Antonie van Leeuwenhoek (2014) 106:615–621 DOI 10.1007/s10482-014-0232-2

ORIGINAL PAPER

Motiliproteus sediminis gen. nov., sp. nov., isolated from coastal sediment

Zong-Jie Wang • Zhi-Hong Xie • Chao Wang • Zong-Jun Du • Guan-Jun Chen

Received: 3 April 2014 / Accepted: 4 July 2014 / Published online: 20 July 2014 Ó Springer International Publishing Switzerland 2014

Abstract A novel Gram-stain-negative, rod-to- demonstrated that the novel isolate was 93.3 % similar spiral-shaped, oxidase- and catalase- positive and to the type strain of Neptunomonas antarctica, 93.2 % facultatively aerobic bacterium, designated HS6T, was to Neptunomonas japonicum and 93.1 % to Marino- isolated from marine sediment of Yellow Sea, China. bacterium rhizophilum, the closest cultivated rela- It can reduce nitrate to nitrite and grow well in marine tives. The polar lipid profile of the novel strain broth 2216 (MB, Hope Biol-Technology Co., Ltd) consisted of phosphatidylethanolamine, phosphatidyl- with an optimal temperature for growth of 30–33 °C glycerol and some other unknown lipids. Major

(range 12–45 °C) and in the presence of 2–3 % (w/v) cellular fatty acids were summed feature 3 (C16:1 NaCl (range 0.5–7 %, w/v). The pH range for growth x7c/iso-C15:0 2-OH), C18:1 x7c and C16:0 and the main was pH 6.2–9.0, with an optimum at 6.5–7.0. Phylo- respiratory quinone was Q-8. The DNA G?C content genetic analysis based on 16S rRNA gene sequences of strain HS6T was 61.2 mol %. Based on the phylogenetic, physiological and biochemical charac- teristics, strain HS6T represents a novel genus and The GenBank accession number for the 16S rRNA gene T species and the name Motiliproteus sediminis gen. sequence of Motiliproteus sediminis HS6 is KF953945. nov., sp. nov., is proposed. The type strain is HS6T T T Electronic supplementary material The online version of (=ATCC BAA-2613 =CICC 10858 ). this article (doi:10.1007/s10482-014-0232-2) contains supple- mentary material, which is available to authorized users. Keywords Motiliproteus sediminis gen. nov., sp. nov. Polyphasic taxonomy 16S rRNA gene Marine Z.-J. Wang C. Wang Z.-J. Du (&) G.-J. Chen College of Marine Science, Shandong University at bacteria Coastal sediment Weihai, Weihai 264209, China e-mail: [email protected] G.-J. Chen e-mail: [email protected] Introduction

Z.-H. Xie Key Laboratory of Coastal Biology and Utilization, The family Oceanospirillaceae within the phylum Yantai Institute of Coastal Zone Research, Chinese Proteobacteria was described by Garrity et al. (2005) Academy of Sciences, Yantai 264003, China in Bergey’s Manual of Systematic Bacteriology.It contains 18 genera (LPSN, http://www.bacterio.net/- Z.-J. Du G.-J. Chen State key Laboratory of Microbial Technology, Shandong classifphyla.html) at the time of writing and most of University, Jinan 250100, China them are strictly respiratory (except for Neptunomonas 123 616 Antonie van Leeuwenhoek (2014) 106:615–621 which gives weak fermentation reactions) aquatic (Murray et al. 1994). The effects of different NaCl organisms of primarily marine origin. The type genus concentrations were assessed by modified MA (sea- of the family is Oceanospirillum (Hylemon et al. water replaced with distilled water) containing differ- 1973) with O. linum as the type species. The family ent concentrations of NaCl (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, Oceanospirillaceae has a close relationship with the 9, and 10 %, w/v). Growth at different temperature genus Marinobacterium (Gonza´lez et al. 1997), a was tested on MA at 4, 8, 12, 17, 24, 28, 30, 33, 37, 42 member of family Alteromonadaceae. In the present and 45 °C. To test the effect of pH on growth, MB was study, the novel isolate was characterized by pheno- adjusted to different pH with MES (pH, 5.5, 6.0, 6.2), typic and phylogenetic analyses and is proposed to be PIPES (pH, 6.5, 7.0), HEPES (pH, 7.5, 8.0), Tricine a member of novel species in a new genus of family (pH, 8.5) and CAPSO (pH, 9.0, 9.5) buffers and each at Oceanospirillaceae. The novel genus forms a cluster a concentration of 20 mM and then measured the with Marinobacterium which supports the view of value of OD600. Anaerobic growth was determined at Kim et al. (2007) that the genus Marinobacterium 33 °C in an anaerobic chamber on MA with or without should be reclassified into the order Oceanospirillales. 0.1 % NaNO3 for 5–7 days. Catalase activity was detected by bubble production in 3 % (v/v) H2O2 solution and oxidase activity was evaluated by using Materials and methods commercial oxidase reagent (bioMe´rieux). Hydrolysis of starch, lipid and algin was tested on MA supple- Organism, maintenance and cultural conditions mented with 0.2 % (w/v) soluble starch, 1 % Tween 80 (v/v) and 2 % sodium alginate respectively (Cowan During the study of diversity of the multi-drug and Steel 1965). Tests for other physiological or resistant bacteria in coastal marine environments, a biochemical characteristics were performed using API novel heterotrophic, facultatively aerobic, olive to 20E, API 20NE, API ZYM kits, API 50CH and Biolog brown, motile, Gram-negative bacterial strain HS6T according to the manufacturer’s instructions except was isolated on marine agar 2216 (MA, Hope Biol- that the salinity was adjusted to 3 %. All the API and Technology Co., Ltd) at 28 °C. The sample was Biolog tests were performed at least twice. Suscepti- collected from the coastal of Yellow Sea (121°705700E, bility to antibiotics was tested using filter-paper discs 37°3705300N) around Yantai, China. For isolation of containing various antibiotics on cultures incubated at bacterial strains, 1 g wet sediment was blended to 33 °C on MA (since strain HS6T showed poor growth 99 mL sterilized seawater with glass beads and shaken on Mueller–Hinton agar) for 2–3 days. vigorously. The suspension was serially diluted to 10-6 with sterilized seawater and 0.1 mL aliquots of Determination of G?C content of DNA, 16S each dilution were spread onto MA. The plates were rRNA gene sequencing and phylogenetic analysis incubated at 28 °C for 5–7 days. Strain HS6T was isolated and then stored at -80 °C in sterile 1 % (w/v) Genomic DNA of strain HS6T was obtained from 60 h saline supplemented with 15 % (v/v) glycerol. The old cultures on MA using a genomic DNA extraction strain was routinely cultivated at 33 °C on MA or MB. kit (Sangon, China) and the DNA G?C content was determined by HPLC (Mesbah et al. 1989). The 16S Morphological, physiological and biochemical rRNA gene of strain HS6T was amplified from the analysis genomic DNA by PCR with the universal primers 27F and 1492R (Lane 1991). PCR products were purified The morphological and physiological features of using a PCR product purification kit (Tiangen Biotech HS6T were examined after incubation at 33 °C for Co., Ltd, Beijing, China) and then ligated to the vector 2–3 days on MA. The transmission electron micro- of pGM-T (Tiangen Biotech Co., Ltd, Beijing, China). scope was used to observe the cell size, morphology Sequencing was performed by Shanghai Sunny Bio- and flagella. Meanwhile, the light microscopy (E600; technology Co., Ltd, China. To identify the taxonomic Nikon) was used as supplement. Motility was assessed status of HS6T, a near complete sequence (1,466 bp) with the method of hanging-drop. Gram reaction was was obtained and submitted to GenBank/EMBL/ examined following the standard Gram procedure DDBJ datebase to search for similar sequences using 123 Antonie van Leeuwenhoek (2014) 106:615–621 617

BLAST algorithm. EzTaxon server (http://eztaxone. to the closest phylogenetic neighbors as shown in ezbiocloud.net/; Kim et al. 2012) was used to achieve Table 1. It characteristically showed bipolar flagella the similarity values of sequences. Phylogenetic ana- which is different from most of the members of family lysis was performed using MEGA version 5.2 (Tamura Oceanospirillaceae and there were 1–2 flagella et al. 2011) after multiple alignments of data by attached to the polar or subpolar of the HS6T CLUSTAL W (Larkin et al. 2007) and manual edition (Fig. 1). The characteristics of morphology of HS6T (to remove gaps at 30 and 50 ends and ambiguous are significant different from the relative genera bases) using BioEdit version 7.0 (Hall 1999). The shown in Table 1 and this can distinguish it from phylogenetic trees were constructed using the neigh- other genera partially. bor-joining (NJ), maximum-likelihood (ML) and Growth was found to occur in 0.5–7 % (w/v) NaCl maximum-parsimony (MP) algorithms. Bootstrap (optimum 2–3 %), at 15–45 °C (optimum 30–33 °C) values were calculated based on 1,000 replicates. and at pH 6.2–9.0 (optimum pH 6.5–7.0). There was no visible colonies formed under anaerobic conditions

Chemotaxonomic analysis on MA supplemented with 0.1 % (w/v) NaNO3. But it can reduce nitrate to nitrite with or without oxygen The cells cultured on MA at 33 °C for 3 days (end of which can distinguish HS6T from most of the members the logarithmic phase) were used to determine fatty in the family Oceanospirillaceae. The strain was acids. The method was described by Sasser (1990) positive for oxidase and catalase activities and nega- previously. And then the extract was separated using tive for hydrolysis of starch, Tween 80 and algin. The Sherlock Microbial Identification System (MIS) positive of gelatinase also can distinguish the new (MIDI, Microbial ID, Newark, DE 19711 U.S.A.). isolate from the genera Marinobacterium, Neptuno- Peaks were automatically integrated and fatty acid monas, Neptuniibacter and Nitrincola. Other cultural, names and percentages calculated by the MIS Stan- physiological and biochemical characteristics of the dard Software (Microbial ID). Respiratory lipoqui- novel strain will be given in the species description. nones were extracted from freeze dried cells and the And the different characteristics that can distinguish methods were described by Tindall (1990a, b). They HS6T from its closest phylogenetic relatives were were separated into different classes by thin layer displayed in Table 1. chromatography on silica gel and removed from the plate and further analyzed by HPLC. Polar lipids were Molecular phylogenetic analysis separated by two dimensional silica gel thin layer chromatography. Total lipid material was detected The nearly full-length (1,466 bp) 16S rRNA gene using molybdatophosphoric acid and specific func- sequence of strain HS6T was obtained and the tional groups detected using spray reagents specific for comparison revealed that strain HS6T had highest defined functional groups, Full details are given in similarity to the type strain of Neptunomonas antarc- Tindall (2007). All procedures of analysis were carried tica (93.3 %), followed by Neptunomonas japonica out by the Identification Service, DSMZ, Braun- (93.2 %) (Miyazaki et al. 2008) and Marinobacterium schweig, Germany. rhizophilum (93.1 %) (Kim et al. 2008). Addition to these, the similarities with other relative neigbour- hoods were all below 93 %. That suggested the strain Results HS6T may represent a novel genus in family Ocean- ospirillaceae. The further studies of phylogenetic trees Phenotypic characteristics (Fig. 2) proved that. In the NJ tree, the strain HS6T formed a separate Strain HS6T can form circular, viscid and olive to branch with the genera Marinobacterium, Neptuno- brown (the color would change over time) colonies monas, Neptuniibacter and Nitrincola. Although the whose diameter were about 1.5 mm after 60 h incu- genus Marinobacterium belongs to the family Alter- bation on MA at 33 °C. The cells of the strain were omonadaceae according to NCBI (http://www.ncbi. found to be Gram-negative, rod-to-spiral-shaped, nlm.nih.gov/) and LPSN, we still insisted the strain motile and 0.5–0.8 9 1.5–6.0 lm in size, in contrast HS6T should be classified to the family 123 618 Antonie van Leeuwenhoek (2014) 106:615–621

Table 1 Phenotypic comparison of HS6T and related genera Characteristic HS6T Neptunomonas Marinobacterium Neptuniibacter Nitrincola

Cell shape rods to spiral rods rods rods to bent rods rods Size (lm) 0.5–0.8 9 1.5–6.0 0.6–1.0 9 1.2–3.0 0.3–0.8 9 0.6–3.0 0.2–0.8 9 1.5–2.3 0.5 9 1.3–1.6 Flagella Bipolar Single Polar Single Polar Single Polarb Single Polar Growth at 4 °C– ?a -a –– 40 °C ?-a ?a –ND a O2 metabolism F F An An F Hydrolysis of Starch – -a -a –– Tween 80 – -a ?a –ND Gelatin ?-a -a –– Nitrate Reduction ??a -a –– G?C Content (%) 61.2 43.6–48.2 54.9–62.5 46.6–54.2 47.4 Quinone(s) Q8/Q7 Q8/Q9b Q8b Q8 ND HS6T (this study); Neptunomonas (Hedlund et al. 1999; Miyazaki et al. 2008; Zhang et al. 2010; Lee et al. 2012; Liu et al. 2013); Marinobacterium (Baumann et al. 1983; Gonza´lez et al. 1997; Chang et al. 2007; Kim et al. 2007; Kim et al. 2008; Huo et al. 2009; Kim et al. 2009a; Kim et al. 2009b; Kim et al. 2010; Chimetto et al. 2011); Neptuniibacter (Arahal et al. 2007; Chen et al. 2012); Nitrincola (Dimitriu et al. 2005) ? positive, - negative, F facultatively aerobic, An strictly anaerobic a [50 % negative/positive b The information is incomplete because some papers missed the relevant information

Oceanospirillaceae. Besides the higher similarity of 16S rRNA gene sequence and phenotypic character- istics, the EzTaxon also classified the genus Marino- bacterium to the family of Oceanospirillaceae. And we also added the 16S rRNA gene sequences of families Alterononadaceae and Shewanellaceae to our phylogenetic tree, they formed a separate cluster which is far from the clusters including the Marino- bacterium. The result supported the view of Kim et al. (2007) that the genus Marinobacterium should be reclassified into the order Oceanospirillales. The ML tree and the MP tree were similar and all of them supported our conclusion—the strain HS6T may rep- resent a novel genus of the family Oceanospirillaceae. The DNA G?C content of the novel isolate was 61.2 mol % which is similar to Marinobacterium but much higher than other relatives (Table 1).

Chemotaxonomic characteristics

The predominant respiratory quinone detected in T Fig. 1 Transmission electron micrograph of HS6T. Cells grown strain HS6 was Q-8 (85 %) which is compatible on MA at 33 °C for 12 h. Bar = 1.0 lm with the most members of family Oceanospirillaceae 123 Antonie van Leeuwenhoek (2014) 106:615–621 619

T 73 Marinobacterium coralli CAIM 1449 (GU183820) 79 Marinobacterium sediminicola JCM 15524T (EU573966) 97 Marinobacterium maritimum KCTC 22254T (EU399548) 96 Marinobacterium stanieri ATCC 27130T (AB021367) Marinobacterium georgiense ATCC 700074T (U58339) 78 100 Marinobacterium halophilum DSM 17586T (AY563030) Marinobacterium jannaschii ATCC 27135T (AB006765) Marinobacterium nitratireducens JCM 15523T (EU573965) Marinobacterium rhizophilum DSM 18822T (EF192391) 81 Nitrincola lacisaponensis ATCC BAA-920T (AY567473) Marinobacterium marisflavi KCTC 12757T (EF468717) T 52 Marinobacterium litorale KCTC 12756 (DQ917760) 99 Marinobacterium lutimaris DSM 22012T (FJ230839) Neptuniibacter caesariensis CCUG 52065T (AY136116) 98 Neptuniibacter halophilus BCRC 80079T (GQ131677) Neptunomonas naphthovorans ATCC 700637T (AF053734) Neptunomonas concharum JCM 17730T (JF748732) 76 86 Neptunomonas antarctica KACC 14056T (FJ713802) 67 Neptunomonas japonica DSM 18939T (AB288092) 70 97 Neptunomonas qingdaonensis KCTC 23686T (JF747202) Motiliproteus sediminis HS6T (KF953945) 63 Oceanospirillum linum ATCC 11336T (M22365) Oleispira antarctica DSM 14852T (AJ426420) 100 T 95 Bermanella marisrubri CCUG 52064 (AY136131) 97 Oceaniserpentilla haliotis DSM 19503T (AM747817) Thalassolituus oleivorans DSM 14913T (AJ431699) T 99 Oceanobacter kriegii ATCC 27133 (AB006767) 99 Oleibacter marinus NBRC 105760T (FJ167390) Shewanella putrefaciens ATCC 8071T (X81623) Alishewanella fetalis ATCC BAA-284T (AF144407) Catenovulum agarivorans DSM 23111T (GQ262000) 53 T 100 Agarivorans albus NBRC 102603 (AB076561) 65 Aliagarivorans marinus BCRC 17888T (FJ167390) Bowmanella denitrificans BCRC 17491T (DQ343294) T 74 Aestuariibacter salexigens DSM 15300 (AY207502) 99 Glaciecola punicea ATCC 700756T (U85853) 55 Alteromonas macleodii DSM 6062T (Y18228) 88 Salinimonas chungwhensis DSM 16280T (AY553295)

0.01

Fig. 2 Neighbour-joining phylogenetic tree based on 16S used to root the tree. Bootstrap values ([50 %) based on 1,000 rRNA gene sequences of HS6T and its closely relatives. Type replications were shown at branching points. Bar, 0.01 strains of families Alteromonadaceae and Shewanellaceae were substitutions per nucleotide position

T (Garrity et al. 2005; Zhang et al. 2010; Miyazaki et al. in strain HS6 were summed feature 3 (C16:1 x7c/iso- 2008 and Arahal et al. 2007) and the genus Marino- C15:0 2-OH,34.7 %), C18:1 x7c (24.1 %), C16:0 bacterium (Kim et al. 2008). A minor amount of Q-7 (21.3 %), C10:0 3-OH (5.0 %), C18:1 x6c (4.1 %), (11 %) was also detected, which seems to be less C14:0 3-OH/iso-C16:1 (3.8 %), C12:0 (2.4 %) and C14:0 abundant or absent in close relatives. The remaining (1.0 %). The components of fatty acids of HS6T were 4 % was difficult to identify. The existence of Q-7 can similar to its close relatives, however relatives distinguish HS6T from the genera shown in Table 1. proportions of individual and components differ The major fatty acids (relative amount [ 1 %) found considerably. The polar lipids found in strain HS6T 123 620 Antonie van Leeuwenhoek (2014) 106:615–621 were very complex. Phosphatidylethanolamine (PE), esterase (C4), esterase lipase (C8), leucine arylami- phosphatidylglycerol (PG) and an unknown phospho- dase, valine arylamidase and naphthol-AS-BI-phos- lipid (PL2) were major polar lipids. Two unknown phohydrolase but negative for lipase (C14), cystine phospholipids (PL1, PL4) and an unknown amino- arylamidase, trypsin, a-chymotrypsin, a-and b-galac- phospholipid (PN) were moderate and some other tosidase, a-and b-glucosidase, b-glucuronidase, unknown lipids (AP, L1-3, PL3, PL5) were minor N-acetyl-b-glucosaminidase, a-mannosidase and a- components. The detailed information are displayed in fucosidase. Arginine dihydrolase, citrate utilization supplementary (Fig. S1). and gelatin hydrolase are positive according to the API 20E tests. Lysine decarboxylase, ornithine decarbox- Polyphasic taxonomic conclusion ylase, urease and tryptophan deaminase are negative.

Indole, acetoin (Voges–Proskauer reaction) and H2S The results of the phenotypic analysis, molecular are not produced. Acid can be produced from D-ribose, phylogenetic analysis and chemotaxonomic analysis L-xylose, L-sorbose, esculin, D-lyxose, D-tagatose, all support the view that strain HS6T represents a novel potassium gluconate, potassium 2-ketogluconate and genus and species of the family Oceanospirillaceae,for potassium 5-ketogluconate and can be weakly pro- which the name Motiliproteus sediminis is proposed. duced from D/L-arabinose and D-lactose (bovine origin) according to API 50CH test results. Myo-inositol, Description of Motiliproteus gen. nov gelatin, L-arginine, L-aspartic acid, L-histidine, glucu- ronamide, a-keto-glutaric acid are utilized and Motiliproteus (Mo.ti.li.pro’teus. L. adj. motilis, sucrose, D-glucose-6-phosphate, D-fructose-6-phos- motile; L. masc. n. Proteus, a mythical figure able phate, D-glucuronic acid, D/L-malic acid, b-hydroxy- to assume different forms; N.L. masc. n. D,L-butyric acid, a-keto-butyric acid are weakly uti- Motiliproteus, a motile bacteria of various forms) lized as sole sources of carbon and energy according to Biolog GNIII MicroPlates assays. Cells are susceptible Cells are Gram-negative, rod-to-spiral-shaped, facul- to streptomycin, ampicillin, penicillin G and chloram- tatively aerobic and motile with bipolar flagella. Both phenicol, moderately susceptible to vancomycin, gen- catalase and oxidase are positive. No growth occurs in tamycin and resistant to clindamycin. The major the absence of salt. Nitrate can be reduced to nitrite cellular fatty acids are summed feature 3 (C16:1 x7c/ with or without oxygen. The major cellular fatty acids iso-C15:0 2-OH), C18:1 x7c and C16:0. Major respiratory are C16:1 x7c/iso-C15:0 2-OH, C18:1 x7c and C16:0 and quinone is Q-8. The major polar lipids present include the main ubiquinone system is Q-8. phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and an unknown phospholipid (PL2). The DNA Description of Motiliproteus sediminis sp. nov G?C content of the type strain is 61.2 mol %. The type strain, HS6T (=ATCC BAA-2613T=CICC Motiliproteus sediminis (se.di.mi’nis. L. n. 10858T) was isolated from marine sediment of Yellow sedimeninis, sediment; L. gen. n. sediminis, Sea, China. of sediment) Acknowledgments This work was supported by the National Besides the characteristics described above, the fol- Natural Science Foundation of China (31370057, 31290231, 31370108) and the China Ocean Mineral Resources R & D lowing characteristics are also observed for the type Association (COMRA) Special Foundation (DY125-15-T-05). Z. J. species. Cells are 0.5–0.8 lm in diameters and Wang acknowledges grant of Graduate Innovation Foundation of 1.5–6.0 lm in length. Forms circular, viscid and olive Shandong University at WeiHai, GIFSDUWH yjs12037. to brown (the color would change over time) colonies on MA after 60 h incubation at 33 °C. Growth occurs References in 0.5–7 % (w/v) NaCl (optimum 2–3 %), at 15–45 °C (optimum 30–33 °C) and at pH 6.2–9.0 (optimum Arahal DR, Lekunberri I, Gonza´lez JM, Pascual J, Pujalte MJ, Pedro´s-Alio´ C, Pinhassi J (2007) Neptuniibacter caesari- 6.5–7.0). Does not hydrolyse starch, Tween-80, algi- ensis gen. nov., sp. nov., a novel marine genome- nate and cellulose. According to the API ZYM kits, sequenced gammaproteobacterium. Int J Syst Evol cells are positive for acid and alkaline phosphatase, Microbiol 57:1000–1006 123 Antonie van Leeuwenhoek (2014) 106:615–621 621

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