the method of characterization. Ausubel et al. (1995) and the 16S rRNA gene was amplified by PCR using primers that have been described previously Deep-sea water was sampled with Niskin bottles attached (Liu & Shao, 2005). Sequences of related taxa were to a conductivity, temperature and depth (CTD) circular obtained from the GenBank database. Phylogenetic analysis was performed using MEGA version 5.0 (Tamura et al., 3Rui Shao and Qiliang Lai contributed equally to this work. 2011). Distances (distance options determined according Abbreviation: DDH, DNA–DNA hybridization. to the Kimura two-parameter model) and clustering with The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene the neighbour-joining (Saitou & Nei, 1987), maximum- sequence of Zunongwangia atlantica 22II14-10F7T is JQ844757. likelihood (Felsenstein, 1981) and minimum evolution Two supplementary figures and a supplementary table are available with methods (Rzhetsky & Nei, 1992, 1993) were determined by the online version of this paper. using bootstrap values based on 1000 replications.

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A nearly full-length 16S rRNA gene sequence (1490 nt) of below standard criteria for classifying strains as representing strain 22II14-10F7T was determined. As shown in Fig. 1, the same species (95–96 %) (Richter & Rossello´-Mo´ra, 2009). the phylogenetic tree based on 16S rRNA gene sequences This proved that strain 22II14-10F7T represented a novel showed that strain 22II14-10F7T and Z. profunda SM-A87T species of the genus Zunongwangia. The DNA–DNA formed an independent monophyletic cluster, with high hybridization (DDH) estimate value was analysed using the bootstrap support (100 %). The two strains shared 16S genome to genome distance calculator (GGDC2.0) (Auch rRNA sequence similarity of 97.3 %. The sequences of type et al., 2010a, b; Meier-Kolthoff et al., 2013). The DDH T , estimate value between strain 22II14-10F7 and Z. profunda strains of other species had 94.0 % similarity to strain T T SM-A87 was 27.20 %±2.43 %. This result confirmed that 22II14-10F7 . This high similarity strongly confirmed that T strain 22II14-10F7T belonged to the genus Zunongwangia. strain 22II14-10F7 represented a novel species. Gram staining was performed using a Gram stain kit (Hangzhou The average nucleotide identity (ANI) between two genomes Tianhe MiReagent) according to the manufacturer’s instruc- was calculated using JSpecies (V1.2.1) as described by Richter tions. The cell size, morphology and flagellation pattern were & Rossello´-Mo´ra (2009). The draft genome sequences of the observed by transmission electron microscopy (JEM-1230; novel strain 22II14-10F7T (GenBank accession number JEOL) using cells negatively stained with phosphotungstic T JQ844757.1) and Z. profunda SM-A87 (NC_014041) (Qin acid grown on marine agar at 28 uC for 1 day. Cell motility et al., 2010) were obtained from the NCBI database. The ANI was observed by the hanging-drop method (Skerman, 1967). value using MUMer between two strains was 85.6 %, which is Catalase and oxidase activities and hydrolysis of Tweens 20,

60 Gaetbulibacter marinus IMCC1914T (EF108219) 0.01 Meridianimaribacter flavus NH57NT (FJ360684) Bizionia echini KMM 6177T (FJ716799) Corallibacter vietnamensis KMM 6217T (HQ257254) Mesoflavibacter zeaxanthinifaciens TD-ZX30T (AB265181) 90 Formosa algae KMM 3553T (AY228461) Winogradskyella rapida SCB36T (U64013) 64 JW-26T (EU221275) 54 Tamlana agarivorans 95 Algibacter mikhailovii LMG 23988T (AM491809) Ulvibacter antarcticus IMCC3101T (EF554364) Leeuwenhoekiella marinoflava LMG 1345T (AF203475) Aquimarina agarilytica ZC1T (FJ750453) DSW-1T (DQ003276) 83 Dokdonia donghaensis 100 Krokinobacter genikus Cos-13T (AB198086) 50 Salinimicrobium catena HY1T (DQ640642) Gillisia limnaea LMG 21470T (AJ440991) 100 Zunongwangia atlantica 22II14-10F7T (JQ844757)

Zunongwangia profunda SM-A87T (CP001650)

Mesonia algae KMM 3909T (AF536383)

Salegentibacter salegens DSM 5424T (M92279) Gramella echinicola KMM 6050T (AY608409) Psychroflexus torquis ACAM 623T (U85881) DSM 16809T (FR733714) 51 Persicivirga xylanidelens 100 Sandarakinotalea sediminis CKA-5T (AB206954) 100 Nonlabens tegetincola UST030701-324T (AY987349)

Fig. 1. Neighbour-joining tree showing the phylogenetic positions of strain 22II14-10F7T and representatives of some other related taxa based on 16S rRNA gene sequences. Filled circles indicate nodes that were also recovered in maximum-likelihood and minimum-evolution trees based on the same sequences. Bootstrap values (expressed as percentages of 1000 replications)

are shown at branch points. Bar, 0.01 nt substitution rate (Knuc) units. Downloaded from www.microbiologyresearch.org by http://ijs.sgmjournals.org 17 IP: 137.108.70.7 On: Mon, 04 Jul 2016 10:01:16 R. Shao and others

40, 80 and starch were tested according to the standard extracted using the standard protocol of MIDI (Sherlock methods (Dong & Cai, 2001). The optimal growth Microbial Identification System, version 6.0B). The fatty temperature was determined over a temperature range of acids were analysed by GC (6850; Agilent Technologies) 4–55 uC in marine broth 2216. The pH range for growth was and identified by using the TSBA6.0 database of the determined in marine broth 2216 adjusted to pH 2–12, at Microbial Identification System (Sasser, 1990). The fatty 1 pH unit intervals, with citrate/phosphate (pH 2.0–7.0), acids profile of 22II14-10F7T was produced in parallel with Tris/HCl (pH 8.0–9.0), or sodium carbonate/sodium bicar- that for Z. profunda SM-A87T in this study. The results for bonate (pH 10.0–12.0) buffers. Tolerance to NaCl was tested both strains are shown in Table S1, available in IJSEM using LB broth with NaCl concentrations of 0, 0.5, 1, 2, 3, 4, Online. The major fatty acids in both strains were iso- 5, 6, 7, 8, 9, 10, 12, 15, 18 or 20 % (w/v). Anaerobic growth C15 : 0, anteiso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, summed was examined on marine agar 2216 supplemented with feature 3 (C16 : 1v7c/v6c) and summed feature 9 (iso- 21 nitrate (1 g l ) incubated in a jar with the Anoxomat Mark C17 : 1v9c or C16 : 0 10-methyl), which accounted for .72 % II Anaerobic System (Mart Microbiology). Microaerobic of the total fatty acids. Both strains only have a small growth (O2, 6 %) was examined by incubation on marine difference in the content of iso-C15 : 1 G. The major fatty agar 2216 in a jar with the Anoxomat Mark II. The acids profile of Z. profunda SM-A87T was similar to that degradation of oil was determined in artificial seawater reported by (Qin et al., 2007), but they differed in the medium (ASM) at a concentration of 0.5 % (w/v) according presence/absence of C15 : 0. to the method of Liu & Shao (2005). The presence of flexirubin-type pigments was examined using 20 % KOH Analyses of respiratory quinones and polar lipids were (w/v) as described by Bernardet et al., (2002). Other carried out by the Identification Service of the DSMZ biochemical tests were carried out using API 20NE and (Braunschweig, Germany). The quinones were extracted API ZYM strips (bioMe´rieux) according to the manufac- according to a previously described method (Tindall, turer’s instructions, except for adjusting the NaCl concen- 1990a, b). Respiratory lipoquinones were separated into tration in all tests to 3.0 %. Z. profunda SM-A87T was tested their different classes (e.g. menaquinones, ubiquinones) by at the same time for comparison. These results are given in TLC on silica gel, then further analysed by HPLC. Polar the species description and Table 1. lipids were extracted from 100 mg freeze-dried cellular material using a chloroform/methanol/0.3 % aqueous NaCl Fatty acids in whole cells grown on marine agar 2216 mixture [1 : 2 : 0.8 (by vol.)] (Bligh & Dyer, 1959). Polar medium at 28 uC for 48 h were saponified, methylated and lipids were separated by two-dimensional silica gel TLC and then identified according to a previously described method (Tindall et al., 2007). The major respiratory Table 1. Physiological characteristics of strain 22II14-10F7T quinone of the strain 22II14-10F7T was determined to be and Zunongwangia profunda SM-A87T MK-6 (95 %); some minor unidentified components were

T T present. This trait is in accordance with the properties of Z. Strains: 1, 22II14-10F7 ;2,Z. profunda SM-A87 .+, Positive; W, profunda SM-A87T. The polar lipid profile of strain 22II14- weakly positive; 2, negative. The characteristics of the type strain Z. T profunda SM-A87T were analysed at the same time as those of strain 10F7 consisted of phosphatidylethanolamine (PE), two 22II14-10F7T. The other characteristics tested using API 20NE and aminolipids (AL1 and AL2) and five unknown lipids (L1– L5) (Fig. S1). It was similar to that of Z. profunda SM- API ZYM, and susceptibility to antibiotics were the same for both T strains. A87 , which only has one identified phospholipid (phos- phatidylethanolamine) (Qin et al., 2007). Characteristic 1 2 Antibiotic susceptibility tests were performed by disc Growth with: diffusion methods as described by Shieh et al. (2003). T T 0 % NaCl 2 + Strain 22II14-10F7 and Z. profunda SM-A87 were tested 15 % NaCl + 2 at the same time in this study. Both of them were sensitive Hydrolysis of: to carbenicillin (100 mg per disc; OXOID), chloromycetin Gelatin 2 + (30), ciprofloxacin (5), clindamycin (2), erythromycin Tween 80 2 + (15), lincomycin (2), minomycin (30), ofloxacin (5), Malic acid W + piperacillin (100) and rifampicin (5) and resistant to API ZYM ampicillin (10), cefalexin (30), cefazolin (30), cefobid (30), a-Mannosidase 2 + co-trimoxazole (25), gentamicin (10), kanamycin (30), Lipase (C14) W 2 metronidazole (5), oxacillin (1), penicillin G (10), poly- + Esterase (C4) W myxin B (30 IU), rocephin (30), streptomycin (10), Susceptibility to: vancomycin (30) and vibramycin (30). The different + Cephradin, tetracycline 2 susceptibility patterns to three antibiotics of the two Norfloxacin + 2 strains are shown in Table 1. DNA G+C content (mol%)* 35.5 36.2 The DNA G+C contents of the novel isolate 22II14-10F7T *Data from genome sequence information. was 35.5 mol% according to the draft genome sequence. It

Downloaded from www.microbiologyresearch.org by 18 International Journal of Systematic and Evolutionary Microbiology 64 IP: 137.108.70.7 On: Mon, 04 Jul 2016 10:01:16 Zunongwangia atlantica sp. nov. is close to that for Z. profunda SM-A87T (36.2 %) acid, potassium gluconate and trisodium citrate. API ZYM according to its genome data (Qin et al., 2010). test strip results are positive for alkaline phosphatase, acid phosphatase, cystine aminopeptidase, esterase (C4), Strain 22II14-10F7T was Gram-stain-negative, non-pig- esterase lipase (C8), leucine aminopeptidase, naphthol- mented, rod-shaped and non-motile (Fig S2). It could not AS-BI-phosphoamidase, trypsin, valine aminopeptidase, grow under anaerobic conditions. It was unable to degrade a-chymotrypsin, a-galactosidase, a-glucosidase, b-glucosi- oil. The alkane monoxygenase gene was not detected in the dase, b-galactosidase and N-acetyl-b-glucosaminidase; draft genome sequence. The differences in physiological, weakly positive for lipase (C14) and b-glucuronidase; biochemical and chemotaxonomic characteristics between negative for a-mannosidase and a-fucosidase. Table 1 strain 22II14-10F7T and Z. profunda SM-A87T are given in shows the characteristics used to distinguish strain 22II14- Table 1. The high 16S rRNA similarity between strain 22II14- 10F7T from Z. profunda SM-A87T. 10F7T and Z. profunda SM-A87T strongly indicates that strain 22II14-10F7T belongs to the genus Zunongwangia.But The type strain, 22II14-10F7T (5CGMCC1.12470T5LMG strain 22II14-10F7T can be differentiated from Z. profunda 27421T5MCCC 1A06481T) was isolated from the deep-sea SM-A87T on the basis of the data on physiological and water of the Atlantic Ocean. The G+C content of the DNA chemotaxonomic characteristics shown in Table 1. On the of the type strain is 35.5 mol%. basis of the data described above, strain 22II14-10F7T should be classified as representing a novel species of the genus Zunongwangia,forwhichanameZunongwangia atlantica sp. Acknowledgements nov. is proposed. This work was financially supported by China Ocean Mineral Resources Research and Development Association (COMRA) pro- Description of Zunongwangia atlantica sp. nov. gram (no. DY125-15-R-01), the Public Welfare Project of the State Oceanic Administration (SOA) (201005032) and the National Zunongwangia atlantica (at.lan.ti9ca. L. fem. adj. atlantica Infrastructure of Natural Resources for Science and Technology referring to the Atlantic Ocean, where the strain was isolated). Program of China (no. NIMR-2011-9; NIMR-2012-9). Cells are Gram-stain-negative rod-shaped, 0.7–0.8 mm wide and 1.7–1.8 mm long and non-motile. Positive for References oxidase, catalase, b-galactosidase, b-glucosidase (aesculin Auch, A. F., Klenk, H. P. & Go¨ ker, M. 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