International Journal of Systematic and Evolutionary Microbiology (2015), 65, 2908–2912 DOI 10.1099/ijs.0.000354

Mariniphaga sediminis sp. nov., isolated from coastal sediment Feng-Qing Wang,1 Qi-Yao Shen,1 Guan-Jun Chen1,2 and Zong-Jun Du1,2

Correspondence 1College of Marine Science, Shandong University at Weihai, Weihai 264209, PR China Zong-Jun Du 2State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, PR China [email protected]

A Gram-stain-negative and facultatively anaerobic bacterium, SY21T, was isolated from marine sediments of the coastal area in Weihai, China (1228 09 370 E378 319 330 N). Cells of strain SY21T were 0.3–0.5 mm wide and 1.5–2.5 mm long, catalase- and oxidase-positive. Colonies on 2216E agar were transparent, beige- to pale-brown-pigmented, and approximately 0.5 mm in diameter. Growth occurred optimally at 33–37 8C, pH 7.0–7.5 and in the presence of 2–3 % (w/v) NaCl. Phylogenetic analysis of the 16S rRNA gene indicated that strain SY21T was a member of the genus Mariniphaga within the family Prolixibacteraceae. The closest described neighbour in terms of 16S rRNA gene sequences identity was Mariniphaga anaerophila Fu11-5T (94.7 %). The major respiratory quinone of strain SY21T was MK-7, and the dominant

fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and anteiso-C15 : 0.The major polar lipids were phosphatidylethanolamine, aminolipid and an unidentified lipid, and the DNA G+C content was 37.9 mol%. The distinct phylogenetic position and phenotypic traits distinguished the novel isolate from M. anaerophila Fu11-5T. Phenotypic and genotypic analysis indicated that strain SY21T could be assigned to the genus Mariniphaga. The name Mariniphaga sediminis sp. nov. is proposed, with the type strain SY21T (5KCTC 42260T5MCCC 1H00107T).

As a large group, of the phylum , of a novel species of the phylum Bacteroidetes, represented which can utilize dissolved organic matter in the marine eco- by SY21T, based on phenotypic and chemotaxonomic system, especially by degrading various polymers, such as properties, and a detailed phylogenetic analysis of the 16S cellulose, chitin and pectin (Kirchman, 2002), play an rRNA gene sequences. important role in the ecosystem. In phytoplankton bloom, The marine sediment samples were collected from marine Bacteroidetes are dominant in absorbing algae-derived sediments from the same site of the coastal area in Weihai, organic matter and can promote carbon, nitrogen and China (1228 09 370 E378 319 330 N), using a sterile plastic sulfur cycling (Giovannoni & Rappe´, 2000). Owing to their pipette at a depth of ¢5 cm. The samples were pooled as a significant role in ecological cycles, there has been growing single sample and stored on ice under transportation to the interest in Bacteroidetes. At the time of writing, the order laboratory. The sample was then processed using the enrich- (Krieg et al., 2011) comprised the families ment culture technique, as described previously (Du et al., Bacteroidaceae, , Porphyromonadaceae, 2014), and incubated at 28 8C in a 500 ml sealed glass Prevotellaceae, Rikenellaceae, Prolixibacteraceae and bottle (completely filled with medium). After 2 months of Marinifilaceae. Recently, the genus Mariniphaga was classi- incubation, 1 ml of liquid medium from the sealed glass fiedinthefamilyProlixibacteraceaeasanovelgenustoaccom- bottle was diluted with 9 ml of sterilized saline solution modate Mariniphaga anaerophila Fu11-5T by Iino et al. and serially diluted up to 10-fold for inoculum preparation. (2014). The plates were incubated at 28 8C for 3–7 days after spread- ing the diluted sample (100 ml) on 2216E agar (Hopebio). In our study, a coastal sediment sample was collected in the A tiny, transparent, beige-pigmented colony was isolated course of an investigation of Bacteroidetes in the coastal and inoculated on 2216E agar to achieve purity. The strain, area of Weihai. We describe the isolation and identification designated SY21T, was routinely incubated on 2216E agar at 33 8C and stored at 280 8C in 15 % (v/v) glycerol with The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene 1 % (w/v) NaCl. M. anaerophila JCM 18693T, obtained sequence of Mariniphaga sediminis SY21T is KP172528. from the Japan Collection of Microorganisms, was used A supplementary figure is available with the online Supplementary as the reference strain for physiological tests and analysis Material. of morphological, phylogenetic and chemotaxonomic

Downloaded from www.microbiologyresearch.org by 2908 000354 G 2015 IUMS Printed in Great Britain IP: 165.246.197.214 On: Wed, 07 Oct 2015 13:09:06 Mariniphaga sediminis sp. nov. characteristics (except polar lipid analysis). Unless otherwise version 3.0; TaKaRa) was used to determine the DNA G+C stated, the samples of M. anaerophila JCM 18693T and SY21T content via HPLC as described by Tamaoka & Komagata were incubated at 33 8C on 2216E agar. (1984) and Mesbah et al. (1989). lDNA (Takara) was used as a standard. The 16S rRNA gene was amplified with two uni- Cell size, morphology and motility of strain SY21T were versal primers, 27f (59-AGAGTTTGATCMTGGCTCAG-39) established by light microscopy (Ci-L; Nikon) after cultiva- and 1492r (59-TACGGYTACCTTGTTACGAC-39). The tion for 72 h on 2216E agar. The gliding motility of strain PCR products were ligated into the pGM-T vector (Tiangen) SY21T cultured on 2216E agar was detected as described by and cloned according to the method of Liu et al. (2014). Bowman (2000). Gram reaction, reduction of nitrate, for- Sequencing reactions were carried out using an ABI BigDye mation of spores and hydrolysis of starch, agar, gelatin, 3.1 sequencing kit (Applied BioSystems) and an automated Tween 80 and CM-cellulose were detected according to the DNA sequencer (model ABI3730; Applied BioSystems). methods of Smibert & Krieg (1994) and Dong & Cai The 16S rRNA gene sequence of strain SY21T was compared (2001). The optimal growth temperature of strain SY21T with available sequences of species with validly published was determined after incubation on 2216E agar and shaking names from NCBI BLASTN. Multiple alignments with in 2216E liquid medium (Hopebio) at 4, 10, 15, 20, 25, 28, 30, sequences of strain SY21T and the most closely related taxa 33, 37, 40 and 45 8C, respectively. Tolerance to NaCl was were carried out using CLUSTAL_X (version 1.81) (Thompson tested on 2216E agar and in 2216E liquid medium with 0– et al., 1997). A phylogenetic tree was reconstructed by 10 % (w/v) NaCl (seawater in 2216E agar and 2216E the neighbour-joining method using MEGA (version 6.0) liquid medium was replaced with artificial seawater: 3.2 g 21 21 21 21 (Tamura et al., 2013). The maximum-likelihood algorithm MgSO4 l , 2.2 g MgCl2 l , 1.2 g CaCl2 l , 0.7 g KCl l , 2 (Felsenstein, 1981) was included to estimate and verify the 0.2 g NaHCO l 1). The novel isolate was shaken in 2216E 3 taxonomic positions of the novel isolate and reference strains liquid medium at pH 5.5 to 10.0. The pH was adjusted by on the tree. adding MES (pH 5.5 and 6.0), PIPES (pH 6.5 and 7.0), HEPES (pH 7.5 and 8.0), Tricine (pH 8.5) or CAPSO Cells of strain SY21T were harvested at the late-exponential (pH 9.0, 9.5 and 10.0) at a concentration of 20 mM. Oxidase growth phase in 2216E liquid medium at 33 8C for character- activity was tested using an oxidase reagent kit (bioMe´rieux) ization of isoprenoid quinones, cellular fatty acids and polar according to the manufacturer’s instructions. Catalase lipids. Isoprenoid quinones were extracted with chloroform/ activity was determined by pouring 3 % H2O2 solution on methanol (2 : 1, v/v) from lyophilized cells (300 mg) and bacterial colonies and observing bubble production. Anaero- purified using HPLC (Minnikin et al., 1984). Analysis of bic growth of strain SY21T was determined after incubation fatty acid composition was carried out using the microbial on 2216E agar at 33 8C for 2 weeks with or without 0.1 % identification system (MIDI) according to the method of (w/v) NaNO3 in an anaerobic jar. To determine the utiliz- Sasser (1990). Polar lipids were extracted from 100 mg ation of cysteine as an alternative electron donor with freeze-dried cell material using a chloroform/methanol/ oxygen, strain SY21T and M. anaerophila JCM 18693T were 0.3 % (w/v) aqueous NaCl mixture (1 : 2 : 0.8, by vol.), shaken, respectively, in test tubes (156150 mm) using the modified after Bligh & Dyer (1959), recovered into the method of Iino et al. (2014). Fermentative growth on chloroform phase by adjusting the chloroform/methanol/ sugars was tested using GXSm medium (Iino et al., 2014) 0.3 % (w/v) aqueous NaCl mixture to a ratio of 1 : 1 : 0.9 with addition of each sugar in place of D-glucose. (by vol.) and separated by two-dimensional silica gel TLC M. anaerophila JCM 18693T was used as a positive control. (no. 818 135; Macherey-Nagel). The first direction was Bacterial growth was followed by determining the increase developed in chloroform/methanol/water (65 : 25 : 4, by in turbidity at 660 nm using a spectrophotometer. Acid vol.), and the second in chloroform/methanol/acetic acid/ production was determined using the API 50CHB system water (80 : 12 : 15 : 4, by vol.). Total lipid material was (bioMe´rieux). Biolog GEN III MicroPlates were used to detected using molybdatophosphoric acid and specific func- determine the utilization of organic substrates as sole tional groups were detected using spray reagents specific for carbon and energy sources. Various biochemical and defined functional groups (Tindall et al., 2007). Polar lipid additional enzyme activities were assayed using API 20E analysis was performed by the Identification Service of the and API ZYM kits (bioMe´rieux). Tests using Biolog GEN DSMZ, Braunschweig, Germany. III MicroPlates and API kits were conducted according to Colonies on 2216E agar were transparent, beige- to pale- the manufacturers’ instructions, except that salinity was brown-pigmented and around 0.5 mm in diameter after adjusted to 3 % (w/v) NaCl. Since strain SY21T showed 72 h of incubation at 33 8C. Cells of strain SY21T were poor growth on Iso-Sensitest agar (Oxoid) and Mueller– approximately 0.3–0.5 mm wide and 1.5–2.5 mmlong, Hinton agar (Hopebio), susceptibility to antibiotics was Gram-stain-negative, short rods, non-gliding and non- assessed on 2216E agar using the Kirby–Bauer disc diffusion motile. No spores were evident. Growth was observed at method, according to Du et al. (2014), followed by the proto- 15–40 8C and in the presence of 1–7 % (w/v) NaCl. The pH cols of the Clinical and Laboratory Standards Institute (CLSI, range for cell growth was pH 6.0–9.5. Optimal growth was 2012). detected at 33–37 8C, pH 7.0–7.5 and 2–3 % (w/v) NaCl. Genomic DNA obtained with a commercial gene extraction Starch, agar, gelatin, Tween 80 and CM-cellulose were not kit (MiniBEST Bacteria Genomic DNA Extraction kit hydrolysed. Strain SY21T was facultatively anaerobic, negative

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Table 1. Differential characteristics of strain SY21T and Mari- The almost full-length 16S rRNA gene sequence (1443 bp) niphaga anaerophila JCM 18693T of strain SY21T was obtained. The 16S rRNA gene sequence T T T similarity was 94.7 % to M. anaerophila Fu11-5 , which is Strains: 1, SY21 ;2,M. anaerophila JCM 18693 . Data from this study lower than the borderline value used for definition of bac- unless indicated otherwise. Both strains were Gram-stain-negative, terial species (i.e. 97 %) as proposed by Stackebrandt & positive for indole production and negative for nitrate reduction, Goebel (1994). As shown in Fig. 1, strain SY21T clustered + 2 motility, spore formation and hydrolysis of agar. , Positive; , with M. anaerophila Fu11-5T and formed a branch separate negative. from other type strains. A phylogenetic tree reconstructed Characteristic 1 2 with the maximum-likelihood method (data not shown) had similar topological structure. Colony colour Beige- to pale- Beige The major respiratory quinone of strain SY21T was MK-7, brown compatible with M. anaerophila Fu11-5T and most other Cell size (mm) 0.3–0.561.5–2.5 0.5–0.661.9–6.9 members of the family Prolixibacteraceae. The following Growth range: . Temperature (8C) 15–40 20–42 compositions of cellular fatty acids ( 5 %) were obtained: NaCl (%, w/v) 1–7 0–9 iso-C15 : 0 (20.6 %), iso-C17 : 0 3-OH (13.5 %), anteiso- pH 6.0–9.5 6.0–8.5 C15 : 0 (11.3 %), iso-C16 : 0 (9.2 %), C15 : 0 (6.5 %) and iso- Catalase + 2 C16 : 0 3-OH (5.9 %). The major fatty acids were consistent T Oxidase + 2 with those of M. anaerophila Fu11-5 , except for some differ- Hydrolysis of starch 2 + ences in the proportions and types of fatty acids. The DNA T Acid production from 2 + G+C content of strain SY21 was 37.9 mol%, which could T utilization of D-glucose be distinguished from that of M. anaerophila Fu11-5 Enzymic activities (41.7 mol%) (Iino et al., 2014). The major polar lipids of b-Fucosidase + 2 strain SY21T were phosphatidylethanolamine, aminolipid N-Acetylglucosaminidase + 2 and an unidentified lipid. Minor amounts of other aminoli- Esterase (C4) 2 + pids, unknown polar lipids, phospholipid and glycolipid Esterase lipase (C8) 2 + were additionally found (Fig. S1, available in the online a-Glucosidase 2 + Supplementary Material). Both of the strains, SY21T DNA G+C content (mol%) 37.9 41.7* and Fu11-5T, contained phosphatidylethanolamine as the major polar lipid. However, strain SY21T contained several *Data taken from Iino et al. (2014). kinds of lipid that were not present in strain Fu11-5T. The polar lipid pattern of M. anaerophila Fu11-5T comprised only phosphatidylethanolamine. As a result, there were differences between strain SY21T and Fu11-5T in major polar lipid components. for reduction of nitrate, and positive for catalase and oxidase. Further data on the morphological, physiological and bio- Chemotaxonomic examination, together with phylogenetic chemical characteristics of strain SY21T are presented in the analysis, effectively revealed that strain SY21T is affiliated species description and Table 1. with the genus Mariniphaga. Despite common features,

T 99 Mariniphaga sediminis SY21 (KP172528) 0.02 99 Mariniphaga anaerophila DSM 26910T (AB921558) 100 Tangfeifania diversioriginum G22T (JQ683777) 84 Draconibacterium orientale FH5T (JQ683778)

Prolixibacter bellariivorans F2T (AY918928)

Sunxiuqinia elliptica DQHS-4T (GQ200190)

T 99 Meniscus glaucopis ATCC 29398 (GU269545) 83 Mangrovibacterium diazotrophicum SCSIO N0430T (JX983191)

Parabacteroides distasonis JCM 5825T (AB238922)

Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strain SY21T and some other phylogenetically related taxa. Bootstrap values (expressed as percentages of 1000 replications) of .50 % are shown at branching nodes. Bar, 0.02 substitutions per nucleotide position.

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T strain SY21 displayed substantial differences from anteiso-C15 : 0,andthemajorpolarlipidsarephosphatidyletha- M. anaerophila Fu11-5T. For example, strain SY21T could nolamine, aminolipid and an unidentified lipid. not grow without NaCl and was positive for catalase and oxi- The type strain, SY21T (5KCTC 42260T5MCCC 1H00107T), dase, unlike M. anaerophila Fu11-5T. Further differences was isolated from a sediment sample collected from the were observed in terms of enzyme activities (Table 1). coastal area of Weihai, China. The Genomic DNA G+C con- Strain SY21T could also be distinguished based on antibiotic T tent of the type strain is 37.9 mol%. sensitivity. Moreover, strain SY21 did not utilize D-glucose to produce acid. In view of the collective phenotypic, phys- iological and chemotaxonomic characteristics and phyloge- netic position, strain SY21T appears to represent a novel Acknowledgements species of the genus Mariniphaga, for which the name This work was supported by the National Natural Science Foundation Mariniphaga sediminis sp. nov. is proposed. of China (31370057, 31290231), the National Science and Technology Major Project of China (2013ZX10004217), and the China Ocean Mineral Resources R&D Association Special Foundation (DY125- Description of Mariniphaga sediminis sp. nov 15-T-05). Mariniphaga sediminis (se.di.mi9nis. L. n. sedimen -inis sedi- ment; L. gen. n. sediminis of sediment). References Cells are 0.3–0.5 mm in width and 1.5–2.5 mm in length. Colo- nies on 2216E agar are transparent, beige- to pale-brown-pig- Bligh, E. G. & Dyer, W. J. (1959). A rapid method of total lipid 37 mented, and approximately 0.5 mm in diameter after 72 h of extraction and purification. 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